G J Wellenberg
email: g.wellenberg@gddeventer.com

F T Bouwkamp

P J V D Wolf

W A J M Swart

M J Mombarg

A L W De Gee

F T Bouwkamp¹

P J V D Wolf¹

W A J M Swart¹

A L W De

A study on the severity and relevance of porcine circovirus type 2 infections in Dutch fattening pigs with respiratory diseases

Keywords: Porcine circovirus type 2, respiratory disease, lungs, swine

A study on the severity and relevance of porcine circovirus type 2 infections in Dutch fattening pigs with respiratory diseases

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

INTRODUCTION

Porcine circovirus type-2 associated diseases (PCVAD) are an important concern to swine producers around the world. In addition to post-weaning multisystemic wasting syndrome (PMWS), porcine circovirus type 2 (PCV2) has also been associated with porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC) and proliferative and necrotizing pneumonia (PNP) [START_REF] References Allan | Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland[END_REF][START_REF] Opriessnig | Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2[END_REF]Wellenberg et al., 2004;[START_REF] Opriessnig | Comparison of Cytokine Profiles in pigs singularly or co-infected with PCV2, or Mycoplasma hyopneumoniae comparison of cytokine profiles in pigs singularly or co-infected with PCV2, or Mycoplasma hyopneumoniae[END_REF][START_REF] Grau-Roma | Detection of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, swine influenza virus and Aujeszky's disease virus in cases of porcine proliferative and necrotizing pneumonia (PNP) in Spain[END_REF][START_REF] Opriessnig | Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis and intervention strategies[END_REF]. In the US, PCV2 associated pneumonia was the most common presentation of PCV2 associated diseases [START_REF] Opriessnig | Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis and intervention strategies[END_REF].

PCV2 has often been associated with lung lesions in pigs co-infected with PRRSV, swine influenza viruses (SIV) or Mycoplasma hyopneumoniae. These agents appeared to create a synergistic environment that resulted in increased amounts of PCV2 and unusual severe respiratory disease outbreaks. However, there have also been reports on uncomplicated PCV2 pneumonia cases in western Canada [START_REF] Opriessnig | Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis and intervention strategies[END_REF]. These lesions included necrotizing and ulcerative bronchiolitis, granulomatous inflammation in the alveolar septa and mixed inflammation and fibroplasias in the lamina propria and peribronchiolar areas.

More insight is warranted on the role of PCV2 as etiological agent in porcine respiratory diseases such as PRDC and PNP in endemic situations. In addition, little or no information is available about whether PCV2 can be associated with pneumonia in pigs from Dutch swine herds, as described for US and western Canada.

The purpose of this study was to get more insight in the severity and relevance of PCV2 infections in Dutch pigs with symptoms of respiratory distress in an endemic situation (situation with no or low prevalence of PMWS: [START_REF] Wellenberg | Spread of post-weaning multisystemic wasting disease (PMWS) in Europe[END_REF]. In studies on the association between PCV2 infections and respiratory disease, the role of other concurrent co-infections can not be excluded and therefore, this study was not only focused on the detection of PCV2 infections, but also on the presence of other infectious agents associated to respiratory diseases in swine.

The results of this study may be used to gather more evidence on the hypothesis that PCV2 plays a role as etiological agent in respiratory diseases such as PRDC and PNP.

A c c e p t e d M a n u s c r i p t 4

MATERIALS EN METHODS

This study was divided in two parts: A and B. In part A, blood samples were examined for the presence of antibodies directed against pathogens associated with porcine lung lesions. In part B, pigs from 8 herds, with high percentages of lung lesions (pneumonia) at slaughter ("high"), and pigs from 8 herds, having low percentages of pneumonia at slaughter, were examined. These pigs, from both groups of herds, were selected on showings signs of respiratory distress, and further examined post mortem.

Part A

Herds

In total, 29 commercial fattening farms, with no signs of PMWS and with varying percentages of pneumonia and pleurisy at slaughter, were involved in this part of the study. The farms were recruited all over The Netherlands, and not from certain districts. In the selection of herds we did not take into consideration management systems, herd size, hygiene, breeding lines, feed and medications. For each herd, the following technical data of 2006 were recorded (mean values); the number of slaughtered pigs, growth rate (grams/day), feed conversion (feed to gain; kg/kg), mortality percentage, percentage of slaughter pigs showing macroscopic lung lesions and pleuritis at slaughter.

In addition, the "2006 over-all slaughterhouse percentage" on recorded pneumonia and pleuritis in slaughtered pigs were recorded for each slaughterhouse. The relative pneumonia and pleuritis scores of each herd were calculated by dividing the percentages of pneumonia and pleuritis found at slaughter with the "2006 overall slaughterhouse percentage" of the corresponding slaughterhouse. This was done to correct for the "slaughterhouse effect" during our statistical calculations, as differences between slaughterhouses in determining and registering abnormalities in lungs and pleura were considerable.

Animals and blood samples (cross-sectional)

A c c e p t e d M a n u s c r i p t 5 Blood samples (n=30, except for one herd n=18 and another herd n=20) were collected crosssectionally from slaughter pigs of the 29 herds. In general, 10 blood samples were collected from pigs in the age of 10 -12 weeks (0 -2 weeks after entering the fattening unit), 10 blood samples from pigs at the age of 16 weeks and 10 blood samples from pigs at the end of the finishing period (between 20 -22 weeks of age). Blood samples were taken at random, so without any prior selection based on clinical signs. Pigs were not vaccinated against swine influenza virus (SIV), Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae and Haemophilus parasuis. Three herds used PRRS-MLV vaccination.

Blood samples were sent to the laboratory and serum samples were examined using routine procedures. All sera (in total n=858) were examined for the presence of antibodies directed against PCV2 IgM and IgG antibodies (Ingezim PCV-ELISA, Madrid, Spain), to determine whether the PCV2 infection was in an acute or chronic phase. Serum samples were tested also for antibodies against porcine reproductive and respiratory syndrome viruses (PRRSV; by Idexx HerdChek PRRS 2XR ELISA, Westbrook Maine, USA), Mycoplasma hyopneumoniae (HerdChek M hyo, Idexx, Westbrook Maine, USA), swine influenza viruses (SIV)-H1N1, -H1N2 and -H3N2 by haemagglutination inhibition (HI) tests, and Actinobacillus pleuropneumoniae (Chekit-App-ApxIV ELISA, Idexx, Westbrook Maine, USA). Antibody tests were performed according to the descriptions of the manufacturer (Idexx, Westbrook Maine, USA). In 3 herds, using a PRRS-MLV vaccine, serum samples with S/P ratio <2.0 were considered to be negative for infection with field strains of PRRSV.

Part B

Herds and animals

Based on the results from part A, 8 "high" and 8 "low" herds were selected. The 8 "high" herds were defined as the herds, described in part A, having high percentages of lung lesions (pneumonia) at slaughter, and the 8 "low" herds had low percentages of pneumonia at slaughter.

A c c e p t e d

M a n u s c r i p t 6 For both the "high" and "low" herds, 3 pigs showing signs of respiratory distress (e.g. dyspnoea)

were selected, and further examined after necropsy. In situations with no clear respiratory distress, 16 weeks old pigs were selected. All pigs (n=48) were selected by one and the same veterinarian.

During the visits, the same veterinarian recorded age and clinical signs of each selected pig. None of the pigs was treated with antimicrobials within one week prior to necropsy.

Necropsy and examinations for lung pathogens

At necropsy, all euthanized pigs were examined for macroscopic lesions, in particular lung lesions.

Lung tissue samples from "high" and "low" pigs were microscopically examined for histopathological lesions using a haematoxylin-eosin staining, and for the presence of bacteria and viruses including PCV2 viral DNA load. From each affected lung, one sample was taken from the transition area between macroscopically affected (with lesions) and unaffected tissue. A second sample, or one sample in case of absence of macroscopic lesions, was taken from each animal from a macroscopically unaffected area of the lung. For each pig, the same area of the lung was selected (the cranial ventral part of the same selected lobe).

Statistical analyses

Serological data from part A were subjected to generalized ordinal regression analysis in

RESULTS

Part A

The overall technical data are presented in Table 1. Data from pigs from 29 herds were collected but three herds were excluded in the data presented in Table 1. Their data sets were not complete (the slaughterhouse percentages of 2006 were not available).

The mean number of slaughtered pigs per herd in 2006 was 1651 and this number varied between herds from 669 to 7600. The recorded lung lesions and pleuritis in slaughtered pigs varied per herd between, 0.1 -17.7% and 0.6 -35.8%, respectively. The relative lung lesion and pleuritis cases per herd varied between 4 -495%, and 5 -284% (corrected for the "2006 slaughterhouses percentages").

Table 1

Serological data part A

Table 2 presents the overall serological data. It shows that, in all 29 herds, antibodies were detected against PCV2, SIV-H1N1, SIV-H3N2 and A. pleuropneumoniae. A. pleuropneumoniae and SIV antibodies in 10-12 week old pigs were considered to be maternal antibodies.

PCV2-IgM antibodies were already detected in pigs from 4 herds around the age of 10 -12 weeks, but more often in pigs around the age 16 weeks; namely in pigs from 23 (79%) of the 29 examined herds.

In 24% of the examined herds, PCV2-IgM was detected in 20 -22 week old pigs, while PCV2-IgG was detected in 20 -22 week old pigs from all herds. PRRSV antibodies were detected in 25 (86%) of the herds.

Table 2

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

Statistical analyses of the data from part A

In total, data from 26 herds were used for statistical analyses. From 3 herds the slaughterhouse percentages of 2006 were not available. Three categories were made for both the outcome variables; a) the percentage of affected lungs at the slaughterhouse, and b) the percentage of affected pleura at the slaughterhouse. The distribution of the three categories and the respective percentage affected lungs and pleura over the three categories are represented in Table 3 for affected lungs and for affected pleura. The categories were defined in such a way that three categories of approximately the same number of observations were formed.

The dataset is furthermore subdivided in data from young finishers (10 -12 weeks), middle aged finishers (16 weeks) and old finishers (20 -22 weeks). Taken together, this means that for each of the 3 age groups statistical analyses were performed for each of the two outcome variables, so six analysis. In every part of the analysis e.g. the variables IgG and IgM antibodies against PCV2 and the slaughterhouse reference percentages for either lungs or pleura were fixed in the statistical models.

The following variables were inserted in the statistical models; growth rates (in grams/day), feed conversion, percentage dead loss, IgG and IgM antibodies against PCV2, antibodies against A.

pleuropneumoniae, Mycoplasma hyopneumoniae, PRRSV (ELISA S/P-ratios), H1N1, H1N2, and H3N2 antibody titres, and SIV seroconversion on herd level (seroconversion was defined as an increase of at least 2 titre steps; 0=no seroconversion, 1=seroconversion between 10 -12 and 16 weeks, or between 16 and 20 -22 weeks, 2=seroconversion between 10 -12 and 16 weeks and between 16 and 20 -22 weeks as well).

Part B

The 8 "high" herds had high percentages of pigs with lung lesions (pneumonia) at slaughter (mean of the relative percentages of pigs with lung lesions was 157%; range 130% -189%), and for the 8 "low" herds, the mean of the relative percentages of pigs with lung lesions at slaughter was 48% (range 14% -74%).

Post-mortem examinations

The mean age of "high"-and "low" pigs with respiratory distress at necropsy was 18.0 and 17.3 weeks, respectively (range between 13 and 24 weeks). Macroscopic lesions (pneumonia, catarrhal pneumonia) and/or histopathological lung lesions (interstitial pneumonia, chronic forms of pneumonia from "high" herds, and in pigs (n=17) from "low" herds as well (Table 4). In Table 4, for each "high"

and "low" pig, the macroscopic and histopathological lesions, PCV2 DNA loads in macroscopic affected and unaffected lungs, SIV/PRRSV, pathogenic bacteria (Arcanobacterium pyogenes, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Pasteurella multocida, Haemophilus parasuis, M. pleuropneumoniae), and Streptococcus spp / E. coli detection in lungs are presented.

Table 4

Statistical analyses of the data from part B

Results of the macroscopic and histopathological examinations, and the detection of the different lung pathogens, were analysed statistically per animal (Table 5). The presented results in Table 5 are the proportions of positive animals for "high" and "low", the P-values for the difference in proportion between those groups and whether the corresponding variable was found more or less often in pigs from "high" herds compared to pigs from "low" herds. The following variables were found significantly more often in "high" than in "low" pigs: lobus apicalis pneumonia, presence of PCV2 in macroscopically unaffected lungs, presence of SIV, presence of Pasteurella multicoda and presence of M. hyopneumoniae. In addition, high PCV2 DNA loads (>10 4 PCV2. DNA copies/mg) were found in lungs of 14 (58%) "high", and in 7 (29%) of the "low" pigs (P=0.13).

The mean PCV2 DNA copy load in macroscopically affected lungs from "high" pigs was also higher than the mean PCV2 DNA copy load in macroscopically affected lungs from "low" pigs, although this difference for macroscopically affected lungs was not significant. The differences in the mean log 10 PCV2 DNA copies/mg values between "high" and "low" pigs was 1.6 for the macroscopically affected parts of the lungs and 2,0 for the macroscopically unaffected parts of the lungs.

Interstitial pneumonia, Streptococcus spp and a-pathogenic bacteria were found significantly more often in "low"pigs.

After the multivariate analysis on data of all 16 herds (8 "high" and 8 "low" herds) two variables remained, namely PCV2 from unaffected lungs with an Odds ratio of 8.7 (95%

PCV2 and co-infections

In one "low" pig with pneumonia (no 9224), the PCV2 DNA load in affected lungs was high (>10 6 copies/mg) while no other viral or bacterial pathogens were detected. In all other lungs with macroscopic and/or histopathological lesions from "high" and "low" pigs where PCV2 was detected, PCV2 was found in combination with other lung pathogens, such as Mycoplasma hyopneumoniae, P. multocida, A. pleuropneumoniae, SIV or PRRSV.

In 11 of the 19 affected lungs (with macroscopic or histopatholocial lesions) from "high" pigs, high PCV2 DNA loads of >10 4 DNA copies/mg were found in combination with one or more other lung pathogens, while this was found only in 5 of the 17 affected lungs from "low" pigs (P=0.02) (Table 4).

Table 5

A c c e p t e d M a n u s c r i p t 13

DISCUSSION

In Europe, the economic losses in swine industry related to respiratory diseases are very high. PRRSV, SIV, Mycoplasma hyopneumoniae, Pasteurella multocida and Actinobacillus pleuropneumoniae, are all known contributors to this disease complex. PCV2 also has been found in association with porcine respiratory diseases, but its role as aetiological agent is still not clear [START_REF] References Allan | Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland[END_REF][START_REF] Opriessnig | Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2[END_REF][START_REF] Grau-Roma | Detection of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, swine influenza virus and Aujeszky's disease virus in cases of porcine proliferative and necrotizing pneumonia (PNP) in Spain[END_REF][START_REF] Opriessnig | Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis and intervention strategies[END_REF]. More insights are warranted on the role of PCV2 as etiological agent in porcine respiratory diseases, especially in herds without clinical signs of PMWS. In PMWS cases the load of PCV2 in lung tissue can be very high [START_REF] Segalés | First report of post-weaning multisystemic wasting syndrome in pigs in Spain[END_REF][START_REF] Segalés | Postweaning multisystemic wasting syndrome (PMWS) in pigs[END_REF][START_REF] Chae | Postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology[END_REF], but in many European countries the number of PMWS cases has been decreasing within the last 5 years [START_REF] Wellenberg | Spread of post-weaning multisystemic wasting disease (PMWS) in Europe[END_REF].

In part A of this study, the PCV2 IgM data show interesting results. These data show that the detection of IgM antibodies against PCV2 in pigs of 16 and 20 -22 weeks of age correlates with reduced percentages of pleuritis or lung lesions at slaughter. This indicates that fattening pigs older than 16 weeks are probably more capable in coping with the effects of PCV2 infections than piglets in the age of 10 -12 weeks. PCV2 infections in young fattening pigs are probably associated with an increased probability of finding lung lesions and pleuritis at slaughter.

The serological data on A. pleuropneumoniae in 20 -22 weeks old pigs were associated with pleuritis scores at slaughter (OR 5, P=0.015). It is well known that A. pleuropneumoniae is able to cause pleuropneumoniae in fattening pigs.

Finding antibodies against SIV-type H1N1 in 10 -12 weeks old pigs reduces the probability of pleuritis at slaughter (OR 0.39, P=0.033). These antibodies, at the age of 10 -12 weeks, are probably maternal antibodies that protect young fattening pigs against H1N1 infections.

Growth, feed conversion and percentage dead loss are not explanatory variables for the percentage lung lesions or pleuritis at slaughter themselves, but they are the result of (amongst others) the disease status of a herd. As such, they may reflect the disease situation in the herds. Daily weight gain (growth), which is an important factor in the economic viability of the pig industry, was significantly reduced in herds with medium or high levels of pleuritis at slaughter. Differences in daily weight gain were up to 56 grams / animal / day between the highest and the lowest category affected pleura.

A c c e p t e d M a n u s c r i p t 14

The fact that feed conversion was not found to be significantly related to pleuritis at slaughter can probably be explained by the fact that these sick pigs have reduced appetite, resulting in less growth but not in a significant change in feed conversion. In addition, for young and middle aged fattening pigs, the percentage dead loss was also found to increase the probability of finding lung lesions at slaughter.

A reduction in growth and a higher mortality rate in herds with high percentages (>10%) of pleuritis at slaughter, represent in the current Dutch situation a reduction in economic value of more than € 4.per produced finisher. Four Euro is currently a substantial part of the net profit per raised pig

In part B of this study, we selected pigs with respiratory distress from both the "high" and the "low" herds. Due to this selection, there was no statistical difference in the number of pigs with pathological lung lesions between "high" (n=19) and "low" herds (n=17). This indicates that the selection of affected pigs, based on signs of respiratory distress, in "high" and "low" herds was done properly. We found lobus apicalis pneumonia, PCV2 in macroscopically unaffected lungs, Pasteurella multocida, M. hyopneumoniae and swine influenza viruses significantly more often in lungs from pigs of herds with high percentages of pneumonia at slaughter age than in herds with low percentages.

The assumption that PCV2 infected pigs are more susceptible to secondary bacterial infections [START_REF] Dorr | Epidemiologic assessment of porcine circovirus type 2 co-infection with other pathogens in swine[END_REF], is supported by the fact that in affected lungs (with macroscopic or histopatholocial lesions) high PCV2 DNA loads of >10 4 DNA copies/mg were found often in combination with one or more other lung pathogens such as Pasteurella multocida, Mycoplasma hyopneumoniae, and swine influenza viruses. Pathogens like PRRSV, swine influenza viruses, and Mycoplasma hyopneumoniae have been associated with the porcine respiratory disease complex (PRDC). PRDC is a complex disease in which PCV2 was suggested to play an important role too [START_REF] Kim | Association of porcine circovirus 2 with porcine respiratory disease complex[END_REF][START_REF] Harding | The clinical expression and emergence of porcine circovirus 2[END_REF][START_REF] Opriessnig | Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis and intervention strategies[END_REF]. In recent studies with pigs in Korea and the United States, co-infections of PCV2 with one or more pathogens were detected in 85% and 98.1% of the examined pigs [START_REF] Kim | Association of porcine circovirus 2 with porcine respiratory disease complex[END_REF][START_REF] Opriessnig | Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis and intervention strategies[END_REF]. They also concluded that co-infections with other viral and bacterial pathogens severely worsened the clinical outcome of porcine circovirus type-2 (associated) diseases.

A c c e p t e d M a n u s c r i p t 15

The serology data of part A on A. pleuropneumoniae are not confirmed by the results from part B, the post-mortem examinations of pigs from "high" and "low" herds. The difference in results between these two parts of this study on A. pleuropneumoniae can possibly be explained that for both the "high" and "low" herds clinically affected animals were selected for post-mortem examination, and the fact that an A. pleuropneumoniae infection gives clear clinical signs. In the acute phase of respiratory distress, the number of A. pleuropneumoniae infected "high" pigs was not higher than the number of A. pleuropneumoniae infected "low" pigs (P=0.68).

In conclusion, the fact that PCV2-IgM in 16 week and 20 -22 week old pigs was associated with a reduced probability of pneumonia and pleuritis at slaughter, and that high PCV2 loads in combination with Pasteurella multocida, Mycoplasma hyopneumoniae, and swine influenza were found more often in affected lungs from pigs of herds with high percentages of pneumonia and pleurisy at slaughter, support the hypothesis that PCV2 plays a role in lung lesions and pleuritis in fattening pigs, not only in herds with high percentages of PMWS, but also in herds with no or low percentages of PMWS cases.

  Virological and bacteriological examinations were performed on all examined lungs. The following techniques were performed for viral examinations; a quantitative real-time PCR for PCV2, real-time PCR tests for EU-and US-type PRRSV, and the influenza-A viruses H1N1, H1N2 and H3N2 (Wellenberg et al., 2004; Van Maanen et al., 2006). The amount of PCV2 DNA was examined in each lung at two different areas (see above). This was done to determine the difference in PCV2 viral DNA load in affected and unaffected parts of lungs within the same "high" or "low" pig. Lung samples were examined for the presence of Actinobacillus pleuropneumoniae, Pasteurella multocida, Haemophilus parasuis and Streptococcus spp by cultivation techniques, and for Mycoplasma hyopneumoniae by quantitative real-time PCR (Dubosson et al., 2004).

  Stata version 10 using the program Gologit2 (Williams, 2006) Outcome variables are respectively the A c c e p t e d M a n u s c r i p t 7 percentage of affected lungs, and the percentage of affected pleura, both subdivided in three categories. The next model is used: odds-ratios for transition from category k to category k+1 The model is a combination of the ordered logistic regression model (ologit, Stata 10) and the multinomial logistic regression model (mlogit, Stata 10). If possible ordered logistic model is used, assuming β i1 = β i2 If this assumption is invalid the multinomial model is used, implicating that for some i β i1 ≠ β i2 The explanatory variables IgM and IgG antibodies against PCV2 as well as the relative percentage of affected lungs and affected pleura at the slaughterhouse are forced into the model. For all other explanatory variables a backwards elimination procedure was used, with a P-value of 0.05. The hierarchical nature of the problem is addressed by correcting the standard errors of all estimated odds ratios for within herd correlation.For part B first a univariate analysis was done. The proportions test (using Stata version 10, prtest) was used to look for significant differences between high herds and low herds concerning the macroscopic and histopathological findings, including data from bacteriological and virological examinations (n=48). A P-value of <0.05 was chosen to indicate a significant difference. In a second stage a multivariate analysis was done using the logistic model backwards elimination procedure was used, with a P-value of 0.05.

Figure

  Figure 1, Figure 2.

  pleuropneumoniae, necrotizing and ulcerative pneumonia) were found in pigs (n=19)

Figure 1 .

 1 Figure 1. Box-and-whisker-plot of growth by three categories of percentage pleuritis at slaughter

Figure 2 .

 2 Figure 2. Box-and-whisker-plot of percentage dead loss by three categories of percentage affected

  Figure 1.

Figures

  Figures 1-2

  Figure 2.

Table 3

 3 Results of the statistical analysis of Part A (Stata 10, Gologit2 procedure) showed that pigs at 16 weeks of age with IgM antibodies against PCV2 had a lower probability of having pleuritis at

	Old

slaughter (OR 0.34, P<0.000). Pigs, in the age category of 20 -22 weeks, with IgM antibodies against PCV2 also had a lower probability of having pneumonia at slaughter (OR 0.29, P=0.032). Young finishers (pigs of 10 -12 weeks) with an antibody titre against SIV-H1N1 showed a lower probability A c c e p t e d M a n u s c r i p t 10 of having pleuritis at slaughter (OR 0.39, P=0.033). At herd level, pigs of 16 weeks of age with a seroconversion for SIV-H1N1 showed a higher probability of pleuritis (OR 1.50, P=0.017).

shows the box-and- whisker-plot of growth per day for the three pleuritis categories. The difference in growth between pIeuritis category 1 and 2 was 56 grams per day more for category 1 (category 1 had a mean of 805 and category 2 had a mean of 749 grams/day). This is a significant difference (Statistix for Windows, version 8.0, Two sample T Test, P=0.0054). Pigs in the age of 10 -12 weeks and 16 weeks with a higher percentage dead loss at herd level had a higher probability of having pneumonia at slaughter: OR 3.27, P=0.009 and OR 2.85, P=0.010, respectively.

Table 1 :

 1 Technical data of the farms in 2006 (n=26)

		Mean value	Variation
	Number of slaughtered pigs	1651	669 -7600
	Daily weight gain	772	689 -855
	Mortality rate (%)	2.6	0.9 -5.9
	Relative lung score	124%	4 -495%
	Relative pleuritis score	96%	5 -284%

Table 2 :

 2 Percentages of farms and pigs (between brackets) with antibodies at the three different moments of sampling (

n= 29 herds and 818 sera).

			Age of the pigs
	Agent	10-12 weeks	16 weeks	20-22 weeks
	PCV2 IgM	14% (4%)	79% (36%)	24% (11%)
	PCV2 IgG PRRSV SIV H1N1 SIV H1N2 SIV H3N2 M. hyopneumoniae A. pleuropneumoniae	31% (6%) 69% (29%) 97% (23%) 76% (14%) 100% (47%) 7% (1%) 96% (44%)	83% (55%) 72% (52%) 86% (34%) 69% (22%) 100% (34%) 31% (7%) 59% (27%) M a n u s c r i p t 100% (93%) 86% (72%) 93% (65%) 79% (43%) 100% (64%) 52% (25%) 72% (42%)
	A c c e p t e d
				21

Table 3 : Descriptive statistics of the three categories with the percentages of affected lungs and pleura at slaughterhouse.

 3

	Number of observations	Mean	Standard	Minimum	Maximum
		(n = 752)	(%)	deviation	(%)	(%)
				(SD)
	Category "Lung"
	1 (low)	209	2.0	1.2	0.1	3.5
	2 (medium)	268	6.2	1.8	4.0	9.0
	3 (high)	275	13.9	2.8	10.1	17.7
	Category "Pleura"
	1 (low)	240	2.2	1.1	0.6	3.7
	2 (medium)	255	13.3	6.2	4.1	20.9
	3 (high)	257	27.2	4.6	21.5	35.8

Table 4 :

 4 Macroscopic and histopathological lesions, PCV2 DNA loads in macroscopic affected and 459 unaffected lungs, SIV/PRRSV pathogenic bacteria and Streptococcus spp / E. coli detection in lungs

	460
	461	of "high" and "low" pigs.
		"high" herd	Animal ID	Macroscopic lesions	Histopathological	lesions	PCV2 DNA copies/mg (log10)	PCV2 DNA copies/mg (log10)	SIV and/or PRRSV	Pathogenic bacteria	Streptococcus spp and/or E.coli
							affected lung unaffected
								lung
		3	61395		1		2,81	2,30	1
			60178		1		0	2,30	1
			64815				5,15	0
		7	3838	1			2,68	3,51	1	1
			3640		1		7,15	7,26		1
			29				-	5,51
		8	1312				1,73	0
			1284				0	1,79
			1344	1			2,76	2,34		1
		13	4450	1			4,52	4,91	1	1
			1454	1			5,38	5,87	1	1
			68	1			7,34	7,41	1	1
		18	2101	1			3,54	3,45		1
			2348	1			7,15	6,76		1
			2113	1			5,68	6,00	1	1
		21	2818		1		6,23	6,48	1
			2268	1			4,91	4,72	1	1
			2222	1			6	6	1	1
		23	73202				4,11	4,15	1
			71033		1		5,62	5,48	1
			525	1			5,51	6,11	1	1
		31	596		1		0	0	1
			639	1			0	0	1	1
			682	1			0	0	1	1
	462
	463

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


Sandra Martín-Peláez

Adele Costabile

Lesley Hoyles

Robert A Rastall

Glenn R Gibson

Roberto M La Ragione

Martin J Woodward

Enric Mateu

Susana M Martín-Orúe

Evaluation of the inclusion of a mixture of organic acids or lactulose into the feed of pigs experimentally challenged with Salmonella Typhimurium

Keywords: Pig, Organic acids, Lactulose, Salmonellosis, DGGE, FISH

A c c e p t e d M a n u s c r i p t 2 Abstract A mixture of organic acids and lactulose for preventing or reducing colonization of the gut by Salmonella Typhimurium was evaluated in pigs. A total of 63 4-week-old commercial piglets were randomly distributed into three different experimental dietary groups: a plain diet without additives (PD) and the same diet supplemented with either 0.4 % (w/v) formic acid and 0.4 % lactic acid (w/v) (AC) or 1 % (w/v) lactulose (LC).

After 7 days of adaptation, two-thirds of the pigs (14 from each diet) were challenged with a 2 mL oral dose of 10 8 CFU/mL of Salmonella Typhimurium, leaving the remaining animals unchallenged (UC). After 4 and 10 days post-challenge, pigs were euthanized and the ileum and caecum content were aseptically sampled to (a) quantify lactic, formic, and short-chain fatty acids (SCFA), (b) quantify bacterial populations and Salmonella by fluorescence in situ hybridization and (c) qualitatively analyse bacterial populations through denaturing gradient gel electrophoresis (DGGE). Modification of fermentation products and counts of some of the bacterial groups analysed in the challenged pigs receiving the treatments AC and LC were minimal. Treatments only influenced the bacterial diversity after 10 days post-challenge, with AC generating a lower number of DGGE bands than UC (P<0.05). Neither the inclusion of a mixture of 0.4 % (w/v) formic and 0.4 % (w/v) lactic acids nor of 1 % (w/v) lactulose in the feed influenced numbers of Salmonella in the ileum and caecum of experimentally challenged pigs.

A c c e p t e d M a n u s c r i p t 3

Introduction

In 2007 salmonellosis still remained the second most commonly reported zoonotic disease in the EU with a rate of 31.1 cases per population of 100,000. As in previous years, S. Enteritidis and S. Typhimurium were the most frequently reported serovars (81% of all known serovars in human cases). Salmonella was most often found in fresh poultry (S. Enteritidis) and pig meat (S. Typhimurium) (European Food safety Authority, 2007). In the case of the pig meat, the route of infection requires asymptomatic infected pigs from the farm, which serve as a source of contamination of carcasses in the slaughter chain [START_REF] Widders | Investigation of Salmonella contamination of pigs in Australia[END_REF].

In order to reduce the incidence of Salmonella in pigs on farms, dietary interventions are being considered. One of these is the inclusion of organic acids (OA) in the feed of the pigs. Various studies have shown the potential of some OA to reduce the prevalence of the pathogen in the pigs when added to water [START_REF] Van Der Wolf | Study plan and preliminary results of the intervention in the Salmonella status of finishing herds by adding organic acids to the drink water of finishers[END_REF] or feed (Jorgensen et al., 1999;[START_REF] Creus | Effect of acidified feed on the prevalence of Salmonella in market-age pigs[END_REF]. The use of prebiotic compounds has also been proposed as a possible strategy to reduce Salmonella prevalence in pigs (Wiemer et al., 1999). Lactulose (a synthetic disaccharide) has been shown to elicit a prebiotic effect (increased counts of bifidobacteria and lactobacilli) in pigs (Konstantinov et al., 2004) and has been proposed as feed additive (1 %) to prevent salmonellosis in fattening pigs (Wiemer, 1999). In vitro experiments have also shown potential inhibitory effects of lactulose on Salmonella Typhimurium (Martín-Peláez et al., 2008).

The aim of the present study was to investigate the potential of a mixture of either lactic and formic acids or lactulose to prevent or reduce the colonization of the gut by Salmonella Typhimurium in pigs.

A c c e p t e d M a n u s c r i p t 4

Materials and methods

Strain

The Salmonella Typhimurium phagetype U302 strain used in the study was obtained from the stock NI-3 belonging to the Veterinary Laboratory on Infectious diseases (UAB) which had been originally isolated from a clinical case of porcine salmonellosis and was shown to be resistant to the antibiotics: ampicillin, chloramphenicol and gentamicin. The bacteria were grown on tryptic soy agar (TSA, Oxoid, UK) for 24 h at 37°C. Afterwards three tubes containing brain-heart infusion broth (BHI, Oxoid, UK) (3 mL/tube) were inoculated with one colony from the TSA plate (1 colony/tube) and incubated for 24 h at 37°C. The day of the challenge, the contents of the three tubes were mixed, diluted with PBS (1:20, v/v) and used for challenge. The placebo was prepared by mixing 2.5 mL of BHI broth plus 47.5 mL of PBS (Oxoid, UK). Doses (2 mL) consisted of either the placebo or the inoculum dispensed in 2.5 mL syringes. One millilitre of inoculum and 1 millilitre of placebo were kept for cultivation and counting. The challenge dose was 1 × 10 8 CFU.

All procedures involving the treatment, housing, husbandry and euthanasia of animals received prior approval from the Animal Protocol Review Committee of the Universitat Autònoma de Barcelona (Spain) and of the Veterinary Laboratories Agency (United Kingdom), and were carried out according to European Union Guidelines (The Council of the European Communities, 1986).

Animals and diets

A total of 63 4-week-old Large White male piglets (7.5±0.2 kg body weight)

were used for the study. Animals were weaned at 3 weeks of age and obtained from a high-health status farm, free from all major pathogens of swine including Salmonella.

A c c e p t e d M a n u s c r i p t 5 Sows (n=24) and piglets were tested serologically (Svanovir Salmonella Ab ELISA, Svanova, Uppsala, Sweden) and bacteriologically (faeces) for Salmonella prior to inclusion in the experimental infections. All animals were negative using both tests.

Diets consisted of either the plain diet (Table 1) without additives (PD), the plain diet with 0.4 % (w/v) formic acid and 0.4 % (w/v) lactic acid added (AC) or the plain diet with 1 % (w/v) lactulose added as syrup (15 mg/kg) (LC).

Experimental design

Pigs were transported from the weaning farm to the biosafety level 3 facilities at Centre de Recerca en Sanitat Animal (CReSA), ear-tagged and distributed into three separate boxes (1, 2 and 3) with three compartments each, one for each diet (AC, LC and PD; 7 animals per compartment). Animals were evenly distributed to get a similar mean live weight between compartments. Each compartment was physically separated from the others by means of a solid partition. Once distributed, the animals were provided with feed and water ad libitum for 18 days. After 7 days of adaptation, each pig from boxes 1 and 2 was challenged with an oral dose of 2ml of inoculum as previously described, whereas each pig from box 3 (UC) was dosed with 2 mL of a placebo. In order to ensure that stomach contained feed before challenge, the pigs were not be fed from 20:00 h until next day early in the morning, approximately 1 h before challenge. All animals were clinically examined from days 1 to 10 post-challenge and body temperature was recorded. Faeces (about 5 grams) were collected directly from the rectum daily from day 0 to day 4 post-challenge and then on days 7 and 10 postchallenge. On days 4 and 10 post-challenge, three randomly selected piglets from each subgroup were sedated with azaperona (2 mg/kg, intra-muscular) and then euthanized by an intravenous overdose of sodium thiopental (200 mg/kg live weight). Mesenteric A c c e p t e d M a n u s c r i p t 6 lymph nodes were also taken at that time for bacteriological analysis. The rest of animals (one per compartment) were also euthanized during the 11 PC day but they were not sampled. They were included in the study in prevention of occasional deaths.

At post-mortem ileal and caecal contents were aseptically sampled for analysis of lactic acid and SCFA by adding 2 mL of digesta to 2 mL of a preservative solution (0.0074 M HgCl 2 , 0.344 M H 3 PO 4 , 0.0172 M C 6 H 12 O 2 in distilled water), for FISH analysis by adding 0.5 mL of digesta to 1.5 mL of 4 % (w/v) paraformaldehyde and for DGGE analysis by adding 0.25 mL of digesta to a tube containing 1.75 mL of buffer ASL (QIAmp® DNA Stool Mini Kit, Qiagen Ltd, West Sussex, UK).

Analytical procedures

SCFA concentrations in caecal samples were analysed by gas-liquid chromatography using the method of [START_REF] Richardson | Simultaneous determination of volatile and non-volatile acidic fermentation products of anaerobes by capillary gas chromatography[END_REF] modified by Jensen et al. (1995).

Faecal samples were examined microbiologically both qualitatively and semiquantitatively and lymph nodes samples only qualitatively. For the qualitative analysis, 1 gr of faeces (or 1 gr of minced tissue) was re-suspended on 10 mL of buffered peptone water (BPW, Oxoid, UK) and incubated overnight at 37°C. Then 100 µl of the BPW were transferred to 10 mL of Rappaport-Vassiliadis broth (RVB, Oxoid, UK) and incubated for 48h at 41.5°C. Finally, 10 µl of RVB were streaked onto xylose-lysinetergitol 4 (XLT4, Oxoid, UK) plates and incubated for 24 h at 37°C. Suspected Salmonella colonies were further identified by means of the API 20E system. For the semi-quantitative analysis, once the samples had been re-suspended in BPW and before any incubation, 100 µl of BPW were taken and serially diluted from 10 -1 to 10 -5 in 0.85% sterile saline. One hundred microlitres of the 10 -2 and 10 -5 dilutions were A c c e p t e d M a n u s c r i p t 7 streaked onto XLT4 plates to which chloramphenicol and gentamicin had been added. Salmonella colonies were counted after 24 h of incubation at 37°C. Randomly chosen positive isolates were identified by means of the API20E system (Bio-Mérieux, Barcelona, Spain), serotyped and the antimicrobial susceptibility pattern was determined in an antibiogram to confirm that they corresponded to the original strain used for the challenge. With this scheme, all animals had a double evaluation for each day: positive/negative and, if positive, a score was given as following: 0, for negative animals; 1, for positive animals but with no colonies at 10 -2 (<10 3 CFU/gr); 2 for animals with less than 100 colonies at 10 -2 (<10 5 CFU/gr); 3 for more than 100 colonies at 10 -2 but less than 10 at 10 -5 (10 6 -10 7 CFU/gr); 4, with >10 CFUat 10 -5 (>10 7 CFU/gr).

Processing of samples for FISH analysis, and enumeration of bacteria present in 500-µL ileal and caecal samples were carried out as described by Martín-Peláez et al. (2008). Table 2 gives details of the probes used in this study; all probes were Cy3labelled and synthesized by Sigma Aldrich.

DNA from caecal samples was extracted using the QIAmp® DNA Stool Mini Kit following the manufacturer's instructions. The recommended lysis temperature was increased to 90°C, and afterwards an incubation step with lysozyme (1 mg/mL of 50000 U/mg protein; Sigma) was added (10 mg/mL, 37°C for 30 min) in order to improve lysis of bacterial cells, according to [START_REF] Castillo | Quantification of total bacteria, enterobacteria and lactobacilli populations in pig digesta by real-time PCR[END_REF]. DNA quality was checked by running 5 µL aliquots of the samples on 0.8 % (w/v) ultraPURE agarose (GibcoBRL) gels containing ethidium bromide (0.4 mg/mL), and visualizing the bands using a UV light. The quantity of DNA in each sample was determined using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Fragments (230 bp) derived from the variable V3 region of the 16S rRNA gene were amplified from DGGE was carried out as described by [START_REF] Waldram | Top-down systems biology modelling of host metabotype-microbiome associations in obese rodents[END_REF] but with minor modifications: the V20-HCDC DGGE system (BDH) was used with gels comprising 35-60 % gradients and run at 100 V. Gels were silver-stained according to the method of [START_REF] Sanguinetti | Rapid silver staining and recovery of PCR products on polyacrylamide gels[END_REF]. Gels were scanned using CanonScan (Canon, Surrey, UK), saved as TIFF files (600 dpi) and imported into TotalLab TL120 v2006f (NonLinear Dynamics Ltd, UK) for analyses. Matching calculations were performed based on Jaccard similarity coefficient (matches by the presence/absence of bands). Dendrograms were constructed by using Unweighted Pair-Group Method with Arithmetic mean (UPGMA) analysis. Finally, Shannon index, a measure of diversity, was calculated following the methodology proposed by Magurran (1988).

Statistical analysis

Data from FISH and SCFA were analyzed by ANOVA using the GLM procedure of SAS (v. 9.1., SAS Inst. Inc., Cary, NC, USA). Firstly, results for the UC pigs receiving the three experimental diets (PD, AC, LC) were run separately from the challenged pigs, and by day of sacrifice, according to the following model:

Y i = µ + α i + ε i
Where Y i was the dependent variable, µ was the overall mean, α i was the effect of the diet and ε~ N (0,σ 2 ε ) represented the unexplained random error. As experimental diets did not show consistent effects on UC animals, in a second analysis all the UC animals Treatment means or LS means were assessed by multiple comparison methods using Tukey's correction. Statistical significance was accepted at P<0.05 and differences among means with 0.05<P<0.10 were accepted as representing tendencies to differences.

Results

Challenge of the pigs resulted in mild clinical signs of salmonellosis with slight fever seen from day 1 until day 4 post-challenge and slight diarrhoea (loose or liquid faeces) from day 2 until day 7 post-challenge, normalizing in the following days. Faecal shedding of Salmonella and positive cultures from mesenteric lymph nodes were found in all pigs. The quantitative analysis of faeces (considering all inoculated pigs) showed that the highest Salmonella-score was detected by day 3 post-challenge (P<0.05) with a mean value of 1.9 (pigs with a score higher than 2: 8/14 in groups AC and PD and 7/14 in group LC) and a mean bacterial load of 10 4.36 ± 10 0.72 CFU/gr. Although no significant differences were seen among treatments (P = 0.15), it is interesting to point out that maximum Salmonella scores (4= more than 10 7 CFU/gr) were only seen in groups AC (1 pig, day 3 post-challenge) and PD (3 pigs day 3 post-challenge).

Concentrations of lactic and formic acid and of total SCFA were not consistently modified by the experimental diets in the UC pigs. Total SCFA in ileum were significantly lower (P<0.05) in AC and LC diets compared to PD at day 4 postchallenge (9.09, 9.11 and 17.41 for AC, LC and PD, respectively, SEM: 1.352) and at day 10 post-challenge, AC diets showed higher levels than PD (P=0.004) (12. 16, 4.93, 5.25 for AC, LC and PD, respectively, SEM: 1.055). Regarding SCFA concentrations in With regards to molar percentages of each SCFA, no significant differences were found except for BCFA in the ileum at day 4 post-challenge, with higher proportions (P=0.013) for the AC diet (1.29, 0.30, 0.00 % for AC, LC and PD, respectively, SEM: 0.260).

Table 3 shows the results obtained when data from UC and challenged pigs were included in the same statistical model as one experimental treatment. After 4 days postchallenge treatments tended to influence the amounts of formic acid and SCFA in the ileum of pigs but these differences disappeared after 10 days post-challenge. Treatments AC and LC generated the same amount of total SCFA as UC, but PD generated less than UC (P=0.046). Profiles of the different SCFA were not influenced by treatment. In the caecum, treatments only exerted an effect after 4 days post-challenge. Treatment LC generated lower amounts of SCFA than UC (P=0.032). Formic and lactic acids and the proportions of the different SCFA were not modified by the treatments.

Changes promoted by the different experimental treatments in some of the most relevant microbial groups were examined by FISH analysis of ileal and caecal samples.

No significant differences related to diets were found in UC pigs. Table 4 shows the results obtained when data from UC and challenged pigs were included in the same statistical model. As expected, challenge of the pigs with Salmonella resulted in Sal303 counts in both the ileum and caecum at 4 and 10 days post-challenge. No Salmonella (Sal303) was detected in the UC group or in any challenge group prior to challenge. No differences in Sal303 counts were found in the challenged pigs with any of the diets.

The treatments influenced the levels of different bacterial populations in ileal (Rbro730/Rfla729) and caecal (Bac303, Bif164, Erec482) samples. Only Lab158 and Prop853 were not affected at any time by the treatment in either segment of the GIT were such that the cluster analysis did not show any obvious grouping of profiles.

Discussion

The aim of this study was to investigate if the inclusion of a mixture of OA (lactic and formic acid) or lactulose in feed was able to reduce gut colonization by [START_REF] Creus | Effect of acidified feed on the prevalence of Salmonella in market-age pigs[END_REF] and lactulose had shown promising results against Salmonella Typhimurium compared to other prebiotics in vitro (Martín-Peláez et al., 2008).

The experimental infection aimed to induce mild diarrhoea, in order to be able to be overcome by the natural defences of the animal, defences which would be reinforced by the use of feeding strategies as those tested in our study. Besides the moderate level of the infection, no diet-related differences in numbers of Salmonella (probe Sal303) were found in ileal or caecal samples although in the group of inoculated pigs receiving the diet without additives a higher number of pigs excreting more than 10 7 Salmonella/gr faeces was found.

Regarding the use of OA against Salmonella, one theoretical mechanism by which they are thought to work is by increasing the concentration of OA in the stomach; this would reduce the population of enterobacteria, thereby creating a biological barrier against enterobacteria colonizing the lower parts of the GIT [START_REF] Canibe | Feed physical form and formic acid addition to the feed affect the gastrointestinal ecology and growth performance of growing pigs[END_REF]Hansen et al., 2007). In this study, pH and OA in the stomach were not measured; therefore, no conclusions can be made regarding this potential effect, but if any differences in the concentration of lactic or formic acid were promoted, they were not detected in the ileum. A decrease in species richness in the caecum, as determined by DGGE, was seen with the use of OA, possibly due to the inhibition of bacterial populations not analysed in this study.

Despite the absence of inhibition of Salmonella Typhimurium by the OA used in this study, other authors have found significant effects on Salmonella when using the same mixture. [START_REF] Creus | Effect of acidified feed on the prevalence of Salmonella in market-age pigs[END_REF] using a commercial diet supplemented with 0.4 % lactic acid and 0.4 % formic acid during the last 8-9 weeks in fattening pigs observed 2007), using the same mixture for 5 weeks in fattening pigs, found lower numbers of seropositive pigs compared to a commercial control diet. The lack of effect found in our study compared to the results of these field studies could be explained by several reasons; for instance, differences in the age of the pigs (market age pigs vs piglets) and the length of the treatment (5-9 weeks vs 2 weeks). Young pigs are known to be unable to acidify their stomach contents as efficiently as adult pigs, due to the low capacity to synthesize HCl by gastric parietal cells [START_REF] Cranwell | The development of acid and pepsin (EC 3.4.23.1) secretory capacity in the pig; the effects of age and weaning[END_REF], leading to insufficient killing of microbes. In this sense, inclusion of OA in the diet would help to compensate the insufficient acidification of the stomach. However, some authors have suggested that inclusion of acids in post-weaning diets could have a contrary effect. [START_REF] Bosi | Continuous dietary supply of free calcium formate negatively affects parietal cell population and gastric RNA expression for H + /K + -ATPase in weaning pigs[END_REF] found that supplementation of weaned pigs with calcium formate (1.2 %) decreased the number of HCl-secreting parietal cells and increased somatostatinproducing cells (somatostatin has a suppressive action on HCl secretion). Thus, addition of formate to the feed of weaned pigs could exert a negative feedback on gastric acid secretion. An additional reason that could explain the lack of effects of the OA may be the differences in the way the animals are exposed to the pathogen in the natural environment compared to an experimental infection. Natural infections may arise through continuous exposure to low doses of Salmonella which is probably protected by being bound in faecal, soil or other environmental materials. This situation is difficult to reproduce in experimentally infected animals, which are exposed to a single high dose of Salmonella. In that situation, Salmonella probably has more chance to overwhelm the gastric barrier fortified by the use of in-feed acidifiers. Further studies in which naïve A c c e p t e d M a n u s c r i p t 15 LC did not increase the proportion of butyric acid detected; therefore, an inhibitory effect elicited by butyric acid on Salmonella virulence would not be expected.

Conclusions

Under our study conditions, neither the inclusion of a mixture of 0.4 % formic and 0.4 % lactic acids nor of 1 % lactulose in the feed influenced survival of Salmonella Typhimurium in the ileum and caecum of pigs challenged by direct oral challenge with high doses of Salmonella. More studies with higher doses and/or under different conditions of infection would be needed to fully explore the potential of these compounds. *AC, challenged pigs consuming plain diet with 0.4 % formic acid and 0.4 % lactic acid added; LC, challenged pigs consuming plain diet with 1 % lactulose added; PD, challenged pigs consuming plain diet without additives; UC, unchallenged pigs. †bd, below detection limit (<4.13 Log 10 bacteria/mL sample). a,b Different superscripts within the same column indicate significant differences within the values (P<0.05).

  A c c e p t e d M a n u s c r i p t 8 samples by using the universal primers P2 (5′-ATTACCGCGGCTGCTGG-3′) and P3 (5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAG GCAGCAG-3′) ofMuyzer et al. (1993) according to[START_REF] Waldram | Top-down systems biology modelling of host metabotype-microbiome associations in obese rodents[END_REF]. Primers were synthesized by Sigma-Genosys, UK.

  one single experimental treatment (UC) and analysed together with the challenged animals received the three diets (PD, AC, LC).

  no differences were observed at say 4 post-challenge or 10 post-challenge.

  Figure 2. The high complexity and inter-individual differences in the DGGE profiles

Salmonella

  Typhimurium in experimentally challenged pigs. The mixture of OA assayed had shown positive results against Salmonella in growing-finishing pigs

  Salmonella seroprevalence compared to a control diet and also decreases in the presence of Salmonella in faeces and mesenteric lymph nodes. Similarly, Do Santos et al. (

Fig. 1 .

 1 Fig.1. Box plot of the number of DGGE bands detected in caecal digesta of pigs

Fig. 2 .

 2 Fig.2. Dendrograms generated from DGGE profiles obtained from caecal digesta of

  Figure 1

  Figure 2

Table 2

 2 Probes used for FISH analysis of bacterial populations in ileal and caecal samples

	Short name	Accession	Full name b	Target species	Sequence (5´ to 3´)	Reference
		no. a
	Bif164	pB-00037	S-G-Bif-0164-a-A-18 Most Bifidobacterium spp. and Parascardovia denticolens CATCCGGCATTACCACCC	Langendijk et al., 1995
	Bac303	pB-00031	S-*-Bacto-0303-a-A-	Most Bacteroides sensu stricto and Prevotella spp.; all	CCAATGTGGGGGACCTT	Manz et al., 1996
			17	Parabacteroides; Barnesiella viscericola and
				Odoribacter splanchnicus
	Erec482	pB-00963	S-*-Erec-0482-a-A-19 Most members of Clostridium cluster XIVa;	GCTTCTTAGTCARGTACCG	Franks et al., 1998
				Syntrophococcus sucromutans, [Bacteroides]
				galacturonicus and [Bacteroides] xylanolyticus,
				Lachnospira pectinschiza and Clostridium
				saccharolyticum
	Lab158	ND	S-G-Lab-0158-a-A-20 Most Lactobacillus, Leuconostoc and Weissella spp.;	GGTATTAGCAYCTGTTTCCA Harmsen et al.,
				Lactococcus lactis; all Vagococcus, Enterococcus,
				Melisococcus, Tetragenococcus, Catellicoccus,
				Pediococcus and Paralactobacillus spp.
	Prop853	ND	ND	Clostridium cluster IX	ATTGCGTTAACTCCGGCAC Walker et al., 2005
	Rbro730 c	pB-00558	S-*-Rbro-730-a-A-18 Ruminococcus bromii-like; Clostridium sporosphaeroides	TAAAGCCCAGYAGGCCGC	Harmsen et al.,
				and Clostridium leptum
	Rfla729 c	pB-00557	S-*-Rfla-729-a-A-18 Ruminococcus albus and Ruminococcus flavefaciens	AAAGCCCAGTAAGCCGCC	Harmsen et al.,
	Sal303	ND	L-S-Sal-1713-a-A-18 Different serovars of Salmonella spp.	AATCACTTCACCTACGTG	Nordentoft et al., 1997
	a ND, No information relating to these probes has been deposited in probeBase (http://www.microbial-ecology.net/probebase).

Table 3

 3 Concentrations and molar ratios of various fatty acids found in ileal and caecal samples of pigs challenged or unchallenged with Salmonella Values are given as means and residual standard deviation (RSD) (AC, LC, PD, n=6; UC, n=9). *AC, challenged pigs consuming plain diet with 0.4 % formic acid and 0.4 % lactic acid added; LC, challenged pigs consuming plain diet with 1 % lactulose added; PD, challenged pigs consuming plain diet without additives; UC, unchallenged pigs. †Sum of acetic, propionic, butyric, valeric and BCFA. a,b Different superscripts within the same column indicate significant differences within the values (P<0.05).

	Typhimurium at 4 and 10 days post-challenge.
	Treatment*	Concentration (mM) on		Molar ratio of total SCFA (%)		Concentration (mM)		Molar ratio of total SCFA (%)
		Day 4 post-challenge		on Day 4 post-challenge		on Day 10 post-challenge		on Day 10 post-challenge
		Lactic Formic	Total	Acetic Propionic Butyric Valeric BCFA Lactic Formic	Total	Acetic Propionic Butyric Valeric BCFA
				SCFA †								SCFA †
	Ileum
	AC	18.60 10.47	8.45	77.04	7.03	13.96	1.33		27.99 12.02	8.75	79.57	9.18	8.93	0.97	1.35
	LC	9.03 12.17	8.94	81.00	1.69	15.52	1.22		8.27 11.68	9.09	84.35	1.82	12.3	0.91	0.63
	PD	9.49	6.86	6.24	85.62	2.22	9.86	1.98		23.82 11.26	8.34	83.76	5.14	9.07	1.15	0.87
	UC	13.26 17.20	11.87	83.43	1.60	13.31	1.10		14.41 10.07	7.45	80.43	2.68	13.08	1.61	2.19
	RSD	9.776	7.422	3.720	6.290	5.008	6.165 0.891		25.55	7.132	4.538	6.734	7.360	4.472 0.834	1.120
	P-treatment 0.326	0.092	0.063	0.135	0.200	0.431 0.308		0.532	0.953	0.904	0.150	0.314	0.221 0.361	0.064
	Caecum
	AC	8.85	3.44	73.75 ab 60.22 28.60	7.75	1.76	1.67	7.11	2.66	110.33	56.94 31.23	8.60	2.32	0.92
	LC	12.36	3.78	70.61 b	57.63 31.30	7.74	1.67	1.66	16.64	5.16	98.12	54.84 31.90	9.23	2.90	1.14
	PD	3.41	1.37	80.85 ab 59.36 28.81	7.82	2.47	1.55	19.15	3.93	85.35	59.73 26.40	10.14	2.91	0.82
	UC	9.28	4.11	109.82ª	60.35 27.29	9.52	1.89	0.95	10.59	3.52	128.82	57.07 28.96	10.75	2.40	0.82
	RSD	9.779	3.043	24.34	4.461	3.531	1.984 1.123	0.789 16.99	3.416	32.71	6.845	7.915	2.885 1.328	0.428
	P-treatment 0.472	0.386	0.020	0.677	0.235	0.236 0.615	0.248	0.419	0.660	0.105	0.641	0.624	0.523 0.778	0.519

Table 4

 4 Bacterial populations, as determined by FISH analysis, present in ileal and caecal samples of pigs challenged or unchallenged with Salmonella Typhimurium at 4 and 10 days post-challenge Log 10 bacteria/mL sample on day 4 post-challenge Log 10 bacteria/mL sample on day 10 post-challenge Values are given as LS means and residual standard deviations (RSD) (AC, LC, PD, n=6; UC, n=9).

	Treatment*
		Bac303 Bif	Erec	Lab	Prop	Rbro	Sal303 Bac303 Bif	Erec	Lab	Prop	Rbro	Sal303
	Ileum
	AC	5.57	5.37	5.58	6.80	7.46	4.88	5.91	5.37 b	5.48	5.33	6.57	6.86	4.93 ab 5.53
	LC	5.73	5.15	5.69	6.62	7.04	4.88	5.73	5.46 ab	5.55	5.09	6.76	6.99	4.61 b	5.52
	PD	5.62	5.27	5.63	6.41	7.15	5.04	5.57	5.31 b	5..49 4.77	6.65	6.60	5.07 a	5.59
	UC	5.11	5.10	5.67	7.01	7.01	4.88	bd †	5.77 a	5.67	5.34	6.54	6.90	5.19 a	bd †
	RSD	0.482	0.270 0.309 0.520 0.377 0.465 0.289	0.303	0.259 0.409 0.474 0.537 0.184 0.107
	P-treatment 0.097	0.271 0.946 0.183 0.154 0.912 0.167	0.035	0.468 0.120 0.835 0.713 <0.001 0.601
	Cecum
	AC	8.24 ab	5.35 b 8.21 ab 7.15	8.05	7.04	7.92	8.11	5.19	7.94	7.18	7.82	7.21	7.73
	LC	8.32 a	6.11 a	8.13 b	6.90	8.15	7.09	7.92	8.00	5.16	7.92	6.91	7.79	6.78	7.70
	PD	8.20 ab	5.87 ab 8.32 ab 6.96	8.14	7.09	8.01	8.07	5.66	8.00	6.96	7.72	6.99	7.81
	UC	8.02 b	5.43 b 8.40 a	7.45	8.09	7.09	bd †	8.06	5.68	8.06	6.91	7.82	7.11	bd †
	RSD	0.181	0.369 0.156 0.502 0.142 0.233 0.131	0.173	0.482 0.181 0.504 0.250 0.300 0.101
	P-treatment 0.025	0.003 0.018 0.174 0.588 0.967 0.421	0.755	0.101 0.523 0.732 0.923 0.095 0.302

Acknowledgements

The Universitat Autònoma de Barcelona was financially supported by a Ministerio de Ciencia y Tecnología project (AGL2003-08370-C02-01) from which derived a grant received by Sandra Martín-Peláez. The authors thank Mr Simone Maccaferri for his help with DGGE analyses and Miss Teresa Ayats for her help in the laboratory.

A c c e p t e d M a n u s c r i p t 14 animals are mixed with deliberately inoculated animals would be useful in interrogating the power of OA to protect against naturally acquired infection.

The inclusion of lactulose in the feed did not influence Salmonella Typhimurium survival compared to PD. This lack of effect could be related to a lack of stimulation of fermentative activity in the ileum or caecum, as indicated by the scarce variations found in the amounts and patterns of SCFA produced. This absence of increases in the fermentative activity could be related to the rapid fermentability of lactulose found by other authors (Kamphues et al., 2007), implying that lactulose would be almost completely fermented before reaching the large intestine. In addition, a lactulosesplitting capacity of the pig's small intestine mucosa has been indentified (Harju, 1986).

It is possible that the dose of lactulose used in this study (1 %, w/v), although recommended by other authors to prevent Escherichia coli infection [START_REF] Sutton | Effects of dietary carbohydrates and organic acid additions on pathogenic E. coli and other microorganisms in the weaning pig[END_REF] or salmonellosis in fattening pigs (Wiemer, 1999), was insufficient.

Another study involving fattening pigs used higher doses of lactulose (2.8 %, w/v) and did not demonstrate any protective effect of lactulose against Salmonella, with the authors attributing this to an insufficient dose used, based on the absence of significant changes in total SCFA and lactic acid along the gut (Kamphues et al., 2007).

The inclusion of lactulose did not reduce the invasive capacity of Salmonella, since all pigs were Salmonella-positive in mesenteric lymph nodes.It is known that an increase in a specific SCFA, such as butyric acid, can downregulate the expression of virulence genes of Salmonella [START_REF] Cherrington | Organic acids: chemistry, antibacterial activity and practical applications[END_REF]. [START_REF] Gantois | Butyrate specifically down-regulates A c c e p t e d M a n u s c r i p t 19[END_REF] speculated that modifications of the gut microbiota to produce more butyric acid could reduce invasive infections by Salmonella serovars in farm animals. Previous studies in vitro have shown increases in the percentage of butyric acid formed with the fermentation of lactulose (Martín-Peláez et al., 2008). However, in the present study, A c c e p t e d M a n u s c r i p b Probe designation according to [START_REF] Alm | The oligonucleotide probe database[END_REF]. This information was retrieved from probeBase. c These probes were used together in equimolar concentrations (both at 50 ng/µL). Formamide (20 %) was included in the hybridization buffer.