Accepted Manuscript

Title: Use of a live attenuated Salmonella enterica serovar Typhimurium

vaccine on farrow-to-finish pig farms

Author: L. De Ridder, D. Maes, J. Dewulf, P. Butaye, F. Pasmans, F. Boyen, F.

Haesebrouck, Y. Van der Stede

PII: S1090-0233(14)00381-5

DOI: http://dx.doi.org/doi: 10.1016/j.tvjl.2014.09.012

Reference: YTVJL 4277

To appear in: The Veterinary Journal

Accepted date: 13-9-2014

Please cite this article as: L. De Ridder, D. Maes, J. Dewulf, P. Butaye, F. Pasmans, F. Boyen, F.

Haesebrouck, Y. Van der Stede, Use of a live attenuated Salmonella enterica serovar

Typhimurium vaccine on farrow-to-finish pig farms, The Veterinary Journal (2014),

http://dx.doi.org/doi: 10.1016/j.tvjl.2014.09.012.

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Use of a live attenuated Salmonella enterica serovar Typhimurium vaccine on farrow-to-1

finish pig farms 2 3

4

L. De Ridder a,b

, D. Maes b,

*, J. Dewulf b, P. Butaye

a,c, F. Pasmans

c, F. Boyen

c, F. 5

Haesebrouck c, Y. Van der Stede

a,d 6

7 a Unit of Co-ordination Veterinary Diagnose-Epidemiology and Risk Analysis, CODA-8

CERVA, Groeselenberg 99, 1180 Ukkel, Belgium 9 b Department of Obstetrics, Reproduction and Herd health, Faculty of Veterinary Medicine, 10

Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium 11 c Department of Pathology, Bacteriology and Avian diseases, Faculty of Veterinary Medicine, 12

Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium 13 d Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, 14

Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium 15

16

17

18

19

* Corresponding author. Tel.: +32 9 2647542. 20

E-mail address: Dominiek.Maes@ugent.be (D. Maes). 21

22

Page 1 of 22

23

Highlights 24

Salmonella enterica infection in pigs is economically important and poses a risk for 25

salmonellosis in humans. 26

The usefulness of an attenuated S. enterica serovar Typhimurium (S. Typhimurium) 27

strain to control S. enterica infections was investigated in three farrow-to-finish pig 28

herds infected with S. Typhimurium. 29

Within each herd, 120 piglets were orally vaccinated at 3 and 24 days of age, and 120 30

piglets served as unvaccinated controls. At specific time points, faeces, ileocaecal 31

lymph nodes caecal contents were examined for S. enterica by isolation and serum 32

was analysed for antibodies against S. enterica by ELISA. 33

All pigs were weighed at suckling and slaughter age to obtain complementary data on 34

the daily weight gain. 35

Prior to slaughter, there were significantly less S. enterica-excreting pigs, a 36

significantly lower S. enterica-specific mean antibody titre and a significantly higher 37

mean daily weight gain in vaccinated animals compared to unvaccinated controls. 38

In two herds, there were significantly lower proportions of S. enterica-positive 39

ileocaecal lymph nodes and caecal contents at slaughter between the vaccinated and 40

control groups, but this difference was not significant across all three herds. 41

S. enterica with the same auxotrophic characteristics and genotype as the vaccine 42

strain was isolated from several samples of faeces, ileocaecal lymph node and caecal 43

contents from vaccinated pigs. 44

These findings indicate that vaccination with an attenuated S. Typhimurium strain 45

reduces S. enterica shedding in S. enterica problem herds, but without consistent 46

Page 2 of 22

reduction of S. enterica loads and with possible persistence of the vaccine strain in 47

tissues. 48

49

Abstract 50

Salmonella enterica infection in pigs is economically important and poses a zoonotic 51

risk. In this study, the efficacy of an attenuated S. enterica serovar Typhimurium strain was 52

evaluated in three farrow-to-finish pig herds. In each herd, 120 piglets were vaccinated orally 53

at 3 and 24 days of age, while 120 piglets served as unvaccinated controls. Faeces, ileocaecal 54

lymph nodes and caecal contents were examined for S. Typhimurium by isolation and serum 55

was analysed for antibodies against S. Typhimurium by ELISA. All pigs were weighed at pre-56

weaning and slaughter to determine daily weight gain. In vaccinated pigs prior to slaughter, 57

significantly fewer animals excreted S. enterica, there was a significantly lower S. enterica-58

specific mean antibody titre and there was a significantly higher mean daily weight gain 59

compared to unvaccinated controls. In two herds, there were significantly lower proportions 60

of S. enterica positive ileocaecal lymph nodes and caecal contents at slaughter between the 61

vaccinated and control groups, but this difference was not significant across all three herds. S. 62

enterica with the same auxotrophic characteristics and genotype as the vaccine strain was 63

isolated from several samples of faeces, ileocaecal lymph nodes and caecal contents from 64

vaccinated pigs. These findings indicate that vaccination with an attenuated S. Typhimurium 65

strain reduces S. enterica shedding, but the reduction is not consistent and the vaccine strain 66

may persist in tissues. 67

68

Keywords: Salmonella enterica serovar Typhimurium; Pigs; Live attenuated vaccine; Field 69

trial70

Page 3 of 22

Introduction 71

The largest number of reported food-borne outbreaks in the EU is currently caused by 72

Salmonella enterica (26.6% of all outbreaks) (EFSA, 2013). In 2011, approximately 56.8% of 73

human salmonellosis cases were related to pigs and their products (EFSA, 2013). Although 74

most infected pigs do not exhibit clinical signs, S. enterica infections may reduce weight gain 75

(Boyen et al., 2009; Davis et al., 2010). Biosecurity measures may help to decrease levels of 76

S. enterica on pig farms (Lo Fo Wong et al., 2004a; Baptista et al., 2010), but they are often 77

insufficient as stand-alone measures. 78

79

Most vaccination studies against S. enterica in pigs have reported beneficial effects 80

(Springer et al., 2001; Roesler et al., 2004; Lindner et al., 2007; Selke et al., 2007; Rösler et 81

al., 2010; Leyman et al., 2012; Farzan and Friendship, 2010; Arguello et al., 2013; De Ridder 82

et al., 2013). However, most of these studies were conducted with relatively small numbers of 83

pre-weaning (Rösler et al., 2010) or weaned piglets (Springer et al., 2001; Roesler et al., 2004; 84

Selke et al., 2007; Leyman et al., 2012; De Ridder et al., 2013) and used a challenge infection 85

protocol. Their relevance for field conditions needs to be verified with large numbers of 86

finisher pigs (Lindner et al., 2007; Farzan and Friendship, 2010; Arguello et al., 2013). 87

88

Currently, the only S. enterica vaccine available commercially for use in pigs in the EU 89

is a live S. enterica serovar Typhimurium vaccine (Salmoporc, IDT Biologika), authorised 90

only in Germany and Poland. This vaccine has been evaluated on a large scale in sows and 91

weaned piglets at 3 and 6 weeks of age (Lindner et al., 2007). In a small scale experiment, 92

administration of two doses of this vaccine to pre-weaning piglets at 3 and 24 days of age 93

significantly reduced organ colonisation and faecal excretion (Rösler et al., 2010). This early 94

vaccination scheme at 3 and 24 days of age minimises labour, but its efficacy needs to be 95

Page 4 of 22

confirmed by large scale studies monitoring pigs to slaughter age. In addition, the possible 96

interference of this vaccination protocol with S. enterica serosurveillance requires 97

investigation. In the present study, we evaluated S. enterica-specific antibody titres in serum, 98

the presence of S. enterica in faeces and tissues, and weight gain of pigs after oral 99

immunisation with this commercially available live attenuated S. Typhimurium vaccine at 3 100

and 24 days of age. 101

102

Materials and methods 103

Herd selection and experimental animals 104

Three pig herds (A, B and C) were selected based on the following inclusion criteria: (1) 105

a high S. enterica-specific antibody level in finishing pigs as determined by the Belgian 106

national Salmonella Surveillance Programme, in accordance with EU Regulation number 107

2160/2003 (Van der Stede et al., 2008); (2) being a closed farrow-to-finish herd; (3) the 108

feasibility of monitoring piglets from birth to slaughter; and (4) agreement by the farmer to 109

apply the same management practices during the study as used previously, with the exception 110

of the use of antimicrobial agents, which was not allowed from 5 days before vaccination 111

until 5 days after vaccination. 112

113

The numbers of sows in herds A, B and C were 200, 180 and 300, and they practised a 114

3, 1 and 2 week batch production system, respectively. The three herds had a conventional 115

health status and did not experience substantial specific clinical disease problems. Pigs in all 116

three herds were vaccinated against Mycoplasma hyopneumoniae and pigs in herd B were 117

vaccinated against porcine circovirus type 2. Anthelmintic treatment was applied every 5 to 6 118

weeks in the fattening unit. Fully slatted floors were present in the nursery and fattening units 119

of all three herds. The pens held 25-30 pigs in the nursery units and 10-15 pigs in the 120

Page 5 of 22

fattening units. Weaned piglets received non-medicated liquid feed and fattening pigs 121

received liquid feed (herd A) or meal (herds B and C). 122

123

On each farm, 20 pregnant sows (Topigs20) were selected 3 days after farrowing, and 124

their piglets were randomly allocated at litter level to a vaccinated or unvaccinated group. All 125

pre-weaning piglets from each sow were included in the study and monitored until slaughter 126

age. The sows from the piglets in each group had the same parity distribution. The study was 127

approved by the ethical committee of the Faculty of Veterinary Medicine, Ghent University, 128

Belgium (EC 2011/196, date of approval January 2012). 129

130

Vaccination and sampling design 131

Details of the study design are summarised in Fig. 1. In each herd, ~120 piglets from 10 132

litters were vaccinated orally at 3 and 24 days of age with an attenuated histidine-adenine 133

auxotrophic S. Typhimurium strain (Salmoporc, IDT Biologika) (V, vaccination group), while 134

120 piglets from 10 other litters were unvaccinated (C, control group). Transfer of piglets 135

from one sow to another was not allowed after 3 days of age. All piglets were weaned at 24 136

days of age. From weaning until slaughter age, the pigs in the V and C groups were kept 137

separately on the farm, during transportation to the abattoir and in the lairage. 138

139

Within each herd, rectal faeces and blood were collected serially from 30 randomly 140

selected pigs per group. Blood was collected into plain tubes (Sarstedt 15 mL). At slaughter, 141

caecal contents and ileocaecal lymph nodes were collected from the same 30 pigs, while 142

ileocaecal lymph nodes were collected from an additional 40 randomly selected slaughter 143

pigs. All pigs were weighed to calculate the daily weight gain (DWG) between 3 days and 29 144

weeks of age. 145

Page 6 of 22

146

Bacteriology 147

S. enterica was isolated from ~20 g faecal samples (rectal and caecal contents) using the 148

ISO 6579 Annex D method (ISO, 2007). Samples were diluted 1:10 in buffered peptone water 149

(BPW, Bio-Rad) and incubated for 16-20 h at 37 °C. A quantity of 100 μL from every pre-150

enrichment solution was spotted onto one modified semi-solid Rappaport-Vassiliadis plate 151

(MSRV, Bio-Rad) and incubated for 46-50 h at 41 °C. A loopful from the edge of a typical 152

migration zone on each MSRV plate was streaked onto a xylose lysine deoxycholate agar 153

plate (XLD, Bio-Rad) and a brilliant green agar plate (BGA, Lab M). Both plates were 154

incubated at 37 °C for 21-27 h. When S. enterica presumptive colonies were present on both 155

the XLD and BGA plates, one colony was inoculated in triple sugar iron agar (TSI, Bio-Rad), 156

sorbitol agar (Becton Dickinson) and lysine decarboxylase broth (Oxoid), and incubated for 157

18-24 h at 37 °C. 158

159

Ileocaecal lymph nodes (~10-13 g) were flamed briefly to decontaminate the surface, 160

then sliced, diluted 1:10 in BPW and homogenised with a stomacher blender (BagMixer, 161

Interscience) for 1 min. Samples were then processed as for faecal samples. 162

163

Isolates from vaccinated pigs were tested using the S. enterica Diagnostic Kit, produced 164

by the manufacturer of the vaccine (IDT Biologika). This kit distinguishes wild type S. 165

enterica strains from the vaccine strain through the use of two fluid media (A and B). Since 166

the vaccine strain is histidine-adenine auxotrophic, it only grows in medium B, which 167

contains histidine and adenine, whereas wild type strains grow in both media. In addition, 168

isolates were typed by multiple locus variable number tandem repeat analysis (MLVA) 169

(Larsson et al., 2009) and pulsed field gel electrophoresis (PFGE) (Peters et al., 2009). 170

Page 7 of 22

171

Serology 172

After coagulation, blood samples were centrifuged for 5 min at 1500 g to collect serum, 173

which was diluted 20-fold and analysed for S. enterica-specific antibodies with a commercial 174

ELISA kit based on lipopolysaccharide (LPS) O-antigens of serogroups B, C1 and D 175

(HerdChek Swine Salmonella, IDEXX Laboratories). Results are expressed as a sample to 176

positive ratio (S:P); samples with S:P ratios ≥ 0.25 (OD% ≥ 10) were defined as positive. 177

178

Statistical analysis 179

The number of positive faecal samples was compared between vaccinated and control 180

animals by means of generalised estimating equations (GEE; Proc Genmod in SAS 9.2). A 181

logit link function, a binomial distribution and an exchangeable correlation structure were 182

used; correlation is the same among any two observations from the same animal. Mortality 183

and isolation data at slaughter were analysed using the Pearson χ2 test in SAS 9.2. In these 184

analyses, samples containing the vaccine strain were considered to be negative for S. enterica. 185

186

The differences in S:P ratios between vaccinated and control animals were analysed by 187

repeated measures analysis of variance (ANOVA) using the Proc Mixed procedure with 188

lsmeans and slice options in SAS 9.2. Time and group (vaccinated and control) were included 189

in the model as fixed effects, and the herd as a random effect. An unstructured covariance 190

structure was selected, based on the lowest Akaike Information Criterion (AIC) value. To 191

adjust for the multiple comparisons in this procedure, the Ryan-Einot-Gabriel-Welch 192

(REGWQ) test was used. P values < 0.05 were considered to be significant (two-sided tests). 193

194

Page 8 of 22

The DWG between 3 days and 29 weeks of age for each pig (excluding pigs that died 195

before slaughter) was normally distributed and was compared between the vaccinated and 196

control groups using the Proc Mixed procedure with the lsmeans option and taking into 197

account the herd effect (SAS 9.2). 198

199

Results 200

Some animals in both groups were lost for follow-up due to mortality (runts, physical 201

abnormalities or idiopathic causes) or lost ear tags (Fig. 1). There was no significant 202

difference in mortality between groups V (50/347, 14.4%) and C (59/362, 16.3%; P = 0.90). 203

204

Bacteriology of faecal samples collected on-farm 205

The proportions of S. enterica positive faecal samples collected on-farm are shown for 206

the three sampling occasions in Fig. 2. Some data are missing, due to an empty rectum or lost 207

ear tags (Fig. 1). Across all herds, the proportions of pigs excreting S. enterica did not differ 208

significantly between groups V and C at 10 weeks of age (9/99, 9.1% vs. 11/98, 11.2%, 209

respectively; P = 0.46) and 16 weeks of age (9/89, 10.1% vs. 11/82, 13.4%, respectively; 210

P = 0.50). However, 2 weeks prior to slaughter (at 29 weeks of age), this proportion was 211

significantly different between V (7/79, 8.9%) and C (22/79, 27.8%; P < 0.01). 212

213

When analysing each herd separately, the proportion of pigs excreting S. enterica was 214

also lower in herd B (1/29, 3.4% for V vs. 12/29, 41.4% for C; P < 0.01) and herd C (0/22, 215

0.0% for V vs. 6/22, 27.3% for C; P < 0.01), whereas this was not the case in herd A (6/28, 216

21.4% for V vs. 4/28, 14.3% for C; P = 0.49). 217

218

Bacteriology of faecal samples collected at the abattoir 219

Page 9 of 22

The proportions of S. enterica positive samples from ileocaecal lymph nodes and caecal 220

contents collected at the abattoir are presented in Table 1; some data are missing, due to lost 221

ear tags (Fig. 1). Over all herds, there were no significant differences in the proportions of 222

samples positive by isolation for S. enterica between groups V and C for ileocaecal lymph 223

nodes (73/201, 36.3% vs. 72/180, 40.0%, respectively; P = 0.46) and caecal contents (37/121, 224

30.6% vs. 49/115, 42.6%, respectively; P = 0.06). 225

226

When analysing each herd separately, significant differences were found in the 227

proportions of samples positive by isolation for S. enterica between groups V and C in two 228

herds: (1) in lymph nodes from herd B (37/64, 57.8% for V vs. 47/61, 77.0% for C; P < 0.03); 229

and (2) in caecal contents from herd C (9/49, 18.4% for V vs. 24/44, 54.5% for C; P < 0.01). 230

231

Detection of the vaccine strain of Salmonella enterica 232

The proportions of samples putatively positive for the S. enterica vaccine strain in each 233

herd are presented in Table 2. Using the Salmonella Diagnostic Kit, 3/99 (3.0%), 2/89 (2.2%) 234

and 3/79 (3.8%) isolates from faecal samples of vaccinated pigs were histidine-adenine 235

auxotrophic at 10, 16 and 29 weeks of age, respectively. Histidine-adenine auxotrophic 236

isolates were detected in 10/121 (8.3%) samples of caecal contents and 4/201 (2.0%) samples 237

of lymph nodes. All histidine-adenine auxotrophic isolates belonged to the same MLVA type 238

and had the same PFGE profile as the vaccine strain. 239

240

Serology 241

S. enterica-specific antibody levels are shown for each group, herd and sampling 242

occasion in Fig. 2. At 3 days of age, prior to vaccination, mean ± (standard deviation, SD) S:P 243

ratios across all herds were comparable between groups V (1.50 ± 0.87; n = 120) and C 244

Page 10 of 22

(1.53 ± 0.79; n = 119; P = 0.73). In herd A, there was a significant difference in mean (± SD) 245

S:P ratios between groups V (2.11 ± 0.68; n = 40) and C (1.75 ± 0.58; n = 40; P < 0.03). 246

247

S:P ratios in groups V and C were three- to five-fold lower at 10 weeks of age than at 3 248

days of age (P < 0.001). The mean (± SD) S:P ratio was significantly higher in group V 249

(0.49 ± 0.51; n = 100) than group C (0.31 ± 0.46; n = 102; P < 0.01). This was mainly due to 250

the significant differences in mean (± SD) S:P ratios between groups V and C in herds A (V: 251

0.43 ± 0.33; n = 35 vs. C: 0.18 ± 0.21; n = 36; P < 0.02) and C (V: 0.71 ± 0.70; n = 33 vs. C: 252

0.09 ± 0.21; n = 37; P < 0.01). In herd B, the mean (± SD) S:P ratio was 0.32 ± 0.36 (n = 32) 253

for V and 0.66 ± 0.66 (n = 29) for C (P < 0.01). 254

255

At 29 weeks of age, the mean (± SD) S:P ratio across all herds was significantly higher 256

in group C (1.07 ± 0.55; n = 89 for V vs. 1.52 ± 0.68; n = 87 for C; P < 0.01). This outcome 257

was mainly attributable to the results obtained in herds A (0.94 ± 0.39 for V vs. 1.53 ± 0.55 258

for C; P < 0.01) and C (0.86 ± 0.54 for V vs. 1.69 ± 0.71 for C; P < 0.01). On farm B, there 259

was no significant difference in mean (± SD) S:P ratios between groups V (1.39 ± 0.58) and C 260

(1.34 ± 0.75; P = 0.71). 261

262

Daily weight gain 263

Over all three herds, a significantly higher DWG (P < 0.01) was observed in group V 264

(n = 289; DWG 546.8 g, 95% confidence interval, CI, 537.7-555.8 g) compared to group C 265

(n = 302; DWG, 509.3 g, 95% CI 499.4-519.2 g; Table 3). This result was consistent in each 266

herd; differences in mean DWG between groups V and C were 29.6 g in herd A, 37.2 g in 267

herd B and 38.9 g in herd C (Table 3; P < 0.01). 268

269

Page 11 of 22

Discussion 270

In this study, a live vaccine administered orally to piglets at 3 and 24 days of age 271

resulted in lower S. enterica excretion in faeces and lower serum antibody levels in vaccinated 272

pigs than in control animals prior to slaughter. An increased DWG in vaccinated animals was 273

also evident. Apart from these beneficial effects, some limitations of the vaccine under field 274

conditions were found. 275

276

Although there was considerable variation among the three herds, significantly fewer 277

vaccinated pigs overall excreted S. enterica prior to slaughter compared to control pigs. This 278

is important, since finishing pigs may introduce S. enterica into the abattoir environment 279

(Letellier et al., 2009). In the vaccinated group, there was no significant difference in the 280

proportion of pigs excreting S. Typhimurium at 10 and 16 weeks, whereas at 29 weeks the 281

percentage was significantly lower; a possible explanation is that the level of infection with S. 282

Typhimurium in the herds included in the study was higher at 29 weeks than at 10 and 16 283

weeks, allowing more opportunity for improvement due to vaccination. A significantly lower 284

S. enterica-specific antibody level was observed before slaughter in vaccinated animals across 285

all herds, suggesting decreased S. enterica infection pressure at this stage of growth (Lo Fo 286

Wong et al., 2004b; Baptista et al., 2009). 287

288

The variable results among the three herds might be due to several reasons. Under field 289

conditions, pigs are infected at different points in time, with a herd-dependent and even batch-290

dependent variability in both infection pressure and host response (Beloeil et al., 2003; Lo Fo 291

Wong et al., 2004b; Rostagno et al., 2012). The presence of herd-specific S. enterica strains 292

might have affected vaccination (Van Parys et al., 2013). The degree of separation of V and C 293

groups was most stringent in herd C (separated rooms), followed by herd A (separated pens, 294

Page 12 of 22

vaccinated pens grouped together) and herd B (separated pens, vaccinated pens dispersed 295

among unvaccinated ones). Antibody titres and faecal excretion of S. enterica in herd C were 296

significantly reduced in vaccinated pigs at finishing, in contrast with herds A and B. This 297

suggests that the best vaccination results will be obtained when all pigs in a herd (or 298

shed/barn) are vaccinated (Arguello et al., 2013). Finally, different maternal antibody levels in 299

piglets at the time of vaccination might have contributed to herd variation. 300

301

Piglets were vaccinated at 3 and 24 days of age to induce protection against possible 302

infection in the nursery period. However, most pigs seroconverted to S. enterica at the start of 303

the fattening period, similar to other studies (Beloeil et al., 2003). Nevertheless, vaccination at 304

an early age has some practical advantages, since piglets are normally handled at this time and 305

can be picked up easily to apply the vaccine orally. 306

307

Rösler et al. (2010) did not demonstrate a negative effect of maternal antibodies on S. 308

enterica vaccination using the same vaccine in sucking pigs. However, in the present study, 309

the herd with the highest maternal S. enterica-specific antibody levels in pigs at first 310

vaccination was the only herd without significantly decreased S. enterica excretion in the 311

vaccinated pigs at finishing stage. Thus, maternal immunity in this herd might have interfered 312

with oral vaccination. 313

314

In a previous study, the vaccine strain could not be detected in the lymph nodes of 315

slaughter pigs that had been vaccinated at 21 and 42 days of age (Lindner et al., 2007). 316

Therefore, persistence of the vaccine strain was not expected. However, the present study 317

indicated that S. enterica isolates similar to the vaccine strain could be detected in faeces 318

throughout the fattening period in all three herds and in the lymph nodes and caecal contents 319

Page 13 of 22

at slaughter. This finding might have important implications for bacteriological monitoring 320

programmes. It is uncertain whether the histidine-adenine auxotrophic S. Typhimurium 321

vaccine strain used in the present study is pathogenic for human beings. 322

323

The DWG was significantly higher in vaccinated pigs, suggesting that vaccination is 324

associated with a higher feed conversion ratio; however, the feed conversion ratio could not 325

be determined in the present study, since feed intake could not be recorded reliably. 326

327

Conclusions 328

The early vaccination scheme with a current commercial live attenuated S. 329

Typhimurium vaccine in pig herds with a high level of S. enterica infection reduced faecal 330

excretion and was associated with lower S. enterica-specific antibody titres at slaughter age 331

when compared to unvaccinated pigs. This vaccine could be a useful tool in the control of S. 332

enterica infection. However, the effects were variable between herds. Furthermore, there were 333

indications that the vaccine strain could persist until slaughter age. 334

335

Conflict of interest statement 336

None of the authors of this paper has a financial or personal relationship with other 337

people or organisations that could inappropriately influence or bias the content of the paper. 338

339

Acknowledgements 340

The authors thank the Federal Public Service of Health, Food Chain Safety and 341

Environment for funding this project (RT-09/05) and the participating farmers for their 342

cooperation. The assistance of veterinary colleagues and students in sampling, and the help of 343

Page 14 of 22

H. Vander Veken, M. Van Hessche and A. Lucchina in performing the diagnostic kit test, 344

MLVA and PFGE are gratefully acknowledged. 345

346

References 347

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by a Salmonella Typhimurium inactivated vaccine in Salmonella-infected finishing pig 349

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Farzan, A., Friendship, R.M., 2010. A clinical field trial to evaluate the efficacy of 383

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Diseases 36, 465-471. 451

452

453

Figure legends 454

455

Fig.1. Flowchart of the study design in the three farrow-to-finish herds. In total, 456

approximately 700 pigs were selected; half of them were vaccinated, whereas the other half 457

were unvaccinated and used as controls. Treatment pigs were vaccinated orally at 3 and 24 458

days of age with an attenuated Salmonella enterica serovar Typhimurium strain (Salmoporc, 459

IDT Biologika). Blood and faeces were collected from 90 vaccinated and 90 control pigs. 460

Sera were analysed by ELISA (HerdChek Swine Salmonella, IDEXX Laboratories). 461

Salmonella isolation (ISO 6579 Annex D) was performed on faeces. At slaughter, ileocaecal 462

lymph nodes (210 vaccinated and 210 control pigs) and caecal content (105 vaccinated and 463

105 control pigs) were collected for Salmonella isolation (ISO 6579 Annex D. All study pigs 464

were weighed at 3 days and 29 weeks of age to calculate daily weight gain (DWG). * Extra 465

data were gathered initially to account for possible mortality losses later. ° Some data were 466

missing due to identification (loss of ear tags) or sampling (empty rectum) problems. 467

468

Fig. 2. Pigs excreting Salmonella enterica (%) (A, C, E) and S. enterica-specific antibody 469

levels in blood (mean S:P ratio) (B, D, F) per group and herd at 3 days of age, 10 weeks of 470

Page 17 of 22

age, 16 weeks of age and 29 weeks of age (2 weeks prior to slaughter). Error bars represent 471

the 95% confidence interval. Asterisks (*) below the graphs indicate a significant difference 472

(P < 0.05) between the control (black) and vaccinated (grey) groups in herd A (A, B), herd B 473

(C, D) and herd C (E, F). 474

475

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Table 1 476

Proportion of slaughter samples positive for Salmonella enterica wild type strains for each vaccination and 477

control group in herds A, B and C in ileocaecal lymph nodes and caecal content. 478

479

Herd A Herd B Herd C Herds A+B+C

Vaccinated Control Vaccinated Control Vaccinated Control Vaccinated Control

Ileocaecal lymph nodes 25/66 (38%) 14/63 (22%) 37/64 (58%) a 47/61 (77%) b 11/71 (16%) 11/56 (20%) 73/201 (36%) 72/180 (40%)

Caecal content 6/39 (15%) 5/37 (14%) 22/33 (67%) 20/34 (59%) 9/49 (18%) a 24/44 (55%) b 37/121 (31%) 49/115 (43%)

480

Proportions in a row, emboldened and with different letters in superscript, are significantly different between the Vaccinated and Control 481

groups (P < 0.05). 482

483

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Table 2 484

Proportion of samples from vaccinated pigs, which possibly contain the Salmonella enterica serovar 485

Typhimurium vaccine strain in faeces at 10, 16 and 29 weeks of age, and in ileocaecal lymph nodes 486

and caecal contents at slaughter. 487

488

Herd

Faeces Ileocaecal lymph

nodes Caecal contents

10 weeks 16 weeks 29 weeks

A 2/35 (5.7%) 0/32 (0.0%) 0/28 (0.0%) 1/66 (1.5%) 2/39 (5.1%)

B 1/30 (3.3%) 1/28 (3.6%) 0/29 (0.0%) 0/64 (0.0%) 2/33 (6.1%)

C 0/34 (0.0%) 1/29 (3.4%) 3/22 (13.6%) 3/71 (4.2%) 6/49 (12.2%)

A+B+C 3/99 (3.0%) 2/89 (2.2%) 3/79 (3.8%) 4/201 (2.0%) 10/121 (8.3%)

489

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Table 3 490

Daily weight gain (DWG; g) between 3 days and 29 weeks of age in pigs in the Vaccinated and 491

Control groups for each of the three farrow-to-finish pig herds and over all herds. 492

493

Herd Mean DWG (95% confidence interval) Difference DWG (P)

Vaccinated Control

A 593.0 (578.8-607.3) 563.4 (549.0-577.9) 29.6 (< 0.01)

B 504.3 (488.9-519.8) 467.1 (452.5-481.7) 37.2 (< 0.01)

C 535.6 (520.9-550.4) 496.7 (482.3-511.0) 38.9 (< 0.01)

A+B+C 546.8 (537.7-555.8) 509.3 (499.4-519.2) 37.5 (< 0.01)

494

495

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496

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