Prevalence of border disease virus in Spanish lambs

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Prevalence of border disease virus in Spanish lambs

B. Valdazo-González, M. Álvarez, T. Sandvik

To cite this version:

B. Valdazo-González, M. Álvarez, T. Sandvik. Prevalence of border disease virus in Spanish lambs.

Veterinary Microbiology, Elsevier, 2008, 128 (3-4), pp.269. �10.1016/j.vetmic.2007.10.032�. �hal-

00532353�

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Title: Prevalence of border disease virus in Spanish lambs Authors: B. Valdazo-Gonz´alez, M. ´ Alvarez, T. Sandvik

PII: S0378-1135(07)00536-6

DOI: doi:10.1016/j.vetmic.2007.10.032

Reference: VETMIC 3872

To appear in: VETMIC Received date: 9-8-2007 Revised date: 27-10-2007 Accepted date: 31-10-2007

Please cite this article as: Valdazo-Gonz´alez, B., Alvarez, M., Sandvik, T., ´ Prevalence of border disease virus in Spanish lambs, Veterinary Microbiology (2007), doi:10.1016/j.vetmic.2007.10.032

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Prevalence of border disease virus in Spanish lambs

B. Valdazo-González

¹

*, M. Álvarez

¹

, and T. Sandvik

²

1

Departamento de Sanidad Animal, Universidad de León, 24071, León, Spain

2

Virology Department, Veterinary Laboratories Agency, Addlestone, Surrey, UK

*

Corresponding author: Present address: Subdirección de Investigación y Tecnología Instituto Tecnológico Agrario de Castilla y León Carretera de Burgos, Km. 119.

47071. Valladolid. Spain Tel.: +34 983 317 688 Fax: +34 983 414 780 E-mail address: ita-valgonbe@itacyl.es

b.valdazo.gonzalez@gmail.com

Manuscript

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Abstract 1

The prevalence of border disease virus (BDV) viraemia in Spanish lambs was determined 2

from 2,089 sera randomly collected at two slaughterhouses in 2001 and 2003, as well as in 3

126 sera obtained in 2004 from a fattening unit with an acute disease problem. BDV was 4

detected with an indirect peroxidase monolayer assay (IPMA), and for the fattening unit sera 5

also by an antigen ELISA. A subset of sera was additionally tested for BDV antibodies. The 6

BDV prevalence in the slaughterhouse sera was 0.24%, whereas 7.1% of randomly selected 7

and 38.6% of sera from clinically affected lambs in the fattening unit were virus positive.

8 Pestivirus antibodies were found in 17.6% of the slaughterhouse sera and 28.6% of those from 9

randomly selected lambs in the fattening unit. In total, 33 virus isolates and 3 antigen positive 10

samples were identified. Genetic typing of 5’-UTR sequences classified all 36 pestiviruses as 11

of BDV type 4. This shows that from a low BDV prevalence in apparently healthy lambs in 12

the entire sheep population, clinical problems associated with BDV can develop when 13

viraemic sheep are brought into intense rearing units.

14 15

Key words: Border disease virus; lamb; prevalence; IPMA; genetic typing.

16

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1. Introduction 17

Border disease (BD) is the clinical outcome of pestivirus infection in sheep. The signs 18

are very variable and can include: barren ewes, abortions, the birth of lambs with tremor and 19

other neurological signs, abnormal fleece or body conformation, or small offspring with poor 20

growth rate and viability (Nettleton, 2000). Besides the important economical losses caused as 21

primary pathogens, pestiviruses may compromise the normal immune response to other 22

pathogens and increase the severity of other infections in sheep (Hussin and Woldehiwet, 23

1994). The key for pestivirus control is the prompt identification and removal of persistently 24

infected (PI) individuals, combined with improved biosecurity measures to avoid reinfection.

25 PI animals have survived transplacental infection with a non-cytopathogenic (ncp) biotype of 26

pestivirus before onset of immunological competence, and usually remain virus positive and 27

antibody negative throughout their lives. Their survival rate is lower than normal animals, but 28

some can survive for years (Nettleton et al., 1992).

29 Three virus species in genus Pestivirus are known to infect naturally and produce BD 30

in sheep: BD virus (BDV), bovine viral diarrhoea virus (BVDV) type 1 and 2 (Vilcek et al., 31

1997; Sullivan et al., 1997; Pratelli et al., 2001). Thus for identification of a virus causing BD, 32

antigenic or genetic typing of virus isolates, or comparative virus neutralisation studies are 33

required (Paton et al., 1995). For genetic typing, analysis of the conserved 5´-untranslated 34

region (5´-UTR) of the genome or the N

^pro

gene are most commonly used (Vilcek et al., 1994;

35 Vilcek et al., 1997; Valdazo-González et al., 2007). Molecular characterization of 36

pestiviruses isolated from Spanish sheep has so far only identified BDV (Hurtado et al., 2003;

37 Valdazo-Gonzalez et al., 2006; 2007; Berriatua et al., 2006).

38 Pestivirus infections in sheep have been reported worldwide. Serological studies have 39

shown seroprevalences ranging between 5 and 50% for various countries and regions within 40

countries ( Nettleton, 2000). In Northern Spain, serological surveys have shown four to 21%

41 of the adult sheep and 10 to 93% of the sheep flocks to be pestivirus seropositive (Álvarez et

42

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al., 1989; Mainar-Jaime and Vázquez-Boland, 1999; Berriatua et al., 2006). Several studies 43

worldwide have reported prevalences of PI or viraemic sheep ranging from 0.3% to 20% in 44

flocks with clinical BD (Buonavoglia et al., 1994; Braun et al., 2002; Berriatua et al., 2004;

45 Valdazo-Gonzalez et al., 2006). In contrast, few investigations for BDV have been carried out 46

in sera from randomly sampled sheep flocks; however two such found no evidence of 47

pestiviruses in adult sheep (Hyera et al., 1991; Heckert et al., 1994).

48 Spain is the second largest sheep meat producing country within the European Union, 49

responsible for 24-25% of the meat production from 32% of the sheep slaughtered 50

(Anonymous, 2001-2003). The Spanish sheep meat demand is for light weight carcasses.

51 Hence, approximately 96% of slaughtered sheep are 25-45 days old lambs (suckling lamb), or 52

lambs weaned and fed ad libitum in the originating flock or fattening units up to 70-100 days 53

of age (young light lambs). This complicates studies on the prevalence of pestivirus viraemia 54

in Spanish sheep, in that a large proportion of the population, that in facts is most likely to be 55

BDV positive, is only available for sampling a relatively short time after birth, with 56

difficulties in obtaining convalescent samples to clarify differential diagnostic issues of 57

persistently versus acutely infected animals.

58 The aim of this study was to determine the prevalence of pestiviruses in Spanish 59

lambs. In samples obtained at slaughter, such data would provide background prevalence 60

information in a subpopulation of healthy lambs previously not investigated for pestiviruses.

61 Furthermore, samples from a fattening unit that was suffering an outbreak of disease were 62

also included, to assess the potential impact of BDV in lambs managed in an intense 63

production unit.

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2. Material and methods 65

2.1. Sample collection 66

Plain blood samples were obtained from two different slaughterhouses in the Province 67

of Leon, and a fattening unit in the Province of Teruel. The slaughterhouses were visited 68

mostly at the peak time for time for slaughter of sheep, in December 2001 (A) and 2003 (B).

69 Blood was collected during the bleeding, from 10 % of randomly selected lambs in each batch 70

from every supplier of animals (farmer, fattening unit, dealer or other trading agents).

71 Altogether around 7-8 % of all lambs slaughtered during these months were sampled. The 72

sampled lambs came from dairy and meat flocks in western Castile and Leon (central northern 73

Spain) and from Extremadura (western Spain). Of a total of 2,089 sera, 1,198 samples were 74

collected at slaughterhouse A whereas 891 came from slaughterhouse B. Of these, 1,285 were 75

from suckling and 804 from young light lambs, respectively (Table 1).

76 From the fattening unit, clotted blood samples from 126 lambs were received in March 77

2004. This 50,000 capacity unit, which contained only 15,000 animals at the moment of 78

sampling, was divided in three buildings (entrance, classification and fattening), each with 79

pens for 250-300 lambs. Animal supplies came from dairy and meat flocks from the whole of 80

Spain, southern Portugal, and occasionally from southwestern France. Lambs were supplied 81

to the unit at a minimum age of 45 days, and were sent to slaughter at approximately 100 days 82

old. Prophylactic routine treatment at arrival included desparasitation, methaphylaxis and 83

vaccination against Clostridium spp, Mannheimia haemolytica, Pasteurella multocida and 84

Branhamella ovis. Of the 126 submitted blood samples, 56 had been collected randomly from 85

different pens, whilst the remaining 70 samples were from lambs transferred to a quarantine 86

area after presenting signs of severe enteric and/or respiratory disease.

87 These disease problems began as a diarrhoea-associated increase in mortality (>5%) in 88

December 2003. Animals from different breed, origin and pens were affected, mostly in the 89

fattening building, but also at the entry. Desparasitation failure and changes in feed were

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found to be involved and solved. Other control measures such as the early slaughter of lambs 91

from most affected pens were set up. However, the problem reappeared and intensified with 92

the arrival of new lambs. Lambs with diarrhoea responded favourably to antibiotic treatment.

93 Those not treated died in 2 days time in a deteriorated condition. Post-mortem examinations 94

showed lesions mostly in the gastrointestinal tract and the mesenteric lymph nodes. Many 95

animals also presented pneumonic lesions from which Mannheimia haemolytica and 96

Pasteurella multocida were isolated.

97 The blood samples were transported to the laboratory at ambient temperature, where 98

they were centrifuged at 1,200 g for 10 minutes. Serum was removed and stored at -20ºC until 99

analysed.

100 With an estimated prevalence of viraemia of 0.29% reported in calves (Wittum et al., 101

2001), the slaughterhouse sampling size would determinate the virus prevalence for each 102

slaughterhouse and lamb age group with 95% confidence level at an admissible error less than 103

±0.6% (Win Episcope computer package, Frankena et al., 1990). For the 56 randomly 104

collected samples from the fattening unit, the admissible error for a 95% confidence level 105

estimate of the virus prevalence was ±1.4%.

106 107

2.2. Virus detection 108

All sera were tested using an indirect peroxidase monolayer assay (IPMA) microtiter 109

virus detection method as described (Valdazo-Gonzalez et al., 2006). Briefly, 96-well 110

microtiter plate cultures of secondary lamb kidney cells proven free from pestivirus 111

contamination were inoculated with 10 μl of test serum at the time of seeding, incubated for 7 112

days before fixing and immunostaining with the NS3-specific monoclonal antibody (mAb) 113

WB103 (VLA, Weybridge, UK) as primary antibody. Test sera were considered virus positive 114

if the characteristic cytoplasmatic staining could be detected microscopically, along with the 115

expected staining for positive, negative and cell controls included on each plate.

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IPMA positive sera were subjected to virus isolation in secondary lamb kidney cell 117

cultures as described (Valdazo-Gonzalez et al., 2006). Stocks of viruses were prepared and 118

stored at -80ºC.

119 Additionally, the 126 sera from the fattening unit were tested by a commercial BVDV 120

antigen capture ELISA also recommended for detection of the E

^rns

glycoprotein in ovine 121

serum samples (BVD-VIRUS III; Bommeli, Switzerland), according to the manufacturer’s 122

instructions.

123 124

2.3. Serological testing 125

All fattening unit sera, plus a subset (10%, as recommended by Nettleton et al, 2000) 126

of 210 randomly selected sera from the slaughterhouses were tested for neutralising 127

antibodies in a standard virus neutralization test (VNT) using the cytopathogenic Moredun 128

BDV strain as challenge virus. Sera were titrated in two-fold dilutions starting at 1:8 up to 129

1:512, and geometric mean titres (GMT) was calculated as described (Valdazo-Gonzalez et 130

al., 2006).

131 132

2.4. Statistical analysis 133

Demonstration of association between pestivirus status and type of lamb, location or 134

clinical status was carried out with the Yates-corrected Chi-square test. GMTs were subjected 135

to analysis of variance. Significance was considered for alpha = 5% (P<0.05) for a two-tailed 136

test. Data were analysed using the Epi Info, Version 6.04d (CDC). Odd ratios (OR) and 95%

137 confidence intervals (CI) were based on Cornfield's approximation or exact limits where 138

appropriate (StatXact Version 6.0, Cytel Software Corporation, Cambridge, MA).

139 140

2.5. Genetic typing

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Viral RNA was extracted from cell culture supernatants, or directly from serum if 142

virus isolation attempts had been negative. Nucleic acid extraction, reverse transcription- 143

polymerase chain reaction (RT- PCR) amplification of a 288 nt fragment of the 5’-UTR, 144

electrophoresis and identification of DNA bands was carried out as described (Valdazo- 145

Gonzalez et al., 2006; 2007). Positive and negative controls were included to validate the 146

different stages. PCR products were purified and cycle sequenced in both directions with the 147

324 and 326 primers using the ABI PRISM Big Dye Terminator Cycle Sequencing Kit 148

(Amersham), and analysed on an ABI PRISM 310 Genetic Analyzer. Computer-assisted 149

analysis of the nucleotide sequences was done as described by Valdazo-González et al. (2006) 150

except that the SeqMan II program from the DNASTAR program package (DNASTAR Inc., 151

USA) was used for assembly and proof reading of sequence track files.

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3. Results 153

3.1. Virus and antibody testing 154

Five of the slaughterhouse sera were virus positive, which corresponds to an over-all 155

prevalence of 0.24% (Table 1). This prevalence remained approximately at the same level for 156

the two years sampled, at 0.25 and 0.22%, respectively, and with no significant differences 157

for the two lamb age groups. For the individual batches of lambs from different suppliers that 158

the five virus positive animals came from, the prevalence ranged from 1% (95% CI: 0.00, 159

2.95) to 8.33% (95% CI: 0.00, 23.97) in suckling lambs, and 2.04% (95% CI: 0.00, 6.00) to 160

3.70% (95% CI: 0.00, 10.83) in young light lambs.

161 Antibodies to BDV were found in 17.6 % of the analysed slaughterhouse sera. A 162

statistically significant lower seroprevalence was found in young light lambs compared to 163

suckling lambs in slaughterhouse B (OR: 11.84; 95% CI: 1.65, 523.30), and also to young 164

light lambs from slaughterhouse A (OR: 8.97; 95% CI: 1.16, 409.60).

165 The GMTs were slightly higher in suckling lambs from slaughterhouse A (60.1 versus 166

48.5), though no statistically significant differences among the different groups were found.

167 None of the virus positive sera were antibody positive.

168 Of the 56 randomly collected sera from the fattening unit, four (7.14%) were virus 169

positive (Table 2) Of the 70 diseased lambs, 27 (38.6%) were virus or antigen positive. Of the 170

latter, 24 were positive by IPMA and 3 more with the antigen ELISA. None of the 171

virus/antigen positive sera scored antibody positive. The frequency of virus positive samples 172

was significantly higher in sera from diseased lambs (OR: 8.04; 95% CI: 2.53, 34.07) than in 173

randomly collected sera. In contrast no association was found between clinical status and 174

seropositivity or GMT, although both were higher in randomly collected sera although both 175

were higher in sera from randomly collected than diseased lambs (28.6% versus 14.3%; 56.2 176

versus 42.2; Table 2).

177

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3.2. Genetic typing 179

In total, 33 ncp pestivirus isolates were obtained from IPMA positive sera, whilst none 180

of the three antigen ELISA positive samples were virus isolation positive. However, the latter 181

three were positive by RT-PCR, giving a total of 36 amplicons that were included for 182

genotyping (Table 3). Aligning of partial 5’-UTR sequences with matching reference 183

sequences showed that the 36 sequences were closely related (seven were identical) to those 184

described by Valdazo-González et al. (2006, 2007), and therefore the same nomenclature was 185

assigned. Thirty-three out of 36 clustered within BDV subtype 4a, whilst the remaining three 186

were allocated to BDV subtype 4b (Fig. 1.).

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4. Discussion 188

This is the first large-scale study to determine the prevalence of pestivirus viraemia in 189

randomly sampled lambs in Spain, as well as to investigate for a potential pestiviral cause of 190

disease in feedlot lambs. Animals of different age, from multiple sheep breeds, types of 191

production (meat/milk) and districts representing the main sheep rearing regions of Spain 192

were included.

193 In the slaughterhouse sera, an over-all prevalence of viraemia of 0.24% was found.

194 This prevalence remained surprisingly constant in both lamb age groups (suckling and young 195

light lambs) as well as year of collection (slaughterhouse), and shows that pestivirus infection 196

appears to be endemic in sheep flocks that supplies the Spanish lamb meat market. This is 197

also supported by the antibody prevalences found in the subsets of slaughterhouse sera tested 198

for BDV neutralising antibodies, which match previously reported regional seroprevalence 199

studies of the adult sheep population in Spain (Álvarez et al., 1989; Mainar-Jaime and 200

Vázquez-Boland, 1999; Berriatua et al., 2006). Theoretically, maternal antibodies might have 201

lowered the diagnostic sensitivity of the IPMA when used for samples from suckling lambs 202

(Anonymous, 2004). This does not appear to have been the case since nearly similar 203

pestivirus prevalence figures were seen for the older age group of lambs. An assay 204

independent of maternal antibodies such as the RT-PCR might have been used alternatively, 205

but since the RT-PCR may give positive results several weeks after acutely infected animals 206

have seroconverted (Sandvik et al., 2000), it would not have been better for estimating the 207

prevalence of PI lambs, which the relatively lower analytical sensitivity of the IPMA makes it 208

better suited for (Saliki and Dubovi, 2004).

209 In other countries, even lower BDV prevalences have been reported. Virological 210

surveys in randomly selected ovine populations in Canada and Tanzania failed to isolate any 211

pestivirus from sheep sera, despite serological findings showed the infection was present 212

(Hyera et al., 1991; Heckert et al., 1994). The negative virus detection results may have been

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caused by sampling of a smaller group of older animals, among which fewer PI animals may 214

have survived, and, in one of the studies, the use of bovine cell cultures for virus isolation. A 215

mail survey among stud sheep farmers in Victoria (Australia) reported that 0.03% of the ewes 216

had produced lambs with BD-like symptoms (Lim and Carnegie, 1984); but this observation 217

might well match our findings for the slaughterhouse sera, since not all viraemic lambs show 218

clinical signs of infection ( Nettleton, 2000).

219 The prevalence of viraemic lambs was substantially higher in the fattening unit.

220 Among the 56 randomly sampled lambs, the prevalence matched the highest seen in an 221

individual batch of slaughterhouse lambs that contained a viraemic animal. Still this is not 222

unique among healthy lambs, Bonniwell et al. (Bonniwell et al., 1987) isolated pestiviruses 223

from 4 out of 24 (16.7%) lambs in an apparently healthy flock just before being marketed, and 224

Monies et al. (Monies et al., 2004) isolated BDV from 4 out of 15 (26.7%) lambs from a 225

breeding ewe flock with multiple origins.

226 Among the diseased lambs in the fattening unit, the virus prevalence was also 227

statistically significantly higher than among the randomly sampled lambs, at 38.6%. The 228

BDV antibody prevalence was also lower in this group of lambs, around half the percentage 229

obtained for randomly sampled lambs. Even if the antibody prevalence is calculated for only 230

the non-viraemic lambs in this group, the percentage was still substantially lower (23.3%) 231

than among the non-viraemic healthy lambs (30.8%). The exact contribution to the clinical 232

disease from BDV cannot be determined with certainty from the available data. However, 233

when both the lower seroprevalence and higher number of virus positive sick lambs are 234

considered, as well as additional observations from autopsy of some of the diseased lambs, in 235

which a high parasitic burden was seen (García Marín, J.F., 2004, personal communication), 236

it is likely that the pestivirus infection was playing a significant causative role in this outbreak 237

of disease. The fattening unit production system in Spain is analogous to cattle feedlot 238

operations in the USA, where pestiviruses are known to exacerbate other infections through

239

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immunosuppression (Campbell, 2004). Despite some effect of the antibacterial and -parasitic 240

prophylactic treatment on arrival, this would not limit horizontal spread of pestiviruses.

241 Acute infection with pestiviruses in healthy lambs is often asymptomatic, resulting in 242

a short viraemia with leucopenia before seroconversion ( Nettleton, 2000). Occasionally more 243

severe disease, killing up to 50% of young lambs has been seen (Chappuis et al., 1986), which 244

has been attributed to the virulence of the infecting virus strain. The reported over-all 245

mortality and observed morbidity ratios at the fattening unit does not indicate a highly 246

virulent virus strain was involved. The sequence data showed that the same virus was present 247

in both four clinically healthy lambs and in several of the diseased lamb; this could indicate 248

that this virus was of low virulence, but since it is cannot be known for sure if the healthy 249

lambs were PIs and the diseased lambs acutely infected, it is not possible to draw a definite 250

conclusion on the virulence of this virus from the sequence data alone.

251 When assessed for diagnosis of BVDV in cattle, most samples found positive by both 252

IPMA and antigen ELISAs tend to have come from PIs, which is explained by both the higher 253

virus titres and the constant viraemia, as compared to acutely infected animals (Sandvik, 254

2005). No similar data on the correlation of IPMA or antigen ELISA positivity and PI is 255

available for sheep. Definitive diagnosis of PI would require detection of virus but no 256

pestivirus antibodies in a convalescent blood sample, but unfortunately no such sample was 257

available from the initially sampled lambs in the fattening unit. It can only be speculated to 258

what extent the BDV positive lambs in the fattening unit were acutely or persistently infected, 259

but the sequence data suggest that both categories of infection were present, and that 260

horizontal spread from a number of PI lambs may have triggered the disease outbreak.

261 All 36 pestivirus 5’-UTR sequences described in the present study were classified as 262

BDV type 4, mainly BDV-4a, but some also as BDV-4b. These findings match previous 263

studies on genotyping of Spanish ovine pestiviruses (Hurtado et al., 2003; Valdazo-Gonzalez 264

et al., 2006; 2007; Berriatua et al., 2006), in that BDV type 4 appears to be unique in Spanish

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sheep, as other types of BDV are in other countries (Becher et al., 1994; Ridpath and Bolin, 266

1997; Vilcek et al., 1998; Becher et al., 2003; Thabti et al., 2005; Stalder et al., 2005;

267 Willoughby et al., 2006). Furthermore, no BVDVs were identified among the 36 pestivirus 268

sequences, unlike findings from the USA (Sullivan et al., 1997), UK, Sweden ( Vilcek et al., 269

1997), Italy (Pratelli et al., 2001), Ireland (Graham et al., 2001) and Norway (Sandvik et al., 270

2002), where either both BDV and BVDV have been seen in sheep (UK and USA), or only 271

BVDV (Sweden, Italy, Ireland and Norway).

272 The genetically homogenous nature of Spanish ovine pestiviruses may also have 273

implications for potential prophylactic measures aiming at reducing clinical losses due to BD 274

in Spain. Whereas genetical similarity often also means antigenic relatedness, it does not say 275

anything about the antigenic differences between separate groups of pestiviruses. Hence it 276

remains to be seen to what extent e.g. BVDVs, which have been used for manufacture of 277

numerous BVD vaccines, might be able to induce antibodies that would cross react with 278

BDV-4 if used in sheep. The optimal approach for control of BD in Spain would be 279

identification and elimination of PI animals in the breeding flocks. For this purpose, it would 280

be very convenient if samples from the current official small ruminant brucellosis eradication 281

programme could be used also for pestivirus diagnosis. At the fattening unit level, isolation 282

and pestivirus screening of lambs presenting enteric and/or respiratory disease would indicate 283

if a given problem might be due to BDV, but effective treatment may be difficult.

284 Prophylactic use of anti-BDV antibody positive serum might to some extent prevent clinical 285

disease in susceptible lambs, but would be less effective in acutely infected lambs if the 286

immunosuppressive effect already has set in.

287 288

5. Conclusion 289

A low but relatively constant prevalence of BDV at 0.24% was seen is sera from two 290

different age groups of lambs collected in two Spanish slaughterhouses in 2001 and 2003. In

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sera from a fattening unit, 7.1% of randomly selected and 38.6% of clinically affected lambs 292

were BDV positive. BDV-4 was the only detected pestivirus. This shows that from a low 293

BDV prevalence in apparently healthy lambs, clinical problems associated with BDV can 294

develop in intense rearing units.

295 296

Acknowledgements 297

We thank G. M. Fernández Bayón, for technical assistance. The cooperation of the proprietor, 298

veterinarians and other staff at the slaughterhouses and the fattening unit is also greatly 299

appreciated. M3 and M96 sequencing was carried out by BVG as a visiting scientist at 300

Institute of Virology, Hannover (Germany) under the supervision of Dr. Irene Greiser-Wilke.

301 Special thanks to M. M. Fernández Criado (DVM) for valuable information about the 302

fattening unit. BVG was the recipient of a pre-doctoral fellowship co-financed by ‘Junta de 303

Castilla y León’ (Autonomous Community government) and the European Social Fund. TS 304

was supported by project OD0345 funded by Defra.

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414 415

Legends to Tables 416

417

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Table 1. Prevalence of pestiviruses and pestivirus neutralising antibodies in sera from 418

suckling (age 25-45 days) and young light lambs (age 70-100 days) sampled in two Spanish 419

slaughterhouses.

420 421

Table 2. Prevalence of pestiviruses and pestivirus neutralising antibodies in sera from 45 to 422

100 days old lambs sampled in a Spanish fattening unit.

423 424

Table 3. Details of the 36 border disease virus isolates or amplicons obtained from samples 425

analysed in the present study.

426 427

Legends to Figures:

428 429

Figure 1: Neighbour-joining phylogenetic tree generated using 244-247 nucleotides of the 5´- 430

UTR of 36 ovine pestiviruses (labelled in bold italic typeface) collected in Spain, using 431

BVDV-2 as outgroup. Reference sequences from most of the currently identified groups of 432

genus Pestivirus were obtained from the GenBank database; their accession numbers have 433

previously been listed in Valdazo-González et al. (2006, 2007) except for 0501209-052GI 434

described by Berriatua et al. (2006). The groups BDV-1, -2 and -3 groups were proposed by 435

Becher et al. (2003), whilst BDV-4 with subgroups a and b was proposed by Valdazo- 436

González et al. (2006, 2007). The numbers on the branches show the confidence values (in 437

percent) for the grouping obtained by analysis of 1000 bootstrap replicates. Bar indicates 0.1 438

nucleotide substitution per site.

439

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1 Table 1. Prevalence of pestiviruses and pestivirus neutralising antibodies in sera from suckling (age 25-45 days) and young light lambs (age 70- 1

100 days) sampled in two Spanish slaughterhouses.

2 Virus detection (IPMA) Antibody detection (VNT)

N n % 95% CI N n % 95% CI GMT 95% CI

Slaughterhouse A (2001)

Suckling lambs 731 2 0.27 0.00, 0.65 74 11 14.86 6.76, 22.97 60.09 27.59, 92.60 Young light lambs 467 1 0.21 0.00, 0.63 46 10 21.74 9.82, 33.66 48.50 24.62, 72,38 Total 1198 3 0.25 0.00, 0.53 120 21 17.50 10.70, 24.30 54.26 26.67, 81.86

Slaughterhouse B (2003)

Suckling lambs 554 1 0.18 0.00, 0.53 56 15 26.79 15.19, 38.38 48.50 18.02, 78.99 Young light lambs 337 1 0.30 0.00, 0.88 34 1 2.94 0.00, 8.62 128 -

Total 891 2 0.22 0.00, 0.54 90 16 17.78 9.88, 25. 68 51.54 21.94, 81.13

Total 2,089 5 0.24 0.03, 0.45 210 37 17.62 12.47, 22.77 53.07 25.11, 81.02

3 N = Number of sera analysed; n = Number of positive sera; CI = confidence interval 4

IPMA = indirect peroxidase monolayer assay; GMT = geometric mean titre 5

Table

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Accepted Manuscript

2 Table 2. Prevalence of pestiviruses and pestivirus neutralising antibodies in sera from 45 to 100 days old lambs sampled in a Spanish fattening 6

unit.

7 Virus detection Antibody detection

N n

^a

% 95% CI n

^b

% 95% IC N n

^c

% 95% CI GMT 95% CI

Randomly collected lambs 56 4 7.14 0.40, 13.89 4 7.14 0.40, 13.89 56 16 28.57 16.74, 40.40 56.20 29.62, 82.78 Diseased lambs 70 24 34.29 23.17, 45.41 27 38.57 27.17, 49.97 70 10 14.29 6.09, 22.48 42.22 19.51, 64.94 8

N = Number of sera analysed; n

^{a, b, c}

= Number of positive sera by (a) IPMA; (b) IPMA + Ag ELISA; (c) VNT 9

CI = confidence interval; GMT = geometric mean titre

10

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Table 3. Details of the 36 border disease virus isolates or amplicons obtained from samples 11

analysed in the present study.

12 13

Name Source Year of

collection Virus

isolation Genetic

Typing Reference

LE31C2 S A 2001 + BDV-4a Valdazo-González et al. (2006, 2007) M3 S A 2001 + BDV-4a Valdazo-González et al. 2007 M96 S A 2001 + BDV-4a Valdazo-González et al. 2007