Clinical and laboratorial findings in pregnant ewes and their progeny infected with Border disease virus (BDV-4 genotype)

Research in Veterinary Science 86 (2009) 345–352

Contents lists available at ScienceDirect

Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc

Clinical and laboratorial findings in pregnant ewes and their progeny infected with Border disease virus (BDV-4 genotype) A.L. García-Pérez *, E. Minguijón, L. Estévez, J.F. Barandika, G. Aduriz, R.A. Juste, A. Hurtado NEIKER-Instituto Vasco de Investigación y Desarrollo Agrario, Department of Animal Health and Production, Berreaga 1, 48160 Derio, Bizkaia, Spain

a r t i c l e

i n f o

Article history: Accepted 2 July 2008

Keywords: Border disease virus Pestivirus Experimental infection RT-PCR ELISA

a b s t r a c t To evaluate the pathogenicity of local isolates of ovine pestiviruses (BDV-4 genotype), 13 virus- and antibody-negative, artificially inseminated pregnant ewes were challenged on days 108 (5 ewes), 76 (5 ewes) and 55 of pregnancy (3 ewes) with 2 ml of ovine pestivirus containing 106 TCID50. Viraemia was detected by RT-PCR from 2 to 15 days pi in most ewes. No abortion due to the infection was observed but the number of stillbirths was high (32%), and bodyweight at lambing was significantly reduced compared to the experimental flock of origin used as control. Clinical symptoms in live lambs consisted on tremors, gait anomalies and inability to stand unaided. Skeletal abnormalities (brachygnathia, prognathia, arthrogryposis) were present in 44% of the lambs. Only 20% of the lambs were clinically normal. RT-PCR was a very sensitive technique compared to antigen ELISA in detecting viral presence in experimentally infected ewes and their progeny. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Border Disease is a congenital infection of newborn lambs caused by a pestivirus antigenically related to bovine viral diarrhoea (BVD) and hog cholera viruses. Affected lambs have various abnormalities including pigmented straight fleece, clonic muscular tremors, growth retardation, skeletal malformation, weakness and low neonatal survival rate. Border disease viral infection in adult sheep is transient, and generally causes seroconversion within 30 to 40 days of infection (Sweasey et al., 1979; Barlow et al., 1980), and after exposure, the ewes remain immune. The disease is of economic importance due to the reproductive failure and the abortions caused by the infection, and the low survival rate and low carcass scores of affected lambs. Border disease is widely distributed and has been reported from several regions in Spain (Mainar-Jaime and Vazquez-Boland, 1999; Valdazo-González et al., 2006), and Border Disease virus (BDV) was detected in 16% of the flocks with problems of abortions in the Basque Country, Northern Spain (Oporto et al., 2006). Furthermore, a study carried out in bulk-tank milk (BTM) from 154 flocks from the same area (Berriatua et al., 2006) revealed a high seroprevalence and a 9% of RT-PCR-positive BTM samples, suggesting the presence of persistently infected animals (PI) in several flocks at sampling. Phylogenetic analysis of the 50 NCR of BDV from seven infected flocks showed that all except one clustered within the group formed by BDV type C strains from a previous study carried out in the region (Hurtado et al., 2003), suggest* Corresponding author. Tel.: +34 94 4034312; fax: +34 94 4034310. E-mail address: [email protected] (A.L. García-Pérez). 0034-5288/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.07.004

ing that BDV strains infecting most Basque flocks have a common origin. More recently, ovine isolates from other Spanish regions (Valdazo-González et al., 2007) were incorporated into this group, which was renamed as BDV type 4 (Arnal et al., 2004). The particular antigenic differences regarding mAb reactivity patterns of these isolates suggested that novel BDV type 4 might be unique to the Iberian Peninsula and bordering districts (Valdazo-González et al., 2007). Several experimental infections with pestiviruses have already been carried out in pregnant ewes (Hussin and Woldehiwet, 1994; Scherer et al., 2001), and differences related to viral strain has been suggested. Thus, the aim of this study was to evaluate the pathogenicity of these local isolates (BDV-4 genotype) as abortifacient agents in pregnant ewes and to identify the main symptoms that should be expected in ovine commercial flocks in natural conditions and potentially in other susceptible ruminants. 2. Materials and methods 2.1. Ewes and experimental design Thirty 2–4 years old Latxa (Black Faced variety) ewes were selected from the experimental flock of NEIKER in Arkaute (Alava, Northern Spain). This flock (138 ewes) was BDV-free as demonstrated by regular serology testing, and the selected ewes were confirmed negative to BDV and sero-antibodies before the experiment started. Ewes were treated to synchronize oestrus and were subsequently artificially inseminated (AI) using freshly collected and diluted semen. Forty-five days after AI the ewes were submitted to ultrasound scanning. AI yielded 13 virus- and antibody-negative

346

A.L. García-Pérez et al. / Research in Veterinary Science 86 (2009) 345–352

pregnant ewes. The experimental infection (EI) was carried out at the Biosecurity level 3 facilities of NEIKER, in Derio (Bizkaia) and started once all permissions on experimental animal welfare were obtained from local authorities. On arrival the sheep were wormed and injected with enterotoxemia vaccine. After an adaptation period, ewes were challenged with ovine pestivirus (0502234 strain) on day 108 (5 ewes, Group A), on day 76 (5 ewes, Group B) and at 55 day of pregnancy (3 ewes, Group C) by intramuscular inoculation into the hind leg muscles of 2 ml of a virus suspension containing 106 TCID50. 2.2. Viral strain The 0502234 strain of ncp pestivirus was isolated from leukocytes of a PI ewe from a commercial flock from the region. Presence of virus in the PI was determined by antigen detection in blood by ELISA and RT-PCR as previously described (Hurtado et al., 2003). Sequencing of the 50 NCR and Npro (Dubois et al., in press) classified the 0502234 strain (GenBank Accession Number EU711348) as belonging to BDV-4 genotype (formerly BDV type C) grouping with other local strains (Hurtado et al., 2003; Berriatua et al., 2006). The virus was isolated at the laboratory of CReSA (Dra. Rosa Rosell, Universidad Autónoma de Barcelona, Spain). Leucocytes were inoculated on confluent monolayer of permanent Sheep Fetal Thymoid Cells (SFT-R) (kindly provided by the Federal Institute for Virus Diseases of Animals, Island of Riems, Germany) and titrated by immune-peroxidase-monolayer-assay with a polyclonal antibody produced at CReSA that identifies all strains of the four pestiviruses genotypes tested (reference and field strains). 2.3. Clinical examination and sample collection Behaviour and clinical signs of infected ewes were observed every day before and after inoculation, until lambing. Rectal temperatures were taken daily until 15 day post-infection (pi), and then every 2–3 days until lambing. Blood samples for haematology, serology and virology were taken immediately before EI and daily until 15-day pi and then weekly until lambing. Ewes were also inspected daily for signs of abortion or lambing. The lambing process was closely supervised, and lambs born alive were assessed clinically soon after birth, particularly for tremor, hairiness of fleece (associated with BDV in several breeds), gait abnormalities, consciousness and position. After bodyweight recording, and blood and serum extraction for haematological, serological and virological studies, all lambs were killed within 24 h of birth with sodium pentobarbitate. Examination of gross morphology was carried out using standard procedures, including crown-rump length (from the forehead to the base of the tail), and tibia and radius measurement. Colostrum intake was recorded. Brain, lungs, spleen, liver and kidneys were removed and weighed. Tissues from all the lambs and aborted foetuses were collected for pathological and molecular studies (data not shown). Once the lambing period finished the ewes were necropsied and tissue samples were kept for further pathological examination (data not shown). 2.4. Haematology Haematological analyses of experimental ewes and live lambs were performed with an electronic counter (Cell Dyn 3700, Abbott Cientifica S.A., Barcelona, Spain). Leukocyte differential counting was carried out by thin blood smears stained with Giemsa and analysed under an oil-immersion objective, differentiating at least 100 cells.

2.5. Serological tests Samples of plasma from experimental ewes, stillbirths and lambs, and also foetal fluids, were used to detect anti- BDV p125/ p80 antibodies using a commercial blocking ELISA (Institut Pourquier; France). The inhibition percentage (IP) in individual plasma samples was calculated as:

IP ¼ ODS=ODN  100 where OD is the optical density of the negative (N) control, and of the sample (S). Samples with %IP P 50, 40–50%, and 640%, were considered negative, weakly positive and positive for BDV antibodies, respectively. 2.6. Virological examination The follow-up of viraemia in experimental ewes, and the detection of BDV in blood of lambs, stillbirths and foetuses, was done by ELISA and RT-PCR. Blood samples (5 ml) were collected in EDTA tubes and diluted (1:2) in sterile ammonium chloride (8.7 g/l). After incubation at room temperature for 15 min, the mixture was centrifuged at 3000g for 10 min. The pellet of leucocytes was washed twice with sterile cold PBS, and stored in 2 aliquots at 80 °C until further processing. One aliquot of leucocytes was used to detect BDV p125/p80 antigen using a commercial indirect sandwich ELISA (Synbiotics; France) following the recommendations of the manufacturer. Samples showing an OD P 120%, 120–80%, or 680% of the positive control were considered positive, weakly positive and negative for BDV antigen, respectively. The second aliquot was processed for RNA extraction using QIAamp Viral RNA Mini kit (Qiagen) following the manufacturer’s procedure, and including a negative control of extraction for each batch of 10 samples. One-tube RT-PCR tests were then carried out as described elsewhere (Vilcek et al., 1994) using the panpestivirus primer pair 324/326 that targets the highly conserved 50 NCR region, and adding 50 ng of RNA template in each reaction. 2.7. Other laboratorial analysis Tissues and placentas of stillbirths were examined for the presence of other abortifacient pathogens. The presence of Chlamydia was determined by modified Ziehl–Nielsen staining and confirmed by antigen detection (Clear-view Chlamydia, Unipath; United Kingdom). Bacterial cultures of stomach content, liver and placenta were carried out on different media: general media (Columbia agar supplemented with 5% sheep blood, bio-Mérieux; France, and McConkey agar, Difco; EEUU), and selective media for Brucella spp. (Farrel medium, Barnavet; Spain), Yersinia spp. (CIN agar, bio-Mérieux; France), and Campylobacter spp. (Campylosel, bioMérieux; France). Isolated bacteria were identified biochemically using conventional tests. The presence of Toxoplasma DNA was evaluated by a single tube nested PCR assay targeting the multicopy 18S-5.8S rRNA internal transcribed spacer (Hurtado et al., 2001). In order to confirm the absence of copper congenital deficiency in lambs, 2 ml of ewes’ sera were analysed by atomic absorption spectrometry (Varian, SpectrAA 220 FS). 2.8. Statistical analysis Differences between the groups of ewes and lambs were analysed by ANOVA; the paired t-test was used. A P value of less than 0.05 was considered significant. The analysis was performed in SAS 8.0 (SAS Institute Inc., Cary, NC, USA) software.

347

A.L. García-Pérez et al. / Research in Veterinary Science 86 (2009) 345–352

evident serological differences were observed among the three groups of ewes. Pestivirus antigen was not detected by ELISA in any of the ewes during the experiment. However BDV was detected by RT-PCR from day 2 pi to day 15 pi in all except two of the ewes, which remained negative throughout the study. Duration of viraemia varied between animals, ranging from 1 to 10 days. BDV was not detected after day 15 pi (Table 1).

3. Results 3.1. Study of infection in ewes 3.1.1. Clinical observations All ewes remained normal during the adaptation period before inoculation. No obvious clinical symptoms were observed during the experimental phase. Eight of the inoculated animals had a moderate and transient rise in body temperature from day 5 pi to day 12 pi (Table 1). It was a short-term hyperthermia, with a maximum of three consecutive days with temperatures above 40.5 °C. No changes in food consumption, alertness or consistency of faeces were observed.

3.1.4. Lambing performance Lambing took place between 146 and 155 days after AI (Table 1), except for one ewe from group A which on day 135 started showing signs of pregnancy toxaemia, and the abortion had to be provoked therapeutically (3 foetuses). The remaining ewes (12) carried the pregnancy to term and delivered a total of 22 lambs (5 singles, 8 twins and 9 triplets), though 7 of them were born dead (Table 1). Bacteriological analyses were negative for other abortifacient agents such as Brucella, Salmonella, Listeria, Campylobacter, Yersinia, and Chlamydophila. Toxoplasma gondii DNA was neither detected in the tissues (brain) of stillbirths, foetuses nor lambs. The average number of lambs per ewe was 1.83, with a prolificacy of 1.8 in group A (taking into account the 3 foetuses), 2.2 in group B and 1.7 in group C. The average birth weights of lambs from group A was 3.798 kg, 2.661 kg in group B and 2.680 kg in group C. In spite of notorious differences in prolificacy between groups B and C, no differences were observed in live weight at lambing between these groups (P > 0.05).

3.1.2. Haematological parameters A significant decrease in the total number of leucocytes was observed from day 2 pi (Table 1), reaching the mean lowest value of 2240 cells/mm3 at day 4 pi, from an initial mean value of 4230 cells/mm3. This decrease was statistically significant from day 2 to day 5 pi compared to values before inoculation. Counts returned to pre-inoculation values on day 12 pi. Red blood parameters (erythrocyte count, packed cell volume, and haemoglobin) were within the normal ranges along the experiment. 3.1.3. Antibodies and viral detection Antibodies to BDV were first detected at day 11 pi (1 ewe), and all ewes seroconverted by day 35 pi as demonstrated by ELISA. No

Table 1 Data about hyperthermia, leucopoenia and viraemia in experimental ewes Group

Sheep no.

Fever: day pi

Lowest WBC/ mm3 (day pi)

Viraemia: day pi

Days gestation

Lambing

No. lamb

Clinical condition & gross findings

ELISA Ab (serum)

ELISA Ag (blood)

RT-PCR (blood)

A

77,781*

–

3500 (4)

–

135

Triple

86,348 87,632

– 6

1420 (4) 4160 (5)

14 5

147 148

Single Double

89,395

12

1480 (5)

4

149

Double

1 2 3 4 5 6 7

Neg Neg Neg Neg Neg Posit Neg

Neg Neg Neg Posit Neg Neg Neg

Neg Neg Neg Posit Posit Neg Posit

82,327

12

2790 (5)

–

150

Single

8 9

Foetus. autolysis Foetus. autolysis Foetus. autolysis Alive, apparently normal Alive, weakness Alive, apparently normal Alive, tremor, unable to stand, weakness Alive, unable to stand, weakness Alive, tremor, brachygnathism % Positive group A

Posit NE 25

Neg NE 13

Posit Posit 56

87,650

6–7

1650 (5)

5–13

146

Triple

77,776

12

1370 (8)

2

148

Triple

87,630

–

2610 (7)

8–15

152

Double

Neg Neg Neg Posit Posit Posit Posit Posit

Posit Neg Posit Neg Neg Neg Neg Neg

NE NE Posit Posit Posit Posit Neg Neg

86,330

5–7

1120 (6)

5–14

155

Single

18

NE

NE

Neg

77,765

–

2730 (2)

5–10

155

Double

19

Neg

Neg

Posit

Neg 50

Posit 30

Posit 67

Neg NE Neg Neg Neg

Posit NE Posit Posit Neg

Posit Posit Posit Posit NE

0

75

100

B

10 11 12 13 14 15 16 17

20 C

77,770 82,314 82,338

6 – 6

1390 (4) 1490 (2) 2510 (5)

6–15 9–12 5–11

147 155 151

Single Single Triple

21 22 23 24 25

Dead Dead. autholysis Alive, arthrogryposis Dead, brachygnathism Dead, brachygnathism Dead, brachygnathism Alive, arthrogryposis, tremor Alive, hyperflexion of tarsal joint, arthrogryposis, tremor Alive, tremor, hyperextension of carpal and tarsal joint Alive, unable to stand, brachygnathism Alive, tremor, brachygnathism % Positive group B Alive, weakness Alive, apparently normal Alive, unable to stand, tremor Dead, arthrogryposis Dead, prognathism, other malformations. Autolysis % Positive group C

Summary of lambing, with details about clinical condition, gross findings at necropsy and ELISA (antibodies and antigen) and RT–PCR results from serum or blood samples (leucocytes). NE: Not estimated (results of ELISA – antibodies and antigen – in lambs who fed colostrums not included; Not included results of RT–PCR from dead lambs without cardiac blood samples available). (day pi.: day post-inoculation; *Ewe with pregnancy toxaemia, abortion on day 135 pi).

348

A.L. García-Pérez et al. / Research in Veterinary Science 86 (2009) 345–352

Bodyweights of single lambs, twins and triplets in each experimental group are shown in Table 2. The birth weights of the 5 single lambs averaged 4.309 kg, for the twins mean birth weight was 3.169 kg and 2.062 kg for the triplets (P < 0.05) (Table 2). Stillbirths had a reduced weight (1.984 kg) compared to live lambs (3.437 kg). Mortality was mostly associated to triple lambs. Comparison of lambing performance results of experimentally infected animals with those of non-infected adult ewes from the source flock (acting as non-infected negative control) showed lower mortality at lambing among non-infected ewes (2%), and a significantly higher bodyweight of live lambs (single, twins and triplets) (Table 2). Ewes from group A had a shorter gestational period (148.5 days) compared to ewes from group B (150 days) or group C (151 days) (P = 0.0035). The mean gestation time for the triplets was shorter (148 days) compared to single or double lambing (151 days) (P = 0.0026). There was a significant effect of the type of lambing and the infection group on the mean gestation time (P = 0.0196). Thus, among double lambs, those born in group A had a shorter gestation time (148.5 days) compared to those from group B (153.5 days) (P = 0.0018). The same effect was observed for single lambs, being the gestation time shorter in group A (148.5 days) than in group B (155 days) (P = 0.0125). Finally, triplets from group C had a longer gestational period (151 days) compared to triplets from group B (147 days) (P = 0.0085). 3.2. Clinical observations in stillbirths and lambs 3.2.1. Clinical symptoms and gross pathology Condition of the lambs, body conformation anomalies and other symptoms are shown in Table 3. Seven lambs were born dead (32% mortality) and among the 15 alive, 47% (7/15) showed weakness and some of them (27%, 4/15) were unable to stand unaided. Three of the lambs (20%) exhibited no clinical symptoms but the remaining showed varying degrees of tonic-clonic muscular tremor (47%, 7/15) or gait abnormalities (36%, 4/11). Nervous symptoms as tremors were apparent in 7 lambs, varying from rhythmic contractions of the muscles of the hind legs to fine trembling of the head and tail. In addition, ear auricle asymmetry caused by a unilateral drop was observed in 33% of lambs (17%, 50% and 33% in groups A, B and C, respectively). Table 2 Lambing performance results and lambs’ bodyweights Experimental animals Total

Dead full-term lambs Live lambs Totala

Bodyweight single (g) Bodyweight twins (g) Bodyweight triplets (g) Bodyweight live lambs (g) Bodyweight dead lambs (g)

Group A

Group B

Group C

Source flockb (noninfected)

n

%

n

%

n

%

n

%

n

%

7

32

0

0

5

45

2

40

3

2

15 22

68

6 6

100

6 11

55

3 5

60

121 124

98

n

Mean

n

Mean

n

Mean

n

Mean

n

Mean

5

4309

2

4950

1

4325

2

3661

39

5032

8

3169

4

3222

4

3116

–

–

67

4135

9

2062

–

–

6

2080

3

2025

15

3120

15

3437

6

3798

6

3107

3

3377

121

4096

7

1984

–

5

2125

2

1633

3

NE

NE, not estimated. a Only full-term lambs were considered (the 3 foetuses from one ewe with pregnancy toxaemia are not included). b Only adult ewes, older than 2 years-old are considered.

Four lambs exhibited gait abnormalities, as hyperextension of carpal and tarsal joint, hyperflexion of tarsal joint or erratic gait especially in the hind limbs. Congenital hypocupraemia was excluded in lambs as copper levels in their dams were within normal ranges (between 65 and 158 mg/100 ml). No obvious fleece changes were observed. Several lambs had dark or black pigmentation of fleece on the back of the neck, but this was not considered abnormal in Latxa Black Faced breed. Skeletal dysmorphogenesis characterised by arthrogryposis with stiffness (Fig. 1a) or lateral deviation of joints (Fig. 1b), brachygnathia (Fig. 1c) or prognathia (Fig. 1d) were commonly observed (44%, 11/25) (Table 3). Other relevant findings at necropsy included hydronephrosis and jaundice observed in 36% (9/25) and 12% (3/25) of the lambs, respectively. Other gross findings like cerebellar hypoplasia, small cysts in the septum pellucidum, and other malformations (fused eyelids, delay in eruption of incisor teeth, tongue and soft palate fused) were observed in a scarce number of lambs (Table 3). Other intracranial malformations including hydrocephalus, porencephaly and hydranencephaly were not macroscopically appreciated. Inoculation of ewes at day 55 and day 76 of gestation (groups C and B) resulted in lambs more severely affected than those from group A in terms of clinical symptoms or macroscopic lesions. Thus, gait and skeletal abnormalities were present in 80% (4/5) and 73% (8/11) of the lambs from group B, respectively, whereas in group A gait abnormalities were not observed and only 11% (1/9) of the lambs showed skeletal lesions (P < 0.05). No differences were observed between the degree of affectation of lambs from groups B and C. Inoculation of ewes at day 108 of gestation (group A) did not lead to mortality of the lambs, compared to 45% and 40% of stillbirths in groups B and C, respectively (Table 2). Also, lambs from group A presented clinical symptoms and gross lesions at a lower percentage (33%, 3/9) (Table 3).

Table 3 Summary of clinical symptoms (live lambs), and gross pathology (live and dead lambs) including several morphological parameters Clinical condition of lambs (live)

All

Group A

Group B

Group C

Pos/anala

Pos/anala

Pos/anala

Pos/anala

3/15 4/15 7/15 7/15 4/11 11/15

2/6 2/6 3/6 2/6 0/4 4/6

0/6 1/6 2/6 4/6 4/5 5/6

1/3 1/3 2/3 1/3 0/2 2/3

11/25 4/25 1/25 6/25

1/9 0/9 0/9 1/9

8/11 3/11 0/11 5/11

2/5 1/5 1/5 0/5

3/22 1/22 1/25 3/25 9/25 10/25

0/6 0/6 0/9 0/9 3/9 6/9

2/11 0/11 0/11 2/11 5/11 2/11

1/5 1/5 1/5 1/5 1/5 2/5

Morphological parameters of lambs

Anal/ Mean

Anal/ Mean

Anal/ Mean

Anal/ Mean

Crow-anus length (cm) Tibia length (cm) Proportion tibia/crow-anus length Radius length (cm) Whole brain weight (g) Proportion brain/bodyweight

22/44.2 22/10.2 22/0.23

6/46.9 6/10.8 6/0.23

11/43.6 11/10.1 11/0.23

5/42.7 5/9.4 5/0.22

22/7.8 20/47.5 20/1.86

6/8.1 4/49.1 4/1.22

11/7.9 11/46.0 11/2.01

5/7.3 5/45.0 5/2.04

Apparently normal Unable to stand Weakness Nervous symptoms (tremors) Gait abnormalities Leucopoenia (linfopoenia) Gross pathology Skeletal abnormalities Arthrogryposis Prognathism Brachygnathism Other observations Cyst in Septum pellucidum Cerebelar hypoplasia Other malformations Icterus Hydronephrosis No gross lesions

a (Pos/anal: number of animals presenting symptoms or lesions/number of animals examined).

A.L. García-Pérez et al. / Research in Veterinary Science 86 (2009) 345–352

349

Fig. 1. Skeletal dysmorphogenesis was characterised by arthogryposis with stiffness (a) or lateral deviation of joints (b), brachygnathia (c) or prognathia (d).

3.2.2. Morphological parameters Data on morphological parameters are shown in Table 3. The analysis of the crown-anus length showed statistical differences between type of lambing (P = 0.0017), being the size of single lambs (50.6 cm) significantly larger than that of twins (45.5 cm) and triplets (39.6 cm). There were also differences between experimental groups (P = 0.0223), with significantly bigger size in lambs from group A (46.9 cm) compared to lambs from group C (42.7 cm) (P < 0.05) (Table 3). No significant differences were apparent between groups with regard to organ weights, with the exception of brain expressed as percentage of the total body weight, which represented a greater percentage in lambs from groups B and C with regard to those from group A (Table 3). In general there was a positive and significant high correlation between the body weight and the brain, lungs, spleen, liver and kidneys weight, with the exception of brain or kidney weight from lambs of group C, which did not show correlation with body weight. Significant differences were observed between groups for tibia and radius length, being smaller among lambs from group C (P < 0.05). 3.2.3. Haematological parameters A severe leucopaenia was observed in 11 out of the 15 live lambs (73%, 11/15), with a minimum of 314 WBC/mm3. Significant differences were not observed between groups (Table 3). Red blood parameters (erythrocytes count, haematocrit, and haemoglobin) and platelets were within the normal ranges. 3.2.4. Antibodies and viral detection in lambs All 22 lambs and 3 fetuses, were subjected to serological and virological studies (Table 1). Blood samples were collected within a few hours after birth, but 3 lambs had sucked from their dams before blood samples collection. As colostral antibodies can mask

the presence of virus, samples from these animals were excluded from the antibody and antigen ELISA analysis. A total of 2 (25%) and 5 (50%) lambs from groups A and B, respectively, showed antibodies by ELISA, reflecting a prenatal humoral response. No antibodies were detected in lambs from group C. Viral antigen was detected by antigen ELISA in 7 lambs (32%), which accounted for 13, 30 and 75% of the lambs from groups A, B and C, respectively (Table 1). RT-PCR improved significantly the detection of virus in lambs, confirming its presence in 56%, 67% and 100% of lambs from groups A, B and C, respectively (Table 1). A good correlation between techniques (antigen ELISA and RT-PCR) was observed in lambs from group C, in which all animals processed by both techniques (3) showed identical results. In general, RT-PCR was more sensitive than antigen ELISA detecting viraemia in lambs and stillbirths. In none of the five animals that showed any degree of autolysis, was antigen or RNA detected in blood samples.

4. Discussion The infection of non-immune pregnant ewes with a local strain of ovine pestivirus between days 55 and 108 of gestation produced a high proportion of stillbirths and lambs with significantly lower liveweight, nervous symptoms and gait and skeletal abnormalities. In addition, 76% of the newborn lambs had a marked leucopaenia indicating a status of immunosuppression susceptible of predisposing to concurrent diseases in the post-lambing period (such as those produced by Escherichia coli, Criptosporidium sp., Mamheimia haemolytica, etc), which could significantly contribute to the cost of BDV infection. In this study, most severely affected lambs were born from ewes infected at the beginning and middle of the gestational period. Other authors have also found that the time of viral exposition determines the clinical presentation of the offspring,

350

A.L. García-Pérez et al. / Research in Veterinary Science 86 (2009) 345–352

being those lambs infected from 16 to 90 days of gestation the most severely affected (Sawyer et al., 1991; Scherer et al., 2001). The pathological study in experimental lambs is still in progress and the results cannot be fully evaluated yet, but Anderson et al. (1987) stated that the severity of clinical signs in lambs was directly correlated with the severity of lesions in their central nervous system. A relevant finding of this study was the lack of abortions related to BD infection, which could be explained by the infection time, the dose of inoculum, the virulence of the strain, the immune status of the ewes, or the lack of concurrent factors like secondary infections that often occur in commercial flocks (Hussin and Woldehiwet, 1994; Nettleton, 2000). Infection in the first 60 days of gestation before the onset of immunological competence makes virus replication uncontrollable leading to foetus death or to the birth of viremic PI lambs. In the present study, lambs from ewes infected on day 55 of gestation were born at term without antibodies in precalostral blood and viraemic. If infection had occurred earlier, embryonic losses and abortions would have probably occurred as observed by other authors (Gardiner and Barlow, 1972; Winkler et al., 1975; Sweasey et al., 1979). However, it is difficult to evaluate these studies since different inoculums and infection routes were used. The infection route used in this study (intramuscular) had been successfully used in initial works with BDV (Winkler et al., 1975; Richardson et al., 1976; Loken, 1987; Caffrey et al., 1997), proving that the mechanical spread of the virus by injection is possible. In fact, several outbreaks of BDV have been associated with vaccines contaminated with pestiviruses (Loken et al., 1991). In addition, we used an inoculum of known potency, and it was possible to produce lambs whose clinical symptoms ranged from severe to mild. Experiments done by Loken (1987) using a similar dose and similar timing of infection, resulted in abortion. Nevertheless, Richardson et al. (1976) demonstrated that the severity of the induced disease in the foetus varies with the infective dose to which the dam is exposed. There are also evidences showing that different strains of BDV may cause different pathological effects and variable clinical diseases (Vantsis et al., 1979; Plant et al., 1983; Nettleton et al., 1992). In this sense, Bonniwell et al. (1987) reported an outbreak of BD in lambs in which the typical signs of tremors and abnormalities in fleece were not present, and clinical signs were restricted to abortion and weakness of newborn lambs. In a flock naturally infected with the same type of virus used in the present work (BDV-4 genotype), Valdazo-González et al. (2006) reported the lack of abortions or clinical symptoms associated to BDV. However, in another two flocks the same authors found a broad spectrum of symptoms, including barren ewes, abortions, stillbirths, neonatal mortality and symptoms in lambs, but surprisingly tremor and malformations were not present in all flocks naturally infected with BDV-4 genotype. These authors emphasised the difficulty of BD diagnosis based only on clinical signs. Two hypotheses could explain the absence of abortion in the experimental ewes and the presence of barren ewes and/or abortions in commercial flocks infected with the same virus (type 4a): (i) other concurrent factors are inducing abortions in commercial flocks when BDV-4 genotype is present, or (ii) a virus-host adaptation has taken place. The concurrent presence of an immunosuppressant agent that increases the pathogenic effects of pestiviruses could explain our results. A tick-borne bacterium like Anaplasma phagocytophilum, transmitted by Ixodes ricinus ticks, which are very abundant in the Basque Country (Barandika et al., 2006), has been shown to have a broad distribution in Basque dairy-sheep flocks, and has been associated with abortion in an important number of flocks (García Pérez et al., 2003). Also, another secondary bacterium, like Arcanobacterium pyogenes, is commonly isolated in placenta and foetal tissues with BD diagnosis

(Diagnostic Service of NEIKER, unpublished results). The highly controlled environmental conditions in which experimental infection was carried out (P3 bio-safety) could explain the absence of abortions due to these pathogens that are common in commercial flocks. The alternative hypothesis based on a host-parasite adaptation cannot be assessed in this study but would fit with the distinct genetic characteristics of the 4a virus group that might represent an adaptative association between Iberian virus strain and sheep breeds. The significant mortality observed in the present study provided evidence of the reproduction failure associated to the infection. However, in other experimental and natural infections, mortality rates were higher ranging between 45% and 49% (Nettleton et al., 1992; Physick-Sheard et al., 1980), and nervous symptoms, skeletal involvement, and intracranial malformations have been reported in different degrees, sometimes in a higher rate than that observed in the present work (Nettleton et al., 1992; Campbell et al., 1995; Barlow, 1980; Harkness et al., 1977; Plant et al., 1983; Richardson et al., 1976; Jeffrey and Roeder, 1987). But again, the differences in viral doses and inoculation routes make difficult to establish comparisons between studies. In this work, significant differences were apparent between experimental lambs and non-infected lambs (flock of origin) with regard to bodyweight. In addition, several body organs (brain, kidney) in lambs infected at 55 days of gestation tended to represent a greater percentage of total bodyweight, and showed shorter tibia and radium and shorter crown-anus lengths, suggesting variations in the development of the carcass and generalised skeletal growth retardation (Richardson et al., 1976; Sweasey et al., 1979; Physick-Sheard et al., 1980; Caffrey et al., 1997). Nevertheless, it is necessary to complete the microscopic pathology to evaluate the intensity of viral effect. Fleece changes are only detectable in smooth-coated breeds (Nettleton, 2000; Hussin and Woldehiwet, 1994). Due to the rough fleece of the Latxa breed clinically affected lambs did not show fleece changes, and similar findings were also observed in other Spanish breeds of sheep also affected by BDV-4 genotype (Valdazo-González et al., 2006). Infection of pregnant ewes with BDV is difficult to detect as the only signs observed are low grade of pyrexia and leucopaenia of short duration (Shaw et al., 1969; Ward, 1971; Vantsis et al., 1979). The ewes of this experiment only showed a short hyperthermia and leucopaenia, between days 2 and 12 pi, period in which viraemia was detected by RT-PCR in most of the ewes. The results of this study have shown that RT-PCR is more sensitive than antigen ELISA in detecting acutely and transiently infected animals. Similar results have been shown by several authors working with bovine and porcine pestiviruses (Horner et al., 1995; Sandvik, 2005; Kaden et al., 1999), but no studies had been done in sheep comparing diagnostic techniques. Comparison of results of the same techniques on blood samples from experimental lambs again showed the higher sensitivity of RT-PCR, which detected viral RNA in a 68% of the lambs versus 32% of detection with antigen ELISA test. These observations led us to think that ELISA would detect only high viraemias or PI animals. As colostral antibodies can mask the presence of virus, one additional advantage of RT-PCR is the detection of the virus even in the presence of maternal colostral antibodies (Zimmer et al., 2004; Sandvik, 2005). In this study, three lambs had ingested colostrum and two of them were virus positive by RT-PCR, indicating the suitability of this technique to be used in laboratory diagnosis. In 4 lambs viral RNA was detected in the presence of serum antibodies. This finding might be a reflection of the high virus content in these lambs, also observed by other authors (Gard et al., 1976; Campbell et al., 1995). Seven seronegative and viremic lambs (confirmed by antigen ELISA and RT-PCR) were candidates to be considered as PI lambs. Although consecu-

A.L. García-Pérez et al. / Research in Veterinary Science 86 (2009) 345–352

tive samples would have been necessary to prove that these animals were PI, the absence of antibodies certainly suggested it. Sweasey et al. (1979) estimated great economic losses of pestivirus infection in experimental animals in terms of growth retardation, and calculations of Sharp and Rawson (1986) in commercial flocks resulted in a potential reduction of income in excess of 20%. It is difficult to extrapolate these data to other areas because management conditions are different, but taking into account that in the present study the production losses (lambs) were 10 times higher among the experimental infected animals than in the BDVfree flock of origin, these losses would account for about 3000 euros in a standard flock of 250 ewes in the Basque Country. In addition, Berriatua et al. (2006) found in this area a high prevalence of BD in sheep flocks (86%). Therefore, to avoid undesirable effects of viral infection an effective control strategy has to be implemented. The present work provides a better knowledge of the clinical and laboratorial features of the novel group of BDV (BDV-4 genotype) affecting sheep in Spain (Valdazo-González et al., 2007; Hurtado et al., 2003). Our results have shown a significant panel of clinical, haematological and virological infection indicators that demonstrate similarities with other ovine pestivirus. Further studies are required to characterise the pathological changes associated to viral infection in different phases of the gestational period.

Acknowledgements This work was supported by funding from FEDER and INIA (research grant RTA04-057) and from the Department of Agriculture, Fisheries and Food of the Basque Government. We acknowledge the technical assistance of Inés Povedano and Beatriz Oporto, and the assessment and valuable help of Ina Beltran de Heredia in all procedures related to artificial insemination of experimental sheep. We want to thank the collaboration of HIPRA laboratories (Enric March). References Anderson, C.A., Sawyer, M., Higgins, R.J., East, N., Osburn, B.I., 1987. Experimentally induced ovine border disease: extensive hypomyelination with minimal viral antigen in neonatal spinal cord. American Journal of Veterinary Research 48, 499–503. Arnal, M.C., Fernández de Luco, D., Riba, L., Maley, M., Gilray, J., Willoughby, K., Vilcek, S., Nettleton, F., 2004. A novel pestivirus associated with deaths in Pyrenean chamois (Rupicapra pyrenaica pyrenaica). Journal of General Virology 85, 3653–3657. Barandika, J.F., Berriatua, E., Barral, M., Juste, R.A., Anda, P., García-Pérez, A.L., 2006. Distribution and risk factors associated with ixodidae tick species in vegetation in the Basque Country in Spain. Medical and Veterinary Entomology 20, 177– 188. Barlow, R.M., 1980. Morphogenesis of hydranencephaly and other intracranial malformations in progeny of pregnant ewes infected with pestiviruses. Journal of Comparative Pathology 90, 87–98. Barlow, R.M., Vantsis, J.T., Gardiner, A.C., Rennie, J.C., Herring, J.A., Scott, F.M., 1980. Mechanisms of natural transmission of border disease. Journal of Comparative Pathology 90, 57–65. Berriatua, E., Barandika, J.F., Aduriz, G., Hurtado, A., Estevez, L., Atxaerandio, R., García-Pérez, A.L., 2006. Flock-prevalence of border disease virus infection in Basque dairy-sheep estimated by bulk-tank milk analysis. Veterinary Microbiology 118, 37–46. Bonniwell, M.A., Nettleton, P.F., Gardiner, A.C., Barlow, R.M., Gilmour, J.S., 1987. Border disease without nervous signs or fleece changes. Veterinary Record 120, 246–249. Caffrey, J.F., Dudgeon, A.M., Donnelly, W.J., Sheahan, B.J., Atkins, G.J., 1997. Morphometric analysis of growth retardation in fetal lambs following experimental infection of pregnant ewes with border disease virus. Research in Veterinary Science 62, 245–248. Campbell, J.R., Radostits, O.M., Wolfe, J.T., Janzen, E.D., 1995. An outbreak of border disease in a sheep flock. Canadian Veterinary Journal 36, 307–309. Dubois, E., Russo, P., Prigent, M., Thiéry, R., in press. Genetic characterization of ovine pestiviruses isolated in France, between 1985 and 2006. Veterinary Microbiology. doi:10.1016/j.vetmic.2008.01.002. García Pérez, A.L., Barandika, J., Oporto, B., Povedano, I., Juste, R.A., 2003. Anaplasma phagocytophila as an abortifacient in sheep farms from Northern Spain. Annals of New York Academy of Sciences 990, 429–432.

351

Gard, G.P., Acland, H.M., Plant, J.W., 1976. A mucosal disease virus as a cause of abortion, hairy birth coat and unthriftiness in sheep. 2. Observations on lambs surviving for longer than seven days. Australian Veterinary Journal 52, 64– 68. Gardiner, A.C., Barlow, R.M., 1972. Experiments in border disease. 3. Some epidemiological considerations with reference to the experimental disease. Journal of Comparative Pathology 82, 29–35. Harkness, J.W., King, A.A., Terlecki, S., Sands, J.J., 1977. Border disease of sheep: isolation of the virus in tissue culture and experimental reproduction of the disease. Veterinary Record 100, 71–72. Horner, G.W., Tham, K.M., Orr, D., Ralston, J., Rowe, S., Houghton, T., 1995. Comparison of an antigen capture enzyme-linked assay with reverse transcription–polymerase chain reaction and cell culture immunoperoxidase tests for the diagnosis of ruminant pestivirus infections. Veterinary Microbiology 43, 75–84. Hurtado, A., Aduriz, G., Moreno, B., Barandika, J., García-Pérez, A.L., 2001. Single tube nested PCR for the detection of Toxoplasma gondii in fetal tissues from naturally aborted ewes. Veterinary Parasitology 102, 17–27. Hurtado, A., García-Pérez, A.L., Aduriz, G., Juste, R.A., 2003. Genetic diversity of ruminant pestiviruses from Spain. Virus Research 92, 67–73. Hussin, A.A., Woldehiwet, Z., 1994. Border disease virus: a review. Veterinary Bulletin 64, 1131–1151. Jeffrey, M., Roeder, P.L., 1987. Variable nature of border disease on a single farm: clinical and pathological description of affected sheep. Research in Veterinary Science 43, 22–27. Kaden, V., Steyer, H., Strebelow, G., Lange, E., Hübert, P., Steinhagen, P., 1999. Detection of low-virulent classical swine fever virus in blood of experimentally infected animals: comparison of different methods. Acta Virologica 43, 373– 380. Loken, T., 1987. Experimentally-induced border disease in goats. Journal of Comparative Pathology 97, 85–89. Loken, T., Krogsrud, J., Bjerkas, I., 1991. Outbreaks of border disease in goats induced by a pestivirus-contaminated orf vaccine, with virus transmission to sheep and cattle. Journal of Comparative Pathology 104, 195–209. Mainar-Jaime, R.C., Vazquez-Boland, J.A., 1999. Associations of veterinary services and farmer characteristics with the prevalences of brucellosis and border disease in small ruminants in Spain. Preventive Veterinary Medicine 40, 193– 205. Nettleton, P.F., 2000. Border disease. In: Martin, W.B., Aitken, I.D. (Eds.), Diseases of Sheep, pp. 95–101. Nettleton, P.F., Gilmour, J.S., Herring, J.A., Sinclair, J.A., 1992. The production and survival of lambs persistently infected with border disease virus. Comparative Immunology, Microbiology and Infectious Diseases 15, 179–188. Oporto, B., Barandika, J.F., Hurtado, A., Moreno, B., Aduriz, G., García-Pérez, A.L., 2006. Incidence of ovine abortion by Coxiella burnetii in Northern Spain. Annals of New York Academy of Sciences 1078, 498–501. Physick-Sheard, P.W., Hopkins, J.B., O’Connor, R.D., 1980. A border disease-like syndrome in a southern Ontario sheep flock. Canadian Veterinary Journal 21, 53–60. Plant, J.W., Acland, H.M., Gard, G.P., Walker, K.H., 1983. Clinical variations of border disease in sheep according to the source of the inoculum. Veterinary Record 113, 58–60. Richardson, C., Hebert, C.N., Done, J.T., 1976. Experimental Border disease in sheep: dose-response effect. British Veterinary Journal 132, 202– 208. Sandvik, T., 2005. Selection and use of laboratory diagnostic assays in BVD control programmes. Preventive Veterinary Medicine 72, 3–16. Sawyer, M.M., Schore, C.E., Osburn, B.I., 1991. Border disease of sheep-aspects for diagnostic and epidemiologic consideration Archives of Virology. Supplementum 3, 97–100. Scherer, C.F., Flores, E.F., Weiblen, R., Caron, L., Irigoyen, L.F., Neves, J.P., Maciel, M.N., 2001. Experimental infection of pregnant ewes with bovine viral diarrhea virus type-2 (BVDV-2): effects on the pregnancy and fetus. Veterinary Microbiology 79, 285–299. Sharp, M.W., Rawson, B.C., 1986. The cost of Border disease infection in a commercial flock. Veterinary Record 119, 128–130. Shaw, I.G., Winkler, C.E., Gibbons, D.F., Terlecki, S., Hebert, C.N., Patterson, D.S., Done, J.T., 1969. Border disease of sheep: vaccination of ewes before mating. Veterinary Record 84, 147–148. Sweasey, D., Patterson, D.S., Richardson, C., Harkness, J.W., Shaw, I.G., Williams, W.W., 1979. Border disease: a sequential study of surviving lambs and an assessment of its effect on profitability. Veterinary Record 104, 447– 450. Valdazo-González, B., Alvarez-Martinez, M., Greiser-Wilke, I., 2006. Genetic typing and prevalence of Border disease virus (BDV) in small ruminant flocks in Spain. Veterinary Microbiology 117, 141–153. Valdazo-González, B., Alvarez-Martinez, M., Sandvik, T., 2007. Genetic and antigenic typing of border disease virus isolates in sheep from the Iberian Peninsula. Veterinary Journal 174, 316–324. Vantsis, J.T., Linklater, K.A., Rennie, J.C., Barlow, R.M., 1979. Experimental challenge infection of ewes following a field outbreak of Border disease. Journal of Comparative Pathology 89, 331–339. Vilcek, S., Herring, A.J., Herring, J.A., Nettleton, P.F., Lowings, J.P., Paton, D.J., 1994. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Archives of Virology 136, 309–323.

352

A.L. García-Pérez et al. / Research in Veterinary Science 86 (2009) 345–352

Ward, G.M., 1971. Experimental infection of pregnant sheep with bovine viral diarrhea-mucosal disease virus. The Cornell Veterinarian 61, 179–191. Winkler, C.E., Gibbons, D.F., Shaw, I.G., 1975. Observations on the experimental transmissibility of Border disease in sheep. British Veterinary Journal 131, 32– 39.

Zimmer, G.M., Van Maanen, C., De Goey, I., Brinkhof, J., Wentink, G.H., 2004. The effect of maternal antibodies on the detection of bovine virus diarrhoea virus in peripheral blood samples. Veterinary Microbiology 100, 145–149.