Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch
Year: 2014
Sheep persistently infected with Border disease readily transmit virus to calves seronegative to BVD virus
Braun, Ueli ; Reichle, S F ; Reichert, C ; Hässig, Michael ; Stalder, H P ; Bachofen, Claudia ; Peterhans, E
Abstract: Bovine viral diarrhea- and Border disease viruses of sheep belong to the highly diverse genus pestivirus of the Flaviviridae. Ruminant pestiviruses may infect a wide range of domestic and wild cloven-hooved mammals (artiodactyla). Due to its economic importance, programs to eradicate bovine viral diarrhea are a high priority in the cattle industry. By contrast, Border disease is not a target of eradication, although the Border disease virus is known to be capable of also infecting cattle. In this work, we compared single dose experimental inoculation of calves with Border disease virus with co-mingling of calves with sheep persistently infected with this virus. As indicated by seroconversion, infection was achieved only in one out of seven calves with a dose of Border disease virus that was previously shown to be successful in calves inoculated with BVD virus. By contrast, all calves kept together with persistently infected sheep readily became infected with Border disease virus. The ease of viral transmission from sheep to cattle and the antigenic similarity of bovine and ovine pestiviruses may become a problem for demonstrating freedom of BVD by serology in the cattle population.
DOI: https://doi.org/10.1016/j.vetmic.2013.11.004
Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-98718 Journal Article Accepted Version
Originally published at: Braun, Ueli; Reichle, S F; Reichert, C; Hässig, Michael; Stalder, H P; Bachofen, Claudia; Peterhans, E (2014). Sheep persistently infected with Border disease readily transmit virus to calves seronegative to BVD virus. Veterinary Microbiology, 168(1):98-104. DOI: https://doi.org/10.1016/j.vetmic.2013.11.004 1 Sheep persistently infected with Border disease readily transmit
2 virus to calves seronegative to BVD virus
3
4 U. Braun (a), S.F. Reichle (a), C. Reichert (a), M. Hässig (a), H.P. Stalder
5 (b), C. Bachofen (b), E. Peterhans (b)(c)
6
7 (a) Department of Farm Animals, University of Zurich, Winterthurer-
8 strasse 260, CH-8057 Zurich, Switzerland; (b) Institute of Veterinary
9 Virology, University of Bern, Länggass-Str. 122, P.O. Box 8644, CH-
10 3001 Bern, Switzerland
11
12
13
14 (c) Correspondence to be addressed to:
15 Institute of Veterinary Virology
16 University of Bern
17 Länggass-Str. 122
18 P.O. Box 8644
19 CH-3001 BERN, Switzerland
20 e-mail: [email protected]
21
1
22 Abstract
23
24 Bovine viral diarrhea- and Border disease viruses of sheep belong to the highly
25 diverse genus pestivirus of the Flaviviridae. Ruminant pestiviruses may infect a wide
26 range of domestic and wild cloven-hooved mammals (artiodactyla). Due to its
27 economic importance, programs to eradicate bovine viral diarrhea are a high priority
28 in the cattle industry. By contrast, Border disease is not a target of eradication,
29 although the Border disease virus is known to be capable of also infecting cattle. In
30 this work, we compared single dose experimental inoculation of calves with Border
31 disease virus with co-mingling of calves with sheep persistently infected with this
32 virus. As indicated by seroconversion, infection was achieved only in one out of
33 seven calves with a dose of Border disease virus that was previously shown to be
34 successful in calves inoculated with BVD virus. By contrast, all calves kept together
35 with persistently infected sheep readily became infected with Border disease virus.
36 The ease of viral transmission from sheep to cattle and the antigenic similarity of
37 bovine and ovine pestiviruses may become a problem for demonstrating freedom of
38 BVD by serology in the cattle population.
39
40
41 Keywords
42 Pestivirus, Border disease virus, Bovine viral diarrhea virus (BVD virus), BVD
43 eradication, sheep, cattle, infection
44
2
45 Introduction
46
47 Originally, the pestiviruses of ruminants were differentiated according to the host
48 species from which they were isolated and referred to as bovine viral diarrhea virus
49 (BVD virus) and Border disease virus of sheep (Carlsson 1991, Carlsson and Belak
50 1994, Campell et al. 1995, Paton et al. 1997; for reviews, see Nettleton and
51 Entrican, 1995; Nettleton et al., 1998; Løken 1995). More recently, nucleotide
52 sequencing documented the wide genetic heterogeneity and taxonomic position of
53 these viruses (Becher et al., 1997; 2003; Vilcek et al., 2005; reviewed by Becher and
54 Thiel, 2011) and confirmed that ruminant pestiviruses may not only jump the species
55 barrier between bovines and sheep, but also infect other ruminants, including wild
56 species. (reviewed by Passler and Walz, 2010). In domestic animals, the inter-
57 species infection appears to occur particularly in the direction of cattle to sheep
58 (Løken 1995; Carlsson and Belak 1994, Krametter-Frötscher et al. 2008; Danuser et
59 al., 2009). In contrast, infections of cattle with the sheep pestivirus have been noted
60 less frequently (Becher et al., 1997; Cranwell et al. 2007; Krametter-Frötscher et al.
61 2008; Hornberg et al. 2009). It was shown that calves kept together with sheep
62 seroconverted and that the antibodies were directed against Border disease virus
63 (Krametter-Frötscher et al. 2008). Information on the clinical effects of infection with
64 Border disease virus in cattle is scarce. By contrast, Border disease virus was shown
65 to cause severe infections in chamois in the Spanish Pyrenees (Marco et al., 2007,
66 2011). In this work we compared the effects of co-mingling of calves with sheep
67 persistently infected with Border disease virus vs. inoculation of calves with a single
68 virus dose.
69
3
70 Animals, materials and methods
71
72 Sheep persistently infected with Border disease virus (PI sheep) and
73 characterization of viruses.
74 Two female one-year-old sheep persistently infected with Border disease virus were
75 used in this study. Sheep #1, of the white alpine breed, was initially taken to the
76 clinic for investigation at two days of age with clinical signs typical of border disease,
77 i.e. ataxia, tremor and hairy fleece. Sheep #2, of the black-brown alpine breed, was
78 transferred to the clinic at three months of age with head tremor and ataxia.
79 Interestingly, the clinical signs of infection gradually decreased in both animals and
80 had disappeared to almost undetectable levels when the sheep were brought in
81 contact with the calves. Both animals tested positive for pestivirus antigen by
82 immunohistological investigation of skin biopsies as described previously (Arquint,
83 2003; Thür et al, 1997). Over a period of one year, leucocytes, and intermittently also
84 nasal swabs, of sheep #1 tested positive for pestiviral RNA by RT-PCR. Leucocytes,
85 epilated hair, urine, muzzle and nasal swabs of sheep #2 were positive for viral RNA.
86 Both animals were negative for pestivirus antibody. Virus was repeatedly isolated
87 from leucocytes of both animals in cultured sheep synovial membrane cells.
88 Sequencing showed that the viruses isolated belong to the species Border disease
89 virus of the pestivirus genus and may form a new genetic cluster (Figure 1, marked
90 by arrow). The virus isolated from sheep # 1 was labeled CH-R4786 and that of
91 sheep # 2 CH-R4785. After passaging twice both viruses we decided to use strain
92 CH-R4786 for single dose inoculation because it grew to a higher titer than strain
93 CH-R4785. Both PI sheep were also tested for ovine herpes virus type 2 (Hüssy et
4
94 al., 2001) because this virus may cause malignant catarrhal fever in cattle (reviewed
95 by Russell et al., 2009). Both animals were found to be positive.
96
97 All animal experiments were approved by the Veterinary Office of the Canton of
98 Zurich.
99
100 Pestiviral status of the calves before initiation of the experiments.
101 The two experiments consisted of a quarantine period followed by the infection
102 phase. For logistic reasons, the quarantine was set at 17 days for the orally
103 inoculated calves and at 30 days for the calves exposed to PI sheep. Before
104 purchase and at the end of the quarantine period all animals tested negative for
105 pestivirus antigen and antibodies in blood samples and for pestivirus antigen in skin
106 biopsies (Thür et al., 1997).
107
108 Inoculation of calves with Border disease virus and co-mingling of calves with
109 PI sheep.
110 Seven calves (4 male/3 female) were orally inoculated with Border disease virus
111 (see below). The animals were of various breeds (Simmental, Holstein Friesian,
112 Charolais x Braunvieh, Charolais x Holstein Friesian) and were between 87 and 176
113 days old at the beginning of the quarantine period (145.9 [32.65] (mean [SD]). The
114 protocol and virus dose for oral inoculation were the same as previously used for
115 experimental infection of calves with BVD virus (Hilbe et al., 2007). Each animal
116 received a total of 4.8ml of Border disease virus BD4 isolated from PI sheep #1,
117 dispensed in the oropharyngeal cavity by four strokes using a Foxy Plus™ hand
118 sprayer (Birchmeier Corporation, Stetten, Switzerland, www.birchmeier.com). The
5
5 119 total dose per animal of 7x10 TCID50 was verified by backtitration. In the co-mingling
120 experiment, nine male calves of of various breeds (Braunvieh, Simmental, Holstein
121 Friesian) that were between 99 and 156 days old (131 [18.8] (131 + 18.8 days) were
122 kept together with sheep # 1 and # 2 for 72 days.
123
124 Clinical investigations and laboratory tests
125 To avoid contamination strict quarantine rules were observed throughout the
126 experiments, involving separation of the animal groups, changing clothes and
127 disinfection between attending different groups of animals.
128 The animals were subjected to standardized clinical investigation by two of the
129 authors (CR: oral inoculation, SR: co-mingling of calves and PI sheep). The
130 investigations included recording of body temperature, heart beat and respiratory
131 frequencies, lung and digestive tract auscultation, inspection for signs of diarrhea,
132 nasal and ocular discharge and inspection of mucosae. In the oral inoculation
133 experiment, clinical investigations were done on days -9, -6, -3, thereafter daily until
134 day 21, followed by days 25, 28, 32, and 35. Nasal swabs were collected daily on the
135 first 21 days post-infection. Blood samples for virus detection and hematological
136 investigations followed the same schedule as the clinical investigation but continued
137 until the termination of the experiment on day 70. The schedule of antibody
138 determination is seen in Figure 3A and B. Hematological investigations included
139 erythrocyte and differential leucocyte counts, hemoglobin, packed cell volume, mean
140 corpuscular hemoglobin concentration, mean corpuscular hemoglobin, and mean
141 corpuscular volume. In the co-mingling experiment, the calves were subjected to
142 standardized clinical investigation at the beginning and end of the quarantine period.
143 The schedule of clinical and laboratory investigations was as follows: hematology:
6
144 days 0, 3,6,7,8,9,10,11,12,13,14, days 16 through 72: every second day. BVD virus
145 and – serology: 0,2,4,6, thereafter daily until day 20, followed by every second day
146 until the termination of the experiment; nasal swabs: up to day 41 for all calves as for
147 virus and serology, thereafter every second day for calves # 2,5,6, and continued
148 until day 72 for calves # 5 and # 6.
149
150 Detection of pestiviral RNA
151 Quantitative (q) RT-PCR was used to detect viral RNA in the EDTA blood and in the
152 nasal swab medium. RNA was extracted from 150μl of anti-coagulated whole blood
153 using the BioRobot Universal System and the QIAamp virus BioRobot MDx Kit
154 (Qiagen, CH-8694 Hombrechtikon, Switzerland). Viral RNA was quantitated using
155 the Qiagen Cador BVDV RT-PCR kit. Volumes were chosen to give a final dilution
156 factor of 12.5. The test recognizes a genome sequence conserved in pestiviruses
157 and can therefore also be used for the detection of border disease virus (Stalder et
158 al., 2008). Following the evaluation of the raw data the amount of the viral DNA
159 present in the sample was expressed in CT values, whereby CT values of >45 were
160 considered to be negative and those with a CT value of 30-45 were considered to be
161 weakly positive. The RNA isolations and RT-PCR preparations were done in
162 separate laboratories. The testing for viral RNA in the nasal swab medium was done
163 analogously to the method described for the viral RNA testing in the blood.
164
165 Sequencing
166 A genome fragment of 504 nucleotides spanning from the 5‘ untranslated region to
167 the core protein including the Npro was amplified as described previously (Bachofen
168 et al., 2008) and the DNA fragments of the correct length were sequenced by
7
169 Microsynth GmbH (CH-9436 Balgach). The sequence data were analysed by means
170 of the SeqMan (DNASTAR Inc., Madison, USA) and the Clone Manager (Scientific &
171 Educational Software, Cary, USA) softwares. The sequences are deposited in
172 Genbank (CH-R4785: GU244490; CH-R4786: GU244489)
173
174 Serological investigations
175
176 ELISA
177 A biphasic in-house ELISA was used for antibody screening as previously described
178 (Rüfenacht et al., 2000). The antigen of this ELISA contains a high concentration of
179 the conserved immunogenic NS3 protein and only little of the more strain-specific E2
180 protein (Wageck Canal et al., 1998). ELISA microtiter plates (Maxisorp, A/S Nunc,
181 Kamstrub, Denmark) were coated with antigen derived from bovine turbinate cells
182 infected with the cytopathic BVD viral strain R1935/72 (Oregon C24V, subgenotype
183 1a). A horse radish peroxidase labelled goat anti-bovine IgG antibody (light and
184 heavy chain, Kirkegaard and Perry Labs., Gaithersburg, USA) was used to detect
185 bound antibody. To visualize bound labelled antibody, the substrate ABTS (2,2'-
186 azino-di-(3-ethyl benzthiazoline-6- sulphonic acid), Roche Diagnostics) was added
187 and color measured in an ELISA reader at 405nm.
188
189 Serum neutralization test
190 To confirm the specificity of the antibodies we carried out serum neutralization
191 assays using Border disease virus CH-R4786 and BVD virus CH-04-01b. The latter
192 is a BVD-1h virus strain, which is the most frequent subgenogroup found in the
193 Swiss cattle population (Bachofen et al., 2008).
8
194
195 Procedure at the onset of erosive changes at the muzzle and the muzzle cavity
196 Specimens were taken at the onset of erosive changes at the muzzle and the muzzle
197 cavity and investigated for the presence of Border disease virus.
198
199 Statistics
200 The data were collected and evaluated by means of the StatView 5.1 programme
201 (SAS Institute, CHJ-8602 Wangen). The data were descriptively analysed (mean
202 values, standard deviations, frequency distributions). A generalized linear model was
203 also used to check for possible significant changes in the variables during the course
204 of the investigations (programme Stata statistical software, Stata Corp., 2009;
205 Release 101.; College Station, TX, USA: StataCorp LP). P-values of < 0.05 were
206 considered to be significant.
207
208
9
209 Results
210
211 Clinical findings
212 Four out of the seven orally inoculated calves had a reduced appetite on day 2 post-
213 infection whereas this was observed in three out of nine calves on day six after the
214 introduction of PI sheep. Transient increases >39.5°C in body temperature were
215 noted in individual animals of the two groups both during the quarantine and the
216 infection periods. These changes occurred scattered over time. All calves exposed to
217 PI sheep showed erosions of the muzzle mucosa between days 3 and 17, whereas
218 this was the case with four out of seven orally inoculated animals (animal #1: days
219 17-22 and days 29/30; animal #2: days 24-30; animal #5: days 4-29; animal #6: days
220 5-22; animal #7: days 7-15). The erosions were located in the areas of the rostral
221 palate (see Fig. 2A, orally inoculated calf), the gingival area of the incisors and the
222 mucosa of the upper and lower lips (Fig. 3B, calf exposed to PI sheep). The lesions
223 showed a roundish or oblong slightly reddened area and were associated with a loss
224 of integrity of the surface epithelium. The animals often salivated and the edges of
225 the gingiva were inflamed.
226
227 Serology
228 Serum samples obtained at various time points were tested for antibodies using
229 ELISA. In addition, serum samples obtained at the termination of the experiment
230 were assayed by SNT. Sera of all orally inoculated animals remained close (0±2%)
231 to the zero value of positive control until day 28 post-infection and thereafter the sera
232 of individual animals varied between -8% and +9% (Fig 3A). Of the serum samples
233 obtained on day 70 post oral inoculation, only the sample of animal #6 showed a
10
234 neutralizing antibody titer of 422 to Border disease virus CH-R4786, whereas the
235 samples of all other animals were negative (<8). The samples of animals exposed to
236 PI sheep remained negative until day 22. Thereafter, with the exception of calf #4,
237 we noted an increase in the ELISA antibody titers. At the termination of the
238 experiment, six out of nine animals were in the ELISA positive range and three
239 (calves #4, 5 and 6) were in the negative range (Fig 3B). To determine the pestiviral
240 specificity of the antibodies, we compared the SNT titers against Border disease
241 virus CH-R4786 and a BVD 1h virus. Using CH-R4786 as the challenge virus, the
242 titers ranged from 450 in the serum of calf #4 to 3,500 in calf #2. These titers were
243 10-30 times higher than those directed against BVD virus as the challenge, which
244 indicated that the antibodies were indeed directed against Border disease virus
245 (Figure 3C).
246
247 Hematology
248 During the entire experiment, i.e. during quarantine and infection period, none of the
249 calves showed leukopenia or significant changes in the parameters of red blood cells
250 (erythrocyte number, PCV, hemoglobin concentration, MCH, MCHC, MCV).
251
252 Virological investigations
253 Viral RNA was not detected in any of the nasal swabs and blood samples taken from
254 the calves exposed to PI sheep, and the same was true for the orally inoculated
255 calves.
256
257
258
11
259 Discussion
260
261 BVD and Border disease viruses may be viewed as “partial generalists”, infecting not
262 only cattle and sheep, but also a wide array of wildlife species including deer,
263 antelopes, camelids and wildebeest (reviewed in Passler and Walz, 2010). The
264 spread of Border disease virus to cattle is likely to gain in importance because
265 existing BVD eradication programs are run without concomitant efforts to control
266 Border disease (Krametter-Frötscher et al., 2010). Therefore, the focus of this work
267 was on the transmission of Border disease virus from sheep to cattle.
268 In a herd of susceptible bovines exposed to sheep PI with Border disease virus, the
269 time point of transmission of virus may differ between individual animals.
270 Consequently, seroconversion as well as possible clinical signs of infection with
271 Border disease virus may occur scattered over time. To control for this, we
272 compared single dose experimental inoculation with contact exposure to PI sheep.
273 The oral route for inoculation of the calves with Border disease virus and dose of
274 infection were chosen to allow a direct comparison with a previous experiment in
275 which we used BVD virus. As indicated by seroconversion in only one out of seven
276 calves orally inoculated with Border disease virus vs. 6/6 calves inoculated with BVD
277 virus (Hilbe et al., 2007), Border disease virus was less efficient than BVD virus at
278 infecting cattle. The relative inefficiency of Border disease virus correlates with a
279 poor growth of this virus in cultured bovine turbinate cells (unpublished observation).
280 The virus concentrations of the strain of Border disease virus shed by the PI sheep
281 are unknown, but PI sheep readily transmitted Border disease virus to calves kept in
282 the same herd. Interestingly, viral RNA was not detected in blood and nasal swabs in
283 any of the calves. This contrasts with the previous experiment in which we detected
12
284 BVD virus by isolation in cell culture in a majority of the orally inoculated animals
285 (Hilbe et al., 2007). In our hands, real-time RT-PCR is more sensitive than cell
286 culture isolation. Thus, viremia of Border disease virus may be of short duration and
287 low titer in calves. Different from the previous experimental infection with BVD virus
288 (Hilbe et al., 2007), we did not observe fever or changes in the leucocyte numbers in
289 calves that seroconverted to Border disease virus. The same was true also for
290 acutely infected sheep and goats (unpublished observation), which points to a low
291 virulence also in these host species. In agreement with earlier reports, the PI sheep
292 showed neurological signs of Border disease only in the first weeks of their lives
293 (Løken, 1998; Krametter-Frötscher et al., 2010).
294
295 The skin lesions observed in some of the calves in both experiments deserve
296 comment. In contrast to the mucosal disease-like lesions reported in sheep
297 chronically or persistently infected with Border disease virus (Monies et al., 2004;
298 Hilbe et al., 2009), the lesions observed in the calves were mild and transient.
299 Moreover, the lesions were also much less severe than those in acute BVD recently
300 described by Hessman et al. (2012). For several reasons, we are at a loss to explain
301 the cause of these lesions. Firstly, we failed to isolate pestivirus, or to demonstrate
302 pestiviral RNA, in any of the lesions analysed. Secondly, such lesions occurred also
303 in some orally inoculated calves that did not seroconvert to Border disease virus, as
304 assayed by ELISA and SNT. Due to their typical clinical picture, epidemiology and
305 absence from Switzerland we can exclude a range of agents known to cause skin
306 lesions, such as vesicular stomatitis and parapox viruses. All evidence obtained in
307 our and in previous experiments (Torleiv Løken, personal communication) do not
308 support the view that the lesions were directly caused by Border disease virus. Thus,
13
309 additional experiments will be required to determine the cause of these lesions,
310 among them contact experiments between sheep uninfected by Border disease virus
311 and calves originating from the same herds.
312
313 Bovines persistently infected with border disease virus have only rarely been
314 observed in the field (Cranwell et al., 2007; Krametter-Frötscher et al., 2008; Strong
315 et al., 2010). Although contact between bovines and sheep is not uncommon under
316 the prevailing management conditions, we detected only 13 bovines PI with Border
317 disease virus among 7,573 analyzed by sequencing in the course of the Swiss BVD
318 eradication campaign (Bachofen and Stalder, in preparation). The ease of
319 transmission of Border disease from PI sheep to seronegative cattle contrasts with
320 the rarity of bovines found PI with Border disease virus in the field. In addition to a
321 possible difference in the degree of adaptation to sheep vs. cattle as host species, a
322 strong herd immunity to BVD virus may decrease the risk of persistent infection with
323 Border disease virus in cattle. In crossed neutralization assays we previously
324 observed up to tenfold antibody titer differences between representative strains of
325 different BVD-1 virus subgroups (Bachofen et al., 2008) and this study shows up to
326 30-fold antibody titer differences between the homologous Border disease strain and
327 a representative of the most frequent subgroup of BVD virus 1 found in our cattle
328 population (see Figure 3C). Vaccination against BVD virus was very rare before, and
329 has been banned since, the start of the eradication program. This suggests that
330 natural infection of cattle with BVD virus may confer some degree of immunity also to
331 Border disease virus. Before the start of BVD eradication, some 60% of the cattle
332 population was seropositive to BVD virus (Rüfenacht et al., 2000), as compared to
333 some 20% of the sheep seropositive to Border disease virus (Schaller et al., 2000;
14
334 Danuser et al., 2007) . However, as the eradication program progresses, herd
335 immunity to BVD virus decreases and more bovines will become susceptible for
336 Border disease virus. While the biological consequences for a seronegative cattle
337 population are difficult to judge at this time, infections with Border disease clearly
338 pose a diagnostic problem for BVD eradication. Specifically, demonstration of
339 freedom from BVD is based on absence of antibodies to BVD in the cattle
340 population. To our knowledge, there exists no simple test that clearly differentiates,
341 on the species level, antibodies to BVD virus from antibodies to Border disease
342 virus. As indicated above and shown in the literature, both BVD- and Border disease
343 viruses are antigenically highly diverse, with the antibodies to individual virus strains
344 cross-reactive to varying degrees (Nettleton et al., 2008; Becher et al., 2003;
345 Bachofen et al. 2008; Ridpath et al., 2010; Strong et al., 2010). The SNT clearly
346 shows the difference in titer between antibodies to the Border disease virus strain
347 used in our experiment and a representative strain of the most prevalent BVD virus
348 subgroup present in the cattle population (Fig. 3C). However, due to its high
349 resolution between antibodies to different subgroups of BVD virus (Bachofen et al.,
350 2008), an SNT using just one strain of BVD-1 virus is not representative for the entire
351 species. Moreover, the SNT is unsuitable for mass testing of sera because it is work-
352 intensive and depends on cultured cells. The development of a simple test that
353 allows a clear-cut and efficient differentiation of antibodies to BVD- and Border
354 disease viruses is a high priority in BVD eradication. The ELISA used in our study is
355 clearly less sensitive for detecting antibodies to Border disease virus than the SNT
356 (compare Figs 3B and 3C). This ELISA detects mainly antibodies to the
357 nonstructural protein NS3 of a BVD-1 virus (Wageck-Canal et al., 1998). Although
358 significantly more conserved than E2, the main target protein of neutralizing
15
359 antibodies, the derived amino acid sequences of the NS3 of BVD- and Border
360 disease viruses are different between, and heterogeneous within, the two species of
361 pestivirus (Stalder, unpublished). Crossed ELISAs of immune sera to the different
362 subgroups of these two virus species would show if NS3 is a suitable antigen for
363 differentiation.
364
365 From the practical viewpoint of BVD eradication programs, it seems advisable to
366 minimize contact between sheep and cattle because of the complications due to
367 spillover of Border disease virus from sheep. This would also reduce the risk of
368 malignant catarrhal fever in bovines, a fatal disease which is caused by ovine herpes
369 virus-2, a virus carried virtually by all sheep (for a review, see Russell et al., 2009).
370
371
372 Acknowledgements
373 We thank Drs. T. Løken and Matthias Schweizer for helpful comments and Mrs. Ruth
374 Parham for linguistic improvements of the manuscript. This work obtained financial
375 support from the Swiss Federal Veterinary Office and the Cantons of Zurich and
376 Bern.
377
378 Conflict of interest statement
379 The authors declare no conflicts of interest
16
380 Figure captions
381
382 Figure 1. Phylogenetic analysis of selected pestiviruses based on Npro.
383 The tree was constructed from the complete Npro coding sequences except for the
384 two strains Aydinb/04 (EU930014) and Burdur (EU930014) (Shortening all
385 sequences to 420 nucleotides did not change the structure of the tree). Sequences
386 were obtained from Genbank and analyzed as described (Peterhans et al., 2010).
387 Please note that the taxonomic position of some of the atypical pestiviruses and of
388 some Border disease viruses is open, e.g. the HoBi like, Chamois and Turkey
389 viruses. The position of the Border disease viruses used in this work is indicated by
390 an arrow. Genbank accession #: CH-R4785: GU244490 (Npro), JQ994199 (5’UTR);
391 CH-R4786: GU244489 (Npro), JQ994200(5’UTR).
392
393 Figure 2. Lesions.
394 A: erosion in the rostral palate of orally inoculated calf #7, B: erosions in calf #3
395 exposed to PI sheep.
396
397 Figure 3. Antibody response in calves.
398 A: Antibody response of orally inoculated calves; B: calves exposed to sheep
399 persistently infected with Border disease; C: comparison of neutralizing antibody titer
400 to Border disease virus CH-R4786 strain and BVD virus strain CH-04-01b of
401 subgenogroup 1h.
402
17
403 References
404
405 Arquint, A. 2003. Immunhistologische Untersuchungen an Hautbiopsien bei akuter
406 BVD (Bovine Virus Diarrhoe). Inauguraldissertation, Universität Zürich
407 Bachofen, C., Stalder, H.P., Braun, U., Hilbe, M., Ehrensperger, F., Peterhans, E.
408 2008. Co-existence of genetically and antigenically diverse bovine viral
409 diarrhoea viruses in an endemic situation. Vet. Microbiol. 131, 93-102.
410 Becher, P., Orlich, M., Shannon, A.D., Horner, G., König, M., Thiel, H.-J. 1997.
411 Phylogenetic analysis of pestiviruses from domestic and wild ruminants. J.
412 Gen. Virol. 78, 1357-1356.
413 Becher, P., Ramirez, R.A., Orlich, M., Rosales, S.C., König, M., Schweizer, M.,
414 Stalder, H., Schirrmeier, H., Thiel, H.-J. 2003. Genetic and antigenic
415 characterization of novel pestivirus genotypes: implications for classification.
416 Virology 311, 94-104.
417 Becher, P., Thiel H.J. 2011. Genus Pestivirus (Flaviviridae), p 483-488,
418 in: Tidona, C.A. and Darai, G. (ed), Springer Index of viruses, second ed.,
419 Springer Verlag, Heidelberg, Germany, 2011).
420 Campbell, J.R., Radostits, O.M., Wolfe, T., Janzen, E.D., 1995. An outbreak of
421 border disease in a sheep flock. Can. Vet. J. 36, 307-309.
422 Carlsson, U., 1991. Border disease in sheep caused by transmission of virus from
423 cattle persistently infected with bovine virus diarrhoea virus. Vet. Rec. 128,
424 145-147.
18
425 Carlsson, U., Belak, K., 1994. Border disease virus transmitted to sheep and cattle
426 by a persistently infected ewe: epidemiology and control. Acta Vet. Scand. 35,
427 79-88.
428 Cranwell, M.P., Otter, A., Errington, J., Hogg, R.A., Wakeley, P., Sandvik, T., 2007.
429 Detection of Border disease virus in cattle. Vet. Rec. 161, 211-212.
430 Danuser, R., Vogt, H.-R., Kaufmann, T., Peterhans, E., Zanoni, R. 2009.
431 Seroprevalence anc characterization of pestivirus infections in small
432 ruminants and new world camelids in Switzerland. Schweiz. Arch. Tierheilk.
433 151, 109-117.
434 Hessman, B.E., Sjeklocha, D.B., Fulton, R.W., Ridpath, J.F., Johnson, B.J., McElroy,
435 D.E., 2012. Acute bovine viral diarrhea associated with extensive mucosal
436 lesions, high morbidity and mortality in a commercial feedlot. Journal of
437 Veterinary Diagnostic Investigation 24, 397-404.
438 Hilbe, M., Arquint, A., Schaller, P., Zlinsky, K., Braun, U., Peterhans, E.,
439 Ehrensperger, F., 2007. Immunohistochemical diagnosis of persistent
440 infection with bovine viral diarrhea virus (BVDV) in skin biopsies. Schweiz.
441 Arch. Tierheilk. 149, 377-344.
442 Hilbe, M., Camenisch, U., Braun, U., Peterhans, E., Stalder, H.P., Zlinszky, K.,
443 Ehrensperger, F., 2009. Mucosal lesions in a sheep infected with the Border
444 Disease Virus (BDV). Schweiz. Arch. Tierheilkd. 151, 391-396.
445 Hornberg, A., Fernandez, S.R., Vogl, C., Vilcek, S., Matt, M., Fink, M., Kofer, J.,
446 Schopf, K., 2009. Genetic diversity of pestivirus isolates in cattle from
447 Western Austria. Vet. Microbiol. 135, 205-213.
19
448 Hüssy, D., Stäuber, N., Leutenegger, C.M., Rieder, S., Ackermann, M., 2001.
449 Quantitative fluorogenic PCR assay for measuring ovine herpesvirus 2
450 replication in sheep. Clin. Diagn. Lab. Immunol, 8, 123-128.
451 Krametter-Frötscher, R., Duenser, M., Preyler, B., Theiner, A., Benetka, V., Moestl,
452 K., Baumgartner, W., 2010. Pestivirus infection in sheep and goats in West
453 Austria. Vet. J. 186, 342-346.
454 Krametter-Frötscher, R., Benetka, V., Möstl, K., W. Baumgartner, W., 2008.,
455 Transmission of Border disease virus from sheep to calves a possible risk
456 factor for the Austrian BVD eradication programme in cattle ? Wiener
457 Tierärztliche Monatsschrift 95, 200-203.
458 Løken, T., 1995. Border disease in sheep. Vet. Clin. North. Am. Food Anim. Pract.
459 11, 579-595.
460 Marco, I., Lopez-Olvera, J.R., Rosell, R., Vidal, E., Hurtado, A., Juste, R., Pumarola,
461 M., Lavin, S., 2007. Severe outbreak of disease in the southern chamois
462 (Rupicapra pyrenaica) associated with border disease virus infection. Vet.
463 Microbiol. 120, 33-41.
464 Marco, I., Cabezon, O., Rosell, R., Fernandez-Sirera, L., Allepuz, A., Lavin, S. 2011.,
465 Retrospective study of pestivirus infection in Pyrenean chamois (Rupicapra
466 pyrenaica) and other ungulates in the Pyrenees (NE Spain). Vet. Microbiol.
467 149, 17-22.
468 Monies, R.J., Paton, D.J., Vilcek, S., 2004. Mucosal disease-like lesions in sheep
469 infected with Border disease virus. Vet Rec 155, 765-769.
20
470 Nettleton, P.F., Entrican, G., 1995. Ruminant pestiviruses. Br. Vet. J. 151, 615-642.
471 Nettleton, P.F., Gilray, J.A., Russo, P., Dlissi, E., 1998. Border disease of sheep and
472 goats. Vet. Res. 29, 327-340.
473 Passler, T., Walz, P.H., 2010. Bovine viral diarrhea virus infections in heterologous
474 species. Anim. Health. Res. Rev. 11, 191-205.
475 Paton, D., Gunn, M., Sands, J., Yapp, F., Drew, T., Vilcek, S., Edwards, S., 1997.
476 Establishment of serial persistent infections with bovine viral diarrhoea virus in
477 cattle and sheep and changes in epitope expression related to host species.
478 Arch. Virol. 142, 929-938.
479 Peterhans, E.; Bachofen, C.; Stalder, H.P.; Schwyzer, M. 2010; Cytopathic bovine
480 viral diarrhea viruses (BVDV): emerging pestiviruses doomed to extinction.
481 Vet. Research , 41: 44 (DOI: 10.1051/vetres/2010016)
482 Ridpath, J.F., Fulton, R.W., Kirkland, P.D., J.D. Neill, J.D., 2010. Prevalence and
483 antigenic differences observed between Bovine viral diarrhea virus
484 subgenotypes isolated from cattle in Australia and feedlots in the
485 southwestern United States. J. Vet. Diagn. Invest. 22, 184-191.
486 Russell, G.C., Stewart, J.P., Haig, D.M., 2009. Malignant catarrhal fever: a review.
487 Vet. J. 179, 324-335.
488 Rüfenacht, J., Schaller, P., Audigé, L., Strasser, M., Peterhans, E., 2000. Prevalence
489 of cattle infected with bovine viral diarrhoea virus in Switzerland. Vet. Rec.
490 147, 413-417.
491 Schaller, P., Vogt, H.R., Strasser, M., Nettleton, P.F., Peterhans, E., Zanoni, R.,
21
492 2000. Seroprevalence of maedi-visna and border disease in Switzerland.
493 Schweiz. Arch. Tierheilkd. 142, 145-153.
494 Stalder, H.P., Laue, T., Marquart, H., Werth, C., Gauch, S., Peterhans, E. 2008.
495 BVDV eradication program in Switzerland: Requirements for a virus detection
496 kit. Abstracts 7th Pestivirus Symposium, Uppsala Sweden, 16-19 September
497 2008
498 Strong, R., La Rocca, S.A., Ibata, G., Sandvik, T., 2010. Antigenic and genetic
499 characterisation of border disease viruses isolated from UK cattle. Vet.
500 Microbiol. 141, 208-15.
501 Thür, B., Hilbe, M., Strasser, M., Ehrensperger., F., 1997. Immunohistochemical
502 diagnosis of pestivirus infection associated with bovine and ovine abortion
503 and perinatal death. Am. J. Vet. Res. 58, 1371-1375.
504 Vilcek, S., Durkovic, B., Kolesarova, M., Paton, D.J. 2005. Genetic diversity of
505 BVDV: consequences for classification and molecular epidemiology. Prev.
506 Vet. Med. 72, 31-35.
507 Wageck Canal, C., Strasser, M., Hertig, C., Masuda, A., Peterhans, E. 1998.
508 Detection of antibodies to bovine viral diarrhoea virus (BVDV) and
509 characterization of genomes of BVDV from Brazil. Vet. Microbiol. 63, 85-97.
22