Veterinary Microbiology 141 (2010) 258–267

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Clinical appearance and pathology of cattle persistently infected with

bovine viral diarrhoea virus of different genetic subgroups
Claudia Bachofen a,*, Ueli Braun b, Monika Hilbe c, Felix Ehrensperger c, Hanspeter Stalder a,
Ernst Peterhans a
a
Institute of Veterinary Virology, University of Bern, Länggass-Str. 122, PO Box 8466, CH-3001 Bern, Switzerland
b
Department of Farm Animals, University of Zürich, Winterthurerstr. 260, CH-8057 Zürich, Switzerland
c
Institute of Veterinary Pathology, University of Zürich, Winterthurerstr. 268, CH-8057 Zürich, Switzerland

A R T I C L E I N F O A B S T R A C T

Article history: Bovine viral diarrhoea (BVD) is an economically important cattle disease with a world-
Received 14 May 2009 wide distribution that is caused by BVD virus, a pestivirus of the flaviviridae family. BVD
Received in revised form 9 September 2009 viruses are genetically highly variable. They are classified into two genetic species (BVDV-
Accepted 22 September 2009 1 and -2) that are further divided into numerous subgroups, particularly for BVDV-1. The
complexity of these viruses is also reflected in their interaction with the host animals.
Keywords: Infections are either transient or persistent and can cause a wide spectrum of clinical signs,
Bovine viral diarrhoea virus from no or very mild disease to severe forms, reminiscent of viral haemorrhagic fevers. In
Pestivirus
this work, we have analysed the clinical signs and the pathology of BVD viral infections in a
Clinic
cattle population where different subgroups of BVDV-1 genotype viruses are endemic. In
Pathology
Genetic subgroup
addition, we have examined potential virulence properties of BVDV-1 subgroups during
Virulence persistent infection by comparing the viral subgroups present in clinical cases with those
detected in persistently infected (PI) animals sampled for epidemiological criteria,
irrespective of their health condition. Furthermore, the clinical and postmortem findings
were compared with respect to genetic characteristics of the viruses isolated from these
animals.
Our results indicate that the BVDV positive animals fall roughly into two categories,
depending on the primary organ affected and the age, with lung-centred pathology
occurring mainly in young animals and mucosal pathology predominantly in older
animals. Furthermore, we found a markedly higher proportion of representatives of the
BVDV-1e subgroup in stillborn calves and aborted foetuses originating from epidemically
unrelated cattle herds, suggesting that BVDV-1e may play a special role in prenatal and
perinatal losses.
ß 2009 Elsevier B.V. All rights reserved.

1. Introduction divided into two species, BVDV-1 and -2. BVDV-1 strains
are particularly diverse, with at least 13 different
Bovine viral diarrhoea virus (BVDV), classical swine subgroups defined genetically (Pellerin et al., 1994; Becher
fever virus and border disease virus form the genus et al., 1997, 2003; Vilček et al., 2001, 2004; Falcone et al.,
pestivirus within the flaviviridae family. BVD viruses are 2003; Xu et al., 2006; Jacková et al., 2008) that differ also
antigenically as shown by up to tenfold antibody titer
differences between subgroups in crossed neutralization
* Corresponding author. Tel.: +41 31 631 25 10; fax: +41 31 631 25 34.
tests (Bachofen et al., 2008). Despite their diversity, all BVD
E-mail addresses: [email protected], [email protected] viruses are either of the cytopathic (cp) or the non-
(C. Bachofen). cytopathic (ncp) biotype, as defined by the effect of viral

0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2009.09.022
 C. Bachofen et al. / Veterinary Microbiology 141 (2010) 258–267 259

growth in cultured cells. The two biotypes also differ with were restricted to single outbreaks of identical or very
respect to their effects in infected animals. Only the ncp closely related viral strains that affected several animals
biotype can establish a persistent infection of the foetus if within one herd or in different herds simultaneously, or to
the dam is transiently infected between approximately experimental infections. Hence, the observations may be
days 40 and 120 of its development. Even though such representative for a given viral strain rather than for an
foetuses may develop normally, they remain infected for entire subgroup of BVDV.
life and shed large quantities of virus, thereby assuring the In contrast to transient infections, disease associations
persistence of BVDV in the cattle population even in the between individual BVDV strains or subgroups have not
absence of animals susceptible to transient infection. been investigated in persistent infection.
Different from other persistent viral infections in man and BVD is endemic in Switzerland, with roughly 60% of
animals, BVDV persistently infected (PI) cattle are immu- cattle seropositive and 0.7% persistently infected, with
notolerant to the infecting viral strain (reviewed in seemingly no change in prevalence over a time period of
Peterhans et al., 2006). some 30 years (Bürki et al., 1964; Homberger et al., 1975;
The clinical consequences of infection with BVD virus Rüfenacht et al., 2000; Stalder et al., 2005). In this work, we
are as diverse as the genetic and antigenic properties of have analysed PI animals referred by private veterinary
these viruses. Disease can result from different patho- practitioners to the clinical and pathology departments of
genetical mechanisms, depending on the different types the veterinary hospital of the University of Zürich. Due to
of infection. Most transient infections may take an the endemic nature of BVD, these animals may represent
inapparent or mild course, associated with low-grade the most severe clinical forms of persistent infection,
fever, diarrhoea and coughing. Rarely, however, acutely comparable to the tip of the iceberg.
infected animals may suffer from high grade fever and We have recorded and applied a scoring protocol for the
bleeding in internal organs. This often fatal form of acute clinical and postmortem findings of the diseased animals
infection was reported in outbreaks in the late 80s and and analysed possible correlations between different
early 90s, caused by highly virulent BVD virus-2 strains organ manifestations and the subgroups of persisting
(Ridpath et al., 2000, 2006). Although generally consid- BVDV. To obtain an insight into the possibility of virulence
ered to be of low virulence, BVDV-1, too, may cause similar of BVDV in persistent infection, we have analysed the viral
severe disease signs in acutely infected animals, albeit subgroups present in diseased PI animals as compared to
only rarely (Liebler-Tenorio et al., 2000; Ridpath et al., subgroups present in animals diagnosed BVDV PI inde-
2007). pendently of the clinical status.
In the case of persistent infections, clinical signs in PI
animals can be differentiated pathogenetically into muco-
2. Animals, materials and methods
sal disease (MD) and non-MD cases. The onset of MD is
associated with the appearance of the cytopathic biotype 2.1. Patients and examinations
of BVDV that arises as a result of mutations and/or
recombination events from the persisting ncp virus At the Department of Farm Animals of the University of
(Kümmerer et al., 1998; Tautz et al., 1998; Becher et al., Zurich, all calves less than 3 months of age and chronically
2001). This lethal disease is characterised by mucosal diseased animals are routinely tested for the presence of
lesions, destruction of the lymphoid tissue in the gastro- BVDV. We have retrospectively analysed all 68 clinical
intestinal tract (Liebler et al., 1995) and untreatable reports of PI animals shown at the clinic for ruminants
diarrhoea. The onset of the clinical signs is often acute from first of January 1995 to 31st of December 2003 (group
but a more chronic form, called late onset MD, is also A, Table 1). In addition, the 18 prospective cases from the
described (Liebler-Tenorio et al., 2000; Fritzemeier et al., years 2004 and 2005 were included (group B, Table 1),
1997). yielding 86 clinical case reports. All animals had under-
The clinical signs in PI animals that have not (yet) gone the same thorough clinical examination following a
developed MD can encompass a wide spectrum of standardised protocol as described previously (Braun et al.,
symptoms, ranging from normal health to subclinical 1996). Haematological, biochemical, bacteriological and
disorders, ill-thrift and growth retardation. Chronic or parasitological analyses were performed as described by
recurrent intestinal and/or pulmonary symptoms are Braun et al. (2007).
frequently observed but occasionally dermatological, The BVDV status of 76 animals of groups A and B was
neurological or haematological disorders are the only determined intra vitam by immunohistochemistry (IHC) of
signs of a persistent infection (Braun et al., 1996; Taylor skin biopsies (66 cases) and by antigen-capture ELISA of
et al., 1997). In many cases the clinical and necropsy blood leukocytes (8 cases) (Strasser et al., 1995) or of skin
findings do not allow a clear differentiation between MD biopsies (2 cases) (Idexx HerdCheck BVDV Ag/Serum Plus,
and non-MD cases. The isolation of both biotypes, ncp and Idexx Europe B.V., 1119 NE Schiphol-Rijk, The Nether-
cp, proves a PI animal to suffer from MD. lands). Some animals were repeatedly tested by multiple
Possible reasons for the variable clinical signs seen in methods. Ten animals of groups A and B were only
BVDV infections have been analysed repeatedly and in diagnosed after being euthanised. The BVDV status of all
some cases an impact of the infecting virus strain was animals was confirmed postmortem by IHC of organ
proven (Baule et al., 1997, 2001; Fulton et al., 2002; Jones samples. All samples revealed the staining pattern typical
et al., 2004; Ridpath et al., 2007). However, these studies for persistent infection, as described previously by Hilbe
focused on transient infections. Moreover, the analyses et al. (2007).
260 C. Bachofen et al. / Veterinary Microbiology 141 (2010) 258–267

Table 1
List of cases included in this study.

Group Clinical report Necropsy report Sequence analysis

(A) Retrospective cases 1995–2003 68 67 52

(B) Prospective cases 2004 + 2005 18 18 18
(C) Pathology onlya 1995–2005 – 60 (C.1b: 45; C.2c: 15) 60
(D) Abortions/neonatesd 1995–2005 – 25 25
a
Euthanised or perished animals, referred directly to the Institute of Veterinary Pathology by private practitioners for postmortem analysis of the entire
carcassb or of body parts/organs onlyc. There were no clinical reports available of these cases.
d
Aborted foetuses/foetal body parts and euthanised or perished neonatal calves (<3 days), referred directly to the Institute of Veterinary Pathology by
private practitioners for postmortem analysis. There were no clinical reports available of these cases.

As a result of the unfavourable prognosis, all animals lesion would serve to assess the general impairment of the
were euthanised within 1–22 days after the initial clinical organ.
investigation and sent to the Institute of Veterinary
Pathology for necropsy. A necropsy report was available 2.3. Mucosal index
of 85 of the cases from groups A and B. In addition, we
included in our study 60 necropsy reports of animals that To compare the mucosal alterations between different
had been euthanised or had perished and been submitted PI animals, we used a standardised mucosal (mc) index
by private veterinarians for necropsy to the Institute of calculated for each animal. This index considers the
Veterinary Pathology where they were tested BVD virus- number of affected sections of the alimentary tract (from
positive by IHC of organ samples (group C, Table 1). The nose to large intestines = max. 7 organs; affected = any
reasons for the submission of these carcasses for post- pathological change from the normal tissue integrity) and
mortem analysis were variable and encompassed exam- the severity of the alterations (0 = no alteration, 1 = mild,
ination of herd problems, clarification of ambiguous 2 = moderate, 3 = severe alteration). With respect to the
clinical disease signs or suspicion of inherited disease multiple dispersed lesions typically seen in MD cases
such as spinal muscle atrophy of Swiss Brown calves. No (Liebler-Tenorio et al., 2000) the calculation places more
information on intra vitam examinations and their results weight on the number of affected sections than on the
were available of the animals of group C. severity of the alterations. The mc index may range from 0
All in all, we analysed 145 necropsy reports (groups A, B, to 21.
P
C), 130 of which contained complete data sets (groups A, B, ðmucosal scoresÞ
C.1). In 15 cases, postmortem reports were only available mc index ¼
number of analysed organs
for single organs or body parts (group C.2, Table 1).
 number of organs with lesions
The cases analysed were extended by 25 cases of BVDV
infected neonates (stillbirths and calves younger than 3
days) and aborted foetuses, tested positive by IHC (staining Example:
pattern typical for persistent infection (group D, Table 1)).
Mucosal organ Score

2.2. Recording of reports Muzzle/nose 3 mc index ¼ 97  5 ¼ 6:4

Oral cavity 2
Oesophagus 1
Descriptive information from the standardised clinical Forestomach 0
and necropsy reports was transformed into ordinal data Abomasum 2
(e.g. general health condition, degree of lung alteration) or Small intestines 1
nominal data (e.g. gender) with the help of a specially Large intestines 0
P
(mucosal scores) 9
created evaluation protocol. The evaluation protocol
applied was created in collaboration with clinicians and
pathologists and is based on clinical scoring protocols 2.4. Viral genomic sequences
used previously (Braun et al., 1996, 2005). For the majority
of findings we used a simple 4-level grading: nothing 177 Swiss BVDV strains, isolated between 1990 and
abnormal detected = 0, mild = 1, moderate = 2, severe 2005 were sequenced and phylogenetically analysed by
alteration = 3. The evaluation of all reports was performed Stalder et al. (2005). These viruses originated from animals
by the same person and without any information on the detected to be PI as a result of a cross-sectional
BVDV subgroup isolated from the corresponding PI epidemiological study to determine the prevalence of
animal. The scoring was based on the graduation of the BVDV in Switzerland (Rüfenacht et al., 2000). In addition,
clinical and postmortem findings made by the various viruses were derived from herd analyses performed in
clinicians and pathologists who had initially recorded the order to assess diagnostic tests or to apply molecular
cases. Grading the findings is an essential part of the epidemiology.
standardised protocols used for initial clinical examina- The 155 BVDV isolates of the clinical cases described
tion and necropsy. In those cases where the organ was here were isolated, sequenced and phylogenetically
only partially affected (e.g. an abscess in a single lung analysed for a study published previously (Bachofen
lobe) we considered that the severity and spread of the et al., 2008).
 C. Bachofen et al. / Veterinary Microbiology 141 (2010) 258–267 261

2.5. Statistical analysis Of the 146 cases of groups A, B and C (abortions and
neonates not included since the gender is often not
For all statistical calculations the NCSS/PASS software stated) 78% were females, while for the animals with a
(Kaysville, UT, USA) was used. As the data were not clinical report (groups A and B, n = 86) the proportion of
normally distributed, we used the non-parametric one- females was 90%. Animals of five different breeds and
way ANOVA test (Kruskal–Wallis) to compare medians of diverse crossbreeds were analysed. The majority of cases
three or more groups of patients. If this test revealed a (n = 146) were of the Swiss Brown (67%), Holstein-Frisian
significant over-all difference, pair-wise comparisons were (12%) and Swiss Fleckvieh (11%) breeds. These numbers
performed using the Wilcoxon Rank-Sum test. To compare reflect the over-all breed proportions of the patient
proportions we applied the Chi-square or Fisher’s exact population of the clinic. Single cases of Rhätisches
test. The probability level was set to 95%. The displayed p- Grauvieh, Dexter, Limousin and beef crossbreeds were
values represent the results of the two-tailed hypothesis also represented. The median age of the animals (n = 146)
testing. was 7 months, with a range from 2 days to 6 years.
Animals referred to the clinic (groups A and B, n = 86)
2.6. Determination of biotypes were significantly older (median = 12 months) than those
submitted directly for necropsy (group C, n = 60, median
We tested serum of 14 PI animals from the prospective age = 2 months). Of the animals with a clinical record
cases for the existence of cytopathic BVDV virus as a (n = 86), 26% had an age of 24 months or over and four
hallmark for the onset of MD. The sera were diluted seven animals were older than 3 years (48, 52, 60 and 72
times in tenfold steps in cell culture medium (Earle’s months). The cases originated from 17 of the 26 Swiss
minimal essential medium (MEM)) enriched with 2% foetal cantons. Most of the cases (n = 146) had a history of
bovine serum (FBS). MEM was purchased from Seromed recurrent or untreatable diarrhoea (41%), pneumonia
(Biochrom, Munich, Germany) and FBS from Sigma or (20%) or both conditions together (9%). Neurological
Oxoid GmbH (Wesel, Germany). FBS was free of BVDV and symptoms (10%) were often anamnestically described in
antibody to BVDV as tested by virus isolation and serum necropsy reports of young calves, directly referred for
neutralization test, respectively. Each serum dilution step postmortem examination (group C). Other anamnestic
was distributed to six wells (100 ml) of a 96-well microtiter signs were abortion, lameness due to claw problems,
plate, seeded with primary bovine turbinate cells. After 5 anaemia and ill-thrift. In 45% of the cases (n = 146) the
days of incubation at 37 8C and 5% CO2, 20 ml of super- anamnestic report indicated concomitant health pro-
natant was transferred to a fresh 96-well microtiter plate, blems in the herd. Growth retardation was indicated in
pre-seeded with bovine turbinate cells. After addition of 30% of the cases.
80 ml of fresh MEM, the microtiter plate was incubated as
before and the passaging procedure repeated once more. 3.2. Clinical and postmortem findings
After each passage, the cells were fixed and stained for viral
protein as described by Adler et al. (1997). The cells were The 12 most frequently observed clinical and post-
then microscopically controlled for the presence of a mortem manifestations are shown in Fig. 1. Nearly all
cytopathic effect. animals showed unspecific signs like loss of appetite (94%),
reduction of the general condition (93%) and ruminal
3. Results hypoperistalsis (92%), followed by intestinal disorders like
diarrhoea (69%) and the resulting dehydration (63%).
In order to investigate differences between BVDV Respiratory sounds of different degree were present in
subgroups regarding virulence during persistent infection, 63% of the cases. Mucosal erosions as a more specific
clinical recordings and necropsy reports of PI animals manifestation were found in the oral cavity and on the
infected with viruses of different BVDV-1 subgroups were muzzle of 45% and 36% of the animals, respectively
analysed. We included patients from the clinic for (Fig. 1A). For the determination of the most frequent
ruminants of the Department of Farm Animals (University postmortem findings, only cases with a complete necropsy
Zürich) and additional necropsy reports of animals referred report were included, whereas partial necropsies (e.g.
by private veterinarians directly to the Institute of single organs or body parts) were excluded (group C.2).
Veterinary Pathology (University Zürich) for postmortem Group D was excluded, because the abortions were often in
analysis. a condition that would no longer allow clear postmortem
diagnosis or consisted only of foetal body parts. The most
3.1. Groups of patients and anamneses frequent necropsy findings differed between animals with
clinical reports (groups A and B) and those referred directly
As shown in Table 1, the cases could be separated into for necropsy (group C.1) (Fig. 1B). In the latter, these were
four groups: retrospective cases (group A, n = 68) and pathological changes in the lung, while alterations in one
prospective cases (group B, n = 18) with both a clinical and or more mucosal organs (from muzzle to large intestines)
a necropsy report, cases directly referred for necropsy by were the primary findings in the clinical cases. Generally,
private practitioners without clinical report (group C, except for the lung, a higher proportion of cases referred to
n = 60) and postmortem analyses of abortions and neo- the clinic (groups A and B) showed macroscopic lesions
nates (animals 3 days of age) without clinical report than animals directly referred for a postmortem examina-
(group D, n = 25). tion (group C.1).
262 C. Bachofen et al. / Veterinary Microbiology 141 (2010) 258–267

Fig. 1. (A) The 12 most frequently observed clinical findings in 86 clinically examined BVDV positive animals (68 of group A and 18 of group B, Table 1). (B)
The 12 most frequently observed necropsy findings from PI animals submitted for postmortem examination from the clinic (black bars) or directly from
private practitioners (grey bars). Only necropsies of entire carcasses were included. The animal groups indicated refer to Table 1. aOne or multiple
pathological alteration(s) of the mucosa of the whole alimentary tract, including nose and muzzle. bOne or multiple pathological alteration(s) of the mucosa
of the gastrointestinal tract. cMacroscopic alteration of the mediastinal or mesenterial lymph nodes.

3.3. Haematological and microbiological findings rather upon clinical suspicion. However, with the excep-
tion of gastrointestinal nematodes that were present in
Haematological data were available from 82 of the 86 62% of the 47 cases analysed, parasites and bacteria such as
cases with a clinical report (groups A and B). The most liver fluke, lung worm, Salmonella, Campylobacter and
frequently observed changes were elevated bilirubin levels enterotoxic Escherichia coli were only occasionally con-
(89%) and monocytosis (88%), neutrophilia (in 74%) and firmed (0–10% of analysed cases). Furthermore, all 30
leukocytosis (in 59% of the animals). Decreased leukocyte animals analysed for the presence of ovine herpesvirus-2,
counts were found in 11% of the cases. Alterations in the causative agent of malignant catarrhal fever in cattle,
erythrocyte parameters were frequent and were displayed were negative. Four and five animals were analysed for the
as polycythemia (73% of the animals), decreased cellular presence of rota- and coronavirus, respectively. For each
haemoglobin content (61%) and a decreased erythrocyte virus, one animal tested positive. Bovine herpesvirus-1
volume (59%). that can cause symptoms similar to BVD/MD is eradicated
For groups A and B, intra vitam parasitological and in Switzerland and can be excluded as contributing factor
bacteriological analyses were not performed routinely but to the symptoms observed. For all groups, postmortem
 C. Bachofen et al. / Veterinary Microbiology 141 (2010) 258–267 263

parasitological and bacteriological analyses were per-

formed sporadically, depending on demand and suspicion
and were not included to the study.

3.4. Comparison of mucosal lesions

In many cases, mucosal lesions were observed at

multiple locations of the gastrointestinal tract. To be able
to compare the general degree of mucosal lesions between
groups of animals, it was necessary to condense the
multiple mucosal alterations to one parameter. We used
the number of affected organs and the severity of the single
alterations to calculate an index for each animal, referred
to as mucosal (mc) index (see Section 2 for details). The
highest value that is theoretically possible (= all analysed
organs are severely affected) is 21. The maximal and
minimal mc index values observed from the 130 cases
analysed (complete necropsy reports only: groups A, B and
C.1) was 16 and 0, respectively, with an arithmetic mean of
1.9.
Because intestinal and pulmonary symptoms were the
most frequent findings observed in the BVDV infected
animals, we investigated whether these findings were
correlated. Interestingly, these organ manifestations
tended to exclude each other. As shown in Fig. 2A, animals
with a high mucosal index had significantly less severe
lung alterations than those with a low mucosal index.
Moreover, animals with severe pulmonary manifestations
were significantly younger than those with less severe
pathological changes in the lungs (Fig. 2B). By contrast, the
mucosal index was significantly higher in older animals
than in young ones (Fig. 2C). Linear regression analysis
(Spearman Rank) demonstrated a weak (r = 0.46) but
statistically significant (p = 0.0003) correlation between
the mc index and the age of the animals.

3.5. Correlation between postmortem findings and presence

of cp biotype

The dichotomy between lung and mucosal lesions

pointed to roughly two groups of BVDV infected patients.
In order to investigate if this correlated with the presence
or absence of mucosal disease, we tested serum samples of
14 PI animals of group B for the presence of cytopathic (cp)
virus by passaging different dilutions of the sera three
times on cultured bovine turbinate cells. This analysis was
restricted to 14 animals, since blood samples were only
available from the prospective cases (group B) and young
calves with maternal antibodies had to be excluded due to
inhibition of virus isolation. The results are shown in
Table 2. With one exception (04-06), the cp biotype was
only present in sera of animals with an mc index of 1.7,
suggesting that these patients had suffered from MD. No cp
biotype was observed in animals with an index of 0.9.
Therefore, we decided to set the threshold between MD-
and non-MD cases to 1.0. Extrapolating this finding to the

spread of mucosal lesions (displayed as mucosal index) and (B) with

Fig. 2. (A) Comparison of medians of BVDV positive animals showing respect to the age of the animals. (C) Comparison of different age groups
different degrees of postmortem lung alterations (0 = no alteration, of animals examined in relation to the mucosal index. a67 cases of group
1 = mild, 2 = moderate, 3 = severe alteration) in regard of the severity and A, 18 cases of group B and 45 cases of group C (Table 1).
264 C. Bachofen et al. / Veterinary Microbiology 141 (2010) 258–267

Table 2 persistently infected independent of their health condi-

Comparison of mucosal-indices and BVDV biotypes isolated from sera of
tion.
14 PI animals of group A (Table 1).

PI animal mc index BVDV biotype 3.7. Comparison of BVDV subgroups and manifestations
04-06 0.14 Ncp + cp
05-13 0.14 Ncp 114 cases with a complete necropsy report and a known
05-14 0.14 Ncp BVDV sequence could be included in this part of the study.
04-05 0.57 Ncp
This number is composed of 52 animals of group A, 18 of
05-05 0.86 Ncp
04-07 1.71 Ncp + cp group B and the 45 cases of group C.1. The single animal
05-04 1.71 Ncp + cp harbouring BVDV 1x was excluded from the analysis,
05-01 2.14 Ncp + cp resulting in 114 PI animals. Of these, 7% had harboured
05-03 2.86 Ncp + cp
viruses belonging to subgroup 1b, 34% to 1e and 1h each,
04-01 3.43 Ncp + cp
05-12 4.57 Ncp + cp and 25% to 1k (Fig. 4).
04-04 5.14 Ncp + cp We observed no statistically significant differences in
05-10 5.14 Ncp + cp the associations of viruses of a given subgroup with
05-11 8.57 Ncp + cp mucosal or pulmonary alterations or suspected MD cases
These 14 animals were chosen due to availability of serum and absence of (Fig. 4), nor did we see differences with regard to different
maternal antibodies. age groups, breeds or other clinical, postmortem or
haematological findings (data not shown). However, there
mucosal-indices of all cases, 40% of the animals (52/130) was a tendency for 1h viruses to be more frequently
had suffered from MD. isolated from animals with mucosal problems, presumable
MD cases (mucosal index  1), and thus older animals,
3.6. BVDV genetic subgroup analysis while 1e was the predominant subgroup of BVD virus in
patients with pulmonary alterations and thus younger
Of the 171 cases analysed in this work (groups A, B, C animals (Fig. 4). To broaden the age range of the patients,
and D), the partial genomic sequence of the persisting virus we additionally analysed the proportions of the different
was known from 155 cases (Bachofen et al., 2008). For the BVDV-1 subgroups in the 25 cases of group D (abortions,
18 cases of group B, viral RNA was isolated from blood, stillbirths and neonates). Interestingly, we observed a clear
from the other cases from frozen tissue samples. Phylo- predominance of representatives of subgroup 1e BVD
genetic subgroup assessment was performed based of the viruses in these animals (Fig. 4). The subgroup proportions
50 untranslated region and in some cases additionally on in group D were significantly different from those observed
the Npro coding region. The subgroups present are shown in in animals with mucosal lesions and suspected MD cases
Fig. 3B. The majority of the animals harboured viruses of (mc index  1).
the subgroups BVDV-1e (32%) and 1h (34%), followed by 1k
(24%) and 1b (9%). From one single animal we isolated the 4. Discussion
BVD-1x virus as described previously (Bachofen et al.,
2008). These are (with exception of 1x) the same The spectrum and severity of clinical signs caused by
subgroups as described by Stalder et al. (2005) (Fig. 3A). BVDV infection are highly variable. Factors contributing to
The subgroup 1e was most prominent in the latter (43%) this complexity include two types of infection with
while 1h was the most frequent subgroup in clinical cases entirely different involvement of the immune system
(34%) (Fig. 3). However, statistical analysis revealed no (immune response in transient versus immunotolerance in
significant differences of the proportions of BVDV sub- persistent infection), two different biotypes of virus (non-
groups present in the diseased PI’s and animals detected cytopathic versus cytopathic) and the diverse genetic

Fig. 3. Comparison of the proportions of BVDV-1 subgroups isolated from PI animals diagnosed for epidemiological studies and independent of the health
status (A) and from clinical cases (B). aAll cases of subgroups A, B, C and D where the viral genomic sequence was available.
 C. Bachofen et al. / Veterinary Microbiology 141 (2010) 258–267 265

or possible associations between disease signs and a given

subgroup of BVDV during this type of infection, have to
date not been addressed.
We have investigated these questions in an endemic
situation that was virtually undisturbed by vaccination or
other control measures, both of which might influence the
outcome of infection. In order to obtain information on the
pathogenic potential of BVDV in persistent infection, we
analysed clinical and necropsy findings of diseased PI
animals and compared the BVDV subgroups present in
these animals those isolated from animals diagnosed as PI
independently of the health status.
Since we included prospective as well as retrospective
cases examined over a period of 10 years, the long time
range and the fact that different clinicians and pathologists
examined the animals might influence the comparability
of the raw data. However, the protocol applied to both the
clinical and pathological investigations did not change
Fig. 4. Proportions of the four main Swiss BVDV subgroups isolated from over this time. In addition, the fact that the initial
different groups of PI animals. aOnly complete necropsy reports were examinations were performed by many different clinicians
included: 52 cases of group A, 18 of group B and 45 cases of group C
and pathologists decreases the risk of a systematic bias.
(Table 1) = 115. However, one single animal harboured a virus that did not
belong to any of the four subgroups analysed and was therefore not Moreover, the transformation of nominal into numerical
included (n = 114). b25 cases of group D (Table 1). data was done by one person, using a standardised
protocol.
To obtain a larger number of cases, we analysed
spectrum of BVDV as well as that of the host animals. different groups of patients. While the retro- and
Studies on the virulence of individual strains of BVDV focus prospective cases from the ruminants’ clinic (groups A
on transient infections. Experimental infections and and B) showed similar demographic parameters and
analysis of disease outbreaks in transiently infected postmortem findings (data not shown), we observed
animals characterised by particular signs and severity of significant differences to the animals of group C. Animals
disease clearly suggested an influence of the infecting viral of groups A and B were significantly older and generally
strain on disease manifestation. For example, Baule et al. more severely diseased than the cases directly referred for
(2001) as well as Jones et al. (2004), have shown BVDV-1d postmortem analysis (group C) (Fig. 1B). This might be due
strains to be associated with respiratory disease in acutely to the fact that, generally, the more valuable and thus
infected calves. Furthermore, Ridpath et al. (2007) could primarily older animals with complex or unsuccessfully
experimentally reproduce the clinical symptoms caused by pre-treated health problems are referred for a stay at the
virulent BVDV-1 strains in acutely infected heifers. clinic. The high proportion of females in groups A and B
In persistent infection, in contrast, analysis of a simply reflects the fact that most of these animals originate
potential impact of the viral strain on the clinical picture from dairy farms.
is, for a number of reasons, more difficult. Among them is Interestingly, also the main postmortem findings
the existence of disease signs associated with persistent differed between groups A and B versus group C, with
infection as such, and the occurrence of MD as a form of mucosal alterations playing a prominent role in groups A
infection associated with the emergence of a cytopathic and B and pulmonary lesions in group C (Fig. 1B). Keeping
biotype of BVDV in these immunotolerant animals. in mind the age difference between the groups, we tested
Furthermore, some of the signs of persistent infection the statistical correlation between lung and mucosal
unrelated to MD may be of chronic nature, such as growth manifestations and the age of the animals. Mucosal lesions
retardation and general ill-thrift. However, as shown by were more frequent in older and lung lesions in younger
pluriparous PI cows and PI bulls detected among the animals (Fig. 2); that held also true when the two animal
candidates for artificial insemination stations, clinical groups (A + B and C) were tested separately (data not
signs of persistent infection can be absent in some animals. shown). The reason for the correlation between age and the
Experimental generation of a sufficient number of PI postmortem finding may be related to the higher risk of
animals with different strains of BVDV would be laborious MD onset with increasing age. MD is correlated to the
and require long-term follow-up. In addition, many appearance of the cp virus that arises as a result of
different factors such as time point of maternal infection, mutations or recombination during viral RNA replication
genetic background of the animals and environmental from the persisting ncp virus (Deregt and Loewen, 1995;
factors, e.g. the presence of various other cattle pathogens, Neill and Ridpath, 2001; Lackner et al., 2004). Statistically,
may modulate the outcome. Moreover, the epidemiologi- a longer time period of viral replication may increase the
cal situation – endemic versus non-endemic – may risk of generating mutations leading to a cp biotype. Due to
influence the clinical appearance of infections. It is not the retrospective nature of the study, we were not in a
surprising, therefore, that questions such as the impact of position to fulfill the virological criterion for diagnosing
BVDV genetic properties on the health status of a PI animal, MD by showing the presence of cytopathic and non-
266 C. Bachofen et al. / Veterinary Microbiology 141 (2010) 258–267

cytopathic BVDV in all cases. To overcome this problem we animals (7 months). Since all PI animals are infected as
combined a virological determination of MD of selected foetuses, animals infected with BVDV-1e may die younger
cases (namely the isolation of both biotypes) with a than animals infected with other subgroups and are
standardised pathological investigation (Table 2). The therefore less frequently observed among older animals.
latter was achieved by creating a ‘‘mucosal (mc) index’’, This is supported by the observation of a surprisingly high
comparable to other clinical or pathological indices, e.g. proportion of BVDV-1e strains in abortions, stillbirths and
the psoriasis area severity index (PASI) in human neonates (Fig. 4), which clearly encourages additional
dermatology. Typically, MD leads to mucosal lesions at investigations. We can exclude that a 1e live vaccine may
multiple locations of the digestive tract. Therefore, the mc be responsible, since, as indicated above, vaccines are used
index places more weight on the number of affected organs only very infrequently, and a 1e vaccine is not licensed in
than on the severity of single lesions. This approach also Switzerland. Moreover, we can also exclude that a
minimizes individual differences in rating the severity of particular strain of 1e may cause these abortions because
lesions and favours completeness of the necropsy reports. the sequence data indicate that genetically different
The results of the comparison of the biotype analysis with strains were present in these animals (Bachofen et al.,
the mc index (Table 2) support the validity of the concept. 2008).
Taking all BVD viral sequences of groups A, B, C and D In summary, our study showed that, in an endemic
together, we did not observe statistically significant situation, clinical cases of BVDV PI animals fall roughly in
differences to the BVDV subgroup distribution previously two distinct categories, with lung-centred pathology
described by Stalder et al., 2005 (Fig. 3). With the exception occurring mainly in young animals and mucosal pathology
of the single ‘‘orphan’’ BVD 1x strain, we found the same mainly in older animals. Moreover, even though we did not
four BVD-1 subgroups in the diseased animals and in the PI find evidence for one BVDV subgroup being generally more
animals sampled in the course of an epidemiological virulent during persistent infection, the epidemiologically
survey. Although sampled in the same time period, the two unrelated concentration of 1e subgroup BVD viruses in
large groups of PI animals are not matched with respect to stillborn calves and aborted foetuses provides preliminary
breed, age or gender. On the one hand, using unmatched evidence that viruses of this subgroup may play a special
hosts, only major differences in tropism and virulence role in prenatal and perinatal losses due to BVD virus.
would show up. The results of our study argue against such
major differences between entire subgroups. On the other Acknowledgments
hand, this observation does not exclude that individual
strains may differ in virulence and tropism, as shown for This work was supported by the Swiss National Science
transient infections. Foundation and a grant from Vetsuisse. We thank Matthias
A different aspect concerns possible BVDV subgroup Schweizer and Reto Zanoni for helpful discussions, the
affiliations with the two main clinical and pathological team of the diagnostic unit for technical help, Marcus
patterns observed, i.e. lung- versus gastrointestinal tract- Doherr for statistical support and Ruth Parham for
centred. In this context, the possibility that these patterns linguistic assistance.
might be influenced by the presence (or absence) of other
pathogens needs to be considered. Such pathogens could References
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