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Bovine viral diarrhoea, bovine herpesvirus and parainfluenza-3 virus
infection in three cattle herds in Egypt in 2000
Article in Revue scientifique et technique (International Office of Epizootics) · January 2004
DOI: 10.20506/rst.22.3.1440 · Source: PubMed
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2. Rev. sci. tech. Off. int. Epiz., 2003, 22 (3), 879-892
Summary
This study reported field outbreaks of bovine viral diarrhoea virus (BVDV)
infection, either alone or mixed with bovine herpesvirus-1 (BHV-1) and/or
parainfluenza-3 virus (PI-3V) in Egypt during 2000. In Lower Egypt, young calves in
three cattle herds in El-Minufiya Province, El-Fayoum Province and in
governmental quarantine in El-Behira Province, showed symptoms of enteritis,
either alone or accompanied by respiratory manifestations. The affected herds
were visited and the diseased animals were clinically examined. Many
epidemiological aspects, such as morbidities, mortalities and case fatalities, as
well as the abortive rate, were calculated. Ethylenediamine tetra-acetic acid-
blood samples, sterile nasal swabs and serum samples were obtained for
virological and serological diagnosis. The laboratory investigations revealed that
the main cause of calf mortalities in the three herds was infection with BVDV,
either alone, as on the El-Minufiya farm, or mixed with PI-3V, as on the El-Fayoum
farm, or mixed with both BHV-1 and PI-3V, as in the herd in governmental
quarantine in El-Behira Province. A total of nine dead calves from the three herds
were submitted for thorough post-mortem examination. Tissue samples from
recently dead calves were obtained for immunohistochemical and
histopathological studies. The most prominent histopathological findings were
massive degeneration, necrosis and erosions of the lining epithelium of the
alimentary tract. Most of the lymphoreticular organs were depleted of
lymphocytes. In pneumonic cases, bronchopneumonia and atypical interstitial
pneumonia were evident. The present study suggested that the
immunosuppressive effect of BVDV had predisposed the animals to secondary
infection with BHV-1 and PI-3V. This study concluded that concurrent infection
with BVDV, BHV-1 and PI-3V should be considered as one of the infectious
causes of pneumoenteritis and, subsequently, the high morbidities and mortalities
among young calves in Egypt. Preventive and control measures against these
infectious agents should therefore be adopted. All animals imported into Egypt
should be free from BVDV infection. Control programmes for the detection and
removal of BVDV-persistent cattle should be applied in cattle herds all over the
country.
Keywords
Bovine viral diarrhoea – Clinical symptom – Epidemiology – Immunohistochemistry –
Infectious bovine rhinotracheitis – Parainfluenza-3 virus – Pathological investigation –
Serology – Virological diagnosis.
N.M. Aly (1)
, G.G. Shehab (2)
& I.H.A. Abd El-Rahim (3)
(1) Department of Virology, Animal Health Research Institute, Nadi El-Sayed Street, Dokki, Giza, P.O. Box 264,
Cairo, Egypt
(2) Department of Pathology, Animal Health Research Institute, Nadi El-Sayed Street, Dokki, Giza, P.O. Box 264,
Cairo, Egypt
(3) Department of Animal Medicine, Faculty of Veterinary Medicine, Assiut University, 71526 Assiut, Egypt
Submitted for publication: 14 May 2002
Accepted for publication: 30 May 2003
Bovine viral diarrhoea, bovine herpesvirus and
parainfluenza-3 virus infection in three cattle
herds in Egypt in 2000
© OIE - 2003

3. Introduction
Pneumoenteritis is generally considered to be the main hazard
to calf health (60). Bovine viral diarrhoea virus (BVDV) is a
major pathogen for cattle and contributes to the genesis of a
wide variety of pathology ranging from inapparent or mild
infection to the inevitably fatal syndrome of mucosal disease
(12). The BVDV belongs to the genus Pestivirus of the family
Flaviviridae. Both a non-cytopathic (ncp) and an antigenically
related cytopathic (cp) BVDV can be isolated from persistently
infected animals suffering from mucosal disease (63).
Diseases associated with BVDV infection have been a
controversial subject because of the multiple clinical forms and
manifestations of the infection, the ability of the virus to
suppress the function of the immune system, and the complex
epidemiology of the disease (6). The BVDV causes different
diseases, including: bovine viral diarrhoea (BVD) infection,
which is usually subclinical; mucosal disease, which is usually
fatal; and congenital abnormalities in calves, caused by an
infection of the foetus in midgestation. The BVDV may also
cause reproductive failure and be immunosuppressive (22, 23,
30, 48, 51, 57). The BVDV induces immunosuppresion (1, 10,
11), which allows for secondary infection of the respiratory
tract, and causes significant losses as a result of its interaction
with other pathogens (21, 26, 35).
The bovine herpesvirus-1 (BHV-1) and parainfluenza-3 (PI-3V)
viruses are the principal causes of pneumonia in cattle (37). The
role of BVDV in the bovine respiratory disease complex is
controversial (20). Abo El-Lail (4) described a respiratory
disease in cattle where both PI-3V and BVDV were involved. It
has also been shown experimentally that initial infection with
BVDV impairs the ability of infected calves to eliminate the
BHV-1 from the infection site (52).
The bovine herpesvirus-1 is a double-stranded
deoxyribonucleic acid virus. It is a member of the genus
Varicellovirus within the subfamily Alphaherpesvirinae, which
belongs to the family Herpesviridae (58, 64). BHV-1 is an
economically important pathogen. Infectious rhinotracheitis is
one of the most common clinical symptoms of BHV-1 in cattle
(28, 56). The viral glycoproteins, which are located on the
surface of the virion, play an important role in pathogenesis and
immunity (18, 45, 62). Latency is one of the major problems
associated with the infection of cattle by BHV-1 (39). Infectious
bovine rhinotracheitis (IBR) is characterised by clinical signs of
upper respiratory tract disease, such as a mucopurulent nasal
discharge, and by conjunctivitis. Signs of general illness are
fever, depression, inappetance, abortions and reduced milk
yield. Mortality is low. Many infections run a subclinical course
(49).
The parainfluenza-3 virus is a single stranded ribonucleic acid
virus. It is a member of the genus Paramyxovirus in the
subfamily Paramyxovirinae, which belongs to the family
Paramyxoviridae (8). The PI-3V infection is commonly
subclinical. Clinical disease may not occur until other
pathogens are present or when adverse environmental
conditions precipitate clinical disease (55).
The aim of this study was to identify the infectious agents of the
calf mortalities in the three investigated cattle herds and to
report the epidemiological aspects and clinical manifestations
associated with the outbreaks. The study also aimed to identify
the pathological alterations as well as the corresponding
distribution of the viral antigen in the tissues of the diseased
calves by using the avidin biotin complex (ABC)
immunoperoxidase technique.
Materials and methods
Animals
A total of 157 (9.2%) of the 1,701 young calves in the three
investigated cattle herds, which contained 2,354 animals in
total, showed evidence of gastroenteritis with severe diarrhoea,
either alone, as in Herd 1, or associated with respiratory
manifestations, as in Herds 2 and 3. These herds (Table I) were
as follows.
880 Rev. sci. tech. Off. int. Epiz., 22 (3)
© OIE - 2003
Table I
Data on the three cattle herds included in the study of bovine
viral diarrhoea virus outbreaks in Egypt in 2000
Descriptions Herd 1 Herd 2 Herd 3
Total number of
animals
Date of investigation January February October
2000 2000 2000
Number of animals 542 292 1,520 2,354
Dairy cattle 375 110 0 485
Age groups of calves:
< 1 month 10 12 0 22
> 1 - < 3 months 16 18 0 34
> 3 - < 6 months 28 32 0 60
> 6 - < 12 months 15 50 1,520 1,585
Total number of calves 69 112 1,520 1,701
Herd 1
Herd 1 was a dairy farm known as Delta Misr dairy cattle farm,
located in El-Minufiya Province, Lower Egypt. When the
authors visited the site, in January 2000, there were a total of
542 cattle on this farm, including ninety-eight pregnant cows
and sixty nine young calves, from one week to eight months
old. Liver, spleen and lung tissue samples were obtained from
three aborted foetuses. Ethylenediamine tetra-acetic acid
(EDTA)-blood samples and sterile nasal swabs were collected
from twelve clinically-diseased calves.

4. Herd 2
Herd 2 was a Friesian dairy cattle farm in El-Fayoum Province.
The cattle in this herd were imported from Germany. This dairy
farm was visited in February 2000, when it contained 110 dairy
cattle, seventy pregnant cows, twelve calves less than one
month old, eighteen calves between one to three months old,
thirty-two calves between three to six months old and fifty
calves from six to twelve months old. The authors collected
tissue samples from the liver, spleen and lung of two aborted
foetuses, and EDTA-blood samples and sterile nasal swabs were
obtained from sixteen clinically-ill calves.
Herd 3
This was a herd of 1,520 fattened young calves aged from six to
twelve months, imported from Romania and quarantined at the
governmental animal quarantine in El-Behira Province. This
quarantine facility was visited about three weeks after the
arrival of the calves, by which time there were increasing
numbers of diseased calves due to the rapid spread of an
infection. On the day of investigation, forty eight calves were
suffering from signs of enteritis mixed with respiratory
manifestations. Nineteen sterile nasal swabs and nineteen
EDTA-whole blood samples were collected from nineteen
diseased calves. In addition, tissue samples from the mesenteric
lymph nodes, pulmonary lymph nodes, spleen and lung were
obtained from two calves that had recently died.
Clinical examination
All of the diseased cattle in the three different infected herds
were clinically examined.
Pathological examinations
Nine of the dead calves (two from Herd 1, three from Herd 2
and four from Herd 3) were submitted for thorough post-
mortem examination. Tissue specimens were taken from the
trachea, lungs, bronchial and mesenteric lymph nodes, spleen,
liver, kidneys, rumen, abomasums and parts of the intestines.
These samples were fixed in 10% neutral buffered formalin,
processed routinely, and then sectioned 4 µm-5 µm thick. The
prepared sections were stained with haematoxylin and eosin for
histopathological examination, and by lendrum’s phloxin and
tartrazine for demonstration of viral inclusion bodies (14).
Immunohistochemical studies
The ABC immunoperoxidase technique was used as a rapid
method of confirming the detection of BVDV
, BHV-1 and PI-3V
in the different organs and tissues obtained from nine dead
calves at necropsy. The technique was applied on paraffin
sections, in accordance with the method of Wilchek and Bayer
(73). According to the intensity of the positive reaction, the
results were classified into four degrees from + to ++++.
Virological investigations
Cell culture
Madian Darby bovine kidney (MDBK) cell culture was used to
isolate BVDV from prepared buffy coats, nasal swabs and tissue
samples. The buffy coats and nasal swabs were prepared
according to Abd El-Rahim et al. (3). The MDBK was also used
to isolate BHV-1 from the collected nasal swabs. The EDTA-
blood samples (n 78) were centrifuged at 1,000 xg for 10 min.
The plasma was removed and the buffy coat (leukocytic
fraction) was aspirated with a sterile Pasteur-pipette into a
sterile tube and then 5 ml of minimum essential medium
(MEM) with 2% foetal calf serum (FCS), which had been
proven to be free from both BVDV and BVD antibodies, were
added. The mixtures were left at room temperature for 1 h and
then frozen at –70°C until used.
Each of the 78 sterile nasal swabs was transferred into a sterile
tube and 5 ml of MEM with 2% FCS and antibiotics (800 µg
streptomycin, 800 IU penicillin and 400 IU gentamycin/ml)
were added, they were well mixed and left at room temperature
for 1 h. The swabs were then kept at –70°C until inoculated
onto MDBK cell cultures. Tissue samples from aborted foetuses
and the recently dead calves were ground in MEM with 2%
FCS and antibiotics (800 µg streptomycin, 800 IU penicillin
and 400 IU gentamycin/ml) and centrifuged at 1,000 xg for
10 min. The supernatant fluids were separated and kept at
–70°C until used for BVDV isolation. The prepared leukocytic
fractions, nasal swabs and tissue suspensions were inoculated
onto MDBK cell cultures for isolation of BVDV according to the
method described by Clarke et al. (16).
Indirect immunofluorescent technique
The indirect immunofluorescent (IF) technique was used on
the inoculatd cell cultures with cytopathic effect (CPE) to
identify the cpBVDV
. It was also used on inoculated cell
cultures without CPE to detect ncpBVDV biotype. The IF
technique was carried out according to OIE (World
organisation for animal health) standards (47).
Three blind passages of the tested samples were done on the
inoculated cell cultures. If there was no evidence of any CPE,
the third passage was conducted on MDBK tubes. Thus, tube
cultures including flying cover slips were used to detect
ncpBVDV biotype by indirect IF technique. Both positive and
negative control samples were also included. The tested
samples were considered positive when there was a clear apple
green cytoplasmic fluorescence in the infected cell culture,
while the negative control, non-infected cells, showed no
fluorescence.
Haemagglutination and haemagglutination inhibition
tests
Haemagglutination (HA) and haemagglutination inhibition
(HI) tests were applied, according to the method described by
Rev. sci. tech. Off. int. Epiz., 22 (3) 881
© OIE - 2003

5. © OIE - 2003
Post-mortem examinations of seven calves from Herds 2 and 3
revealed the above-mentioned gross pathological lesions and
also linear haemorrhages in the tracheal and bronchial mucosa.
Multiple patchy congested areas were seen on the pulmonary
lobes.
Immunohistochemical findings
Detection of the viral antigens by ABC immunoperoxidase
technique in tissues collected at autopsy is shown in Tables II,
III and IV and in Figures 1, 2 and 3.
882 Rev. sci. tech. Off. int. Epiz., 22 (3)
Lennette and Schmidt (40), to a total of 78 nasal swabs, in order
to detect PI-3V antigen.
Virus neutralisation test
The virus neutralisation (VN) test was used to identify BHV-1
that had been isolated on MDBK tissue culture. The fixed virus
variable serum method described by Gillespie et al. (29) was
used.
Epidemiological studies
Many epidemiological aspects in the three affected herds were
studied, such as the incriminated sources of infection, the mode
of transmission, age susceptibility, morbidities, mortalities and
the abortive rate.
Results
Clinical symptoms
Inspection of the affected calves in Herd 1 revealed a rise in
body temperature (up to between 40°C and 42°C), anorexia,
severe depression, salivation, and profuse watery diarrhoea
with dehydration. Multiple erosions usually of 1 mm-5 mm in
diameter were present in the mucosa of the muzzle, oral cavity
and tongue. Higher infection and severe clinical manifestations
were observed among young calves from three to twelve
months old. In addition to the previous clinical signs, other
manifestations observed in diseased animals in Herds 2 and 3
were: severe respiratory and ocular manifestations, including an
increase in respiratory rate; serous or mucoid nasal discharge;
respiratory dyspnoea; coughing and sneezing; and
conjunctivitis with ocular discharge. Severe conjunctivitis with
profuse ocular discharge was noticed among affected calves in
Herd 3. In Herds 1 and 2, abortion was observed among
pregnant cows. Congenital malformations were noticed in
Herds 1 and 2, where some newborn calves were weak and
blind, and sometimes had musculoskeletal deformities.
Post-mortem findings
Nine dead calves from the three investigated herds were
subjected to post-mortem examinations. The most prominent
aspect of the post-mortem examinations of two calves from
Herd 1 was the presence of multiple erosions of about 1 mm to
5 mm in diameter on the muzzles, oral mucosa, tongue,
ruminal pillars, abomasums, and small intestines. Congestion
and severe haemorrhages were seen in the mucosal surfaces of
the abomasums, small intestines and ileocecal valve. Necrotic
foci were observed in the hepatic tissues. The bronchial and
mesenteric lymph nodes were extremely congested and
haemorrhagic. The spleen was congested, while the kidneys
were pale in colour.
Table II
The distribution and intensity of avidin biotin complex reaction
against bovine viral diarrhoea (BVD), bovine herpesvirus-1
(BHV-1) and parainfluenza-3 (PI-3) viruses in the tissue samples
obtained from two dead calves in Herd 1
Organs
Distribution and intensity of viral antigens
BVD virus BHV-1 PI-3 virus
Trachea – – –
Lung + – –
Bronchial lymph nodes – – –
Mesenteric lymph nodes + + + – –
Tongue + + + – –
Spleen + + + – –
Rumen + + – –
Abomasum + + – –
Intestines + + + – –
Liver + + + – –
Kidneys + + + – –
Heart + – –
Table III
The location and intensity of avidin biotin complex reaction
against bovine viral diarrhoea (BVD), bovine herpesvirus-1
(BHV-1) and parainfluenza-3 (PI-3) viral antigens in the tissues
obtained from three dead calves in Herd 2
Organs
Distribution and intensity of viral antigens
BVD virus BHV-1 PI-3 virus
Trachea – – +
Lung + – + + +
Bronchial lymph nodes – – + +
Mesenteric lymph nodes + + – –
Tongue + + + – –
Spleen + + – +
Rumen + + – –
Abomasum + + + – –
Intestines + + – +
Liver + + + – +
Kidneys + + – +
Heart + + – +

6. © OIE - 2003
submucosal layer of the trachea (Fig. 3a), the lining bronchial
epithelium, the lymphocytes and macrophages of the spleen,
the bronchial lymph nodes, the lumen of the blood vessels, the
lining endothelial cells of the heart, in between the cardiac
muscle fibers and the lining endothelial cells of the renal blood
vessels, the glomerular capsule and the tuft capillaries, and in
the cytoplasm of the lining tubular epithelium.
The BVDV antigen was detected by immunohistochemical
assay in tissue samples from four dead calves from Herd 3
(Table IV), which were also positive for virus isolation by the
tissue culture method.
Rev. sci. tech. Off. int. Epiz., 22 (3) 883
Table IV
The distribution and intensity of avidin biotin complex reaction
against bovine viral diarrhoea (BVD), bovine herpesvirus-1
(BHV-1) and parainfluenza-3 (PI-3) viral antigens in the tissue
samples collected from four dead calves in Herd 3
Organs
Distribution and intensity of viral antigens
BVD virus BHV-1 virus PI-3 virus
Trachea – + + + + +
Lung – + + + + + +
Bronchial lymph nodes – + + + + +
Mesenteric lymph nodes + + + + –
Tongue + + + – –
Spleen + + + + + + + +
Rumen + + – –
Abomasum + + + – –
Intestines + + + +
Liver + + + + + +
Heart + + + + + +
Kidneys + + + + + +
Fig. 1
Intestinal glands showing dense brown granules against
bovine viral diarrhoea virus antigen in their lining glandular
epithelium (arrows). Avidin biotin complex immunoperoxidase
– counter stained haematoxylin × 400
a) Mesenteric lymph nodes
b) Spleen
Fig. 2
The mesenteric lymph nodes and spleen revealing dense brown
granules against bovine viral diarrhoea virus antigen within the
lymphocytes and macrophages. Avidin biotin complex
immunoperoxidase – counter stained haematoxylin × 400
a) Trachea revealing dense brown granules against bovine herpesvirus-1 antigen inside the
degenerated mucosal epithelium and in the submucosal layer. Avidin biotin complex (ABC)
immunoperoxidase – counter stained haematoxylin × 500
b) Lung showing brown granules against parainfluenza-3 virus in the lining bronchial and
alveolar epithelium. The ABC immunoperoxidase – counter stained haematoxylin × 250
Fig. 3
Detection of viral antigens by ABC immunoperoxidase
technique
An intense positive reaction against BVDV was seen in the basal
layer of the mucosal layer of the tongue, the cytoplasm of the
lining epithelium of most of the villi, the intestinal glands of
both the small and large intestines (Fig. 1), the cytoplasm of the
hepatic and kupfer cells, and within the lymphocytes and
macrophages of both the spleen and the mesenteric lymph
nodes (Figs 2a and 2b).
Meanwhile intense positive reaction against PI-3 was
demonstrated in the lining bronchial and alveolar epithelium
(Fig. 3b). A severe positive reaction against BHV-1 viral antigen
was observed inside the degenerated mucosal epithelium, the
a) b)
a) b)

7. © OIE - 2003
Histopathological studies
Most of the histopathological lesions were confined to the
alimentary and respiratory tracts, the lymphoreticular tissues
and the kidneys. In Herd 1, microscopical examination of the
gastrointestinal tract revealed epithelial degeneration and
necrosis of the lining of the ruminal pillars. The necrotic cells
were sloughed leading to erosions or ulcerations with
inflammatory cell aggregations mixed with fibrinous exudates
associated with haemorrhage and thrombosis of most of the
lymphatics. Examination of the abomasums revealed necrosis
of the glandular epithelial cells in addition to haemorrhage and
dilatation of the gland lumens. In the intestine, pathological
changes were prominent in all parts of the intestines (Figs 4a
and 4b), and included severe damage and necrosis of the
intestinal villi and sloughing of the lining epithelium. The
mucosal glands were cystic. There was severe haemorrhage and
congestion of the submucosal blood vessels. The ileocecal valve
showed depletion in the Peyer’s patches and the villi appeared
atrophied with excessive goblet cells formation. tract. The trachea showed variable degrees of degeneration,
necrosis and epithelial exfoliation. Congestion in the
submucosal blood vessels and haemorrhages associated with
round cell infiltrations in the lamina propria were detected. The
lungs showed proliferative reactions involving the bronchiolar
and alveolar epithelia, (epithelization), so that the lesions were
considered as atypical interstitial pneumonia. Most of the
bronchiolar and alveolar epithelial cells were vacuolated.
Intranuclear acidophelic inclusions were detected in the lining
alveolar epithelium as shown in Figure 6. Most of the bronchi
and alveoli contained a mixture of neutrophils, macrophages
and lymphocytes, with detached epithelial cells and serous
exudates which had almost completely blocked the lumina of
the bronchioles and alveoli with multiple syncytial giant cells.
In addition, vasculitis and thrombosis were observed in most of
the pulmonary blood vessels. Examination of the kidneys
revealed nephritis in the tissue samples collected from all three
investigated cattle herds.
884 Rev. sci. tech. Off. int. Epiz., 22 (3)
a) Duodenum revealing severe necrosis and sloughing of most of the lining epithelium of
the intestinal villi with inflammatory cells infiltration. Haematoxylin and eosin × 250
b) Duodenum showing glandular oedema with separation of the lining epithelium inside
their lumen. Haematoxylin and eosin × 250
Fig. 4
Pathological changes in the intestines
a) Haematoxylin and eosin × 500
b) Phloxin and tartrazin × 1,000
Fig. 5
Liver showing both intranuclear and intracytoplasmic inclusion
bodies in the hepatocytes (arrows)
The hepatic cells showed variable degrees of vacuolar
degeneration and multiple focal areas of necrosis associated
with round cell infiltrations. Both intranuclear and
intracytoplasmic acidophilic inclusion bodies, which were
positive with phloxin and tartrazine stain, were seen in most of
the examined hepatic tissues (Figs 5a and 5b). There was
degeneration of the epithelial lining the bile ducts and fibrosis
in the portal areas. The lymphoid follicles of the bronchial and
mesenteric lymph nodes were markedly depleted, with an
accumulation of a large number of macrophages in the
subcapsular zone. The white pulp of the spleen was also
depleted. There was vasculitis of the small and medium sized
blood vessels.
In Herds 2 and 3, microscopic changes were noticed in the
respiratory system and there were changes in the alimentary
Fig. 6
Lung showing intranuclear acidophelic inclusion bodies in the
lining alveolar epithelium (arrows) associated with massive
degeneration and necrosis of the lining alveolar epithelium.
Haematoxylin and eosin × 500
a) b)
a) b)

8. Virological diagnosis
Isolation and identification of bovine viral diarrhoea
virus
The results of the isolation and identification of both cpBVDV
and ncpBVDV biotypes using the cell culture method and the
immunofluorescent technique are represented in Table V
.
Immunofluorescence positive samples were characterised by
specific intracytoplasmic fluorescence.
Detection of parainfluenza-3 virus antigen
Table VI shows the results of HA and HI tests which were used
to detect PI-3V antigen in nasal swabs collected from the
diseased calves in the three investigated cattle herds.
Demonstration of bovine herpesvirus-1
Both the cell culture method and the VN test were used to
demonstrate and identify BHV-1 isolates in the collected nasal
swabs. Cytopathic effect was observed in MDBK cell culture
after 36 h post-inoculation; this appeared in the form of
rounding of the cells, shrinkage of cell walls and an increase in
granularity, leading to the formation of a bunch of grapes within
72 h-96 h. In the VN test, the isolated virus was neutralised by
reference bovine hyperimmune serum against BHV-1. Virus
was only isolated from Herd 3, in which thirty-two out of forty-
eight nasal swab samples were positive (Table VII).
Epidemiological studies
In 2000, there were outbreaks of severe acute BVD, either
alone, as in Herd 1, or mixed with PI-3V infection, as in Herd 2,
or mixed with both PI-3V and BHV-1 infection, as in Herd 3.
The current study suggested that the immunosuppressive effect
of BVDV predisposed cattle in Herds 2 and 3 to infection with
PI-3V or both PI-3V and BHV-1. Herd 3 had been imported
from Romania just a few weeks before the appearance of clinical
signs, and the stress of this transportation facilitated the
concurrent infection with both PI-3V and BHV-1. Also, the
study suggested that the young calves of Herd 3 (in
governmental quarantine in El-Behira Province) were infected
with BVDV before being imported into Egypt. Morbidity,
mortality, case fatality and abortive rates in the three affected
herds are represented in Table VIII.
Discussion
The BVDV infection in cattle has been reported throughout the
world, and the wide spectrum of clinical syndromes associated
with this positive-stranded and enveloped RNA virus makes it
one of the most important viral pathogens of cattle (17). The
BVDV spreads widely among cattle herds in the world (5, 23,
54, 68, 72). The BVDV was isolated for the first time in Egypt
in 1972 from a calf suffering from severe enteritis (33). The
present study recorded an outbreak of acute BVDV infection in
2000 in three cattle herds in Egypt, with clinical manifestations
Rev. sci. tech. Off. int. Epiz., 22 (3) 885
© OIE - 2003
Table V
Isolation of both cytopathic (CP) and non-cytopathic (NCP) bovine viral diarrhoea virus (BVDV) biotypes
Detection of BVD
Herds Nasal BVDV Buffy BVDV Dead BVDV Aborted BVDV
swabs CP NCP coats CP NCP calves CP NCP foetuses CP NCP
1 14 – 5 14 – 9 – – – 5 – 3(b)
2 16 – 7 16 – 10 – – – 3 – 2(c)
3 48 16 – 48 21 – 4 4 (a)
– – – –
Total 78 16 12 78 21 19 4 4 – 8 – 5
a) tissue samples from the same four calves were also positive by the immune staining technique
b) from lung and spleen
c) from spleen
Table VI
Detection of parainfluenza-3 virus antigen in the three
investigated herds
Haemagglutination and haemagglutination
Herds inhibition tests
Number of examined nasal swabs Positive %
1 14 – (0)
2 16 10 (62.5)
3 48 28 (58.3)
Total 78 38 (48.7)
Table VII
Isolation of bovine herpesvirus-1 from nasal swabs
Herds
Virus neutralisation test
Number of examined nasal swabs Positive %
1 14 – (0)
2 16 – (0)
3 48 32 (66.7)
Total 78 32 (41)

9. of enteritis and congenital malformations. Isolation and
identification of the causative viruses, as well as the detection of
the viral antigens by immunohistochemistry, indicated that the
main cause of calf mortalities in the three herds was severe
acute bovine viral diarrhoea. Concurrent infection with BVDV
and PI-3V was observed in Herd 2. In Herd 3, mixed infection
of BVD, PI-3 and BHV-1 was reported. The BVDV was
widespread throughout the country as determined by the
detection of neutralising BVD antibodies in 99 (37.6%) out of
263 sheep sera in 24 different localities throughout all the
provinces of Egypt (31). In March 1982, an outbreak due to
BVDV infection was reported by Ghaffar and Ata (27) in cattle
and buffalo in Fayoum and Qina Governorates. The BVDV and
BHV-1 are prevalent in the country as indicated by the
detection of antibodies in bovine and buffalo serum samples
collected from some governmental farms in Lower Egypt (32).
The BVDV seems to be transmitted directly from either acutely
or persistently infected cattle to susceptible cattle. Persistently
infected cattle play a more significant role in the transmission
and maintenance of BVDV than acutely infected cattle (13, 67).
For a persistent infection to become established, ncpBVDV
must infect the developing embryo or foetus during the first
four months of gestation (41). Persistently infected-calves
usually have poor viability and suffer early disease and death
with or without signs of diarrhoea (54). This study suggested
that Herds 1 and 2 were suffering from persistent BVDV
infection. Animals from Herd 3 may already have been infected
with BVDV before being imported.
Both CP and NCP biotypes of BVDV are capable of affecting
foetal and neonatal development. The CP biotypes are
frequently associated with embryonic death, abortion, and
malformation; NCP biotypes are associated with
immunotolerance and persistent infection (46). This study
suggested that the malformations observed in newborn calves
in the affected herds were a result of intrauterine infection with
cpBVD biotype.
Clinically, fevers, anorexia, depression, salivation, erosive
stomatitis, abortion, and profuse watery diarrhoea with
dehydration were the most observable signs of acute BVD in the
affected herds. Young calves aged from three to twelve months
were more susceptible to the infection and showed severe
clinical signs. The BVDV infection should be considered when
investigating severe acute outbreaks of enteritis (19) and
abortion in cattle (38). In addition to the above mentioned
clinical signs, severe respiratory and ocular manifestations,
including rapid respiration, mucopurulent nasal discharge,
dyspnoea, coughing and sneezing and conjunctivitis were
observed among diseased animals in Herds 2 and 3.
Accurate diagnosis of BVDV infection depends upon isolating
the virus from nasal swabs or blood or tissue samples from
affected animals in a diagnostic laboratory (2, 6, 21, 32, 34, 61,
71). In this study, laboratory investigations revealed isolation
and identification of ncpBVDV biotype from the nasal swabs,
buffy coats and tissue samples collected from Herds 1 and 2
using the cell culture method and the immunofluorescent
technique, while the CP biotype was detected in the samples
obtained from Herd 3. The indirect peroxidase staining
technique is a practical test for detecting BVDV in infected
herds (43), and has been used previously to diagnose BVDV
infection (34) and IBR (74). In the present study, the ABC
immunoperoxidase method was used as a rapid way of
confirming the demonstration of BVD, BHV-1 and PI-3 viral
antigens in the collected tissue samples. This technique
reflected the involvement of a wide variety of tissues. The
location and intensity of BVD, BHV-1 and PI-3 antigens in the
examined organs were similar to the observations of Hosny et
al. (36) and Shehab et al. (66). The results of the tissue culture
method and the avidin biotin complex-immunoperoxidase
technique for detecting BVDV infection in tissue samples
obtained from 4 dead calves from Herd 3 were correlated.
Histopathological findings of severe acute BVD were observed.
There was severe congestion and haemorrhages throughout the
gastrointestinal tract as seen previously by Ernst et al. (25), Ruth
(61), Mebus et al. (42). Massive degenerative changes were
observed in the liver and kidneys as detected by Barlow et al.
(7), Omran (50), Tyler and Ramsey (69).
The BVDV infections in congregated and stressed cattle may
lead to severe respiratory or enteric disease (9, 59). The current
study suggested that the immunosuppressive effect of BVDV
886 Rev. sci. tech. Off. int. Epiz., 22 (3)
© OIE - 2003
Table VIII
Morbidity, mortality, case fatality and abortive rates in the three affected cattle herds
Epidemiological aspects
Herds
Total number Number of Morbidity Number of Mortality Case fatality Total number Number of Abortive
of calves diseased rate dead calves rate rate of pregnant aborted rate
calves (%) (%) (%) cows cows (%)
1 69 35 50.7 21 30.4 60 98 26 26.5
2 112 48 42.9 32 28.6 66.7 70 14 20
3 1,520 74 4.9 26 1.7 35.1 0 0 0
Total 1,701 157 9.2 79 4.6 50.3 168 40 23.8

10. facilitated the infection of the diseased calves with other viral
agents such as BHV-1 and PI-3V
. This suggestion coincides with
the findings reported by Potgieter et al. (53). The concurrent
infection with the BVDV
, BHV-1 and PI-3V worsened the
clinical disease and the economic losses in the three affected
herds. Field outbreaks of mixed infection by BVDV and other
viruses such as malignant catarrhal fever virus (65) and foot and
mouth virus (36) have been previously reported.
Respiratory and enteric infections are the most costly and
troublesome disease problems in calves in Egypt (32). This
study revealed that the direct economic losses of BVDV
infection (and the indirect losses due to the
immunosuppressive effect of the virus, which facilitates
infection with other infectious agents), are considerable. These
economic losses were estimated at US$13,800, US$15,600 and
US$10,400 in Herds 1, 2 and 3 respectively. The economic
losses associated with BVDV infections are due to suboptimal
reproductive performance due to infertility and embryonic
death, abortion, prenatal growth retardation, stillbirths,
congenital defects, postnatal growth retardation, and death
from mucosal disease (24).
Bovine PI-3V is a common infection of cattle, 60%-90% of
cattle in a herd often having serological evidence of past
infection (8). The PI-3V was detected in the nasal swabs, which
were collected from Herds 2 and 3 using HA and HI tests.
Subclinical viral pneumonia, which is associated with the PI-3V
and is uncomplicated by secondary infection, is usually of
minor importance (55). However, in the current study, severe
respiratory signs were observed among the affected calves of
Herds 2 and 3 as a result of complication of BVDV infection
with PI-3V
, as in Herd 2, or with both PI-3V and BHV-1, as in
Herd 3. These findings agree with the experiment of Castrucci
et al. (15), who found that simultaneous infection of calves with
BVDV and PI-3V induced a clinical response which was
considered of moderate intensity. A different situation was
observed, however, when simultaneous infection was made
with BVDV and BHV-1. In the inoculated calves, BVDV and
BHV-1 together induced a severe clinical response characterised
by a wide variety of signs which included mucoid nasal
discharge, profuse salivation and pronounced diarrhoea.
The BHV-1 was found primarily in large, concentrated cattle
populations, such as feedlots or intensively managed dairy
operations (44). Both the immunosuppressive effect of the
BVDV and PI-3V
, and the effect of being transported over long
distances, predisposed the imported feedlot calves in Herd 3 to
infection with BHV-1. The BHV-1 can be spread through nasal
secretions or aerosol droplets containing the virus. Close
contact among animals is responsible for the high rate of
transmission (70). The present study also suggested that the
crowdedness and close contact of the 1,520 animals in Herd 3
(in the governmental quarantine in Behira Province) played an
important role in rapidly transmitting BHV-1 among the young
imported feedlot calves, which had been imported from
Romania just a few weeks before these investigations.
In cattle infected with BHV-1, the initial lesions which
developed in the upper respiratory tract usually progressed to
broncho and/or lobar pneumonia as a result of secondary
infection, either a viral infection, such as PI-3V, or a bacterial
infection (49). In the present study, it was evident that BHV-1
has a cellular destructive power on the parenchymatous organs
and causes necrosis of hepatic cells, as observed previously by
Shehab et al. (66). The BHV-1 is distributed worldwide, but has
been eradicated from Denmark and Switzerland and control
programmes have started in some other countries (49).
Conclusion
The present study leads us to conclude that:
– concurrent infection with BVDV
, BHV-1 and PI-3V should be
considered as one of the infectious causes of pneumoenteritis
and the subsequent high morbidities and mortalities among
young calves in Egypt. Consequently, preventive and control
measures against these infectious viral agents should be
adopted
– the ABC immunoperoxidase technique is one of the most
rapid and accurate laboratory tests for the diagnosis of different
types of mixed viral infections, such as concurrent infection
with BVDV
, BHV-1 and PI-3V
– both BVDV and BHV-1 have an immunosuppressive effect
and facilitate secondary infection in diseased calves, which
subsequently results in high economic losses
– all animals imported into Egypt must be free from BVDV
infection
– control programmes for detecting and removing BVDV-
persistent cattle should be applied in cattle herds all over the
country.
Rev. sci. tech. Off. int. Epiz., 22 (3) 887
© OIE - 2003
■

11. © OIE - 2003
888 Rev. sci. tech. Off. int. Epiz., 22 (3)
Cas de diarrhée virale bovine et d’infection à l’herpèsvirus bovin et
au virus para-influenza 3 dans trois troupeaux de bovins en Égypte
en 2000
N.M. Aly, G.G. Shehab & I.H.A. Abd El-Rahim
Résumé
Cette étude rapporte la présence de foyers causés par le virus de la diarrhée
virale bovine (BVDV) en Égypte, en 2000. Le virus responsable a été détecté soit
seul, soit conjointement avec l’herpèsvirus bovin de type 1 (BHV-1) et/ou le virus
para-influenza de type 3 (PI-3V). En Basse Égypte, des symptômes d’entérite,
parfois accompagnés de manifestations respiratoires, ont été observés chez de
jeunes veaux dans trois troupeaux, dans les provinces d’El-Minufiya et
d’El-Fayoum, ainsi que dans les installations gouvernementales de quarantaine
de la province d’El-Behira. Les troupeaux contaminés ont été inspectés et les
animaux atteints soumis à un examen clinique. Un grand nombre d’aspects
épidémiologiques (morbidité, mortalité et létalité, par exemple) ont été
déterminés, dont le taux d’avortement. Des échantillons de sang sur EDTA (acide
éthylène diamine tétra-acétique), des frottis nasaux ainsi que des échantillons
sériques ont été prélevés à des fins de diagnostic virologique et sérologique. Les
analyses de laboratoire ont révélé que la principale cause de la mortalité
observée chez les veaux des trois troupeaux était une infection au seul BVDV
(élevage d’El-Minufiya), à la présence simultanée du BVDV et du PI-3V (élevage
d’El-Fayoum) ou à la présence conjointe du BVDV, du BHV-1 et du PI-3V (troupeau
de la station gouvernementale de quarantaine de la province d’El-Behira). Un
examen post-mortem complet a été réalisé sur neuf veaux issus des trois
troupeaux. Des prélèvements de tissus ont été effectués chez les veaux morts
récemment pour examen immunohistochimique et histopathologique. Les
analyses histopathologiques ont principalement mis en évidence une
dégénérescence massive, accompagnée de nécrose et d’érosion de la paroi
épithéliale du tube digestif. La plupart des organes lymphoréticulaires
présentaient un important déficit lymphocytaire. Des signes de
bronchopneumonie et de pneumonie interstitielle atypique étaient manifestes
dans les cas pulmonaires. Selon les auteurs de cette étude, l’effet
immunodépresseur du BVDV aurait prédisposé les animaux à une infection
secondaire au BHV-1 et PI-3V. Cette étude a permis de conclure qu’une infection
opportuniste à BVDV, BHV-1 et PI-3V devait être envisagée comme l’une des
causes de l’infection de pneumoentérite et des taux élevés de morbidité et de
mortalité observés ultérieurement parmi les jeunes veaux d’Égypte. Il convient
dès lors de mettre en place des mesures préventives et de contrôle contre ces
agents infectieux. Tous les animaux importés en Égypte devraient être indemnes
de toute infection au BVDV. Il convient de mettre en place des programmes de
contrôle destinés à dépister et à éliminer les bovins porteurs du BVDV au sein des
troupeaux sur l’ensemble du territoire.
Mots-clés
Diagnostic virologique – Diarrhée virale bovine – Épidémiologie – Examen pathologique –
Immunohistochimie – Rhinotrachéite infectieuse bovine – Sérologie – Symptôme clinique
– Virus para-influenza de type 3.
■

12. © OIE - 2003
Rev. sci. tech. Off. int. Epiz., 22 (3) 889
Diarrea viral bovina e infección por el herpesvirus bovino y el
virus de la parainfluenza 3 en tres rebaños vacunos de Egipto en el
año 2000
N.M. Aly, G.G. Shehab & I.H.A. Abd El-Rahim
Resumen
Los autores describen el estudio de una serie de brotes infecciosos que se
declararon en 2000 en Egipto, causados por el virus de la diarrea viral bovina
(VDVB), ya fuera solo o acompañado del herpesvirus bovino 1 (HVB-1) y/o el virus
parainfluenza 3 (VPI-3). En las tierras bajas egipcias, y concretamente en las
provincias de El-Minufiya y el El-Fayoum y las instalaciones oficiales de
cuarentena de la provincia de El-Behira, se observaron síntomas de enteritis en
tres rebaños de jóvenes terneras, que a veces se presentaban solos y otras
veces acompañados de disfunción respiratoria. Se efectuaron inspecciones de
los rebaños afectados y análisis clínicos de los animales enfermos. Se calcularon
numerosos parámetros epidemiológicos, tales como las tasas de morbilidad,
mortalidad y letalidad y el índice de abortos. Para proceder al diagnóstico
virológico y serológico, se recogieron muestras de sangre en ácido etilendiamino
tetracético, muestras de exudado nasal en hisopos estériles y muestras de suero.
Los análisis de laboratorio revelaron que, en los tres rebaños, la principal causa
de mortalidad era la infección por el VDVB, ya fuera solo (como en la explotación
de El-Minufiya) o combinado con el VPI-3 (como en la granja de El-Fayoum) o con
éste y el HVB-1 (como en el rebaño de las instalaciones de cuarentena de la
provincia de El-Behira). Un total de nueve terneras de los tres rebaños fueron
sometidas a inspección post-mortem exhaustiva. Se extrajeron muestras
tisulares de terneras muertas poco tiempo antes para someterlas a análisis
inmunohistoquímico e histopatológico. Desde el punto de vista histopatológico,
las observaciones más destacadas fueron una degeneración muy extendida,
zonas de necrosis y erosión del epitelio de revestimiento del tracto digestivo. La
mayor parte de los órganos linforreticulares habían perdido los linfocitos. Los
animales neumónicos presentaban signos evidentes de bronconeumonía y
neumonía intersticial atípica. Del estudio se desprende que los efectos
inmunosupresores del VDVB habían predispuesto a los animales a contraer
infecciones secundarias por el HVB-1 y el VPI-3, lo que a su vez lleva a concluir
que es preciso tener en cuenta que las infecciones oportunistas por el VDVB, el
HVB-1 o el VPI-3 constituyen una de las causas infecciosas de neumoenteritis y,
ulteriormente, de las elevadas tasas de morbilidad y mortalidad entre las terneras
jóvenes de Egipto. De ahí la necesidad de adoptar medidas preventivas y de
control contra esos agentes infecciosos. Todos los animales que Egipto importe
deben estar libres de la infección por el VDVB. Por otra parte, los rebaños
bovinos de todo el país deben estar sujetos a programas de control para detectar
y eliminar los ejemplares que presenten infección persistente por el VDVB.
Palabras clave
Análisis patológico – Diagnóstico virológico – Diarrea viral bovina – Epidemiología –
Inmunohistoquímica – Rinotraqueítis infecciosa bovina – Serología – Síntomatología
clínica – Virus de la parainfluenza 3.
■

13. © OIE - 2003
890 Rev. sci. tech. Off. int. Epiz., 22 (3)
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