Isolation and Characterization of BoHV-1 from Seropositive Cows after Inducing Artificial Stress in West Bengal, India
Infectious Bovine Rhinotracheitis (BoHV-1) is the most important emerging disease of cattle in India. With an aim to reactivate BoHV-1 from latently infected sero-positive cattle for molecular characteristics of the isolates prevalent in tropical and sub-tropical countries like India and further epidemiological investigations on IBR infections this study had been conducted. Artificial stress with dexamethasone at the dose rate of 0.1 mg kg-1 body weight for 5 consecutive days was induced in BoHV-1 sero-positive cows. Then isolation from nasal swabs was attempted in Madin Darby Bovine Kidney (MDBK) cell line to find out the prevalent strain in India. The virus was isolated from all the three cows. All the three isolates were typed as BoHV-1.2 (Strain India 4, India 5 and India 6). The reactivation obtained in this study with dexamethasone suggests the usefulness of BoHV-1 cow latency model for epidemiological investigations on BoHV-1 infections in tropical and sub-tropical countries like India, Pakistan etc.
to cite this article:
Tapabrata Saha, Chanchal Guha, Dhruba Chakraborty, Biplab Pal, Ujjwal Biswas, Amaresh Chatterjee, Patricia Koenig and Martin Beer, 2013. Isolation and Characterization of BoHV-1 from Seropositive Cows after Inducing Artificial Stress in West Bengal, India. Pakistan Journal of Biological Sciences, 16: 720-725.
Received: October 04, 2012;
Accepted: January 16, 2013;
Published: April 04, 2013
Extensive artificial insemination in cattle since few decades in India has
not only facilitated the exchange of genetic characteristic both nationally
and internationally but also increased their susceptibility to various diseases.
The most important among them is Infectious Bovine Rhinotrachietis (IBR) caused
by BoHV-1 (Gibbs and Rweyemamu, 1977; Kilari
et al., 2000; Dhand et al., 2002).
IBR seropositive cows in different states of India were regularly reported by
different scientists like Mehrotra and Rajya (1981),
Gill et al. (1987), Satyanarayana
and SuriBabu, (1987), Vaid et al. (1991),
Suresh et al. (1999), Chinchkar
et al. (2002), Dhand et al., 2002),
Rajesh et al. (2003) and others. SuriBabu
et al. (1984) and Mohan et al.(1989)
indicated the presence of IBR in the bovine population in most of the organized
farms in India. Like most herpesviruses, IBR virus becomes latent (trigeminal
ganglia in case of respiratory infection and sacral ganglion after genital infection)
following a primary infection with field virus or vaccination with an attenuated
strain as mentioned by Davies and Carmichael (1973)
and Radostits et al. (2000). Stressful situation,
such as transport, parturition, high and low ambient temperature (specially
in pure and cross-breeds), high milk yield and artificial stress induced by
steroid injection cause reactivation of the latent virus from the ganglia and
consequently intermittent shedding of virus into the environment thereby act
as a potent source of infection to other healthy cattle. In India, the first
report of IBR was made long ago by Mehrotra et al.
(1976) as an outbreak of kerato-conjunctivitis in cross breed calves. There
were previous records regarding reactivation of BoHV-1 with stress, most of
which were after experimental inoculation of the virus in animal and thereafter
inducing stress with steroids and isolation of the reactivated virus (Pastoret
et al., 1980; Ackermann et al., 1982;
Rock et al., 1992; Hage
et al., 1998; Six et al., 2001).
But the present study was aimed in isolation of BoHV-1 by artificial stress
only from seropositive cattle which were naturally infected with field strain
virus. Dexamethasone being an immunosuppressant was injected in the sero-positive
cattle to induce stress. Further characterization of the isolated virus was
done in International Reference Laboratory. Although Saha
et al. (2010) had isolated BoHV-I (Strain India 3) from nasal discharge
of cattle, there was no record of isolation of BoHV-1 after inducing artificial
stress in cattle in India. Thus this study has a great value, which can provide
valuable clues to epidemiological investigations on BoHV-1 infections in tropical
and sub-tropical countries like India, Pakistan and others.
MATERIALS AND METHODS
In India particularly in state like West Bengal till now no routine vaccination
against Infectious Bovine Rhinotracheitis (Bovine Herpes virus type-1) was practiced
in cattle thereby any BoHV-1 isolate from cattle would be the field strain virus.
Thus in this study dexamethasone being an immunosuppressant was injected in
BoHV-1 seropositive cattle for reactivation and further isolation and characterization
of the field strain virus. This study was conducted during 2006-09.
Seroprevalence analysis: Seroprevalence analysis were conducted by Virus
Neutralization Test (VNT) as the method recommended by OIE
(2009) to detect the BoHV-1 seropositive cattle in an organized cattle farm
of Kolkata, West Bengal, India. Virus neutralization test was performed in Madin
Darby Bovine Kidney (MDBK) cell line in Eagles Minimum Essential Medium
(EMEM) with antibiotics (Penicillin at the rate of 1, 00, 000 I.U. and Streptomycin
at the rate of 100 mg L-1 of medium) and fetal calf serum (10%).The
Standard Strain of Bovine Herpesvirus type-1 (BoHV-1 strain 2204) virus procured
from Central Animal Disease Research and Diagnosis (CADRAD) Centre, Indian Veterinary
Research Institute, Uttar Pradesh at the Institute of Animal Health and Veterinary
Biologicals, Kolkata, was used as standard virus where the entire work was conducted.
The virus was propagated in MDBK cell line and infectivity titer in cell culture
i.e., TCID50 per mL was recorded at regular interval. No vaccination
against IBR (BoHV-1) was undertaken in that farm.
Animal selection and induction of artificial stress: Three dry, non-pregnant,
healthy seropositive cows (Animal identification No. 301, 616 and 665) from
a private cattle farm were selected randomly for this study. To induce artificial
stress dexamethasone was injected intravenously at 0.1 mg kg-1 b.wt.
for 5 consecutive days following the dose applied by Pastoret
et al. (1986) and Six et al. (2001).
Straub and Lorenz (1991) and Pastoret
et al. (1980) had also applied different immunosuppressive drugs
including dexamethasone to latently infected cattle to re-isolate the inoculated
BoHV-1 from them. After injecting dexamethasone for five consecutive days, isolation
of BoHV-1 from the animals was attempted. The cows were observed regularly for
any unusual signs. Paired serum samples were also collected for a possible rise
in antibody titer against BoHV-1 after 21 days.
Isolation and identification of BoHV-1: Collection, processing of the
sample and isolation of the virus was attempted as the method described by OIE
Samples of nasal swab were taken in viral transport medium i.e., Eagles
Minimum Essential Medium (EMEM) with antibiotics (Penicillin at the rate of
1,00,000 I.U. and Streptomycin at the rate of 100 mg L-1 of medium)
and fetal calf serum (5%).
Nasal swabs were collected after dexamethasone injection when the cows
shows nasal secretion indicating immunosuppression. Nasal swabs were collected
with commercially available sterile swabs when the secretion was serous rather
than mucopurulent in nature. Nasal swabs were collected by vigorously rubbing
cotton swabs against mucosal surfaces.
After submerging into viral transport medium, the nasal swabs were transported
to the laboratory in ice packs. In the laboratory, swabs were agitated in the
transport medium to elute virus and left at room temperature for 30 min. The
swabs were removed and the transport medium was clarified by centrifugation
at 1500 g for 10 min. The supernatants were filtered through 0.45 μm filters
and a 100 μL volume of supernatant from the processed swabs was inoculated
into MDBK cell culture monolayers (tissue culture flask of 25 mL volume) and
incubated at 37°C for 2 h. After 2 h adsorption, the cells were rinsed,
maintenance medium was added and the cultures were subsequently incubated at
37°C in an incubator with 5% CO2.
The inoculated cell culture was observed daily for CPE under inverted microscope.
CPE was characterized by grape like clusters of rounded cells gathered around
a hole in the monolayer. In cases, where no CPE was observed within 7 days,
the inoculated cell culture was frozen and thawed thrice, clarified by centrifugation
and the supernatant was used for inoculation into fresh monolayers. In case
of negative to CPE after 7 days with this 1st passage, the sample was regarded
as negative to BoHV-1.
The isolated virus was then freeze dried in glass ampoule for transportation.
The freeze-dried virus samples were sent to the OIE and National Reference Laboratory
for BoHV-1 (Friedrich-Loeffler-Institut, Institute of Diagnostic Virology, Federal
Research Institute for Animal Health, InselRiems, Germany) for confirmation
and subsequent typing in dry ice. Cell culture, immunofluorescence study, real
time PCR, Restriction Fragment Length Polymorphism (RFLP) analysis and sequencing
of the virus sample was performed.
Clinical symptoms of the cattle after artificial stress: Serous nasal
discharge was observed from 7th day in three cows (No. 301 and 616) except in
one animal (No. 665) which showed nasal secretion from the end of 5th day (last
day of dexamethasone injection). Nasal secretion gradually decreased from 10th
day onward. The cows showed mild depression but their appetite was normal. The
animals showed no rise of temperature, which was almost subnormal (below 101°F)
in all the animals. The animals showed mild diarrhea for 3-4 days starting from
8th day and their nasal mucosa were prominently pink. The animal appeared almost
normal but milk production suddenly dropped.
Virus isolation and identification: Herpesvirus was recovered from all
the three seropositive cattle showing the nasal discharge and the duration of
excretion of the virus varies with the individual cattle. The results of virus
isolation are presented in Table 1.
It was observed that the shedding of BoHV-1 virus was prolonged in those animals
whose antibody titers rise comparatively less in the convalescent serum (Table
The isolated virus showed characteristic CPE and was neutralized by respective
convalescent serum. The BoHV-1 reference virus (BoHV-1 strain 2204) was neutralized
to a comparable extent by the same convalescent serum (Kumar
et al., 1994) and there was four fold to seven fold increases in
antibody titers in paired sera samples (Table 2). Electron
microscopy study showed icosahedrons shaped virus (Fig. 1)
of 100 nm in diameter.
In immunofluorescence analysis all the isolates stained clearly and homogenously
positive for BoHV-1 gB, gD, gG, gC and gE. These tests were performed for infected
monolayer as well as for single viral plaques grown under semisolid medium to
exclude the possibility of mixtures of different strains within the samples.
Quantitative real time PCR tests showed distribution of equal amounts of gE
and gB in each of the samples. These findings exclude the concomitance of BoHV-5
and verified the classification as Bovine herpesvirus type 1.
|| BoHV-1 under electron microscope
||Isolation of IBR virus (BoHV-1) from nasal secretions by inducing
artificial stress with dexamethasone in seropositive cattle
To clarify whether the isolates exhibited different capabilities of cell to
cell spread, serial dilutions were grown in cell culture under semisolid medium
and stained for BoHV-1 gB. Diameters of least 30 plaques from each isolate were
determined using a graduated ocular and correlated to the plaque size of BoHV-1
Schönböken which was set as 100%. Plaque sizes were analyzed and no
satisfactory significant difference could be demonstrated among the isolates.
Restriction Fragment Length Polymorphism (RFLP) analysis identified all isolates
as Bovine herpesvirus type 1. All the 3 isolates were assigned to genotype
BoHV-1.2 (Strains India 4, India 5 and India 6).
Nucleotide sequencing: Parts of the regions encoding gH, gB and gC were amplified
and sequenced on a 3130 Genetic Analyzer (ABI). In gH sequence analysis Strains
India 4, 5 and 6 showed identical sequences. In gB sequence analysis all the
3 isolates from India showed an identical sequence in the analyzed 450 base
pair of the gB gene. The sequence was named India
gBcons. In gC sequence analysis
all the 3 isolates from India showed an identical sequence in the analyzed 158
base pair spanning overlapping region of gC gene. The sequence was named India
||Antibody titres against IBR virus (BoHV-1) in paired serum
samples collected at day 0 and day 21 after inducing artificial stress with
dexamethasone in seropositive cattle and isolation of virus
In the present study serous nasal discharge was observed in all the three dexamethasone
treated cows, which is the indication of immunosuppression and thereby liberation
of the virus (De Carlo et al., 2004). The cows
showed mild depression due to immunosuppression but their appetite was normal.
The animals showed no rise of temperature, which was almost subnormal (below
101°F) in all the animals. The animals showed mild diarrhea for 3-4 days,
which corroborates with the findings of De Carlo et
al. (2004). The animal appeared almost normal (Straub
and Lorenz, 1991). Sudden drop of milk production was also been recorded
by Pritchard et al. (1997). All the cows become
normal soon after the course of dexamethasone ended. Since dexamethasone is
not a very long acting immunosuppressive drug, discontinued use results in a
return to normal of the three cows (Pastoret et al.,
Isolation of BoHV-1.2 (Strain India 4, India 5 and India 6) after dexamethasone
injection in seropositive animals underlined that dexamethasone being an immunosuppressant,
mimics natural stress and induces reactivation of the virus from a latent state.
It was observed that there is variation in the recovery of virus in these animals
which is due to the factor that the virus shedding itself is inconsistent. The
level of local antibodies present is so high that even though reactivation occurs,
infectious virus is rapidly neutralized and excretion cannot be detected which
is responsible for variation in isolation of the virus among the cows (Pastoret
et al., 1980).
There was four fold to seven fold increases in antibody titers in paired sera
samples. Since immunosuppressant is short lived, viral antigen present can act
as a booster to the immune response, and therefore this would explain the increase
in antibody titer after dexamethasone treatment (Pastoret
et al., 1980).
Although most BoHV-1.2 strains had been reportedly isolated from genital organ
lesions but in this study all the three BoHV-1.2 (Strain India 4, India 5 and
India 6) isolates were from nasal secretions of dexamethasone injected cattle
which is also significant enough. Although the seroprevalence of BoHV-1 in West
Bengal were 33.97% in 2004-05 (RDDL, 2004), 39.1% in
2005-06 (RDDL, 2005) and 47.92% in 2006-07 (RDDL,
2006) which has an increasing trend but there are no reports regarding isolation
of the BoHV-1 from cows. In this respect isolation of BoHV-1 after inducing
artificial stress with dexamethasone in this region has a great importance regarding
various epidemiological aspects. Moreover these are the first reported isolates
of BoHV-1 after artificial stress in West Bengal as well as in India.
Molecular study of the isolate found to be BoHV-1 genotype i.e., BoHV-1.2.
Sequence analysis confirmed the clustering with known BoHV-1 strains and showed
a clear difference to other ruminant herpes viruses. A regional clustering of
the BoHV-1 isolates for the analyzed gC sequence showed a marginal differences
to known isolates from Europe or the United States of America. Investigation
of further Indian BoHV-1 isolates will be necessary to confirm the significance
of this observation. The analysis of highly conserved genome regions gB did
not reveal significant differences to previously reported BoHV-1.
In tropical and sub-tropical countries animal husbandry development is directed
towards the rearing of cross-bred cattle and they are more susceptible to environmental
stress like heat stress. In BoHV-1 infected cattle, the virus remain latent
for life-long and excreted through secretions (nasal, ocular and vaginal) during
any type of stress and thereby imposes a threat of disseminating the virus to
other cattle (Pistl et al., 2003). As no prevention
and control measures adopted either in organized or unorganized cattle farms
in countries like India, Pakistan, etc. even introduction of latently infected
sero-negative cattle will impose a thread to spread the virus to healthy cattle
(Rola et al., 2005) promptly under stressful
conditions. Several reactivation stimuli can lead to viral re-excretion, which
is responsible for the maintenance of BoHV-1 within a cattle herd. In India
the seoprevalence of IBR is 41.23% in 2003-04 (ADMAS, 2003),
which is alarming, and yet no prevention and control measures have been adopted
by the Government of India. Moreover prevention and control of the disease caused
by BoHV-1 consists of thorough sanitary measures, the DIVA (Differentiating
Infected from Vaccinated Animals) strategy mainly by vaccination with marker
vaccine and isolation of the virus (Pistl et al.,
2003). Thus isolation and characterization of more viruses in countries
like India will contribute to the control and prevention of IBR.
BoHV-1 is a worldwide disseminated pathogen displaying significant differences
in regional incidence and prevalence with regards to the geographical positions
and the breeding managements of the considered regions (Ackermann
and Engels, 2006).Thus reactivation obtained in this study with the BoHV-1
cow latency model and dexamethasone suggests the usefulness of the model for
further studying the molecular characteristics of the BoHV-1 isolates prevalent
in tropical and sub-tropical countries like India, Pakistan etc. which will
provide valuable clues to epidemiological investigations on BoHV-1 infections.
Moreover this model to detect virus may be taken to prevent the introduction
of BoHV-1 infected sero-negative animals in a herd in order to improve the efficacy
of control programmes.
The authors are grateful to the Director and Veterinary Officer of Behala Express
Dairy for giving necessary permission to carry out the study in their cattle
farm. The authors are grateful to the Joint Director, Institute of Animal Health
and Veterinary Biologicals, West Bengal for necessary permission to carry out
this work in their institute. The authors convey their special thanks to the
Director, Indian Institute of Chemical Biology, Jadavpur and the Director, National
Institute of Cholera and Enteric Diseases (NICED), Kolkata for giving necessary
permission for ultra-centrifugation and transmission electron microscopy of
the isolated virus. The authors are in debt to PD Dr. Martin Beer and Dr. Patricia
Koenig of OIE and National Reference Laboratory for Bovine herpesvirus
type 1, Friedrich-Loeffler-Institut, Germany for typing of the virus.
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