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Egypt. J. Comp. Path. & Clinic. Path. Vol. 21 No. 4 (December) 2008; 306 - 318
Isolation and Molecular Characterization of Camel Pox Virus
By
Salem, S.A.H.*; Omayma, A. Shemies*; Nahed, A. Mahmoud* and
Arafa, A.A.**
* Virology Dept., Animal Health Research Institute, Dokki, Giza
** National Laboratory for Veterinary Quality Control on Poultry Production
(NLQP), Dokki, Giza, Egypt.
C
SUMMARY
amel pox one of the economic disease of dromedary infected camels. The skin eruption one of the characteristic lesion which appear
in various stages of the infection. The samples were collected from
camel flock in Southern Sinai [sera and skin lesions (scabs)]. The camel
pox virus (CPV) were isolated on chorioallantoic membrane (CAM), and
Vero cells. The isolated virus was identified by Agar Gel Precipitation
Test (AGPT), electron microscope (E.M.) and Polymerase Chain Reaction (PCR). E.M. considers one of the rapid and specific methods of
laboratory confirmation. E.M. was done on CAM samples with characteristic pock lesions, and detection of a typical brick shape of orthopox
virus in the cytoplasm of the infected cells. Applying PCR technique
considers the faster and more sensitive molecular advanced technique
for diagnosis of CPV.
Referred by
Prof. Dr. Gabr El-Bagoury
Professor of Virology, Fac. Vet. Med.,
(Moshtohor), Banha University
Professor of Virology, Animal Health Research Institute
Prof. Dr. Nawal, M. Ali
of camel- pox (CP), belongs to
family pox-viridae, genus orthopoxvirus. The viral infectious disease has been reported through areas of middle east, Africa, Asia
and southern parts of Russia and
India. Outbreaks of CPV infection
can be responsible for sever economic losses in these countries
(Sophie et al., 2007 and O.I.E.,
2008). The incubation period is
INTRODUCTION
amel-pox was greatly incriminated as one of the major skin disease of camels, it is a
highly transmissible and contagious disease. The disease occurs
more frequently and more severely
in young animals (Gitao, 1997 and
Muhammad et al., 1998). Camelpox virus (CPV) is DNA epitheliotropic virus, the etiological agent
C
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Egypt studies concerning CPV are
somewhat few. Tantawi et al.
(1974) isolated (CPV) for the first
time in Fayoum Governorate, Kenawy et al. (1989) in Sharkia Governorate, Nawal and Ahmed
(1997) and Maysa et al. (1998) in
Matrouh Governorate and Zaitoun
et al. (2000) in Assuit Governorate. The aim of the present
work to deal with camels that suffered from skin lesions suspected
pox virus in Southern Sinia Governorate, Egypt, by trials of virus
isolation on chorioallantoic membrane (CAM) of specific pathogen
free emberyonated chicken eggs,
propagation of the isolates on tissue culture cell line (Vero cells)
and Identification of the isolates by
Agar gel precipitation Test, Electron microscopical technique and
molecular identification by polymerase chain reaction (PCR).
usually 9-13 days. The clinical
manifestations of (CP) range from
in apparent and mild local infections, confined to the skin, to moderate and sever systemic infections
(Wernery and Kaaden, 2002).
The disease is characterized by fever, enlarged lymph nodes and
skin lesions.
In generalized form pox lesions may cover the entire body,
pox lesions can be found in the
mucous membranes of the mouth
and respiratory and digestive tracts
(Kritz, 1982 and Wernery and
Kaaden, 2002). The morbidity
rate of CP is variable and depends
on wether the virus is circulating
in the herd. The incidence of the
disease is higher in males, than females and the mortality rate is
greater in young (25% - 100%)
than in adults (5- 28%), (Mayer
and Zerny, 1990). Transmission is
by either direct contact between
infected and susceptible animals or
indirect infection via a contaminated environment. Virus is secreted in milk, saliva and ocular
and nasal discharge. Dried scabs
shed from the pox lesions contain
live virus for at least 4 months and
contaminate the environment
(Wernery et al., 1997). CPV is
very host specific and dose not infect other animal species. Field reports of mild skin lesions in humans associated with CP have
been made (Coetzer, 2004). In
MATERIALS AND METHODS
(1) Samples:
a- Skin lesions:
A number of camels were reported to have skin lesions in
southern Sinai governorate. Dried
scabs were collected from 5 selected cases showed skin lesions
(at different stages of development) and kept in sterile vials contain glycerol buffer to be used in
this study. The collected scabs
were ground in a sterile mortar according to (Kenawy et al., 1989).
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Egypt. J. Comp. Path. & Clinic. Path. Vol. 21 No. 4 (December) 2008; 306 - 318
(5) virus Identification:
a- Agar Gel precipitation test
(AGPT):
It was carried out according
to (Kitching et al., 1986), the isolated virus was identified as CPV
using Hyper immune serum,
kindly supplied from (AHRI), virology department, Dokki, Cairo.
b- Electron microscopy (E. M):
Samples from CAM Pock lesions were fixed in 3% glutraldhyde + 10% formaline, processed and sectioned for transmission electron microscopy in Electron Microscopical Center of Veterinary Hospital according to
Frances and Anderson (1987).
C- Polymerase chain reaction
(PCR):
The received samples were
tested by PCR, Briefly, DNA was
extracted from each sample by using DNA extraction QiaAmp DNA
kit (Qi A Gen, Valencia, Calif,
USA). Samples were amplified using PCR Reddy Mix PCR master
Mix (Thermo, UK).
PCR assay was done as described by Meyer et al. (1994), allows the detection and differentiation of species of the genus orthopoxvirus because of the size differences of the amplicons. Using the
primer pair: 5`- AAT- ACA- AGGAGG- ATC- T- 3`- and 5`- CTTAAC- TTT- TTC- TTT- CTC- 3`
the gene sequence encoding the Atype inclusion protein (ATIP) will
be amplified. The size of the PCR
b- Serum samples:
5 serum samples were collected from 5 camels with skin lesions and 20 samples were collected from the contact apparently
healthy camels.
(2) Hyperimmune serum:
Hyper immune serum of
camel pox virus was obtained from
animal Health research institute
(AHRI) Virology Department,
Dokki, Cairo.
(3) Camel- pox virus:
Obtained from (AHRI) Virology Department, Dokki. Cairo.
(4) Virus isolation:
a- Specific pathogen free (SPF)
emberyonated chicken eggs
(ECE):
SPF-ECE (11-12) days old
were obtained from National
Laboratory for Veterinary Quality
Control on Poultry Production
(NLQP), Dokki, Giza, Egypt, were
inoculated by the prepared samples
via CAM route according to
(Robinson and Balassu, 1981).
b- African green monkey kidney
cells (Vero):
It was kindly supplied and
propagated at virology department,
(AHRI), Dokki, Cairo.
The CAM suspensions obtained from infected SPF, ECE
with characteristic pock lesions of
(CPV) were inoculated into Vero
cells after filtration through 0.45
μm filter.
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per immure serum.
product, specific for the camelpox
virus (CPV), is 881 bp. The test
was applied on PCR thermal cycles machine (AB1, 2720).
(6) Serological investigation by
AGPT:
All sera collected were
screened against the identified locally isolated CPV from AHRI, Virology department, Dokki. Cairo.
According to method described by
(Tantawi et al., 1974).
(4) Electron microscopy:
The ultrastructure of E. M.
clarified the CPE in nucleus which
appears has uneven distribution of
chromatin which accumulated in
many corners of the nucleus in inner surface of the nuclear membrane. Progeny virus develops in
the cytoplasm in association with
aggregates of dense fibrous or
granular material and some immature particles appear spherical
(Fig., 3). CPV has a typical brick
shaped appearance with irregularly
tubular surface proteins appear in
the cytoplasm of infected cells
(Fig., 4).
RESULTS
(1) Virus propagation on CAM
of SPF-ECE:
The virus was grown on the
CAM and produced characteristic
Pock lesions (circular, opaque,
white enlarged areas) on the 5th
days post inoculation at the second
passage. The virus grow readily at
37ºC Fig (1).
(5) Polymerase Chain Reaction
(PCR):
The five tested samples
showed the characteristic PCR
positive bands of 881 bp size fragment of camel-pox virus (Fig., 5).
(2) Virus propagation on Tissue
culture:
The virus replicate on Vero
cells after 3 blind passages of the
filtrated virus isolated on CAM of
the SPE-ECE and produce the cytopathic effect CPE of (CPV) after
4-5 days post inoculation the CPE
was characterized by cell rounding, aggregation and detachment of
the cell sheet Fig. (2).
(6) Serological investigation by
AGPT:
The AGPT was applied to detect the immune status of the infected and contact apparently
healthy camels.
The results revealed that
100% of infected camels (5 samples out of 5 infected camels) were
positive reactors and 60% of contact camels (12 samples out of 20
contact camels) were positive reactors.
(3) Agar Gel precipitation test
AGPT:
A clear precipitation lines
(positive results) appear between
the isolated virus and the CPV hy309
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Fig. (1): Chorioallantoic membrane showing pock lesions characteristic
for CPV.
A
B
Fig. (2): A- Non infected Vero cells.
B- Infected Vero cells showing CPE characterized by cell rounding, aggregation and detachment of cell sheet.
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Fig. (3): The ultrastructure of EM clarified the CPE in nucleus and cytoplasm (X 10.000).
Fig. (4): The CPV has a typical brick-shaped appearance with irregularly tubular surface proteins appear in the cytoplasm of infected cells (X 100.000).
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Fig. (5): Characteristic PCR positive bands of 881 bp size fragment of
camelpox virus.
Lane 1: 100 bp DNA ladder.
Lane 2: Camelpox positive control
Lane 3: Negative control
Lane 4-8: DNA of suspected samples of camelpox.
sions on the chorioallantoic membrane (CAM) of the infected SPF
fertile eggs. The pock lesions appear clear after the second passages as opaque, white, circular,
enlarged areas with slight thickening in the CAM, most of the inoculated eggs still containing living
embryos 5 days post inoculation
(PI), these coincidence with (Tantawi et al., 1974) who isolated CPV
for the first time in Egypt in Fayoum Governorate and Kenawy et
al. (1989) in Sharkia Governorate,
Chauhan and Kaushik (1987) reported the pock lesions characteristic for (CPV) on CAM as small,
opaque and circular, Kaaden et al.
(1989) reported the same results,
Alhendi et al. (1994) reported that
non of inoculated eggs died until
DISCUSSION
he presence of the various
forms and sizes of skin eruptions on the different parts of the
infected camels rarely associated
with systemic involvement including pyrexia, anorexia and fever
may suggested that the suspected
disease was camel pox (CP). The
complain from camels flock in
southern Sinai during 2008, the
characteristic lesions on the mouth
and nostrils suspect a camel pox
virus infection. They all passed
through the typical stages of pox
viral disease (papule, vesicles, pastular scabs and ulceration) with
elevation of temperature 40-41ºC
in some cases. Such suspecion was
primarily supported by the appearance of the characteristic Pock le-
T
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(Khalafalla et al., 1998) isolated
seven viruses in Vero cells from
outbreaks of CPV in Eastern Sudan, EL-Harrak and Loutfi
(2000) reported that isolated CPV
from dromedaries in moracco was
passaged eight times in Vero cells
and Aboul Soud et al. (2004) studies the growth of CPV in Vero
cells. The viral isolate tested
against hyper-immune serum of
CPV that showed clear precipitation lines, these results were similar to those obtained by Kenawy et
al. (1989), Kalafalla et al. (1998),
Maysa et al. (1998) and Aboul
Soud et al. (2004). The growth of
camel pox virus (CPV) on a cell
culture can be confirmed by Electron microscopical examination
(EM) and polymerase chain reaction (PCR). Transmission electron
microscopy (TEM) is a rapid
method to demonstrate CPV in tissue samples, however it is the best
method for distinguish clinical
cases of orthopoxviruses and
parapox viruses (Munz, 1992).
EM findings proved that orthopox
viral infection was probable cause
of pox in our camels. Which has a
typical brick- shaped appearance
with irregularly arranged tubular
surface proteins which present free
in the cytoplasm of the cells
(Andrews et al., 1987) noted that
E.M. detection of the virus particles in pox lesions was one of the
important diagnostic tools of the
disease. The electron microscopi-
they were all opened, 5 days post
inoculation and CAM showed
whitish lesions. Nawal and Ahmed (1997) showed that the virus
(CPV) grow easly on the CAM of
ECE. (embryonated chicken egg)
and produced pock lesions on the
5th day PI. Maysa et al. (1998)
found that CPV was able to replicate on CAM of ECE producing
pock lesions resembling buffalo
pox virus. Zaitoun et al. (2000)
found that (CPV) a characteristic
pock lesions on CAM of ECE
(multiple, small, opaque and circular). These isolates blindly pass
aged on T. C the specific cells
from our choice were Vero cell
line. The isolates could be filtrated
by 0.45 µm disposable filter the
filterable material were inoculated
on T.C. for cells after 3 blind passages and produce the CPE of
CPV after 4-5 days PI. The CPE
was characterizing at first by cell
rounding, aggregation and finally
detachment of the cell sheet. These
results agree with (Ngugen et al.,
1989) who isolated strains of CPV
which had a strong CPE in different cell lines, (Otterbein et al.,
1996) isolated two CPV strains,
which were serially passage on
Vero cells, Nawal and Ahmed
(1997) reported that the isolated
CPV from Matrouh Governorate
replicated in Vero cells and produce CPE at the 4th day PI. Wernery et al. (1997) isolated CPV in
Vero cells and Dubca cell lines,
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corded by Caroline and Geofrey
(2002), Afanso et al. (2002) and
O.I.E. (2008).
cal pictures were similar to those
obtained by Munz et al. (1986)
and coincidence with that of
Mayer and Zerny (1990). The
distribution and morphological
features of the virus particles similar to those obtained by Kaaden et
al. (1992) and Munz (1992). The
results also agree with that of Pfeffer et al. (1996) and Pfeffer et al.
(1998). CPV was isolated in Saudi
Arabia and identified by E. M.
(Abu El Zein et al., 1999). CPV
can be diagnosed in crusts collected from suspected camels using CAM of infected ECE, tissue
culture propagation and identification by Electron microscope, however these method are laborious
and time consuming, today assays
based on advanced technology that
can serve diagnosis of animal infectious disease like PCR that provide easy to handle tools for virus
diagnosis, requiring only minute
amounts of specimen, we there
fore apply this technique to specific detection of CPV.
The serological investigation
revealed that all sera (5) collected
from camels which had pox infection showed positive results in
AGPT, while 12 serum samples
out of 20 serum samples from
symptomless contact camels gave
positive results. These positive results may be attributed to subclinical infection as these camels in
close contact with diseased camels.
These results similar to those obtained by Tantawi et al., (1974)
who showed the presence of antibodies against CPV in the sera of
camels using AGPT, Chauhan
and Kaushik (1987) who found
that 25.8% of serum samples were
positive reactors, Nawal and Ahmed (1997), applied AGPT and
detect CPV antibodies in 100% of
infected camels sera and 67% in
contact camels sera.
CONCLUSION
ur conclusion that CPV one
of the infectious disease affected camel endemic in Egypt,
from time to time there is a field
problem among camel flocks
mostly in desert governorate like
Matrouh (1997), recently Sinai
(2008). The characteristic lesions
and clinical sings appear on skin
one of diagnostic tools for suspicious of camel pox which supported and confirmed by lab diag-
O
PCR assay was done using
the primer pair: 5'-AAT-ACAAGG-AGG-ATC-T-3' and 5'-CTTACC-TTT-TTC-TTT-CTC-3'. The
gene sequence encoding the Atype inclusion protein (ATIP) will
be amplified. The size of the PCR
product, specific for the camel-pox
virus (CPV), is 881 bp. These results agree with those obtained by
Meyer et al. (1994) and results re314
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nosis include virus isolation and
identification, sero-diagnosis, E.
M. and advanced technology like
PCR.
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