Isolation and Molecular Characterization of Camel Pox Virus By Salem, SAH

Egypt. J. Comp. Path. & Clinic. Path. Vol. 21 No. 4 (December) 2008; 306 - 318

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Referred byReferred by Prof. Dr. Gabr El-Bagoury Professor of Virology, Fac. Vet. Med.,

(Moshtohor), Banha University Prof. Dr. Nawal, M. Ali Professor of Virology, Animal Health Re-

search Institute

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.

SUMMARY

C amel pox one of the economic disease of dromedary infected cam-els. 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 Reac-tion (PCR). E.M. considers one of the rapid and specific methods of laboratory confirmation. E.M. was done on CAM samples with charac-teristic 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.

INTRODUCTION

C amel-pox was greatly in-criminated as one of the ma-

jor skin disease of camels, it is a highly transmissible and conta-gious disease. The disease occurs more frequently and more severely in young animals (Gitao, 1997 and Muhammad et al., 1998). Camel- pox virus (CPV) is DNA epithelio-tropic virus, the etiological agent

of camel- pox (CP), belongs to family pox-viridae, genus ortho-poxvirus. The viral infectious dis-ease has been reported through ar-eas of middle east, Africa, Asia and southern parts of Russia and India. Outbreaks of CPV infection can be responsible for sever eco-nomic losses in these countries (Sophie et al., 2007 and O.I.E., 2008). The incubation period is


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usually 9-13 days. The clinical manifestations of (CP) range from in apparent and mild local infec-tions, confined to the skin, to mod-erate and sever systemic infections (Wernery and Kaaden, 2002). The disease is characterized by fe-ver, enlarged lymph nodes and skin lesions. In generalized form pox le-sions 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 fe-males 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 contami-nated environment. Virus is se-creted 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 in-fect other animal species. Field re-ports of mild skin lesions in hu-mans associated with CP have been made (Coetzer, 2004). In

Egypt studies concerning CPV are somewhat few. Tantawi et al. (1974) isolated (CPV) for the first time in Fayoum Governorate, Ke-nawy et al. (1989) in Sharkia Gov-ernorate, Nawal and Ahmed (1997) and Maysa et al. (1998) in Matrouh Governorate and Zaitoun et al. (2000) in Assuit Gover-norate. The aim of the present work to deal with camels that suf-fered from skin lesions suspected pox virus in Southern Sinia Gover-norate, Egypt, by trials of virus isolation on chorioallantoic mem-brane (CAM) of specific pathogen free emberyonated chicken eggs, propagation of the isolates on tis-sue culture cell line (Vero cells) and Identification of the isolates by Agar gel precipitation Test, Elec-tron microscopical technique and molecular identification by poly-merase chain reaction (PCR). MATERIALS AND METHODS

(1) Samples:

a- Skin lesions: A number of camels were re-ported to have skin lesions in southern Sinai governorate. Dried scabs were collected from 5 se-lected cases showed skin lesions (at different stages of develop-ment) and kept in sterile vials con-tain glycerol buffer to be used in this study. The collected scabs were ground in a sterile mortar ac-cording to (Kenawy et al., 1989).


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b- Serum samples: 5 serum samples were col-lected from 5 camels with skin le-sions and 20 samples were col-lected 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) Virol-ogy 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 ob-tained from infected SPF, ECE with characteristic pock lesions of (CPV) were inoculated into Vero cells after filtration through 0.45 μm filter.

(5) virus Identification: a- Agar Gel precipitation test (AGPT): It was carried out according to (Kitching et al., 1986), the iso-lated virus was identified as CPV using Hyper immune serum, kindly supplied from (AHRI), vi-rology department, Dokki, Cairo.

b- Electron microscopy (E. M): Samples from CAM Pock le-sions were fixed in 3% glu-traldhyde + 10% formaline, proc-essed and sectioned for transmis-sion electron microscopy in Elec-tron Microscopical Center of Vet-erinary 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 us-ing DNA extraction QiaAmp DNA kit (Qi A Gen, Valencia, Calif, USA). Samples were amplified us-ing PCR Reddy Mix PCR master Mix (Thermo, UK). PCR assay was done as de-scribed by Meyer et al. (1994), al-lows the detection and differentia-tion of species of the genus ortho-poxvirus because of the size differ-ences of the amplicons. Using the primer pair: 5`- AAT- ACA- AGG- AGG- ATC- T- 3`- and 5`- CTT- AAC- TTT- TTC- TTT- CTC- 3` the gene sequence encoding the A- type inclusion protein (ATIP) will be amplified. The size of the PCR


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product, specific for the camelpox virus (CPV), is 881 bp. The test was applied on PCR thermal cy-cles machine (AB1, 2720).

(6) Serological investigation by AGPT: All sera collected were screened against the identified lo-cally isolated CPV from AHRI, Vi-rology department, Dokki. Cairo. According to method described by (Tantawi et al., 1974).

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).

(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 cy-topathic effect CPE of (CPV) after 4-5 days post inoculation the CPE was characterized by cell round-ing, aggregation and detachment of the cell sheet Fig. (2).

(3) Agar Gel precipitation test AGPT:

A clear precipitation lines (positive results) appear between the isolated virus and the CPV hy-

per immure serum.

(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 in-ner surface of the nuclear mem-brane. Progeny virus develops in the cytoplasm in association with aggregates of dense fibrous or granular material and some imma-ture 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).

(5) Polymerase Chain Reaction (PCR):

The five tested samples showed the characteristic PCR positive bands of 881 bp size frag-ment of camel-pox virus (Fig., 5).

(6) Serological investigation by AGPT:

The AGPT was applied to de-tect the immune status of the in-fected and contact apparently healthy camels. The results revealed that 100% of infected camels (5 sam-ples out of 5 infected camels) were positive reactors and 60% of con-tact camels (12 samples out of 20 contact camels) were positive reac-tors.


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Fig. (1): Chorioallantoic membrane showing pock lesions characteristic

for CPV.

Fig. (2): A- Non infected Vero cells. B- Infected Vero cells showing CPE characterized by cell round-ing, aggregation and detachment of cell sheet.

A B


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Fig. (3): The ultrastructure of EM clarified the CPE in nucleus and cyto-plasm (X 10.000).

Fig. (4): The CPV has a typical brick-shaped appearance with irregu-larly tubular surface proteins appear in the cytoplasm of in-fected 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.

DISCUSSION

T he presence of the various forms and sizes of skin erup-

tions on the different parts of the infected camels rarely associated with systemic involvement includ-ing 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, pas-tular scabs and ulceration) with elevation of temperature 40-41ºC in some cases. Such suspecion was primarily supported by the appear-ance of the characteristic Pock le-

sions on the chorioallantoic mem-brane (CAM) of the infected SPF fertile eggs. The pock lesions ap-pear clear after the second pas-sages as opaque, white, circular, enlarged areas with slight thicken-ing in the CAM, most of the inocu-lated eggs still containing living embryos 5 days post inoculation (PI), these coincidence with (Tant-awi et al., 1974) who isolated CPV for the first time in Egypt in Fay-oum Governorate and Kenawy et al. (1989) in Sharkia Governorate, Chauhan and Kaushik (1987) re-ported the pock lesions character-istic 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


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they were all opened, 5 days post inoculation and CAM showed whitish lesions. Nawal and Ah-med (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 repli-cate 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 circu-lar). 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 pas-sages 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 differ-ent 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 pro-duce CPE at the 4th day PI. Wern-ery et al. (1997) isolated CPV in Vero cells and Dubca cell lines,

(Khalafalla et al., 1998) isolated seven viruses in Vero cells from outbreaks of CPV in Eastern Su-dan, 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) stud-ies the growth of CPV in Vero cells. The viral isolate tested against hyper-immune serum of CPV that showed clear precipita-tion lines, these results were simi-lar 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 Elec-tron microscopical examination (EM) and polymerase chain reac-tion (PCR). Transmission electron microscopy (TEM) is a rapid method to demonstrate CPV in tis-sue 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 parti-cles in pox lesions was one of the important diagnostic tools of the disease. The electron microscopi-


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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 simi-lar to those obtained by Kaaden et al. (1992) and Munz (1992). The results also agree with that of Pfef-fer 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 col-lected from suspected camels us-ing CAM of infected ECE, tissue culture propagation and identifica-tion by Electron microscope, how-ever these method are laborious and time consuming, today assays based on advanced technology that can serve diagnosis of animal in-fectious disease like PCR that pro-vide easy to handle tools for virus diagnosis, requiring only minute amounts of specimen, we there fore apply this technique to spe-cific detection of CPV.

PCR assay was done using the primer pair: 5'-AAT-ACA-AGG-AGG-ATC-T-3' and 5'-CTT-ACC-TTT-TTC-TTT-CTC-3'. The gene sequence encoding the A-type inclusion protein (ATIP) will be amplified. The size of the PCR product, specific for the camel-pox virus (CPV), is 881 bp. These re-sults agree with those obtained by Meyer et al. (1994) and results re-

corded by Caroline and Geofrey (2002), Afanso et al. (2002) and O.I.E. (2008).

The serological investigation revealed that all sera (5) collected from camels which had pox infec-tion showed positive results in AGPT, while 12 serum samples out of 20 serum samples from symptomless contact camels gave positive results. These positive re-sults may be attributed to subclini-cal infection as these camels in close contact with diseased camels. These results similar to those ob-tained by Tantawi et al., (1974) who showed the presence of anti-bodies 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 Ah-med (1997), applied AGPT and detect CPV antibodies in 100% of infected camels sera and 67% in contact camels sera.

CONCLUSION

O ur conclusion that CPV one of the infectious disease af-

fected 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 suspi-cious of camel pox which sup-ported and confirmed by lab diag-


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nosis include virus isolation and identification, sero-diagnosis, E. M. and advanced technology like PCR.

Although there is a vaccina-tion policy against camel pox, by using local producing attenuated camel pox vaccine supplied by (Veterinary Serum and Vaccine Re-search Institute, there is few cases appear among camel flocks, the further studies must be submitted to determine the role of insect cli-matic condition, nature of soil, ef-ficacy of vaccination and other factors play a role of transmission.

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Isolation and Molecular Characterization of Camel Pox Virus By Salem, SAH