Abstract: In this study skin lesions were collected from camel suffered from Camelpox Virus (CPV) infection from different region of South Sina Governorate and Maruit camel farm of the Desert Research Center. CPV was isolated and propagated on CAM of 9-11 days SPF-ECE for 10 passages resulting in characteristic pock lesion of the CPV and the highest titer was (4.2 log10EID50 mL-1) at the 7th passage, also CPV was isolated and propagated on Vero cell line for 20 passages resulting in the characteristic CPE in the passage No. 3 after 5 days of inoculation and the virus titer increased gradually till reach (4.7 log10TICD50 mL-1) at the 17th passage. The isolated virus was identified as CPV using Virus Neutrization Test (VNT) with Neutrization Index (NI) equal to 2 and the identification confirmed by using the double antibody sandwich ELISA (DAS-Elisa) on antigen prepared from different virus passages on Vero cell line.
INTRODUCTION
Camelpox (CP) is the most frequent infectious viral disease widely reported in camels in Africa and Asia Alhendi et al. (1994). In Egypt, Camelpox virus (Fayoum-71) was firstly isolated in Fayoum during 1971-1972. Tantawi et al. (1974a), in Sharkia Governorate (Kenawy et al., 1989), in Assiut, Bni-Adi and neighboring village (El-Kom and EL-Hawkta) (Zaitoun et al., 2000) and from three Egyptian governorates, Marsa Matroh, Aswan and Giza as (CPV/Aswan/07) (Salem et al., 2008).
CPV could be isolated and propagated on CAM, giving typical pock lesions with a titer of 105.3; Gabery et al. (1997). CPV also can be isolated and propagated on Vero cell line resulting in rounding, aggregation and plaques formation (Davies et al., 1975; Abdel Baky et al., 2006).
The aim of the present study is isolation and characterization of CPV in Egypt from South Sina government and Maruit camel farm of Desert Research Center by amplification and sequence analysis for the (C18L) in order to make a compression with other isolates of CPV.
MATERIALS AND METHODS
Skin lesions: A number of camels were reported to have skin lesions in Southern Sinai Governorate and Maruit camel farm of the Desert Research Center. Dried scabs were collected (at different stages of development) and kept in sterile vials contain transport medium to be used in this study. The collected scabs were ground in a sterile mortar according to Kenawy et al. (1989).
Camelpox virus: Saudi strain of CPV (Jouf-87 strain) was kindly supplied from Pox Vaccine production and Research Department, Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo.
Rabbit and Hyperimmune serum against Camelpox virus: It was prepared at Pox Vaccine Production and Research Dept, Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo using vaccinal strain of CPV (Jouf-78), according to the method described by Aboul Soud (2006).
Embroynated chicken eggs (ECE): Specific Pathogen Free (SPF) Embroynated Chicken Eggs (ECE) Fertile SPF- ECE (9-11 days old) were used for virus propagation via the Chorio-allanoic Membrane (CAM) route of inculcation it was obtained kindly from the new castle disease Department in the Veterinary Serum And Vaccine Research Institute.
African green monkey kidney cell line (vero): It was kindly supplied by Pox Vaccine production and Research department, Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo, using Eagl’s Minimum Essential Medium (MEM) with new born calf serum (10% for growth medium and 2% for maintenance medium).
Propagation and titration of the isolated Camelpox virus in embryonated chicken eggs (ECE): The isolated Camelpox virus was propagated and titrated on the CAM of SPF of 9-11 days old ECE according to Gabery et al. (1997).
Propagation and titeration of the isolated Camelpox virus in Vero cell line: Isolated Camelpox virus was adapted and titrated in Vero cells according to Abdel Baky et al. (2006).
Application of VNT test for identification for the propagated isolated CPV test in vero cell line: VNT was conducted as described by Abdel Baky et al. (2006) to specify the virus isolate as a Camelpox virus.
Application of double antibody sandwich elisa (DASE) for identification for the propagated isolated CPV: Double antibody sandwich elisa was used for identification for the antigen prepared from different passages of the isolated CPV on Vero cell line (passages No. 5, 10, 15).
RESULTS
Propagation and titration of the isolated Camelpox virus on CAM of ECE: Isolated Camelpox virus was inoculated on CAM of SPF- ECE 9-11day old for 10 successive passages resulting in the characteristic pock lesion of CPV as illustrated in Fig. 1.
The titer increased from the 3rd passage reached its beak at the 8th passage (4.2 log10TICD50 mL-1) and still constant after that as shown in Table 1.
Propagation and titration of the isolated Camelpox virus in African green monkey cells (vero cell lines): Isolated CPV inoculatd on Vero cell line for 20 passages, the CPE of the isolated CPV in different passages illustrated in Fig. (2, 3, 4, 5), the titer of the virus was increased (from 3.0 log 10TCID50 mL-1 in the 3rd passage to highest virus titer 4.7 log10TCID50 mL-1 at 17th passage) and the complete CPE of the virus appeared more earlier by the serial successive passages as shown in Table 2.
| Fig. 1: | Pock lesion of the inoculated isolated CPV |
| Table 1: | Titration of the propagated isolated Camelpox virus on CAM of SPF-ECE |
Application of virus neutralization test in vero cell line for the propagated isolated Camelpox virus (Alfa procedure): Vero cell line shows reduction of virus titer with 2log10TICD50 mL with neutralizing index of 2.0. For both isolated virus passages No. (5) and (10) (Table 3). Four Identification of the isolated CPV antigen by Double Antibody Sandwich ELISA (DASE).
The results were calculated on the basis of S/P ratio, the highest dilution of propagated isolated Camelpox virus antigen gave positive reaction was 1/40 at the passage 15, compared to the highest dilution of the Jouf-78 strain CPV antigen give positive reaction at 1/60.
| Fig. 2: | CPE of isolated CPV in vero cells (low power) |
| Fig. 3: | CPE of isolated CPV in vero cells (low power) |
DISCUSSION
CPV was isolated and propagated on CAM of SPF-ECE, the pock lesions increased in numbers and size by successive serial passages with survival of the inoculated chicken embryo post virus inoculation. It indicate the adaptation of the isolated CPV on the ECE, these results are in accordance with Alhendi et al. (1994) and Zaitoun et al. (2000).
The CPV virus titer on the ECE increased from 3rd passage till the 10th passage, the peak titter was reached by the 8th passage reached 4.2 log10 TICD50 mL and then it became fixed at this level till the 10th passage. Similar result obtained by Tantawi et al. (1974b).
Local CPV isolate revealed characteristic CPE on the inoculated Vero cell line after three successive passages as small aggregates, formation of Grape like appearance and detachment of some aggregates with forming of network sheet like, also Intra-cytoplasmic Inclusion Body (ICIB) were detected in intact infected Vero cells.
| Fig. 4: | CPE of isolated CPV in vero cells (high power) |
| Fig. 5: | CPE of isolated CPV in vero cells (high power) |
Similar result obtained by Davies et al. (1975) and Abdel Baky et al. (2006).
The titer of the virus was increased from 3.0 log10TCID50 mL in the 3rd passage till it reached its highest virus titer 4.7 log10TCID50 mL at 17th passage and still constant after this passage as it indicate the complete adaptation of the isolated virus on Vero cell line similar result obtained by Abdel Baky et al. (2006).
Alpha procedure of neutralization test was carried on the isolated propagated virus on Vero cell line at the 5th and 10th passages showing reduction of virus titer with 2 log10TICD50 mL with neutralizing index of 2.0 in the tow passages (No. 5th and No. 10th), showing cross reactivity between the prepared hyper immune sera against CPV (Jouf-78 strain) and the isolated CPV similar results was obtained by Abdel Baky et al. (2006).
| Table 2: | Cytopathogenicity and titer of the of the isolated propagated CPV on vero cell line |
| d: Day, ±: Cell rounding, highly retractile cells and ICIB, +: Small aggregates formation and foci of clear area, ++: Grape like appearance and detachment of some aggregates, +++: Network sheet like, complete CPE (70-80%) and sheet destruction (70-80%) | |
| Table 3: | S/P ratio of ELISA for the antigens obtained from propagated isolated Camelpox virus on vero cell line and jouf-78 strain |
| S/P ratio: Mean optical density of sample -mean optical density of negative sample mean optical density of positive sample- mean optical density of negative sample. S/P ratio>1 is considered as the positive result | |
ELISA results reveled that antigen dilutions 1/20, 1/30 and 1/40 were the end points, respectively and in case of CPV vaccine (Jouf-78 strain) the end points for the antigen dilution which gave positive reactivity was 1/60, this result indicate the cross reactivity between the prepared hyper immune sera against antigen prepared from the CPV (Jouf-78 strain) and the isolated propagated CPV and these results were in accordance with the result of virus neutrization test of the isolated CPV.