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Studies on the adaptation of lumpy skin disease virus (isdv) in cell cultures
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Abstract: This study was planned to propagate the virulent locally isolated strain of LSDV (Ismailia strain) in tissue culture cells and embryonated chicken eggs for obtaining an attenuated virus. This attenuated virus was investigated using serious serological tests and inoculation of susceptible animals to be evaluated as a suitable viral vaccine capable of protecting cattle against the disease in the future. For this purpose the following experiments were done:
1- Propagation of the virulent LSDV (local Ismailia strain) in LT,BK,MDBK,BHKand VERO cells for 5 successive passages. Further propagation of the viru till the 80th passages in MDBK cells and the results revealed that CPE appeared in celss but in LT and MDBK cells the CPE which was clearer and complete after 4 days.
2- Titration the 5th passage of the virus in each type of cell cultures and titers of each 5th passages in each respective type of cell culture were log10(5.3), log10(5.2),log10(4.1) TCID50/ml for LT,BK,MDBK,BHK, and VERO cells respectively.
3-Titration each 10th passage of the virus in MDBK cells revealed the obtained titers to be log10(5.4),log10(5.8),log10(6), log10(6.2),log10(6.4)and log 10(6.4)TCID50/ml the 10th,20th,30th,40ht,50th,60th,70th and 80th passages respectively.
4-Growth curve of the 80th passage of TC adapted virus was studied different hours P.I. to investigate the best time of virus harvestation. Maximum titers were log10(5.6) and log10(6) TCID50/ml for cell free and cell associated virus respectively at 72 hours P.I.
5- Standardization and characterization of the propagated virus in TC was done using SNT, IFAT , histopathological studies and G.P. inoculation. All tests confirmed that the propagated virus (all passages) was LSDV without contamination with other viruses. IFAT on the 80th passage of the virus revealed that the virus appeared in the cytoplasm at 36 hours P.I. close to the nucleus and diffused into the cytoplasm at 72 hours P.I. Histopathological studies revealed that specific ICIB appeared at 48 and 72 hours P.I. as round or oval acidophilic masses surrounded by a halo near the nucleus. Inoculation of various passages of TC adapted virus in G.P. revealed that the adapted virus was immunogenic (all passages) but 60th , 70th and 80th passages were more immunogenic.
6- Studies on the 80th passage of TC adapted virus in ECE indicated that Pock lesions
appeared by the third passage and high titer was obtained by the 10th passage and
equaled to log108.2EID50/ml
7- Titration of each tenth TC virus passages and the 10th ECE passage in cattle showed that high titer and slight local reaction were obtained with the 60th, 70th
and 80th TC passages and the 10th ECE passages.
8- Sterility, safety and potency tests were carried out in cattle to evaluate the 80th and 10th ECE passages. The adapted virus was free from any contaminants and
inoculation of 1/10 and 1/100 virus dilutions show small size skin nodules at the site of inoculation and a slight rise of body temperature at the 7th day P.I. which persisted for 3 days then returned to the normal level.
Safety of the adapted virus was proved by absence of skin oedema, skin nodules and lymphadenitis during the observation of 4 months P.I. Challenge test was done
after 1 month P.I. of the previous dilution of the virus by I/D inoculation of 0.5ml of the virulent virus as well as the controls. The animals inoculated with both
dilutions of TC and ECE adapted viruses remained normal after challenge with no signs of the disease while the controls show classical signs of LSDV infection.
9- Studies on reisolation of the inoculated TC adapted virus from blood, salvia, nasal and lacrimal swabs revealed virus isolation only from blood on the 10th, 11th, 12th and 13th day P.I Titers of the reisolated virus in MDBK cells at these days were log104.5,log104.2,log104 and log104 TCID50/ml respectively. Confirmation of the reisolated virus was done by SNT, IFAT and results assured that the isolated virus was LSDV.
10- Ser-conversion was done on sera collected from cattle inoculated with both TC and ECE adapted viruses (each 10 days till 4 months P.I.). Studies on the
collected sear by SNT, AGPT and solid phase ELISA revealed that antibodies appeared by the 10th day P.I. and increased gradually till reaching the maximum
by 40th and 50th day P.I. and remained stable after 90 days P.I. till 120th day P.I.
11- Neutralizing antibody titers were 8to 19 on the 10th day P.I. for 1/10 and 1/100 virus dilutions of both TC and ECE adapted viruses. Peak titers were 256 to 512
for the same dilutions of viruses on the 40th and 50th day P.I. Neutralizing indices reached the maximum on the 40th , 50th day P.I for 1/10 and 1/100 dilutions oF both viruses and average indices were between 2.5 to 3 for the same days respectively. AGPT revealed and confirmed the results of SNT and a clear precipitin reaction appeared with sera collected on the 30th, 40th, 50th and 60th day P.I.
12-Solid phase ELISA was applied to investigate the level of antibodies in the collected sera and average titers of 80 was obtained on the 10th day P.I for both
virus dilutions. Average titers of both dilutions of TC adapted virus on the 40th and 50th day P.I. were 1280 and 640 while the titers of both dilutions of ECE adapted virus for the same days were 1280 and 640.
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| Publication year |
1995
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| Availability location |
معهد بحوث الامصال واللقاحات البيطرية.
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| Availability number |
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| Organization Name |
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| Country |
Egypt
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| Author(s) from ARC |
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| Agris Categories |
Animal diseases
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AGROVOC
TERMS |
Capripoxvirus.
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| Publication Type |
PhD Thesis
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