الفهرس 
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Abstract
6. SUMMARYLumpy skin disease (LSD) is a viral disease of cattle characterized by the development of nodules upon the skin. OIE classified LSD in livestock industry as “list A” of diseases due to the rapid spread and ability to cause economic losses due to reduction of milk production, infertility and damage of hide of infected animal due to characterized skin nodules. In Egypt it was recorded in infected cows all year-round. Control of the disease without vaccination is extremely difficult in endemic areas. Therefore, the aim of this thesis is to evaluate the efficacy of the live attenuated sheep pox virus vaccine in protecting indigenous cattle against LSD under Egyptian condition by measuring of both acquired humeral and cellular immunity of animals using more accurate device called flow-cytometer.
The study was carried on
Healthy calves
15 apparently healthy calves (6-10 month of age) with average body weights 140-200 Kg, not previously vaccinated against LSDV.
Animals were assigned to 2 groups:
group 1: five healthy cattle male calves were kept as control.
group 2: ten healthy cattle male calves were vaccinated with live attenuated sheep-pox vaccine. Vaccinated by 2 doses with 1 month interval these animals were used for flow-cytometeric analysis of different T cell subsets CD4+, CD8+, γδ T cells expressing CD 25 and B cells expressing CD45 marker at zero, third , fourth , fifth week after vaccination with booster dose. (60-100ml) of whole blood were collected aseptically from each animal in acid citrate dextrose (ACD) to a final concentration 15-20% and within 4-5 hrs , PBMCS were separated using histopaque and suspended in RPMI medium and then counted with trypan blue then cultivated in 3 wells first with PBS as control, second with in activated lumpy skin disease virus as mitogen for stimulation of cells, third with in activated sheep pox virus as mitogen for stimulation of cells in RPMI and incubated in 5% co2 incubator at 370c for 6 days . After that these cells harvested and stained with primary monoclonal Abs and secondary labeled Abs. then these cells was analyzed using flow-cytometery for detection of T-cells subsets % expressing CD25 and B cells expressing CD45 marker along study.
Results revealed that CD4+ ,CD8+, γδ Tcells % expressing CD25 and B cells expressing CD45R0 marker after stimulation of PBMCS culture with in activated LSDV and Sheep pox virus was significantly increased in comparison to non-stimulated culture at zero, third , fourth and fifth week after vaccination with booster dose of live attenuated sheep pox viral vaccine and result also revealed that the percentage of CD4+ ,CD8+, γδ Tcells expressing CD25 and B cell expressing CD45R0 marker after stimulation of PBMCS culture with in activated Sheep pox virus (homologous mitogen) was significantly higher than in case of using in activated LSDV (heterologous mitogen).results also revealed that CD4+ ,CD8+, γδ Tcells % expressing CD25 and B cell expressing CD45R0 marker after stimulation of PBMCS culture with in activated LSDV and Sheep pox virus was significantly increased in the fifth week after the second vaccination than the fourth week than the third week than before vaccination.
And also these ten calves were used to measure humeral immune response using serum neutralization test after 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 weeks of vaccination with live attenuated sheep pox viral vaccine. Results revealed that Abs titers were significantly increased from week to week till the tenth week after vaccination and become protective against lumpy skin disease infection from after the third week post vaccination.
3.1.1.2. Diseased calves
A total number of five calves (10 month of age) with average body weights 140-200 Kg previously infected with LSDV after 40 days from appearance of clinical sings were used to measure cell mediated response using flow-cytometeric analysis of different T cell subsets CD4+, CD8+, γδ T cells expressing CD 25 and B cells expressing CD45 marker, (60-100ml) of whole blood were collected aseptically from each animal in acid citrate dextrose (ACD) to a final concentration 15-20% and within 4-5 hrs , PBMCS were separated using histopaque and suspended in RPMI medium and then counted with trypan blue then cultivated in 3 wells first with PBS as control, second with inactivated lumpy skin disease virus as mitogen for stimulation of cells, third with inactivated sheep pox virus as mitogen for stimulation of cells in RPMI and incubated in 5% co2 incubator at 370c for 6 days . After that these cells harvested and stained with primary monoclonal Abs and secondary labeled Abs. then these cells was analyzed using flow-cytometery for detection of T-cells subsets % expressing CD25 and B cells expressing CD45 marker along study.
Results revealed that Results revealed that CD4+ ,CD8+, γδ Tcells % expressing CD25 and B cell expressing CD45R0 marker after stimulation of PBMCS culture with in activated LSDV and Sheep pox virus was significantly increased in comparison to non-stimulated culture and result also revealed that the percentage of CD4+ ,CD8+, γδ Tcells expressing CD25 and B cell expressing CD45R0 marker after stimulation of PBMCS culture with in activated LSDV (homologous mitogen) was significantly higher than in case of using in activated Sheep pox virus (heterologous mitogen).
And a total number of ten calves (7-10 month of age ) with average body weights 140-200 Kg previously infected with LSDV after 2,3,4,5,6,7,8,9 and 10 weeks from appearance of clinical sings were used to measure humeral immune response using serum neutralization test. Results revealed that Abs titers were significantly increased from week to week till the tenth week after appearance of clinical signs and become protective against lumpy skin disease infection from after the third week post infection. And when compared with those animals vaccinated with live attenuated ShPV vaccine results revealed that Abs titers of infected animals were higher than Abs titers of vaccinated animals among all weeks of the study.
Moreover, from the previous results these tests used for measuring the efficacy of live attenuated sheep pox virus vaccine, so these tests can be used at least instead of challenge test.