ISSN 2320-5407 International Journal of Advanced Research (2016), Volume 4, Issue 4, 212-219

Zeidan S.M 1 , Namaa A. Mohamed 2 , HananM.S.ELZawahry 3 , Saad M.A.A 4 , Olfat E. Nakhla 2 , Afaf A.Abdelwahab 3 , Abeer A. Tammam 3 , El-Dakhly A.T 3 *. 1. Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo,Egypt,Rinder pest like disease department. 2. Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo,Egypt, Sheep pox department. 3. Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo, Egypt, Render pest department. 4. Central Laboratories for Evaluation of Veterinary Biologics, Abbasia, Cairo,Egypt.

The present study was performed to evaluatethe safety and immunogenicity of combined sheep pox (SP) and Peste des Petits ruminants (PPR) vaccine prepared in a lyophilized form. A sheepgroup was vaccinated with the field dose of the prepared combined vaccine subcutaneouslyand the cellular and humeral immune responses of vaccinated sheep were evaluated for six months by different serological tests and compared with the response of eachvaccine alone as a control. It was found that the vaccinated sheep became protected from the 2 nd week post vaccination and keep the protective level till the end of the experiment (24 week). The combined vaccine was found to be safe and potent as evident from sero conversion as well as challenge studies in sheep. We concluded that the use of the prepared combined vaccine component did not interfere with each otherand can be used for economic vaccination strategies.

Introduction:-
Sheep pox virus is a member of genus Capri poxvirus in the family Poxviridae (Kitching, 2004). Sheep pox is a disease of sheep and goats characterized by pyrexia, generalized skin and internal pox lesions, and lymphadenopathy. Sheep pox and goat pox are ancient diseases that are cuffently endemic in the Middle East, the Indian subcontinent, and Central and Northern Africa. Kids and lambs are generally more susceptible than adults (Raox and Bandyopadhy, 2000).
Pest des petites ruminant (PPR) is an acute contagious viral disease of small ruminants characterized by fever, oculonasal discharges, stomatitis, diarrhea and pneumonia with foul offensive breath (Dhar et al, 2002;Ozkul et al, 2002;Asim et al, 2008 and. PPR virus (PPRV) is a Morbillivirus belongs to the Paramyxoviridae family (Barrett et al., 2005). The disease mainly affects goats and sheep, but it is usually reported more severe in goats where it inflicts heavy losses. The morbidity and mortality rate was reach up to 100% in severe cases. (OIE, 2012). In most countries, large scale state vaccination programs are implemented against the 2 diseases, as Vaccination is the best method to prevent the disease in susceptible animals. It carried out by different type of vaccines and the most effective type was the freeze dried live attenuated vaccine (OIE, 2010 and OIE , 2012).
A combined vaccine may be defined as a vaccine that consists of two or more antigens, that is intended to protect either against more than one infectious disease or against an infectious disease caused by different types or serotypes of the same organism (WHO, 2014). The availability of combined vaccines containing protective antigens against the majority of diseases for which universal immunization is recommended due to the simplify of the implementation, increase the acceptance, reduce the global cost of immunization programs and improve disease control, while offering the possibility of disease elimination or even pathogen eradication (Francis, 1999).
The present study was designed to systematically investigate the immunogenicity of combined PPR and SP vaccine under laboratory conditions for possible use of combined vaccine for cost efficient vaccine based diseases control strategy.

Material and method:-
Group-5: was vaccinated with the attenuated SPV vaccine (control positive) Group-6: was vaccinated with the prepared combined lyophilized PPRV-SPV vaccine

Evaluation of the prepared combined vaccine:
The prepared vaccine was subjected to quality control testing including: Sterility tests: These tests include testing the freedom from bacterial; fungal and mycoplasma contaminants according to OIE (2014) Safety test: The safety of prepared combined vaccine was tested through inoculation of 5 sheep with a dose containing 10 5 TCID 50 of each virus (100X field dose of the prepared vaccine) according to OIE (2012).
Virus titration: Both of SPV and PPR viruses was titrated before mixing and lyophelization in Vero cells according to Rao and Malik (1982) and the virus titer was calculated according to Reed and Menuch (1938) as log 10 TCID 50 /ml. SPV was titrated post lyophelization in sheep according to OIE (2010) and expressed as SID 50 .
Potency tests: four groups of sheep , one of them was used as control negative (G-3) the other groups (G4,G5andG6) were vaccinated by inoculated subcutaneously in the ventral aspect of the tail with the field dose of PPR vaccine, SP vaccine and the prepared SP-PPR combined vaccine respectively. The animals were clinically observed daily to detect post-vaccinal reaction, and their cellular and humeral immune responses were evaluated.

For PPR:
The potency of combined vaccine to PPR virus was evaluated by comparing the humeral immune responseof the vaccinated sheep with that of single attenuated PPR vaccine For SP: Challenge test was applied according to OIE (2010); 3 weeks post vaccination on 2 sheep from each of group 3, 5 and 6 with 0.5 ml of the virulent SPV through the intradermal route. The challenged animals were kept in separate isolator for observation for 14 days and any clinical signs were recorded Samples: -Heparinized blood samples were collected for assay of the cellular immunity from vaccinated and control animals before and after vaccination at different intervals (1, 3, 5, 7, 10, 14, 21 and 28 days), .

Evaluation of immune response to the prepared vaccine:
Cellular and humeral immune responses of vaccinated sheep were evaluated for six months by different serological tests.

Evaluation of cellular immune response:
The cellular immunity was evaluated by application of Lymphocyte blastogenesis assay. It was carried out according to Rai et al (1985) and Alvero et al. (2006) using XTT cell viability assay kit (MD biosciences)

Evaluation of humeral immune response: Serum neutralization test (SNT):
It was carried out using the microtitre technique according to Rossiter et al. (1985) where PPR antibody titer was calculated as the reciprocal of the final serum dilution which neutralized and inhibited the CPE of 100 TCID 50 of PPR virus according to Singh et al (1967); while SPV antibody titer was expressed as neutralizing index according to Martin et al. (1975). Indirect ELISA: It was performed to evaluate the humoral immune response according to the method described by Anderson et al (1982) and the results were expressed by serum protective (S/P) ratio.

Results:-
The prepared combined PPR and SP vaccine was subjected to sterility testing and found to be free from foreign contaminants. The prepared combined vaccinewhen tested for safety was found to be safe as there was only moderate nodule at the site of inoculation 4-7 days post inoculation which disappeared gradually and there was no abnormal clinical signs related to the 2 viruses. The titer of PPRV was 10 5.7 TCID50/ml while the titer of SPV was 10 5.8 TCID50/ml and the titer of SPV in combined vaccine after lyophelization when evaluated in sheep as 10 6 SID50/ml. but the titer of PPR in combined vaccine after lyophelization in tissue culture was 10 5.2 TCID50/ml. The potency of the combined vaccine to PPR virus estimated by humeral immune response comparing with the single attenuated PPR vaccine as shown in table (III). The data in that table showed that the combined vaccine was potent.
The potency of the combined vaccine to SP was estimated by challenge test against virulent SPV, where the vaccinated sheep showed only hypersensitivity reaction at the site of inoculation which disappear within 4 days while the non-vaccinated sheep showed local and generalized pox reaction.
Assessment of cellular immune response to vaccinated sheep had been done post vaccination directly and expressed in (table I and II). The data of ELISA test showed that the humeral immune response was detectable by the 1st week post vaccination as shown in table (V and VI).TheS/P ratio of ELISA reading considered protectivewhen reading of sample ≥1;So that the sheep vaccinated with combined vaccine (G6) become protected against PPR on the 2 nd week and S/P ratio reached itspeak on the 4 th week as 1.18 and 1.4 respectively (table V) while the control positive sheep vaccinated with PPRvaccine (G3) become protective in the 3 rd week and S/P ratio reached its peak on 4 th week was 1.28 and 1.33 respectively (table V). The sheep vaccinated with combined vaccine (G6) became protected against SP on the 2 nd week and reached itsspeak on the 3 rd week where S/P ratio was 1.24 and 1.6 respectively (table VI) while the control positive sheep vaccinated with SP vaccine (G5) become protective on 1 st weekand the S/P ratio reached itspeak on 3 rd week as 1.07 and 1.71 respectively (table VI).

Discussion:-
The objective of the present study was to develop a combined vaccine against sheep pox and PPR which is able to protect small ruminants against both PPR and sheep pox viruses. Many successful trials of vaccination of animals with more than one vaccine at the same time were reported between viral vaccines were conducted as Rift Valley Fever (RVF) and sheep pox virus(SPV) vaccines (Taha et al, 1990); PPR and RVF (Mouaz et al, 1998); and recently PPR and goat pox vaccines (Abd El- Razek et al, 2006). All the fore mentioned statements for simultaneous and compound vaccination programs as a way to save costs, time and efforts. In this study we prepared PPR-SPV combined vaccine. It is known that sheep pox immunity depends mainly on the cell-mediated immune response in comparison to the humeral immune response (Kalra andSharma, 1984 andCarn, 1993). The results of cell mediated immunity showed gradual increasing in cellular immuno response to sheep pox virus where it reached its peak on the 10 th day in vaccinated sheep with combined single SP vaccine (table II) The test results of the combination of these two disease vaccines indicated that the prepared vaccine produce good immunogenicity and protection and can be used safely.   1WPV  2WPV  3WPV  4WPV  8WPV  12WPV  16WPV  20WPV  24WPV  G3  0  0  0  0  0  0  0  0  0  G4  2  4  16  32  32  32  32  32  32  G6  2  8  16  64  64  64  64  64 64 PPR antibody titer = the reciprocal of the final serum dilution which neutralized and inhibited the CPE of 100TCID 50 of PPR virus NB: the antibody titer ≥8 is considered protective. WPV= week post vaccination G3= control negative group G4= control positive group given PPR vaccine (Field dose) G6= tested group given combined vaccine (Field dose)