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Latin American Journal of Biotechnology and Life Sciences
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2021.06.03.12
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INVESTIGATION / RESEARCH
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Comparative study between lumpy skin disease virus and sheep pox virus vaccines against recent field isolate of lumpy skin disease virus
 

Nermeen G Shafik1, Heba A Khafagy1, Amal AM1, Ayatollah I Bassiuony2, Farid Fouad Zaki1, Christine A Mikhael2 and Mohamed Samy Abousenna1
Available from: http://dx.doi.org/10.21931/RB/2021.01.03.12
ABSTRACT

Lumpy Skin Disease[AGO1]  (LSD) is a disease of cattle and water buffaloes, caused by the Lumpy Skin Disease Virus (LSDV); there is an antigenic relationship between LSDV, Sheeppox virus (SPPV), and Goat pox virus GTPV[AGO2] ; accordingly, it can be used homologous or heterologous Capripoxvirus strains for vaccination of cattle against LSD. This study compares the efficacy of live attenuated Neethling LSDV vaccine and lives attenuated Romanian SSPV Vaccine against LSDV field isolate using three different batches for each vaccine type. Experimental calf groups were vaccinated with vaccines batches, and after 21 days, serum samples were collected to evaluate humoral immune response by using SNT and commercial ELISA technique, then the vaccinated calves were challenged by virulent LSDV field isolate. The results of SNT for vaccinated calves by LSDV vaccines indicated mean neutralizing antibody titer 1.2, 1.6, and1.5 log10 for the batches 1, 2 and 3 respectively, while vaccinated calves by SPPV vaccines indicated 1.05, 1.05 and 1.5 log10 for the batches 1,2 and 3 respectively; the ELISA mean sample to positive (S/P[AGO3] ) percentage for the vaccine batches 1, 2 and 3 of LSDV were 40, 45 and 42% respectively and for SPPV vaccine batches 1,2 and 3 were 35, 37 and 40 % respectively, the challenge test[AGO4]  indicated mean titer for the groups of calves vaccinated with LSDV vaccine were 4.2, 4.5 and 3.8 log10 and for groups vaccinated with SPPV vaccine were 2.6, 2 and 2.65 log10  respectively, it was concluded that potential using of Neethling LSDV vaccine against LSD is superior for combating and prevention the lumpy skin disease[AGO5] .    
Keywords: Lumpy Skin Disease Virus, Sheep Pox Virus, Challenge test, Serological test             
INTRODUCTION

The genus Capripoxvirus (CaPV) within the subfamily Chordopoxvirinae, family Poxiviridae contains three closely related viruses, namely Sheep pox and lumpy skin disease (LSD), which are diseases of sheep and cattle respectively, in addition to Goat pox virus  which causes goat pox disease. The Virus strains affecting sheep and cattle are host-specific, reflecting in the different geographic distribution of lumpy skin disease 1. All strains of capripoxvirus are antigenically closely related and cross-react serologically 2, because of the antigenic homology among all strains, the probability of using a single vaccine strain like sheep pox virus to protect cattle and sheep 3. Lumpy skin disease clinical signs include persistent fever, widespread skin nodules (lumps), and enlarged peripheral lymph nodes complications, including severe emaciation and death; LSD also has pulmonary form, especially in young calves with a high mortality rate[AGO8] 4.  The disease has high economic impacts as it affects milk production and the leather industry 5.
The disease's first appear ance was in Africa in 1929, reported from Zambia given the name 'Ngamiland Cattle Disease'. The disease continued to spread and resulted in a panzootic; the first appearance of the disease in Egypt was during two outbreaks in Suez and Ismalia governorates in 1989. In early 2006, a severe LSD outbreak struck foreign (imported from Ethiopia) and local cattle in different Egyptian governorates, causing enormous economic losses; the disease also reappeared in 2012 and 2013 6.
Controlling lumpy skin disease depends mainly on vaccination and vector control. Capripoxviruses are cross-reactive within the genus. Consequently, it is possible to protect cattle against LSD using strains of capripoxvirus derived from sheep or goats in the vaccine 7. Live attenuated strains of capripoxvirus vaccines have been explicitly used for the control of LSD 8. According to these, there are three types of vaccine used against lumpy skin disease, attenuated LSDV (Lumpy Skin Disease) vaccines which provide good protection in cattle, attenuated SPPV (Sheep pox virus) vaccines which have been used in cattle against LSDV in those regions where LSD and SPP are both present as it believed in providing partial protection against lumpy skin disease, and Attenuated Gorgan GTPV vaccine containing GTPV Gorgan strain, this vaccine has been used in those countries where GTP and LSD overlap 9, 10. However, it is recommended to carry out evaluation trials using the most susceptible breeds before introducing a vaccine strain for vaccination into the field.
In Egypt, the control of LSD was applied by vaccination of cattle with SPPVusing a Romanian strain for a long time which proved to partially tackle the LSD outbreaks with partial cross-protection with some notices on its duration of immunity as a recommendation for revaccination after 8 months 11, and recently live attenuated LSDV vaccines containing Neethling strain were introduced to be used in Egypt.
The humoral immune response of vaccinated cattle against LSD either by SPPV and LSD vaccines can be evaluated using virus neutralization test (VNT) and ELISA 1, 4, 12; in this study we compare the efficacy of live attenuated lumpy skin disease vaccine and live attenuated sheep pox vaccine against lumpy skin disease virus field isolate, using serological and challenge tests.

MATERIAL AND METHODS

Vaccines
a-      Live attenuated Lumpy skin disease virus vaccine (Neethling strain).
Three batches (one imported and two local batches[AGO13] ).
b-      Live attenuated Sheep pox virus vaccine (Romanian strain).
Three local batches[AGO14] .
These vaccine batches were delivered to Central Laboratory for Evaluation of Veterinary Biologics (CLEVB), Abbassia-Cairo, and evaluated last year for sterility, safety, and potency.

Virus
Lumpy skin disease virus (LSDV):
-          Animal Health Research Institute supplied virulent circulating field strain; the virus was isolated in 2018 13. It was used for the challenge test for vaccinated calves.
-          Tissue culture adapted strain; the virus was adapted on Madin-Darby bovine kidney (MDBK) cells with a titer of 105.5 as shown in Figures (1 & 2); it was used for the serum neutralization test.
Cells
The Reference Strain Bank Department supplied Madin-Darby bovine kidney (MDBK) cells in the Central Laboratory for Evaluation of Veterinary Biologics (CLEVB). It was used for the serum neutralization test.

Animals and vaccination
Twenty four susceptible mixed breed calves 6-12 months old free from specific antibodies against LSD virus were supplied by[AGO15]  CLEVB; these calves were divided into eight groups; each group contains three calves, six groups of them were used for vaccination, and the other two groups kept as positive control and negative control groups. The six vaccinated groups were divided as follow, three groups of them were vaccinated with field dose of the three batches of live attenuated lumpy skin disease vaccine (one group for each vaccine batch), and the other three groups were vaccinated with field dose of the three batches of live attenuated sheep pox virus vaccine (one group for each vaccine batch). After 21 days post-vaccination, serum samples were collected from vaccinated and unvaccinated calves for serological tests (SNT and commercial ELISA).
Challenge of vaccinated calves
After 21 days from vaccination, the vaccinated group,s, and positive control group were challenged with lumpy skin disease virulent virus according to OIE  1; the animals were shaved at the flank region, then six log10 dilutions were prepared for inoculation, each dilution inoculated intradermally at four sites for each shaved part (0.1[AGO16]  ml per each inoculum). The challenged animals were kept[AGO17]  under observation for seven days after the challenge; after that, the titer of the challenge virus was calculated for vaccinated and control animals. The difference in titer between control and vaccinated animals was recorded for each vaccine batch. The negative[AGO18]  control was kept[AGO19]  without vaccination or challenge.
Serum Neutralization Test (SNT)
It measures the humoral immune response against lumpy skin disease Virus for sera of vaccinated calves; it was applied according to the method described in OIE 1, the neutralizing titer was calculated according to Reed and Muench 14.
ELISA IDVIT KIT
ID screen® Capripox Double Antigen Multi-species, REF CPVDA-5A, LOT E83. was used to detect specific antibodies against capripoxvirus. The test was carried out according to the manufacturer's instructions insert. The results of each sample were calculated as aggregate to positive control ratio S/P[AGO20]  percentage (S/P %) in (Formula 1). The samples less than 30% are considered negative, while samples higher than or equal to 30% are considered positive.

Formula 1. Calculation of S/P ratio
RESULTS
The mean titers of the live attenuated lumpy skin disease virus vaccine batches and the live attenuated sheep pox virus vaccine batches on tissue culture were recorded. The titer was expressed as log10 tissue culture infective dose50 (TCID50 )/dose, indicated the titers for the three live attenuated lumpy skin disease virus vaccine batches and the three live attenuated sheep pox virus vaccine batches, as shown in table No.1.


Table 1. The titer of virus vaccine batches on tissue culture.
The humoral immune response against lumpy skin disease virus for the sera collected from vaccinated calves after 21 days SNT and ELISA determined post-vaccination; the lumpy skin disease virus was adapted on MDBK cells for use in serum neutralization test, Figure 1 (A) showing normal MDBK cells and Figure 1 (B) showing the cytopathic effect of lumpy skin disease virus on MDBK cells, the results of SNT for serum samples of calves vaccinated by live attenuated lumpy skin disease virus vaccines and live attenuated sheep pox virus vaccine indicated mean neutralizing antibody titer indicated as shown in table No. (2), while the results of ELISA test were calculated as S/P percentage, the mean percentage for the sera of vaccine batches of live attenuated lumpy skin disease virus vaccines and live attenuated sheep pox virus vaccine batches recorded in table No.3.


Figure 1. (A and B): Adapted Lumpy skin disease effect on MDBK cell line

Table 2.  Evaluation of humoral immune response in the sera of vaccinated calves by using SNT.


Table 3. Evaluation of humoral immune response in the sera of vaccinated calves using ELISA Kit against Capripox.
Vaccine potency in cattle; the challenge of vaccinated groups and positive control group by virulent lumpy skin disease virus was done, and after seven days, the difference in titer between vaccinated and positive control group was calculated for each group (reading the lesion in inoculated sites), the mean titer for the groups of calves vaccinated with live attenuated lumpy skin disease vaccine and for groups vaccinated with local and imported live attenuated sheep pox vaccines as shown in table No. 4.


Table 4: Challenge of vaccinated calves by LSDV.

DISCUSSION

In Egypt and the Middle East, live attenuated sheep pox vaccine is used to protect against LSD; although some researches recorded the effectiveness of this vaccine against LSD 15, there are some other records that it may offer insufficient protection against LSD 16, 17, 18, 19, recently live attenuated LSD vaccine was introduced to be applied in Egypt, but we still need many kinds of research to investigate and compare the efficacy and adverse reactions between live attenuated LSD virus vaccine and other Capripox virus vaccines.
In this study, we compare the efficacy of live attenuated LSDV vaccine and live attenuated sheep pox virus vaccine against LSD in calves as the main host for the disease, using three batches for each vaccine type, showing the vaccines batches titer, evaluate humoral immune response of vaccinated calves (using SNT and ELISA) and challenge vaccinated calves by a virulent strain of LSDV.
The titers of live attenuated LSDV vaccine batches per dose were satisfactory where the recommended field dose for LSDV in the vaccine is log10 3.5 TCID50, and the minimum protective dose is log10 2.0 TCID50. The titers of live attenuated SPPV vaccine batches were also satisfactory, where the recommended dose of sheep pox virus in the vaccine for cattle is log10 3 TCID50 1.
The SNT was carried out to detect the neutralizing antibody titer against LSDV for sera of vaccinated calves after 21 days post-vaccination, the results for live attenuated LSDV vaccine batches indicated that only mean titer for the first batch (1) didn't reach neutralization index 1.5 while the other two batches (2 and 3) were ≥1.5 as recommended by OIE 1, and the results for the live attenuated SPPV vaccine batches indicated that only the batch (3) reached neutralization index ≥1.5 while the other two batches (1 and 2) didn't record the positive titer. These results reflect that the live attenuated LSDV vaccine achieved the best results in titer and number of batches reaching the positive titer, but we must consider if the antibody is low, it doesn't reveal that the animal isn't protected. Neutralization test is used for evaluation [AGO26] of humoral immune response for LSDV as recommended by OIE, as other similar studies applied it for evaluation of serum samples of cattle vaccinated by live attenuated SPPV vaccine against LSDV 15, also applied by Christine 20, who used it in evaluation calves vaccinated by Attenuated Sheep Pox and Inactivated Lumpy Skin Disease Vaccines against Lumpy Skin Disease and used in the evaluation of the cross-protection between Sheep pox and bovine Lumpy skin vaccines in vaccinated sheep 21.
ELISA kit against Capripox is also used in the evaluation of humoral immune response for the sera of vaccinated calves; the results for live attenuated LSDV vaccines batches indicated positive results[AGO27]  ≥30% (as mentioned in ELISA kit) for the three batches, also live attenuated SPPV vaccine indicated results ≥  30%  for the three batches. However, we noticed that the percentages for live attenuated LSDV vaccines batches are higher than percentages of live attenuated SPPV vaccine batches. Although the neutralization test is the standard test[AGO28]  for evaluating the humoral immune response for LSDV as recommended by OIE, studies recommend using commercial ELISA, commercial Capripox Double Antigen ELISA (ID.Vet) was used to evaluating serological tests for detecting antibodies against LSDV and find a strong correlation between VNT/MDBK and ELISA 22; also, the ELISA method and neutralization test were used for detection antibodies against LSDV23.
Challenge test is used for testing the potency of the vaccines. It was carried out in vaccinated calves (as the primary host for LSDV) by calculating the difference in titer of inoculum (virulent LSDV); the results for live attenuated LSDV vaccines batches indicated a protective titer for the three batches where the minimum protective result is >log10 2.5 as recommended by OIE 1, and for the live attenuated SPPV vaccine batches indicated only first. Third batches are protective with border titer limit, but the second batch didn't reach protective titer, and that indicated that live attenuated LSDV vaccine is more protective than live attenuated SPPV vaccine against LSDV. These results agreed with  Hamdi 21, who used virulent LSDV in the challenge of two groups of calves, one group vaccinated with Romania SPPV  vaccine and the other with Neethling LSDV vaccine and reported that Romania SPPV  vaccine provides partial protection against LSDV while Neethling LSDV vaccine provides complete protection against LSDV; also recombinant LSDV vaccine (LSD-Rift Valley F[AGO29] ever.mf vaccine) was used in two groups of calves, one group challenged by virulent LSDV and another group by virulent Rift Valley Fever V[AGO30] irus and recorded that tested vaccine is safe and protective for both diseases24. Neethling vaccine is reported significantly more effective than x10 RM65 Sheep Pox vaccine strain in preventing LSD morbidity25.
CONCLUSION

It was believed that the broad cross-protection within the Capripoxvirus genus allow[AGO31] s using of any Capripoxvirus isolate as a[AGO32] n effective vaccine against LSDV, otherwise and avoiding this confirmation bias, it is clear from the relevant results that the potential user off Neethling LSDV vaccine against LSDV is superior for combating and prevention the Lumpy skin disease.    

Conflict of interest
The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
Ethical approval
Institutional Animal Care and Use Committee at Central Laboratory for Evaluation of Veterinary Biologics acknowledge the research manuscript, and it has been reviewed under our research authority and is deemed compliance with bioethical standards in good faith.
Author Contributions
NGS designed experiments, AAM and MSA the experiments were performed HAK, AAM AIB, CAM, and MSA. Data analysis was accomplished by NGS, FFZ, HAK, AAM, and MSA. AAM,AIB and MSA wrote the manuscript.
Funding
This study received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors
Data Availability
All data generated or analyzed during this study are included in this published article
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Received: 1 May 2021
Accepted: 9 July 2021
Nermeen G Shafik1, Heba A Khafagy1, Amal AM1, Ayatollah I Bassiuony2, Farid Fouad Zaki1, Christine A Mikhael2 and Mohamed Samy Abousenna1
1 Central Laboratory for Evaluation of Veterinary Biologics (CLEVB), Agricultural Research Center, Cairo,  Egypt.
2 Veterinary Serum and Vaccine Research Institute, Pox department, Agricultural Research Center, Cairo,  Egypt.
Corresponding author: Mohamed samy Abousenna and  Aml AM
Mohamedsamy2020@hotmail.com; dr.amal1984@gmail.com
https://orcid.org/0000-0003-2202-9544
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