1. Introduction
A peste des petites ruminant (PPR) is a highly contagious, acute, re-emerging/developing disease in small ruminants. It is caused by a virus from the Paramyxoviridae family, Morbillivirus genus [
1]. PPR is endemic throughout Africa, the Middle East, and large regions of Asia [
2], and is currently reported in more than 70 countries in Africa, the Near and Middle East, as well as Central and East Asia, including Georgia in 2016 and Bulgaria in 2018 [
3,
4,
5]. Due to its impact in April 2015, the Food and Agriculture Organization of the United Nations (FAO) and the Office International des Epizooties (OIE) launched a campaign to eradicate PPR globally by 2030 [
2].
In the Republic of Kazakhstan (RK), the outbreak of PPR was officially recorded among small agricultural ruminants in the Turkestan region in 2003 [
6]. The disease was reported in the country in 2005–2006 and 2014 [
7,
8]. Through the present introduction of PPR, it is highly possible in southeast Kazakhstan through the land boundary with China. PPR has become widespread in China since 2013, which is when the disease was allegedly introduced from the Republic of Tajikistan, according to the European Food Safety Authority (EFSA) and as shown by the results from the phylogenetic analysis [
9].
Sheep pox (SPP) is a viral acute contagious disease caused by the virus from the Capripoxvirus genus, which belongs to the vast family of Poxviridae [
10]. It is endemic in Central and Northern Africa, in the Near and Far East, as well as on the Indian subcontinent [
11,
12]. Over the past three decades, the epizootic situation in sheep and goat pox has been deteriorating in countries in Africa and Eurasia. During this period, the diseases were reported in over 70 countries across the globe [
13]. The outbreak of SPP among small agricultural ruminants in 2019 in the western part of the Republic of Kazakhstan (RK) (Mangistau region) should be noted. Thanks to emergency measures undertaken by the veterinary services, the infection was localized [
14]. Though the available monovalent PPR and SPP vaccines [
15,
16] are effective, the current PPR and SPP epizootic situation in RK requires improvements to be made to the existing prophylactic preparations against these infections. Thus, a new combined vaccine was developed in Kazakhstan to prevent both PPR and SPP simultaneously. The combined vaccines against PPR and SPP were developed earlier and were experimentally used with positive results in India [
17], Cameroon [
18], Morocco [
19,
20], Ethiopia [
21], Egypt [
22], and Russia [
23]. Immunogenicity and the effectiveness of these vaccines are comparable to the same monovalent vaccines against PPR and SPP. Despite the formation of effective immunity, the available publications do not provide data on the assessment of the duration of post-vaccination immunity in animals vaccinated with the combined vaccines developed against these diseases. However, the vaccine strains used for the preparation of existing combined vaccines against PPR and SPP are sufficiently immunogenic and, being part of monovalent vaccines, create immunity in the once-immunized animals for at least 1 year.
Keeping in mind the long-term efficacy of the Nigeria 75/1 strain of the PPR virus (PPRV) and the NISKhI strain of the SPP virus (SPPV) in monovalent vaccines, a combined vaccine against PPR and SPP based on these strains was developed for the first time in the Republic of Kazakhstan.
As is known, the Nigeria 75/1 strain of the PPRV causes persistent immunity in once-immunized animals for up to 3 years, while the NISKhI strain of the SPPV can form protective immunity in the once-vaccinated animals for 1 year. The effectiveness of these strains, which are the main immunogens in the composition of the combined PPR and SPP vaccine developed by us, was a prerequisite for studying the duration of immunity in animals vaccinated with the new combined PPR and SPP vaccine for the Republic of Kazakhstan.
Objective of this work was to assess the duration of immunity in sheep that were vaccinated with a combined vaccine against PPR and SPP developed in the Republic of Kazakhstan.
2. Materials and Methods
2.1. Vaccine Virus
The combined vaccine against PPR and SPP was constructed, as previously described [
24]. For the production of a combined vaccine against PPR and SPP, we used attenuated vaccine strains Nigeria 75/1 PPRV (GenBank: KY628761.1) and NISKhI SPPV (GenBank: AY077834.1), which were grown in the Vero cell line. This combination of strains was tested for the first time. During the production of the combined vaccine, suspensions of the Nigeria 75/1 PPRV and NISKhI SPPV vaccine strains with the same titer (at least 10
6.5 lg TCID
50/mL) were combined in an equal ratio of 1:1. A combination of peptone-sucrose stabilizer (in the final concentration of 3% peptone and 2% sucrose) was added to the prepared combined vaccine fluid of PPRV and SPPV in a ratio of 1:1. Then, the vaccine liquid was divided into aliquots in 2.0 mL ampoules and lyophilized for storage.
The combined vaccine was manufactured at the Research Institute for the Biological Safety Problems (RIBSP), Kazakhstan. The attenuated vaccinal strains Nigeria 75/1 (GenBank: KY628761.1) of PPRV and NISKhI (GenBank: AY077834.1) of SPPV were used in its production. The vaccine was tested for sterility, purity, and identity, according to OIE Terrestrial Code, 2018 (Chapter 1.1.9) (World Organization for Animal Health (OIE), Paris, France, 2018) [
25]. The titer of the vaccine viruses of PPR and SPP in the 1.0 mL dose of the lyophilized combined vaccine was 10
3.0 TCID
50/mL.
2.2. Control Viruses
The virulent strain A (GenBank: AY077833.1) of SPPV, in the form of an organ–tissue lyophilized material, was obtained from the Microbial Collection Laboratory, RIBSP, Kazakhstan [
26].
The PPR field virus strain Kentau-7 was obtained from the Microbial Collection Laboratory, RIBSP, Kazakhstan. This strain was isolated from the pathology material from a goat in the PPR outbreak in the territory of Kazakhstan in 2003 by performing sequential passaging (7 passages) of the supernatant of the affected organ of the fallen goat on a lamb kidney cell culture (LK) [
27].
2.3. Animals
Fine-fleeced sheep of the Kazakh breed, aged 6–12 months, from the farms free of acute infectious diseases and seronegative to PPR and SPP were used to assess immunity duration. A total of 49 sheep were used for the research.
Prior to the experiments, the animals were labeled and held in quarantine for 1 month, with regular thermometry, clinical observation, and analysis of blood sera of specific antibodies to PPR and SPP viruses in SNT, according to OIE Terrestrial Code of 2019 (Chapter 3.7.9) and 2018 (Chapter 3.7.12), respectively [
10,
28]. The animals free of specific antibodies to the PPR and SPP viruses and not vaccinated against these diseases were used in the experiment.
The experiments were carried out in specially equipped ABSL-2 animal facilities. On arrival in the RIBSP animal facilities, sheep were ear-tagged and allowed to acclimatize in the facilities for two weeks prior to the onset of the experiment. Each group was housed in a separate room with no direct contact with each other. Experimental animals had free access to water and feed throughout the experiment.
This study was conducted in accordance with national and international laws and guidelines for the handling of animals. The protocol was approved by the Ethics Committee for Animal Experimentation at the RIBSP, Science Committee of the Ministry of Education and Science (SC MES), RK (Permission number: 3101/14).
2.4. Vaccination
The sheep were randomly divided into three groups as follows: I group (n = 9), II group (n = 20), and III group (n = 20). The experimental sheep were divided into groups using an online random number generator (Randomizer), while all the personnel involved in the research experiment did not have access to data about which group this or that animal belongs to.
Group I sheep (n = 9) were used to evaluate the safety of the developed combination vaccine by subcutaneous vaccination of 6 sheep with a dose containing 105.0 TCID50/mL of each virus in a volume of 2.0 mL. The other 3 sheep were immunized with 2.0 mL of phosphate saline buffer (PBS).
All immunized animals were subjected to a clinical study through regular observations and daily rectal temperature records in order to detect post-vaccination reactions. The formation of inflammatory edemas in the form of infiltrates at the injection site sized up to 1.0 cm in diameter and being self-eliminated within 3–6 days was considered to be a positive post-vaccination reaction.
Sheep from the group II (n = 20) were used to evaluate the duration of immunity in animals post-vaccination with a combined vaccine against PPR and SPP. Sheep of this group inoculated subcutaneously with a single dose (2.0 mL) of vaccine. The titer of the vaccine viruses of PPR and SPP in the 1.0 mL dose of the lyophilized combined vaccine was 103.0 TCID50/mL.
Sheep from group III (n = 20) served as controls (unvaccinated animals) for group II.
All the immunized animals were clinically monitored by regular observations and by recording daily rectal temperatures. Nasal, ocular, oral, and rectal swabs, as well as blood samples were taken from sheep within 14 days post-vaccination (dpv) and analyzed using RT-qPCR to monitor viral load.
2.5. Serum Sample Collection
Blood samples were collected on 0, 7, 14, 21, 30, 90, 180, 270, and 360 dpv for serum antibody estimation. Serum was separated from all the blood samples and heat inactivated at 56 °C for 30 min and stored at −20 °C until further used.
2.6. SNT for PPRV and SPPV
The collected blood sera of immunized sheep were tested for PPR and SPP virus-neutralizing antibodies (VNA) in SNT in Vero cell line, according to the OIE Terrestrial Code, of 2019 (Chapter 3.7.9) and 2018 (Chapter 3.7.12), respectively [
10,
28]. The viral-neutralizing activity of sera was determined by the neutralization index, which was calculated taking into account the difference in logarithmic titers of the control and test sera in accordance with Reed and Muench method [
29]. A neutralizing titer ˃10 is considered positive for PPRV, while an index ≥1.5 is positive for SPPV. The tests were performed at least twice and the average of the two tests was used for subsequent analysis.
2.7. c-ELISA Test for PPRV
At the above-mentioned time periods, the obtained blood sera were additionally analyzed in a competitive ELISA (c-ELISA) (ID Screen®PPR Competition (PPRC-4P), ID.vet, Montpellier, France) for the presence/absence of VNA to the PPRV, according to the manufacturer’s instructions. Then, OD at 450 nm was read. The unit of measurement was an S/N percentage. S/N percentage of ≤50% was considered positive, while 50–60% was considered doubtful. >60% S/N percentage was considered negative for the presence of antibodies against PPRV. All the results were recorded as Mean ± S.E.M.
2.8. RNA Extraction and Detection of PPRV Nucleic Acid in Blood and Swabs Samples
Virus genome RNA was extracted from the collected specimen with the help of the commercial kit for RNA extraction (ID Gene™ Mag Fast Extraction Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions.
The following primers were used to amplify the N gene of the PPRV: NP3 5′-GTC-TCG-GAA-ATC-GCC-TCA-CAG-ACT-3′ and NP4 5′-CCT-CCT-CCT-GGT-CCT-CCA-GAA-TCT-3′ [
30]. The viral genome was assessed using the real-time PCR commercial kit (RT-qPCR) (ID Geneтм Peste des Petits Ruminants Duplex, IDvet genetics, Grabels, France), according to the manufacturer’s instructions in the thermocycling system Rotor-Gene Q (Qiagen, Germany) under the following program: (1) reverse transcription for 10 min at 45 °C, (2) polymerase activation for 10 min at 95 °C, and (3) denaturation/DNA elongation for 15 s at 95 °C/60 s at 60 °C.
2.9. DNA Extraction and Detection of SPPV Nucleic Acid in Blood and Swabs Samples
DNA from oral ad nasal swabs was extracted with the help of the ID Gene™ Mag Fast Extraction Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions.
The following primers were used to identify SPPV: F-primers INS1.1 5-AGA AAC GAG GTC TCG AAG CA-3 and R-primers INS1.2 5-GGA GGT TGC TGG AAA TGT GT-3, chosen from the genes of the membrane protein CD47 of the SPPV [
31]. The viral genome was assessed using real-time PCR using the kit RT-qPCR (ID Gene™ Capripox Virus Triplex qPCR) (IDvet genetics, Grabels, France), according to the manufacturer’s instructions in the thermocycling system Rotor-Gene Q (Qiagen, Germany) under the following program: (1) polymerase activation for 10 min at 95 °C and (2) denaturation/DNA elongation for 15 s at 95 °C/60 s at 60 °C.
2.10. Challenge Studies
The challenges were performed on the 30th, 90th, 180th, 270th, and 360th dpv of sheep.
From 20 vaccinated sheep of group II, 10 sheep were randomly selected to challenge the field strain Kentau-7 of the PPRV and identified as group IIa. The remaining 10 sheep were used to challenge the virulent strain A of the SPPV and were identified as group IIb (
Table 1).
Before the tests, the sheep of these subgroups were transported to separate, specially equipped rooms (ABSL 2), and their rectal temperature was recorded.
Of the 20 sheep of group III, 10 sheep were used to challenge the virulent strain A of the SPPV and were identified as group IIIa, and the remaining 10 sheep were used to challenge the field strain Kentau-7 of the PPRV and were identified as group IIIb (
Table 1).
Before the tests, which were carried out on the 30th, 90th, 180th, 270th, and 360th days, 2 sheep from each control subgroup (IIIa and IIIb) were transported to separate, specially equipped rooms (ABSL 2), and their rectal temperature was recorded.
Vaccinated (IIa) and control (IIIa) animals selected for the challenges for the PPRV were infected with the field strain Kentau-7 of the PPRV subcutaneously in the subscapular region at a dose 1 × 10
5.0 TCID
50/mL (
Table 1).
Vaccinated (IIb) and control (IIIb) animals selected for challenge study for the SPPV were infected intradermal in the area under the tail fold with a virulent strain A of the SPPV at a dose of 1 × 10
4.5 TCID
50/mL (
Table 1).
Control of the experimental animals (vaccinated and intact sheep) were carried out within 14 days post-challenge (dpс), with daily measurement and registration of rectal temperature and for the manifestations of clinical signs of PPRV and SPPV, which were evaluated using the point systems, described earlier [
32,
33]. Nasal, ocular, oral and rectal swabs, as well as blood samples were taken from sheep within 14 dpс and analyzed using RT-qPCR to monitor viral load.
After completing the control trials, the recovering control sheep were treated with antibiotics to avoid secondary infection and sheep that reached clearly defined humane endpoints of a moderate severity were euthanized humanely.
2.11. Statistical Methods
Statistical analysis was carried out with the use of GraphPad Prism version 8.0.1. The results of the serological test, rectal temperatures post sheep vaccination with both vaccinal strains, as well as the difference between groups after the challenge with control viruses were analyzed with the help of bilateral ANOVA tests. A value p ≤ 0.05 was considered statistically significant. The difference in efficacy between the groups was compared using the one-lateral Fisher’s exact test for two proportions at the alpha level of ˂0.05.
4. Discussion
Today, sheep breeding is considered to be one of the most important branches of the agro-industrial complex of Kazakhstan, which in some cases is the only source of the most important types of products: wool, lamb meat, fur, and fur sheepskins. The number of sheep in Kazakhstan is increasing every year, and according to the Bureau of National Statistics of the Agency for Strategic Planning and Reforms of the Republic of Kazakhstan, at the beginning of this year, the number of sheep was 20.05 million heads. However, as has been established, a large concentration of animals in limited areas coming from different epizootic regions, a wide exchange of animals within the country and the import of their highly productive breeding breeds from abroad, difficulties in organizing continuous production, full-fledged feeding and ensuring an optimal microclimate create favorable conditions for the occurrence of mass infectious diseases. These dangerous infectious diseases of animals include PPR and SPP.
PPR and SPP are highly infectious diseases of small ruminants, which, in the event of an outbreak, cause significant economic damage to agriculture. An outbreak of these infections has been registered in countries bordering Kazakhstan, including Russia and China. The epizootic situation that has developed to date for these infections indicates the need to develop a highly effective means of specific prevention against both PPR and SPP. In this regard, we have developed a combined vaccine for the simultaneous prevention of PPR and SPP.
The objective of this work was to assess the duration of immunity in sheep that were vaccinated with a combined vaccine against PPR and SPP developed in the Republic of Kazakhstan.
Previously, the mass vaccination of animals using this type of vaccine against PPR and SPP was reported [
34], where the authors revealed a difference in sensitivity between sheep breeds to the PPRV and SPPV, which affected the level of seropositivity in animals to these viruses.
In our study, when testing SN, the seropositivity of the fine-fleeced Kazakh breed for the SPPV virus reached 95% at 7 dpv and 100% at 14 dpv, while at 14 dpv, the seropositivity to PPRV reached 80% and 100% protection of animals from PPRV, and this was noted at 21 dpv (
Figure 2). The serological response to PPRV obtained using c-ELISA reached 95% at 14 dpv and 100% at 21 dpv (
Figure 3).
Protective titers of NA to SPPV (1.8 log2) developed in vaccinated sheep, one week earlier, as compared to the titers of NA to PPRV (4.1 log2), which were formed on the 14th day of dpv (
Figure 2). Similar results were obtained in the study by Fakri et al. [
21,
22] where the authors observed an early humoral response (after 7 days) to SPPV post-vaccination of animals with a combined vaccine. However, only 90% of the vaccinated animals were protected from SPPV 21 dvp with this vaccine.
Immunity against PPRV formed in animals vaccinated with the combined vaccine persisted for 12 months without a significant decrease in antibody titers (6.8 log2) (
p ˃ 0.05), while SPPV titers persisted for up to 6 months (5.6 log2) (
p ˃ 0.05), and then gradually decreased until the end of the experiment (1.9 log2) (
Figure 2). However, the results of the study on the developed vaccine on a control model demonstrated the ability of this vaccine to induce 100% clinical protection against the field PPRV and against the virulent SPPV until the end of the experiment (12 months) (
Figure 2 and
Figure 3). During control tests, two vaccinated animals, that passed the control test at 30 dpv and 270 dpv, showed a slight increase in body temperature post-challenge with the field strain Kentau-7 of the PPRV, whereas the unvaccinated animals developed typical clinical signs of the disease (
Figure 5). The vaccinated animals had an average clinical score of 0.2 points post-challenge with the field strain Kentau-7 of the PPRV, whereas the unvaccinated animals had an average score of 19.3 points, indicating the high protective activity of the test vaccine against the field strain of PPRV.
As is known in many studies, when evaluating the protection provided by PPR vaccines, vaccinated and control animals are infected intranasally (i/n), as it is believed that PPRV, like other morbilliviruses, initially infects the epithelial cells of the respiratory tract. However, Pope, R.A. et. al. (2013) suggest that the virus is absorbed by the immune cells of the mucous membrane of the respiratory tract, which then transfer the virus to the lymphoid tissues, where the primary replication of the virus occurs and from where the virus enters the bloodstream [
35].
Note that the i/n of infection of animals has additional difficulties associated with the fact that animals shake their heads/sneeze after the introduction of a virulent virus, which can lead to an insufficient dosage of the virus, or to an overdose of the virus, as when sneezing infected animals release the virus into the environment and animals (especially with weaker immunity) can receive an additional dose of the virus. In this regard, we choose the subcutaneous route of infection for PPR, instead of the intranasal.
In this regard, in order to challenge the strain Kentau-7 PPRV, we chose the subcutaneous route of infection, instead of the intranasal. In our studies, pronounced clinical signs of PPR were observed in animals subcutaneously infected with the field strain Kentau-7 PPRV (
Figure 6 and
Figure 7).
Furthermore, three vaccinated sheep post-challenge with the virulent strain A of the SPPV had local reactions, which disappeared independently within 3–4 days, whereas the unvaccinated animals developed typical clinical signs of the SPP (
Figure 9 and
Figure 10). For the vaccinated and control groups of animals post-challenge with the virulent strain A of the SPPV, the average clinical score for the entire study period was calculated, the value of which was 0.2 points for the vaccinated group, and 15.2 points for the control group.
Blood and swab samples were collected from the animals following vaccination and challenge, and RT-qPCR was carried out to detect viral nucleic acids in blood and various body excretions. None of the vaccinated animals showed the presence of PPRV and SPPV in blood samples and nasal, ocular, oral, and rectal swabs collected at regular intervals after vaccination and challenge. However, after challenge, the PPRV and SPPV antigen was detected in the blood and in all types of swabs taken from intact animals (
Figure 4). Similar results were obtained by S. S. Chaudhary et al. (2009), as well as by M. Hosamni et al. (2006), when using a divalent vaccine against PPRV and SPPV/ GPV [
17,
36].
Thus, we conclude that a single immunization of sheep with the recommended dose (103.0 TCID50/mL) of the developed combined vaccine against the PPR and SPP induces immunity against the SPPV on day 7 and against the PPRV on day 14 post-vaccination. The combined vaccine provides reliable protection against two infections simultaneously for 12 months (observation period). At the same time, in sheep vaccinated with this vaccine, immunity against PPRV persists for more than 12 months. This is due to the choice of the vaccine strain Nigeria 75/1, which, as the main component of a live monovalent vaccine, causes strong immunity in vaccinated animals, lasting at least 3 years. Note that the Niskhi vaccine strain is able to cause earlier humoral immunity in vaccinated animals compared to other vaccine strains of the SPPV, which are used to make a combined vaccine and provides 100% protection against SPP for up to 12 months.
The obtained results once again proved the effectiveness and safety of the combined vaccines against PPR and SPP while being used, thus disproving the incompatibility of the vaccine strains of PPRV and SPPV. As has been established, Rajak et al. (2005), in their earlier studies, have shown that the vaccinal PPRV does not influence the immunogenicity of other unrelated antigens, which is confirmed by our results and the results obtained by Fakri et al. (2020c) that demonstrated prolonged compatibility of PPRV and SPPV [
34,
37].
Future studies should examine the effectiveness of the combined vaccine in the field for large vaccination campaigns.