Duration of Protective Immunity in Sheep Vaccinated with a Combined Vaccine Against Peste Des Petits Ruminants and Sheep Pox

Home , Ovine rinderpest, Sheeppox

Article Duration of Protective Immunity in Sheep Vaccinated with a Combined Vaccine against Peste des Petits Ruminants and Sheep Pox

Zhanat Amanova 1,*, Kuandyk Zhugunissov 1 , Kainar Barakbayev 1, Zhanat Kondybaeva 1, Zhanna Sametova 1, Yeraly Shayakhmetov 1, Dastan Kaissenov 1, Kuanysh Dzhekebekov 1, Asankadyr Zhunushov 2, Yergaly Abduraimov 1, Kunsulu Zakarya 1 and Yerbol Bulatov 1

1 Research Institute Biological Safety Problems, Gvardeiskiy 080409, Kazakhstan; [email protected] (K.Z.); [email protected] (K.B.); [email protected] (Z.K.); [email protected] (Z.S.); [email protected] (Y.S.); [email protected] (D.K.); [email protected] (K.D.); [email protected] (Y.A.); [email protected] (K.Z.); [email protected] (Y.B.) 2 Institute of Biotechnology, National Academy of Sciences of the Kyrgyz Republic, Bishkek 720071, Kyrgyzstan; [email protected] * Correspondence: [email protected]; Tel.: +7-(701)-415-63-79

Abstract: In this study, the ability of the combined vaccine against peste des petits ruminants (PPR) (Nigeria strain 75/1) and sheep pox (SPP) (NISKhI strain) to form a protective immune response for 12 months in Kazakh breed fine-fleeced sheep aged 6–12 months was demonstrated. The duration Citation: Amanova, Z.; Zhugunissov, of the protective immunity of immunized sheep from PPR and from SPP was evaluated using K.; Barakbayev, K.; Kondybaeva, Z.; a serum neutralization test (SNT), followed by testing of the resistance of vaccinated sheep to Sametova, Z.; Shayakhmetov, Y.; infection with the field strain Kentau-7 of the PPRV and the virulent strain A of the SPPV. The PPR Kaissenov, D.; Dzhekebekov, K.; antibody response was additionally measured by c-ELISA. A single immunization of sheep with a Zhunushov, A.; Abduraimov, Y.; et al. Duration of Protective Immunity in combined vaccine in a volume of 2.0 mL, containing the PPR and SPP vaccine viruses in the titers of 3.0 Sheep Vaccinated with a Combined 10 TCID50/mL, provided reliable protection of animals from two infections simultaneously for Vaccine against Peste des Petits 12 months (observation period). At the same time, in sheep immunized with the combined vaccine, Ruminants and Sheep Pox. Vaccines antibodies of PPRV persisted for up to 12 months, with slight fluctuations. The combined vaccine 2021, 9, 912. https://doi.org/ induced 100% clinical protection against the field strain of PPRV and the virulent strain of SPPV in 10.3390/vaccines9080912 immunized sheep for up to 12 months, while unvaccinated animals became ill with the manifestation of clinical signs specific to PPRV and SPPV. Academic Editors: Valentin Pérez and Julio Benavides Keywords: combined vaccine; peste des petites ruminants; sheep pox; immunity

Received: 8 July 2021 Accepted: 10 August 2021 Published: 16 August 2021 1. Introduction

Publisher’s Note: MDPI stays neutral A peste des petites ruminant (PPR) is a highly contagious, acute, re-emerging/developing with regard to jurisdictional claims in disease in small ruminants. It is caused by a virus from the Paramyxoviridae family, published maps and institutional affil- Morbillivirus genus [1]. PPR is endemic throughout Africa, the Middle East, and large iations. 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 [3–5]. 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]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. In the Republic of Kazakhstan (RK), the outbreak of PPR was officially recorded This article is an open access article among small agricultural ruminants in the Turkestan region in 2003 [6]. The disease was distributed under the terms and reported in the country in 2005–2006 and 2014 [7,8]. Through the present introduction conditions of the Creative Commons of PPR, it is highly possible in southeast Kazakhstan through the land boundary with Attribution (CC BY) license (https:// China. PPR has become widespread in China since 2013, which is when the disease was creativecommons.org/licenses/by/ allegedly introduced from the Republic of Tajikistan, according to the European Food 4.0/). Safety Authority (EFSA) and as shown by the results from the phylogenetic analysis [9].

Vaccines 2021, 9, 912. https://doi.org/10.3390/vaccines9080912 https://www.mdpi.com/journal/vaccines Vaccines 2021, 9, 912 2 of 18

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 106.5 lg TCID50/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 (Gen- Bank: 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, Vaccines 2021, 9, 912 3 of 18

France, 2018) [25]. The titer of the vaccine viruses of PPR and SPP in the 1.0 mL dose of the 3.0 lyophilized combined vaccine was 10 TCID50/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, Kaza- khstan [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 5.0 vaccine by subcutaneous vaccination of 6 sheep with a dose containing 10 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 3.0 was 10 TCID50/mL. Sheep from group III (n = 20) served as controls (unvaccinated animals) for group II. Vaccines 2021, 9, 912 4 of 18

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 50-GTC-TCG- GAA-ATC-GCC-TCA-CAG-ACT-30 and NP4 50-CCT-CCT-CCT-GGT-CCT-CCA-GAA-TCT- 30 [30]. The viral genome was assessed using the real-time PCR commercial kit (RT-qPCR) (ID GeneтмPeste 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. Vaccines 2021, 9, 912 5 of 18

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 (Table1).

Table 1. Design of the challenges on animals. s/c: subcutaneously; i/d: intradermal.

Dose and Method Challenge Virus Group Number of Animals of Infection

Field strain Kentau-7 Gr- IIa 10 1 × 105.0 TCID /mL, s/c of the PPRV Gr- IIIa 10 50

Virulent strain Gr- IIb 10 1 × 104.5 TCID /mL, i/d A of the SPPV Gr- IIIb 10 50

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 (Table1). 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 5.0 region at a dose 1 × 10 TCID50/mL (Table1). 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 4.5 SPPV at a dose of 1 × 10 TCID50/mL (Table1). 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 dpсand 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.

3. Results 3.1. Safety of the Developed Vaccine The general post-vaccination condition of the vaccinated sheep within 3 weeks was within normal limits. Only two sheep had local reactions in the form of irregularly rounded swellings, 0.5 cm in diameter, pinkish in color, and protruding 3 mm above the surface Vaccines 2021, 9, x 6 of 18

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.

3. Results 3.1. Safety of the Developed Vaccine Vaccines 2021, 9, 912 The general post-vaccination condition of the vaccinated sheep within 3 weeks6 of 18was within normal limits. Only two sheep had local reactions in the form of irregularly rounded swellings, 0.5 cm in diameter, pinkish in color, and protruding 3 mm above the surfaceof the surrounding of the surrounding skin. The skin. inﬁltrates The gradually infiltrates disappeared gradually disappeared within 4 days within without 4 an days withoutincrease an in increase body temperature. in body temperature. The body temperature The body of temperature both vaccinated of both and controlvaccinated sheep and controlremained sheep within remained the normal within range the (38.5–39.8normal range◦C) (Figure (38.5–39.81) for °C) 14 ( days.Figure 1) for 14 days.

FigureFigure 1. 1.BodyBody temperature temperature of of vaccinated sheepsheep when when studying studying the the safety safety of a of combined a combined vaccine vaccine against against PPR and PPR SPP. and The SPP. Thedotted dotted line line in thein the graph graph shows shows the upper the upper limit oflimit the of normal the normal body temperature. body temperature. Rectal temperatures Rectal temperatures of 6 vaccinated of 6 vaccinated animals animalsand 3 and control 3 control animals animals are shown are shown in the graph.in the graph. (*) Local (*) reactions Local reactions that appeared that appeared post vaccination post vaccination in 2 vaccinated in 2 vaccinated sheep sheepexisted existed for 4 for days. 4 days. 3.2. SNT Results for PPRV 3.2. SNT Results for PPRV On the day of vaccination (day 0), all the animals were seronegative for PPRV antibodies.On the After day 7of days, vaccination NAs with (day an 0), average all the titer animals of 1.5 were log2 wasseronegative detected infor the PPRV serum antibodies.samples After of the 7 immunizeddays, NAs sheep, with an which average increased titer toof 4.11.5 log2 log2 on was the detected 14th dpv. in On the day serum 21, samplesthe NAs of titer the reachedimmunized 6.7 log2. sheep, The which peak increased of the average to 4.1 NAs log2 titer on the (7.2 14th log2) dpv. in the On blood day 21, theserum NAs samples titer reached of immunized 6.7 log2. sheep The waspeak recorded of the average on the 30th NAs dpv. titer The (7.2 indicated log2) in NAs the titer blood serumwas maintained samples of up immunized to 360 dpv, withsheep slight was fluctuationsrecorded on (p the> 0.05) 30th (Figure dpv. 2The). Antibodies indicated toNAs titerPPRV was were maintained absent in theup bloodto 360 sera dpv, of with the control slight sheepfluctuations (Figure 2().p ˃ 0.05) (Figure 2). Anti- bodies to PPRV were absent in the blood sera of the control sheep (Figure 2). 3.3. SNT Results for SPPV On the day of vaccination (day 0), all the animals were seronegative for SPPV antibodies. A week after vaccination, the sheep were found to have NAs with an average titer of 1.8 log2. From day 7 to day 21, there was a steady increase in the average NA titers in the blood sera of vaccinated sheep. Thus, on the 14th and 21st dpv, NAs titer in the blood serum of immunized sheep reached 3.0–5.2 log2, respectively. The highest NAs titer (6.0 log2) in the blood sera of immunized sheep was detected at 30 dpv. The developed antibodies were preserved for up to 6 months post vaccination, with insignificant fluctuations (p > 0.05). Further, a steady decrease in the titer of the SPPV was observed, and by the end of the experiment (after 360 days), it was only 1.9 log2 (p < 0.001) (Figure2). Antibodies of SPPV were absent in the blood sera of control sheep (Figure2). VaccinesVaccines 2021, 20219, x , 9, 912 7 of 187 of 18

FigureFigure 2. Neutralizing 2. Neutralizing antibody antibody responses responses in sheep in sheep following following vaccination vaccination with with a a combination combination vaccine.vaccine. BloodBlood serasera of of vaccinatedvaccinated sheep were sheep taken were and taken tested and testedin a SNT in a at SNT 0, 7, at 1 0,4, 7, 21, 14, 30, 21, 90, 30, 180, 90, 180,270, 270, and and 360 360 dpv. dpv. Neutralizing Neutralizing titers titers were were deter- mineddetermined against Nigeria against Nigeria75/1 vaccine 75/1 vaccine strain strainand NISKhI and NISKhI vaccine vaccine strain strain using using specific speciﬁc serasera for for the the PPRV PPRV and and for thefor SPPV.the SPPV. All vaccinatedAll vaccinated sheep sheep (100%) (100%) were were protected protected against against PPRV andand SPPV SPPV at at 21 2 dpv1 dpv and and 14 dpv,14 dpv, respectively. respectively.

3.4. с-. ELISA Test Result for PPR 3.3. SNT Results for SPPV Before vaccination, sheep serum samples were tested for the presence of antibodies, whichOn werethe day found of tovaccination be negative, (day as all 0), the all samples the animals had an were S/N ratioseronegative >150%. After for SPPV being antibodies.vaccinated A week for after only 7vaccination, days, the percentage the sheep of were S/N droppedfound to sharplyhave NAs to 85%, with with an average 45% of titer of the1.8 animalslog2. From being day protected, 7 to day the 21, remaining there was 25% a steady of the increase animals hadin the a titer average of produced NA titers in theantibodies blood sera above of vaccinated the permissible sheep. limit Thus, of the on S/N the value 14th (>60%), and 21st while dpv, in30% NAs of titer the animals, in the blood serumthe c-ELISA of immunized results weresheep questionable reached 3.0 (50–60%).–5.2 log2, However, respectively. at the The 14th highest dpv, 95% NAs of thetiter (6.0 log2)vaccinated in the blood sheep sera were of protected immunized from sheep PPRV, was judging detected by the at antibody 30 dpv. titers, The developed since the an- tibodiesS/N values were werepreserved 50%. The for average up to 6 S/N months value post was 17%vaccination, on the 21st with dpv, insignificant and all animals fluctua- tionswere (p100% ˃ 0.05). protected Further, from a PPRV.steady The decrease lowest S/Nin the % titer value of (12%) the wasSPPV recorded was observed, on the 30th and by dpv. The antibodies produced by day 30 post-vaccination persisted until the end of the the end of the experiment (after 360 days), it was only 1.9 log2 (p ˂ 0.001) (Figure 2). An- experiment (360 days), with slight fluctuations (p > 0.05) (Figure3). tibodies of SPPV were absent in the blood sera of control sheep (Figure 2). 3.5. Detection of Viral Nucleic Acids in Blood and Swabs 3.4. с-. ELISAIn all the Test blood Result samples for PPR and swabs taken from vaccinated sheep (group II) post- vaccination,Before vaccination, no viral nucleic sheep acids serum specific samples to the PPRV were andtested SPPV for were the detected,presence indicating of antibodies, whichthat thewere combined found to vaccine be negative, was safe as to all use the and samples provided had sterile an S/N immunity ratio >150%. against After both being viruses. By contrast, in all the types of samples taken from unvaccinated animals, PPRV vaccinated for only 7 days, the percentage of S/N dropped sharply to 85%, with 45% of and SPPV specific viral nucleic acids were detected. the animals being protected, the remaining 25% of the animals had a titer of produced antibodies above the permissible limit of the S/N value (˃60%), while in 30% of the animals, the c-ELISA results were questionable (50–60%). However, at the 14th dpv, 95% of the vaccinated sheep were protected from PPRV, judging by the antibody titers, since the S/N values were 50%. The average S/N value was 17% on the 21st dpv, and all animals were 100% protected from PPRV. The lowest S/N % value (12%) was recorded on the 30th dpv. The antibodies produced by day 30 post-vaccination persisted until the end of the experiment (360 days), with slight fluctuations (p ˃ 0.05) (Figure 3).

Vaccines 2021, 9, x 8 of 18

Vaccines 2021, 9, 912 8 of 18 Vaccines 2021, 9, x 8 of 18

Figure 3. Evolution of the average percentage of inhibition (with standard deviation) in each group of vaccinated and control sheep during the experiment after a single vaccination. In the present study, the unit of measurement was S/N percentage, and it was assumed that a lesser S/N % meant higher antibody levels. S/N percentage of ≤ 50% was considered positive, while that of 50 ˂ S/N ≤ 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.

Figure 3. EvolutionFigure 3. of Evolution the average3.5. Detection of the percentage average of Viral percentage of Nucleic inhibition Acids of (withinhibition in standardBlood (with and deviation) Swabsstandard deviation) in each group in each of vaccinatedgroup and control sheepof during vaccinated the experiment and controlIn all after sheep the a singleduring blood vaccination. the samples experiment and In the after swabs present a single taken study, vaccination. from the unit vaccinated In of the measurement present sheep was (group S/N II) percentage, andstudy, it was the assumed unit postof measurement that-vaccination, a lesser S/N was no % S/N meant viral percentage, nucleic higher antibody acidsand it specificwas levels. assumed S/N to the percentage that PPRV a lesser and of S/N≤ SPPV50% % meant was were considered detected, higher antibody levels. S/N percentage of ≤ 50% was considered positive, while that of 50 ˂ S/N ≤ positive, while that of 50 < S/Nindicating≤ 60% was that considered the combined doubtful. vaccine >60% was S/N safe percentage to use was and considered provided negative sterile immunity for the 60% was considered doubtful. >60% S/N percentage was considered negative for the presence of presence of antibodies againstagainst PPRV. both All the viruses. results wereBy contrast recorded, in as allMean the± typesS.E.M. of samples taken from unvaccinated antibodies against PPRV. All the results were recorded as Mean ± S.E.M. animals, PPRV and SPPV specific viral nucleic acids were detected. Starting from 2–3 days post-infection with the ﬁeld strain Kentau-7 from the nasal 3.5. Detection of ViralStarting Nucleic from Acids 2– in3 daysBlood post and- Swabsinfection with the field strain Kentau-7 from the nasal swabsswabs of all unvaccinated sheep, sheep, from from 4 4–5–5 days days from from the the blood blood and and ocular ocular swabs swabs in in6 out 6 out In all theofof 1010 blood tested samples sheep, fr from andom 5 5–6 swabs–6 days days takenfrom from oral fromoral swabsswabs vaccinated in in 8 8out out sheepof of 10 10 tested tested(group sheep,sheep, II) as aswell well as as post-vaccination,fromfrom no 6–76– 7 viral daysdays nucleic fromfrom rectalrectal acids swabsswabs specific inin 4 4 to outout the ofof 10PPRV 10 infected infected and sheep, SPPVsheep, viralwere viral nucleic detected,nucleic acid acid speciﬁc specific for indicating thatthefor the the PPRV combinedPPRV was was detected detected vaccine (Figure was(Figure 4 safe). 4). to use and provided sterile immunity against both viruses. By contrast, in all the types of samples taken from unvaccinated animals, PPRV and SPPV specific viral nucleic acids were detected. Starting from 2–3 days post-infection with the field strain Kentau-7 from the nasal swabs of all unvaccinated sheep, from 4–5 days from the blood and ocular swabs in 6 out of 10 tested sheep, from 5–6 days from oral swabs in 8 out of 10 tested sheep, as well as from 6–7 days from rectal swabs in 4 out of 10 infected sheep, viral nucleic acid specific for the PPRV was detected (Figure 4).

FigureFigure 4. 4.PPRV-speciﬁc PPRV-specificRNA RNA andand SPPV-speciﬁcSPPV-specific DNA were me measuredasured by by real real-time-time reverse reverse transcription transcription PCR PCR (RT (RT-qPCR),-qPCR), andand the the amount amount of of viral viral RNARNA andand DNADNA isis expressedexpressed as a Ct, a value that that increases increases as as the the amount amount of of viral viral RNA RNA increases. increases.

Capripoxvirus genomes were ﬁrst detected in the sheep’s blood samples on 5–6 days post-challenge (dpc) with virulent strain A, whereas in most infected animals, viral DNA was ﬁrst detected in nasal, ocular, and oral swabs at 6–7 days. Note that the infectious virus was detected in the nasal and ocular secretions of all unvaccinated sheep, while in Figure 4. PPRV-specific RNA and SPPV-specificoral swabs, DNA were viral me DNAasured was by isolatedreal-time in reverse 7 out transcription of 10 infected PCR sheep. (RT-qPCR), In rectal swab samples, and the amount of viral RNA and DNA is viralexpressed DNA as was a Ct, detected a value that from increases day 7 postas the challenge amount of in viral 7 out RNA of 10increases. sick sheep (Figure4).

Vaccines 2021, 9, x 9 of 18

Capripoxvirus genomes were first detected in the sheep’s blood samples on 5–6 days post-challenge (dpc) with virulent strain A, whereas in most infected animals, viral DNA was first detected in nasal, ocular, and oral swabs at 6–7 days. Note that the infectious virus was detected in the nasal and ocular secretions of all unvaccinated sheep, while in oral swabs, viral DNA was isolated in 7 out of 10 infected sheep. In rectal swab samples, Vaccines 2021, 9, 912 9 of 18 viral DNA was detected from day 7 post challenge in 7 out of 10 sick sheep (Figure 4).

3.6. Clinical Protection Post Challenge with the PPR Field Virus Strain Kentau-7 3.6. ClinicalThe vaccinated Protection sheep Post Challengedid not show with theclinical PPR Fieldsymptoms Virus Strainof PPR Kentau-7, and their rectal tem- peratureThe vaccinateddid not exceed sheep the did standard not show limits clinical (38.5 symptoms–39.8 °С) of, except PPR, and for theirtwo rectalsheep tempera-that un- turederwent did notchallenged exceed the on standard 30 dpv and limits 270 (38.5–39.8 dpv (Figure◦C), except5); at this for twostage, sheep their that clinical underwent score challengedreached up onto 301 (Figure dpv and 6). 270At dpvthe same (Figure time,5); atin this one stage, vaccinated their clinicalsheep that score passed reached the upcontrol to 1 (Figure test 306 ). dpv, At the showed same time,an increase in one vaccinated in body tem sheepperature that passed of 40.2 the °C control for 2 days test ◦ 30post dpv,-challenge, showed anwhich increase then in returned body temperature to normalof (Figure 40.2 C 5). for In 2 daysone vaccinated post-challenge, sheep which that thenpassed returned the control to normal test, 270 (Figure dpv,5 ).the In body one vaccinatedtemperature sheep reached that passed40.3 °C theat 4 control dpc, which test, ◦ 270returned dpv, theto normal body temperature at 6 dpc (Figure reached 5). In 40.3 twoC shee at 4p dpc, that which passed returned the control to normal test at at90 6 dpv, dpc (Figurelocal reactions5). In two were sheep noted that in passed the form the of control wrong test-rounded at 90 dpv, shape local swellings, reactions weremeasuring noted 0.1 in theand form 0.2 cm of wrong-rounded in diameter, pink shape in color, swellings, and protruding measuring 0.1 1 mm and above 0.2 cm the in diameter, surface of pink the insurrounding color, and skin, protruding which 1were mm independently above the surface stopped of the within surrounding 2 dpc. However, skin, which the weresam- independentlyples taken from stopped the local within reactions 2 dpc. of However, vaccinated the sheep, samples as takena result from of the localPCR reactionsanalysis, ofwere vaccinated clean. sheep, as a result of the PCR analysis, were clean.

Figure 5.5. DynamicsDynamics ofof changeschanges inin thethe bodybody temperaturetemperature ofof sheepsheep thatthat areare vaccinated,vaccinated, andand controlcontrol groupsgroups post-challengepost-challenge withwith thethe ﬁeldfield strainstrain Kentau-7Kentau-7 of the PPRV. RectalRectal temperaturestemperatures ofof tenten animalsanimals inin eacheach groupgroup areare shownshown inin thethe graph.graph. The dotted line inin thethe graphgraph showsshows thethe upperupper limitlimit ofof thethe normalnormal bodybody temperature.temperature. (#) Within 14 days,days, inin thethe vaccinatedvaccinated animals, no clinicalclinical symptoms characteristic of PPRV were observed, with the exception of local r reactionseactions in three sheep. (##) Local reactions that appeared post-challenge in vaccinated sheep existed for 2 days. (###) Temperature reactions that (##) Local reactions that appeared post-challenge in vaccinated sheep existed for 2 days. (###) Temperature reactions that appeared post-challenge in vaccinated sheep existed for 2 days. (*) Starting on the 3rd dpc, unvaccinated sheep devel- appeared post-challenge in vaccinated sheep existed for 2 days. (*) Starting on the 3rd dpc, unvaccinated sheep developed oped local reactions in the form of swellings. (**) From 5 to 11 days, the control animals developed clinical symptoms, localsuch reactionsas pyrexia in in the the form range of swellings.of 40.0–40.6 (**) °C From, watery 5 to and 11 days,mucopurulent the control discharge animals developedfrom the eyes clinical and symptoms,nose, hyperemia such asin ◦ pyrexiathe gums, in theand range some ofanimals 40.0–40.6 had C,diarrhea, watery decreased and mucopurulent appetite, dischargeand depression. from the (pp) eyes The and peak nose, of pyrexia hyperemia in unvaccinated in the gums, andanimals some was animals noted had on the diarrhea, 8th dpc decreased. (2 е) On appetite, day 8, 2 andsheep depression. in the control (pp) group The peak were of humanely pyrexia in euthanized unvaccinated post animals challenge was noteddue to onthe the deterioration 8th dpc. (2 ofе) their On day general 8, 2 sheepcondition. in the (***) control During group the specified were humanely time intervals euthanized (from post 10 to challenge 14 days), due8 out to of the 10 deteriorationinfected animals of their remained general alive condition. and began (***) to During recover. the speciﬁed time intervals (from 10 to 14 days), 8 out of 10 infected animals remained alive and began to recover.

Vaccines 2021, 9, 912 10 of 18 Vaccines 2021, 9, x 10 of 18

FigureFigure 6. Assessment 6. Assessm of theent clinical of the signs clinical in vaccinated signs in andvaccinated control sheepand control post challenge sheep post with challenge the ﬁeld strain with Kentau-7 the of the PPRV.field (a) strain Challenge Kentau 30 dpv.-7 of (b the) Challenge PPRV. (a 90) Challenge dpv. (c) Challenge 30 dpv. 180 (b dpv.) Challenge (d) Challenge 90 dpv. 270 (c dpv.) Challenge (e) Challenge 180 360 dpv. VG vaccinateddpv. (d group) Challenge infected 270 with dpv. virulent (e) Challenge SPPV (n = 360 10). dpv. CG control VG vaccinated group infected group with infected virulent with SPPV virulent (n = 2). Data are means ±SPPVstandard (n = 10). errors; CG **** controlp < 0.0001. group infected with virulent SPPV (n = 2). Data are means ± standard errors; **** р < 0.0001.

3.7. Necropsy of Dead and Euthanized Sheep Post Challenge with the Field Strain Kentau-7 of the PPRV. All control animals began to get sick on the 5th dpc, with the development of pyrexia in the range of 40.0 to 40.6 °C. In most infected animals, pyrexia lasted up to 9 days. The peak of pyrexia to 41.1 °C (equal to 4 clinical points) in three infected animals was observed on 8 dpc, after which the temperatures of sick animals began to decrease (Fig- ure 5; Figure 6).

Vaccines 2021, 9, 912 11 of 18

3.7. Necropsy of Dead and Euthanized Sheep Post Challenge with the Field Strain Kentau-7 of the PPRV Vaccines 2021, 9, x All control animals began to get sick on the 5th dpc, with the development of pyrexia11 of 18

in the range of 40.0 to 40.6 ◦C. In most infected animals, pyrexia lasted up to 9 days. The peak of pyrexia to 41.1 ◦C (equal to 4 clinical points) in three infected animals was observed on 8 dpc, after which the temperatures of sick animals began to decrease (Figure5 ; All infected sheep showed clinical signs specific to PPRV in the form of watery dis- Figure6). charge from the eyes and nose and local reactions in the form of swellings, measuring All infected sheep showed clinical signs speciﬁc to PPRV in the form of watery 1.0–2.0 cm in diameter. In two control sheep, watery discharge from the eyes and nose discharge from the eyes and nose and local reactions in the form of swellings, measuring gradually became purulent on 7–8 dpc. In addition, within 4 days of the onset of the fe- 1.0–2.0 cm in diameter. In two control sheep, watery discharge from the eyes and nose graduallyver, the gums became of all purulent the animals on 7–8 became dpc. In hyperemic. addition, within In gene 4 daysral, the of the animals onset ofwere the lethargic fever, theand gums depressed. of all the Additionally, animals became some hyperemic. animals In had general, diarrhea, the animals cough, were but lethargic they were and not depressed.dominant. Additionally,At this stage, some each animalssheep had had a diarrhea, total clinical cough, score but ranging they were from not 10 dominant. to 23. Two Atsheep this stage,that passed each sheep the control had a test total at clinical 90 dpv score and ranging360 dpv fromhad respiratory 10 to 23. Two disorders sheep that in the passedform of the pneumonia control test and at had 90 dpv periodic and 360 coughing dpv had in respiratoryconnection disorderswith this; intheir the total form clinical of pneumoniascores reached and had25–26. periodic However, coughing despite in connectionthe manifestation with this; of their pronounced total clinical clinical scores signs reachedspecific 25–26.to PPRV, However, of the 10 despite control the animals, manifestation 8 animals of pronounced recovered. After clinical conducting signs speciﬁc control totests PPRV, (14 ofdays), the 10the control recovered animals, animals 8 animals were isolated recovered. and After treated conducting with antibiotics control in tests order (14to days),avoid thethe recoveredappearance animals of secondary were isolated infections. and treated Two withsheep antibiotics from the in control order to group avoid (se- thelected appearance on the 90th of secondary and 360th infections. day) were Two humanely sheep from euthanized the control on group the 8th (selected dpc due on theto the 90thdeterioration and 360th of day) their were general humanely condition euthanized; at this onstage, the 8ththey dpc achieved due to clinical the deterioration indicators of of25 their and general26, respectively condition; (Figure at this 6). stage, Forcefully they achieved euthanized clinical sheep indicators were subjected of 25 and to 26,a nec- respectivelyropsy (Figure (Figure 7). 6). Forcefully euthanized sheep were subjected to a necropsy (Figure7).

Figure 7. Internal examination of forcibly euthanized sheep post challenge with the field strain Figure 7. Internal examination of forcibly euthanized sheep post challenge with the ﬁeld strain Kentau-7 of the PPRV. (a) Enlargement of the pharyngeal lymph nodes. (b) Enlargement of the Kentau-7 of the PPRV. (a) Enlargement of the pharyngeal lymph nodes. (b) Enlargement of the pre-scapular lymph node. (c) Enlarged mesenteric lymph node on the incision. (d) Congestive hy- pre-scapular lymph node. (c) Enlarged mesenteric lymph node on the incision. (d) Congestive peremia and isolated spot hemorrhages in the mucous membranes of the larynx and trachea. (e) hyperemiaSpotty hemorrhages and isolated under spot the hemorrhages serous membrane in the mucous of the lungs. membranes (f) Hemorrhages of the larynx under and the trachea. serous (emembrane) Spotty hemorrhages of the liver. under (g) The the mucous serous membrane membrane of of the the lungs. small ( fintestine) Hemorrhages is swollen, under reddened the serous and membranecovered with of the mucus liver. (signs (g) The of mucousacute catarrhal membrane enteritis). of the small(h) The intestine gallbladder is swollen, is full. reddened and covered with mucus (signs of acute catarrhal enteritis). (h) The gallbladder is full. The necropsy of euthanized control animals showed hyperemia and enlargement of The necropsy of euthanized control animals showed hyperemia and enlargement of the pharyngeal (Figure 7a), pre-scapular (Figure 7b) and mesenteric (Figure 7c) limph the pharyngeal (Figure7a), pre-scapular (Figure7b) and mesenteric (Figure7c) limph nodes. nodes. Hemorrhages were found in the tracheal mucosa (Figure 7d), in the lungs (Figure Hemorrhages were found in the tracheal mucosa (Figure7d), in the lungs ( Figure7e ), and 7e), and in the liver (Figure 7f). The mucous membrane of the small intestine was thick- in the liver (Figure7f). The mucous membrane of the small intestine was thickened in places,ened in covered places, with covered gray-brown with gr mucus,ay-brown and mucus, there were and hemorrhages there were allhemorrhages over the surface all over ofthe the surface intestinal of the mucosa intestinal (Figure mucosa7g). The(Figure gallbladder 7g). The was gallbladder ﬁlled (Figure was 7filledh). Samples (Figure 7h). takenSamples from taken the affected from the organs affected (lungs, organs liver, (lungs, and intestines) liver, and were intestines) positive were for PPRVpositive in for RT-qPCRPPRV in studies.RT-qPCR studies.

3.8. Clinical Protection Post Challenge with the Virulent Strain A of the SPPV In immunized sheep post challenge (after 30, 90, 180, 270, and 360 days), no clinical symptoms of smallpox infection were observed, the body temperature of the sheep was within the physiological norm (38.5–39.7 °C) (Figure 8), except for local reactions in three sheep at the site of the introduction of the virulent virus SPP. At the same time, in one vaccinated sheep that passed the control test 30 dpv, the local reaction, equal to one clinical score, was in the form of a speck, pink in color and in 1.0 cm in diameter, which independently disappeared at 3–4 dpс. In two vaccinated sheep that underwent control

Vaccines 2021, 9, 912 12 of 18

vaccinated sheep that passed the control test 30 dpv, the local reaction, equal to one clinical score, was in the form of a speck, pink in color and in 1.0 cm in diameter, which indepen- с dentlytesting disappeared 270 dpv, local at 3–4 reactions dp . In were two in vaccinated the form sheep of wrong that-round underwented shape control swellings, testing 270measuring dpv, local 0.5 reactionscm and 1.0 were cm inin thediameter, form of which wrong-rounded were resolved shape on the swellings, 3nd and measuring 4rd dpс, с 0.5respectively. cm and 1.0 cm in diameter, which were resolved on the 3nd and 4rd dp , respectively.

FigureFigure 8. 8.Dynamics Dynamics ofof changeschanges inin thethe bodybody temperaturetemperature of sheep vaccinated and and control control groups groups post post-challenge-challenge with with the the virulentvirulent strain strain A A of of the the SPPV.SPPV. Rectal Rectal temperaturestemperatures ofof tenten animalsanimals in each group are shown in in the the graph. graph. The The dotted dotted line line in in graphgraph shows shows thetheupper upper limitlimit ofof thethe normalnormal bodybody temperature.temperature. (#) Within 14 14 days, in in the the vaccinated vaccinated animals, animals, no no clinical clinical symptomssymptoms characteristic characteristic ofof SPPVSPPV werewere observed,observed, withwith the exception of local reactions in in three three sheep. sheep. (##) (##) Local Local reactions reactions that appeared post challenge in vaccinated sheep existed for 2–3 days. (*) Starting on the 2nd dpc, unvaccinated sheep that appeared post challenge in vaccinated sheep existed for 2–3 days. (*) Starting on the 2nd dpc, unvaccinated sheep developed local reactions in the form of edema of a dense consistency. (**) From 5 to 12 days, the control animals devel- developedoped clinical local symptoms, reactions insuch the as form watery of edema and mucopurulent of a dense consistency. discharge (**) from From the 5eyes to 12 and days, nose, the papules, control animalshyperemia developed in the clinicaloral cavity, symptoms, decreased such appetite, as watery and and depression. mucopurulent (3 e) discharge On day 9, from 3 sheep the eyes from and the nose, control papules, group hyperemia were humanely in the eu- oral cavity,thanized decreased post-challenge appetite, due and to depression.the deterioration (3 e) Onof their day 9,general 3 sheep condition. from the (pp) control The group peak of were pyrexia humanely in unvaccinated euthanized post-challengeanimals was noted due toon the the deterioration 7th dpc. (***) ofDuring their generalthe specified condition. time intervals (pp) The (from peak of10 pyrexiato 14 days), in unvaccinated 7 out of 10 infected animals ani- was notedmals onremained the 7th alive dpc. and (***) began During to therecover. speciﬁed time intervals (from 10 to 14 days), 7 out of 10 infected animals remained alive and began to recover. In animals infected with the virulent SPPV (strain A), primary clinical lesions began to developIn animals on 2 infected–3 days withdpc, thewit virulenth the development SPPV (strain of A), a local primary reaction clinical in the lesions form began of an to developedema of on a 2–3dense days consistency dpc, with up the to development 3 × 4 – 4 × 5 cm of ain local size reaction. At this stage, in the the form clinical of an edemascore ofof aeach dense sheep consistency increased up by to 1 3–×3 points4 – 4 × (5Figure cm in 9). size. On At day this 5, stage, the body the clinicaltemperature score ofof eachin- sheepfected increasedsheep reache by 1–3d 40.8 points °C. (FigureThe peak9). Onof pyrexia day 5, the of body41.2 °C temperature (equal to 4 of clinical infected points) sheep reachedwas observed 40.8 ◦C. on The the peak 7th dpc of pyrexia in three of sheep 41.2 ◦ C(Figure (equal 9) to. 4The clinical body points) temperature was observed in seven on thesick 7th animals dpc in began three to sheep normalize (Figure on9). the The 11 body–12 dpc temperature (Figure 8). in seven sick animals began to normalizeOn the on 5th the day, 11–12 simultaneously dpc (Figure8 with). the fever, all experimental animals had watery and mucopurulent discharge from the eyes and nose, as well as skin lesions (papules) with a diameter of 1.0–2.0 cm on the inner surface of the skin of the forelimbs and hind limbs, as well as on the skin of the udder. Most of the sick animals (70%) had a loss of appetite, which indicated the presence of hyperemia in oral cavity. At this stage, each sheep had a total clinical score in the range from 10 to 18. Additionally, on day 9, three sheep from the control group (selected on day 30, 180, and 360) were humanely euthanized post-challenge due to the deterioration of their general condition, presenting, respectively, a clinical score of 21, 21, and 20 (Figure 9). In addition to the above clinical signs, shortness of breath, cough, and moderate to severe depression were observed in the euthanized sheep. The corpses of the euthanized sheep were subjected to a necropsy (Figure 10). After conducting control tests (14 days), the recovered animals were isolated and treated with antibiotics in order to avoid the appearance of secondary infections.

Vaccines 2021, 9, 912 13 of 18 Vaccines 2021, 9, x 13 of 18

Figure 9. Assessment of the clinical signs in vaccinated and control sheep after infection with virulent SPPV. (a) Chal- Figure 9. Assessment of the clinical signs in vaccinated and control sheep after infection with virulent SPPV. (a) Challenge lenge 30 dpv. (b) Challenge 90 dpv. (c) Challenge 180 dpv. (d) Challenge 270 dpv. (e) Challenge 360 dpv. VG vaccinated group30 dpv. infected (b) Challenge with virulent 90 dpv. SPPV (c) Challenge (n = 10). 180 CG dpv. control (d) Challengegroup infected 270 dpv. with (e ) virulent Challenge SPPV 360 ( dpv.n = 2). VG Data vaccinated are means group ± standardinfected errors; with virulent **** р < SPPV0.0001 (.n = 10). CG control group infected with virulent SPPV (n = 2). Data are means ± standard errors; **** p < 0.0001.

On the 5th day, simultaneously with the fever, all experimental animals had watery and mucopurulent discharge from the eyes and nose, as well as skin lesions (papules) with a diameter of 1.0–2.0 cm on the inner surface of the skin of the forelimbs and hind limbs, as well as on the skin of the udder. Most of the sick animals (70%) had a loss of appetite, which indicated the presence of hyperemia in oral cavity. At this stage, each sheep had a total clinical score in the range from 10 to 18. Additionally, on day 9, three sheep from the control group (selected on day 30, 180, and 360) were humanely euthanized post-challenge due to the deterioration of their general condition, presenting, respectively, a clinical score of 21, 21, and 20 (Figure9). In addition to the above clinical signs, shortness of breath, cough, and moderate to severe depression were observed in the euthanized sheep. The

Vaccines 2021, 9, 912 14 of 18

corpses of the euthanized sheep were subjected to a necropsy (Figure 10). After conducting Vaccines 2021, 9, x control tests (14 days), the recovered animals were isolated and treated with antibiotics14 of 18 in

order to avoid the appearance of secondary infections.

Figure 10. Internal examination of forcibly euthanized sheep post-challenge with the virulent strain A of the SPPV. (a) Figure 10. Internal examination of forcibly euthanized sheep post-challenge with the virulent strain A of the SPPV. (a) Spotty Spotty hemorrhages in the subcutaneous tissue and musculature. (b) Enlarged mesenteric lymph nodes. (c) Enlargement hemorrhages in the subcutaneous tissue and musculature. (b) Enlarged mesenteric lymph nodes. (c) Enlargement of the of the popliteal lymph nodes. (d) Subcapsular lesions in the tracheal mucosa. (e) Spotty hemorrhages, and lesions in the lungs.popliteal (f) Serous lymph nodes.lesion in (d the) Subcapsular liver. (g) Subcapsular lesions in the lesion tracheal in the mucosa. kidney. ((eh)) SpottyThe gallbladder hemorrhages, is full. and lesions in the lungs. (f) Serous lesion in the liver. (g) Subcapsular lesion in the kidney. (h) The gallbladder is full. 3.9. Necropsy of Dead and Euthanized Sheep Post-Challenge with the Virulent Strain A of the SPPV3.9. Necropsy of Dead and Euthanized Sheep Post-Challenge with the Virulent Strain A of the SPPV During the autopsy of the euthanized sheep, pinpoint hemorrhages were found in the subcutaneousDuring the autopsy tissue and of the musculature euthanized ( sheep,Figure pinpoint10a). The hemorrhages lymph nodes were throughout found in the the bodysubcutaneous were mostly tissue enlarged and musculature and swollen, (Figure especially 10a). The the lymph popliteal nodes lymph throughout nodes the (Figure body 10wereb,c). mostly Small, enlargedwell-defined, and pale swollen, subcapsular especially lesions the popliteal were found lymph in the nodes tracheal (Figure mucosa 10b,c). of Small, well-deﬁned, pale subcapsular lesions were found in the tracheal mucosa of the the autopsy animals (Figure 10d). The lungs were swollen, especially in the dead sheep, autopsy animals (Figure 10d). The lungs were swollen, especially in the dead sheep, and and had spotty hemorrhages under the serous membrane of the lungs and lesions with had spotty hemorrhages under the serous membrane of the lungs and lesions with foci of foci of gray nodules (Figure 10e). Lesions were found in the liver and kidneys (Figure gray nodules (Figure 10e). Lesions were found in the liver and kidneys (Figure 10f,g). The 10f,g). The gallbladder was filled (Figure 10h). The results of the RT-PCR showed that gallbladder was ﬁlled (Figure 10h). The results of the RT-PCR showed that samples taken samples taken from the affected organs (lungs, liver, and kidneys) of euthanized animals from the affected organs (lungs, liver, and kidneys) of euthanized animals were positive were positive for SPPV. for SPPV.

4.4. Discussion Discussion Today,Today, sheep breeding is considered to be one of the most important branches of the agroagro-industrial-industrial complex complex of ofKazakhstan, Kazakhstan, which which in in some some cases cases is the is theonly only source source of the of most the mostimportant important types types of products: of products: wool, wool, lamb lamb meat, meat, fur, fur and, and fur fur sheepskins. sheepskins. The The number number of ofsheep sheep in in Kazakhstan Kazakhstan is is increasing increasing every every year, year, and and according according toto thethe BureauBureau of National StatisticsStatistics of the AgencyAgency forfor StrategicStrategic Planning Planning and and Reforms Reforms of of the the Republic Republic of Kazakhstan,of Kazakh- stan,at the at beginning the beginning of this of year, this the year, number the number of sheep of was sheep 20.05 was million 20.05 heads. million However, heads. How- as has ever,been established,as has been aestablished, large concentration a large concentration of animals in of limited animals areas in cominglimited fromareas differentcoming fromepizootic different regions, epizootic a wide regions, exchange a wide of animals exchange within of animals the country within and the the country import and of their the importhighly of productive their highly breeding productive breeds breeding from abroad,breeds from difficulties abroad, in difficulties organizing in continuousorganizing continuousproduction, production, full-fledged full feeding-fledged and feeding ensuring and an ensuring optimal an microclimate optimal microclimate create favorable create favorableconditions conditions for the occurrence for the occurrence of mass infectiousof mass infectious diseases. diseases. These dangerousThese dangerous infectious in- fectiousdiseases diseases of animals of animals include PPRinclude and PPR SPP. and SPP. PPRPPR and and SPP SPP are are highly infectious diseases diseases of of small ruminan ruminants,ts, which, in the event ofof an an outbreak, cause significant significant economic damage to agriculture. An An outbreak outbreak of of these infectionsinfections has has been been registered registered in in countries bordering Kazakhstan, including including Russia Russia and 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.

Vaccines 2021, 9, 912 15 of 18

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 (Figure2). The serological response to PPRV obtained using c-ELISA reached 95% at 14 dpv and 100% at 21 dpv (Figure3). 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 (Figure2). 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) (Figure2). 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) (Figures2 and3). 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 (Figure5). 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 (Figures6 and7). Vaccines 2021, 9, 912 16 of 18

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 (Figures9 and 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 (Figure4). 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 3.0 (10 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.

5. Conclusions Based on the analysis of the research data we obtained, we can conclude that the developed combined vaccine against PPR and SPP provides reliable protection against these infections in immunized sheep for 12 months. At the same time, vaccinated animals have immunity against PPR for more than 12 months. Large-scale ﬁeld trials using the developed combined vaccine, as well as in increasing the immunogenicity of the NISKhI strain of the SPP virus, need further study. The developed combined vaccine can be an alternative to the monovalent vaccines against PPR and SPP used in Kazakhstan and in Asian countries, which, when used, protect vaccinated animals from PPR and SPP for up to 12 months.

Author Contributions: Conceptualization, K.Z. (Kuandyk Zhugunissov); Data curation, Z.A. and K.Z. (Kuandyk Zhugunissov); Formal analysis, Z.A. and K.Z. (Kuandyk Zhugunissov); Funding acquisition, Y.A. and K.Z. (Kunsulu Zakarya); Investigation, K.B., Z.K., Z.S., Y.S., and D.K.; Method- ology, Z.A., K.B., Z.K., Z.S., Y.S., and K.D.; Project administration, Y.A. and K.Z. (Kunsulu Zakarya); Resources, D.K. and K.D.; Supervision, Y.A. and Y.B.; Validation, K.Z. (Kuandyk Zhugunissov), A.Z., Y.A., and Y.B.; Visualization, Z.A., K.Z. (Kuandyk Zhugunissov), Z.S., Y.S., A.Z., and Y.B.; Writing— original draft, Z.A.; Writing—review and editing, K.Z. (Kuandyk Zhugunissov). All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Science Committee of the Ministry of Education and Science, Republic of Kazakhstan [G 2012/]. Vaccines 2021, 9, 912 17 of 18

Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and 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). Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We thank D.S. Taranov and E.Zh. Alimbayev for their help during the animal experiments. We also thank the computer equipment maintenance operator for technical assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funder SC MES, RK had no role in the development of the study; in the collection, analysis and interpretation of data; in the writing of the report; or in deciding whether to submit the article for publication.

References 1. Amarasinghe, G.K.; Ayllon, M.A.; Bao, Y.; Basler, C.F.; Bavari, S.; Blasdell, K.R.; Briese, T.; Brown, P.A.; Bukreyev, A.; Balkema- Buschmann, A.; et al. Taxonomy of the order Mononegavirales: Update 2019. Arch. Virol. 2019, 164, 1967–1980. [CrossRef] 2. FAO. Global Strategy for the Control and Eradication of PPR Health; FAO: Rome, Italy, 2015; Available online: http://www.fao.org/3/ I4460E/i4460e.pdf (accessed on 5 April 2020). 3. Zhao, H.; Njeumi, F.; Parida, S.; Benfield, C.T.O. Progress towards Eradication of Peste des Petits Ruminants through Vaccination. Viruses 2021, 13, 59. [CrossRef] 4. Banyard, A.C.; Parida, S.; Batten, C.; Oura, C.; Kwiatek, O.; Libeau, G. Global distribution of peste des petits ruminants virus and prospects for improved diagnosis and control. J. Gen. Virol. 2010, 91, 2885–2897. [CrossRef] 5. Parida, S.; Muniraju, M.; Altan, E.; Baazizi, R.; Raj, G.D.; Mahapatra, M. Emergence of PPR and its threat to Europe. Small Rumin. Res. 2016, 142, 16–21. [CrossRef] 6. Abduraimov, Y.O.; Yershebulov, Z.D.; Zhugunisov, K.D.; Ye, A.B.; Nurgaziyev, R.Z.; Ye, D.K. The study of immunobiological properties of the vaccine against ovine rinderpest. Bull ASAU 2016, 2, 121–125. (In Russian) 7. Mischenko, A.V.; Mischenko, V.A.; Melnikov, V.P.; Chernykh, О.Y. Peste des petits ruminants. J. Vet. 2016, 9, 3–9. (In Russian) 8. Mischenko, A.V.; Mischenko, V.A.; Chernykh, O.Y.; Shevkoplyas, V.N.; Krivonos, R.A.; Lysenko, A.A.; Chernov, A.N. Epidemic characteristics of Peste des Petits Ruminants. J. Vet. Vrach. 2018, 6, 40–46. (In Russian) 9. Semakina, V.; Akimova, T.; Irza, A.V.; Mitrofanova, M.N. Peste des petits ruminants in the world and in the countries neighboring the russian federation. J. Vet. Vrach 2019, 1, 43–50. (In Russian) [CrossRef] 10. OIE World Organisation for Animal Health. Sheep pox and goat pox. In OIE Testerial Manual 2018; OIE World Organisation for Animal Health: Paris, France, 2018; Chapter 3.7.12. 11. Bhanuprakash, V.; Hosamani, M.; Singh, R.K. Prospects of control and eradication of capripox from the Indian subcontinent: A perspective. Antivir. Res. 2011, 91, 225–232. [CrossRef][PubMed] 12. Boumart, Z.; Daouam, S.; Belkourati, I.; Rafi, L.; Tuppurainen, E.; Tadlaoui, K.O.; El Harrak, M. Comparative innocuity and efficacy of live and inactivated sheeppox vaccines. BMC Vet. Res. 2016, 12, 133. [CrossRef] 13. Akimov, T.P.; Semakina, V.P. Epizootic situation of smallpox in sheep and goats in the world. J. Vet. Vrach. 2019, 3, 3–8. [CrossRef] 14. World Organization for Animal Health (OIE). Available online: https://www.oie.int/wahis_2/public/wahid.php/Reviewreport/ Review?page_refer=MapFullEventReport&reportid=30374 (accessed on 15 June 2021). 15. Mamadaliev, S.M.; Abduraimov, Y.O.; Ershebulov, Z.D.; Kulmanbetov, K.D.; Taranov, D.S.; Zhugunissov, K.D.; Sametova, Z.Z.; Kipshakpaeva, N.B. Method of Preparation of Peste des Petits. Ruminants. Patent 16916, 2010. Application granted date: 6.11.2008. (In Kazakhstan). 16. Mamadaliev, S.M.; Khairullin, B.M.; Troitsky, T.N.; Abduraimov, Y.O. Method of Manufacturing a Dry Culture Virus Vaccine Against. Sheeppox. Patent 16059, 2009. Application granted date: 2.04.2004.(In Kazakhstan). 17. Chaudhary, S.S.; Pandey, K.D.; Singh, R.P.; Verma, P.C.; Praveen, G. A vero cell derived combined vaccine against sheep pox and peste des petits ruminants for sheep. Vaccine 2009, 27, 2548–2553. [CrossRef] 18. Martrenchar, A.; Zoyem, N.; Diallo, A. Experimental study of vaccine against peste des petits ruminants and capripox infection in goats in northern Cameroon. Small Rumin. Res. 1997, 26, 39–44. [CrossRef] 19. Fakri, F.; Ghzal, F.; Daouam, S.; Elarkam, A.; Douieb, L.; Zouheir, Y.; Omari Tadlaoui, R.; Fassi Fihri, O. Development and field application of a new combined vaccine against peste des petits ruminants and sheeppox. Trials Vaccinol. 2015, 4, 33–37. [CrossRef] 20. Fakri, F.; Ghzal, F.; Daouam, S.; Elarkam, A.; Douieb, L.; Tadlaoui, K.; Fassi-Fihri, O.; Elharrak, M. Field Application of a Combined vaccine against peste des petits ruminants and sheep pox. J. Vaccines Vaccin. 2015, 6.[CrossRef] 21. Ayelet, G.; Fasil, N.; Jenebere, S.; Mekonnen, G.; Teshale, S.; Negussie, H. Study on immunogenicity of combined sheep and goat pox and peste des petits ruminants’ vaccines in small ruminants in Ethiopia. Afr. J. Microbiol. Res. 2012, 6, 7212–7217. 22. Zeidan, Z.; Mohamed, N.; Elzawahr, H.; Saad, S.; Nakhla, O.; Wahab, A.; Tammam, A.; Dakhly, E. Preliminary study for preparation of combined attenuated vaccine against sheeppox and peste des petits ruminants’ viruses. Int. J. Adv. Res. 2016, 4, 212–219. [CrossRef] Vaccines 2021, 9, 912 18 of 18

23. Konstantinov, A.V.; Starov, S.K.; Diev, V.I.; Moroz, N.V.; Kurnenkova, E.V.; Basova, D.K.; Kononov, A.V.; Fedoseyev, K.Y.; Melnikov, V.P. Antigenic and protective properties of associated virus vaccine against sheeppox, goat pox and peste des petits ruminants. Vet. Sci. Today 2017, 3, 28–32. (In Russian) 24. Amanova, Z.T.; Barakbayev, K.B.; Bulatov, Y.A.; Abduraimov, Y.O.; Zakarya, K.D. Improvement of manufacturing technology of combined vaccine against peste des petits ruminants and sheeppox. In Theory and Practice of Veterinary Pharmacy, Ecology and Toxicology in the Agro-Industrial Complex; Lunegov, A.M., Ed.; St. Petersburg State University of Veterinary Medicine: St. Petersburg, Russia, 2021; pp. 19–21. (In Russian) 25. OIE. Tests for sterility and freedom from contamination of biological materials intended for veterinary use. In Testerial Manual 2018; OIE: Paris, France, 2018; Chapter 1.1; pp. 109–122. 26. Mamadaliev, S.M.; Troitsky, T.N.; Mambetaliev, M.; Khairullin, B.M.; Zaytsev, V.L. A Strain of the Sheep Pox Virus Used to Test the Immunogenicity of the Virus. Vaccine. Patent 10105, 2007. Application granted date: 16.04.2001. (In Kazakhstan). 27. Mamadaliev, S.M.; Koshemetov, Z.K.; Nurabaev, S.S.; Orynbayev, M.B.; Kasenov, M.M.; Bulatov, Y.A.; Mambetaliev, M. Strain of Peste des Petits Ruminants Virus, Suitable for Manufacturing Diagnostic Drugs and Monitoring the Immunogenicity of Vaccine. Strains. Patent Kentau-7 PK-4-05D, 2010. Application granted date: 15.09.2008. (In Kazakhstan). 28. OIE. Peste des petits ruminants (infection with peste des petits ruminant’s virus). In Testerial Manual 2018; OIE: Paris, France, 2018; Chapter 3.7.9; pp. 1–16. 29. Reed, L.J.; Muench, H. A simple method of estimating fifty per cent endpoints. Am. J. Epidemiol. 1938, 27, 493–497. [CrossRef] 30. Couacy-Hymann, E.; Roger, F.; Hurard, C.; Guillou, J.P.; Libeau, G.; Diallo, A. Rapid and sensitive detection of peste des petits ruminant’s virus by a polymerase chain reaction assay. J. Virol. Methods 2002, 100, 17–25. [CrossRef] 31. Rao, T.V.; Bandyopadhyay, S.K. A comprehensive review of goat pox and sheep pox and their diagnosis. Anim. Health Res. Rev. 2000, 1, 127–136. [CrossRef] 32. Bamouh, Z.; Fakri, F.; Jazouli, M.; Safini, N.; Tadlaoui, K.O.; Elharrak, M. Peste des petits ruminants pathogenesis on experimental infected goats by the Moroccan 2015 isolate. BMC Vet. Res. 2019, 15, 452. [CrossRef][PubMed] 33. Hamdi, J.; Bamouh, Z.; Jazouli, M.; Alhyane, M.; Safini, N.; Tadlaoui, K.O.; Fihri, O.F.; El Harrak, M. Experimental infection of indigenous North African goats with goatpox virus. Acta Vet. Scand. 2021, 63, 9. [CrossRef][PubMed] 34. Fakri, F.Z.; Embarki, T.; Baha, W.; Tadlaoui, K.O.; Fihri, O.F. Large mass vaccination of small ruminants against Peste des Petits Ruminants and Sheeppox using a combined live attenuated vaccine. J. Vet. Med. Res. 2020, 7, 1200. 35. Pope, R.A.; Parida, S.; Bailey, D.; Brownlie, J.; Barrett, T.; Banyard, A.C. Early Events following Experimental Infection with Peste-Des-Petits Ruminants Virus Suggest Immune Cell Targeting. PLoS ONE 2013, 8, e55830. [CrossRef][PubMed] 36. Hosamani, M.; Singh, S.; Mondal, B.; Sen, A.; Veerakyathappa, B.; Bandyopadhyay, S.; Yada, M.; Singh, R.P. A bivalent vaccine against goat pox and peste des petits ruminants induces protective immune response in goats. Vaccine 2006, 24, 6058–6064. [CrossRef] 37. Rajak, K.K.; Sreenivasa, B.P.; Hosamani, M.; Singh, R.P.; Singh, S.K.; Singh, R.K. Experimental studies on immunosuppressive effects of peste des petits ruminant’s virus in goats. Comp. Immunol. Microbiol. Infect. Dis. 2005, 28, 287–296. [CrossRef]