A Clinical, Pathological, Epidemiological and Molecular Investigation of Recent Outbreaks of Peste des Petits Ruminants Virus in Domestic and Wild Small Ruminants in the Abu Dhabi Emirate, United Arab Emirates

Simple Summary Peste des petits ruminants (PPR) is a contagious disease in domestic small ruminants. The virus can also infect wildlife, with unknown roles in PPR epidemiology. In the United Arab Emirates (UAE), the previously published reports were from wildlife only. This is a first report from the UAE describing PPRV in domestic small ruminants. From four outbreak notifications, the disease is investigated by clinical, pathological, and molecular studies. We found that the clinical and pathological forms of the disease were almost identical in all examined animals and compliant to the classical forms of the disease. Phylogenetic analysis based on the N gene and F gene classified the virus within Asian lineage IV. Abstract (1) Background: Peste des petits ruminants (PPR) is a highly contagious animal disease affecting small ruminants, leading to significant economic losses. There has been little published data on PPR virus (PPRV) infection in the United Arab Emirates (UAE); (2) Methods: four outbreaks reported in goats and Dama gazelle in 2021 were investigated using pathological and molecular testing; (3) Results: The infected animals showed symptoms of dyspnea, oculo-nasal secretions, cough, and diarrhea. Necropsy findings were almost similar in all examined animals and compliant to the classical forms of the disease. Phylogenetic analysis based on N gene and F gene partial sequences revealed a circulation of PPRV Asian lineage IV in the UAE, and these sequences clustered close to the sequences of PPRV from United Arab Emirates, Pakistan, Tajikistan and Iran; (4) Conclusions: PPRV Asian lineage IV is currently circulating in the UAE. To the best of our knowledge, this is a first study describing PPRV in domestic small ruminant in the UAE.


Introduction
The peste des petits ruminants (PPR), as a disease, was first described in West Africa in 1942 [1]. It is economically significant, causing death of small ruminants and affecting was first reported in wildlife in 1986 [17]. Another report also described PPRV in Arabian wildlife in the UAE in 2009, which was caused by the Asian lineage IV virus [18]. In 2022, another study from experimental infection of goats with PPRV originated from Barbary Sheep in the UAE was also described the virus within Asian lineage IV [25]. Since 2005, a total of 62 outbreaks of PPR in small ruminants and wildlife species in the UAE have been reported to the WOAH [31,32]. Despite this, no lineage information for the PPRV in domestic animals in the UAE is available. Additionally, the host range of the PPR virus has expanded to include large ruminants (camels, cattle, and water buffalo) and wild small animals over the past few decades, raising concerns about the ongoing PPR global eradication program, which is primarily focused on the reachable sheep and goat populations [33]. Therefore, the present study aims to characterize PPRV strains currently circulating in the domestic and wild small ruminants in the UAE, and to study the clinical and pathological features of the disease during the response to PPR outbreak notifications within Abu Dhabi Emirate in 2021. The present dominant PPRV lineage in the UAE was further defined based on sequencing and phylogenetic analysis of the F gene and N gene, and its risk factors for occurrence were reviewed.

Epidemiological Data, Necropsy and Sampling
In response to four PPR outbreaks notifications that occurred in Abu Dhabi Emirate in July-September 2021, PPR suspected goats (n = 3) and dama gazelle (n = 1) ( Figure 1) with a history of fever, dyspnoea, oculo-nasal mucopurulent discharges, cough, and diarrhea were received at the Abu Dhabi Agriculture and Food Safety Authority (ADAFSA) veterinary laboratories for necropsy examination and laboratory diagnosis.  Gross lesions were recorded after necropsy examinations, lung and intestine samples were collected for histopathology. In addition, a total of 10 samples, including spleen (n = 3), nasal swabs (n = 2), oral swabs (n = 2), lung (n = 1), intestine (n = 1), and lymph node (n = 1), were taken from three goats and one dama gazelle (Table 1) and shipped on ice to the ADAFSA veterinary laboratories for investigation.  Animals originated from four different breeding farms located in two regions, Al Dhafra (Farm 1) and Al Ain (Farms 2, 3, and 4) within Abu Dhabi Emirate ( Figure 1). Farms (1, 3, and 4) contains sheep, goats, whereas farm 2 contains dama gazelle species. Sheep and goats present in the three farms are from local breeds and were raised for breeding purposes. The origin of the dama gazelle species was unknown (Table 1). Gross lesions were recorded after necropsy examinations, lung and intestine samples were collected for histopathology. In addition, a total of 10 samples, including spleen (n = 3), nasal swabs (n = 2), oral swabs (n = 2), lung (n = 1), intestine (n = 1), and lymph node (n = 1), were taken from three goats and one dama gazelle (Table 1) and shipped on ice to the ADAFSA veterinary laboratories for investigation.

Histopathological Examination
Tissue samples, including intestine and lung from one goat, were fixed in 10% neutral formalin for 24-48 h at room temperature for histopathological examinations following a previously described method [34]. Formalin-fixed tissue samples were processed in an automatic tissue processor (ATP1-220, Triangle Biomedical Sciences, Durham, NC, USA), embedded in paraffin blocks, and cut into 5 µm thick sections. For microscopic examination, sections were stained with Hematoxylin and Eosin (H&E) (Thermo Fisher Scientific, Runcorn, Cheshire, UK). Images were captured using the VisionTek digital microscopy system (DM01, Sakura Finetek, Torrance, CA, USA).

Quantitative Real-Time PCR (RT-qPCR) Based Detection of PPRV
Total RNA extraction from each biological sample was performed using the EZ1 Virus Mini Kit V2.0 (48) (Qiagen, Hilden, Germany) as per the kit instructions. Briefly, 60 µL of the RNA was eluted from 400 µL of the swab or tissue lysate using the Advanced EZ1 instrument (Qiagen in Hilden, Germany). The presence of PPRV was detected and confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) targeting the N gene of PPRV as previously described [35]. The primer sets used were PPRNF forward primer (5 -CACAGCAGAGGAAGCCAAACT-3 ), PPRNR reverse primer (5 -TGTTTTGTGCTGGAGGAAGGA-3 ), and the PPRNP TaqMan probe (FAM-5 -CTCG-GAAATCGCCTCGCAGGCT-3 -BHQ1). The reaction mixture in a total volume of 20 µL, consisted of 12 µL of Master Mix (Roche, Basel, Switzerland), 5 µL tested RNA and 1 µL of each primer (10 pmol/µL) and probe solution (10 pmol/µL), using the Real-time ready RNA Virus Master Kit (Roche). The following thermal profile was applied: an initial reverse transcription at 45 • C for 30 min, followed by reverse transcriptase inactivation and DNA polymerase activation at 95 • C for 5 min and 50 cycles of amplification (15 s at 95 • C and 30 s at 60 • C). The RT-qPCR analysis was performed using the LightCycler ® 2.0 Instrument (Roche, Life Science, Basel, Switzerland).

Amplification of N Gene and F Gene
The QIAGEN OneStep RT-PCR Kit (QIAGEN, Germany) was used to amplify N gene and F gene from a representative sample from each outbreak (shown in bold in Table 1) following the manufacturer's instructions. The N gene (351 bp) was amplified with NP3: 5 -GTCTCGGAAATCGCCTCACAGACT-3 and NP4: 5 -CCTCCTCCTGGTCCTCCAGAA TCT-3 . The PCR thermal profile was set to 95 • C for 10 min for the initial denaturation and polymerase activation, followed by 35 cycles of 94 • C for 30 s, 55 • C for 30 s and 72 • C for 1 min. Amplification of the F gene (448 bp) was carried out using the primers PPRV-F: (5 -AGTACAAAAGATTGCTGATCACAGT-3 ) and PPRV-R: (5 -GGGTCTCGAAGGCTAGG CCCGAATA-3 ) [36]. The PCR thermal profile was set to 50 • C for 30 min for reverse transcription, 95 • C for 15 min for enzyme inactivation, followed by 95 • C for activation of the polymerase followed by 35 cycles of 94 • C for 1 min, 50 • C for 1 min and 72 • C for 2 min. The final extension was conducted at 72 • C for 7 min. The amplicon was visualized in a 1.8% agarose gel.

Sanger Sequencing
ExoSAP-ITTM PCR Product Cleanup Reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to purify the PCR products of the N gene and F gene, and BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Waltham, MA, USA) was utilized to perform bidirectional Sanger sequencing using the same primers used to amplify N gene and F gene and BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Waltham, MA, USA) as previously described [36]. The 20 µL reactions consisted of 9 µL of water, 3.5 µL of 5× Sequencing Buffer, 1 µL of the BigDye Terminator v3.1, 1 µL of 3.2 pmol of each primer, and 5.5 µL of DNA. The BigDye XTerminator TM Purification kit (Applied Biosystems) was used to purify the reaction mixture in accordance with the manufacturer's instructions. Sequencing was performed on a SeqStudio Genetic Analyzer (Applied Biosystems, Waltham, MA, USA) using the 'MediumSeq BDX' run module. The sequence trimming and assembly were performed with CLC Genomic Workbench v.20 (Qiagen, Aarhus, Denmark). The consensus sequences of N gene and F gene were first subjected to NCBI BLAST analysis. These sequences were also deposited in the GenBank database under accession numbers shown in Table 1.

Sequence Alignment and Phylogenetic Analysis
Multiple sequence alignments of 255 bp and 322 bp of the N gene and F gene of the PPRV UAE strains characterized in this study were performed with the ClustalW program [37] impeded in MEGA X. Each sequence of the N gene or F gene was aligned with their corresponding PPRV curated sequences recently published [6] which are available in the PPR Network online page (https://www.ppr-labs-oie-network.org/materials-andprotocols/pprv-sequence-datasets, accessed on 13 December 2022). A newly published sequence from the UAE wildlife (GenBank: OM867572.1) [25] is also included in the phylogenetic tree. The MEGA X program [38] was used to construct the phylogenetic tree using the Maximum Likelihood method and the General Time Reversible model [39], with 1000 Bootstrap confidence.

Outbreak Investigations
Four PPR outbreak notifications were received by ADAFSA epidemiology unit during the period July-September 2021 from Al Ain (3 outbreaks) and Al Dhafra (one outbreak) regions in the Abu Dhabi Emirate ( Figure 1) and (Table 2). Upon outbreaks investigations, animal species affected included goats in three mixed (with sheep) breeding farms and a dama gazelle in one farm. The latter is located within Al Ain region, where high (60%) intensity of small ruminant farms is present. The age of the infected goats varies from less than three months to 1 year old, whereas the infected gazelles were around 1-2 years old. Two infected goats (farms 1 and 3) together with the dama gazelles (farm 2) were unvaccinated against PPR. In contrast, one goat infected (farm 4) was vaccinated seven months before the outbreak. The infected (farms 1 and 3) also had a recent history (3 weeks before the outbreak) of introducing new unvaccinated animals. These were from a farm within Abu Dhabi emirate (farm 1) and from another emirate regarding farm 3. The morbidity rate varies from 5-9% among the infected goat herds in farms (1, 3, and 4), whereas it was 80% in the dama gazelle herd. The highest mortality and case fatality rates of 6.9% and 80% were observed in farm 3, which contains sheep and goats. In contrast, 20% and 25% of mortality and case fatality rates were reported in the infected dama gazelle in farm 2 ( Table 2).

Clinical and Pathological Findings
The clinical signs reported by the field veterinarians for animals presented for necropsy (goats and dama gazelle) were fever, anorexia, serous-mucopurulent, oculonasal discharges, coughing, and diarrhea. At autopsy, the carcasses were fair and dehydrated. All animals exhibited erosive ulcerative lesions with fibrinous pseudo-membranes on the tongue, gum, dental pads, hard palate, and throat mucosa. Nostrils showed mucopurulent discharges and congested mucosa. The lungs showed suppurative bronchopneumonia characterized by consolidation atelectasis, red-dark discoloration of the cranioventral parts (in two goats), congestion and hemorrhage in dama gazelle. The intestine showed hemorrhagic inflammation with hypertrophy of mesenteric lymph nodes seen in all animals (Figures 2 and 3).

Histopathological Analysis
Histological examination of the goat intestine section showed necrosis of the villous tips, vascular changes, and infiltration of inflammatory cells in the lamina propria ( Figure 4A). The broncho-interstitial pneumonia was the main feature of the lung histological results. The lesions were edema, congestion, and hemorrhage, with infiltration of mononuclear cells in the alveoli and the alveolar septa causing thickened interalveolar septa ( Figure 4B).

Histopathological Analysis
Histological examination of the goat intestine section showed necrosis of the villous tips, vascular changes, and infiltration of inflammatory cells in the lamina propria ( Figure  4A). The broncho-interstitial pneumonia was the main feature of the lung histological results. The lesions were edema, congestion, and hemorrhage, with infiltration of mononuclear cells in the alveoli and the alveolar septa causing thickened interalveolar septa (Figure 4B).

Histopathological Analysis
Histological examination of the goat intestine section showed necrosis of the villous tips, vascular changes, and infiltration of inflammatory cells in the lamina propria ( Figure  4A). The broncho-interstitial pneumonia was the main feature of the lung histological results. The lesions were edema, congestion, and hemorrhage, with infiltration of mononuclear cells in the alveoli and the alveolar septa causing thickened interalveolar septa (Figure 4B).

Detection of PPRV by N Gene Real-Time PCR
A total of ten samples (nasal swabs = 2, oral swabs = 2, lung = 1, spleen = 3, lymph node = 1, and intestine = 1) from four different animals (3 Goats and 1 Dama Gazelle) related to four PPR outbreaks were tested positive for PPRV by RT-qPCR. The Ct value for all samples tested ranged between 20 to 25.

Characterization of PPRV Strains and Determination of its Geographical Lineage
The BLAST results of the partial N gene and F gene of sequences obtained in this study, reveal a 99% nucleotide similarity to the PPRV strain from Barbary sheep recently isolated in the UAE (GenBank: OM867572).
Separate phylogenetic trees were constructed using either N gene or F gene sequences of the UAE PPRV strains characterized here (ADAFSA_PPRV1_2021, ADAFSA_PPRV2_2021, ADAFSA_PPRV3_2021 and ADAFSA_PPRV4_2021), and their corresponding curated sequences mentioned in the method section. All UAE-PPRV strains from goats and dama gazelle studied here, were located within the Asian Lineage IV, close to PPRV from United Arab Emirates (GenBank: OM867572.1), Pakistan (GenBank: KC191635) and Tajikistan (GenBank: DQ840198.1) based on N gene sequence analysis and close to PPRV isolated from United Arab Emirates (GenBank: OM867572.1), Iran (GenBank: MN036451) and Pakistan (GenBank: KC191630 and FN996973.1) based on F gene sequence analysis as shown in Figure 5A,B for N gene and F genes respectively.  [39] in MEGAX [38]. Alignments were calculated with ClustalW [37] impeded in MEGAX. Bootstrapping (values indicated in %) was performed with 1000 replicates. The sequences obtained in this study clustered within Asian lineage IV and are marked with red color (numbered from 1-4).
(B) Phylogenetic relationship between PPRV detected in UAE in goats and dama gazelle and other virus isolates sequences described in method section. The tree was constructed based on the F gene (322 bp), using the Maximum Likelihood method and General Time Reversible model [39] in MEGAX [38]. Alignments were calculated with ClustalW [37] impeded in MEGAX. Bootstrapping (values indicated in %) was performed with 1000 replicates. The sequences obtained in this study clustered within Asian lineage IV and are marked in red (numbered from 1-4).
Based on N gene sequence analysis, the UAE-PPRV strains from goats and dama gazelle obtained in this study shares identity between 97% to 98% among them and 98-99% when compared to recently sequence from Barberry sheep detected in UAE [25], shown in Table 3.   [39] in MEGAX [38]. Alignments were calculated with ClustalW [37] impeded in MEGAX.
Bootstrapping (values indicated in %) was performed with 1000 replicates. The sequences obtained in this study clustered within Asian lineage IV and are marked with red color (numbered from 1-4). (B) Phylogenetic relationship between PPRV detected in UAE in goats and dama gazelle and other virus isolates sequences described in method section. The tree was constructed based on the F gene (322 bp), using the Maximum Likelihood method and General Time Reversible model [39] in MEGAX [38]. Alignments were calculated with ClustalW [37] impeded in MEGAX. Bootstrapping (values indicated in %) was performed with 1000 replicates. The sequences obtained in this study clustered within Asian lineage IV and are marked in red (numbered from 1-4).
Based on N gene sequence analysis, the UAE-PPRV strains from goats and dama gazelle obtained in this study shares identity between 97% to 98% among them and 98-99% when compared to recently sequence from Barberry sheep detected in UAE [25], shown in Table 3. Based on F gene sequence analysis, the UAE PPRV strains sequences are 99-100% identical, but share an identity of 98-99% with recently sequenced Barberry sheep detected in UAE [25], shown in Table 4.

Discussion
The PPR Global Control and Eradication Strategy (GCES), released by FAO and WOAH in 2015, aims for global eradication of the disease by 2030 [2]. For a country to proceed from control to eradicate PPR, the GCES follows a technical sequential stepwise approach (stage 1 to stage 4): assessment, control, eradication, and post eradication. The PPR Monitoring and Assessment Tool (PMAT) is a companion tool to the PPR GCES to determine a country's stage and to provide guidance to countries. It takes into consideration the five technical elements identified in the GCES (Diagnostics, Surveillance, PPR Prevention, and Control, Legal framework, and Stakeholder involvement) [40].
The UAE is currently at the eradication stage 2 of the four stages of the progressive stepwise approach for preventing and controlling PPR. To proceed to post eradication stage 4, genome information of PPRV isolates causing new outbreaks should be available [2]. Previous published reports of PPRV in 1986, 2009 and 2022 in UAE were from wildlife only. To our knowledge, the characterization of the virus in domestic small ruminant is first reported in this study.
Since PPRV recently caused epidemics in both domestic livestock and wildlife in the UAE, this research was conducted to better understand its molecular epidemiology. Four PPR outbreaks were documented in the Al Ain and Al Dhafra regions of the Abu Dhabi Emirate between July and September 2021.
Animal species infected included goats in three farms and a dama gazelle in one farm. The age of infected goats in the three infected farms varies from less than three months to one year old. Two infected goats farms were unvaccinated and had a history of introducing new unvaccinated animals from other locations within the UAE, whereas one infected goat was from a vaccinated farm seven months before the report of the outbreak. A young age and animal movement are important risk factors for PPR occurrence [41]. The UAE national animal health plan aimed to control and eradicate PPR by 2025 follows the global PPR control and eradication strategy, which adopts mass vaccination of sheep and goats against PPR to control and eliminate the disease from the globe by 2030. In the Abu Dhabi emirate the PPR vaccine containing the Nig75/1 reference strain is used for one round of annual mass vaccination campaigns targeting adult animals (above three month of age). The program is also assessed by serological post vaccination testing with a good result of immunization coverage. Thus, the animals infected in the vaccinated farm were probably not eligible to be vaccinated (below three months) during the campaign period. Immunity against PPR in vaccinated sheep and goats is likely life-long. However, due to the high rates of population turnover, the coverage of the vaccinated animals may decline by as much as 25% yearly, or even more quickly if vulnerable animals such as newborns are added to the animal holding [42]. Therefore, it is necessary to increase vaccination coverage together with controlling animal movement between susceptible farms.
In our study, the sheep did not show clinical symptoms during the outbreak, therefore they were not examined. The susceptibility of goats to be severely affected by PPR compared to sheep is reported by many authors. Clinically, a significantly higher proportion of goats (90.9%) were reported to be sick compared to only 9.1% of sheep [43,44]. Thus, the above-mentioned report results are in line with those reported in this study.
In this study, one outbreak of PPR was reported in a dama gazelle farm, which was surrounded by a high density of domestic small ruminant farms. Available evidence indicates that PPR does not persist in wildlife populations [45]. Therefore, controlling the disease around wildlife captivities is necessary by increasing vaccination coverage of small ruminants. Moreover, monitoring the condition through surveillance, which includes sheep, goats, wild small ruminants, and a typical PPRV hosts such as camels across the UAE, will help in identifying risks of PPRV across the country. The diagnosis of PPR infection in the UAE was based on clinicopathology of the outbreaks and further confirmed by molecular methods. According to research, PPR in wild small ruminants exhibited symptoms comparable to those seen in infected sheep and goats, such as pneumonia, necrosis, and the depletion of different lymphatic tissues [46].
Search of literature revealed lack of a comprehensive clinical description of PPR in wild small ruminants such as the gazelle. In this investigation, we reported erosive mucosal lesions in the upper digestive and respiratory tracts of dama gazelle, which are typical of PPR in sheep and goats [46]. The small erosions on the tongue of sacrificed gazelles were previously described [47]. The dama gazelle in this investigation did not exhibit the lung lesions that were seen in goats (suppurative bronchopneumonia, which is characterized by consolidation of the cranioventral sections). Microscopic findings of the intestine and lung sections in goats are comparable with those reported previously [7,22].
The phylogenetic analysis based on both the N gene and the F gene classified the virus within Asian lineage IV, which is currently circulating in neighboring countries and globally [8,10]. These UAE-PPRV strains from goats and dama gazelle studied here were located within the Asian Lineage IV, close to PPRV from United Arab Emirates, Pakistan, Tajikistan and Iran for N gene and F genes sequence analysis respectively. However, the way of introduction of the virus from outside the country into UAE remains unclear. As PPRV needs close contact for transmission, these new outbreaks within UAE are likely the result of the introduction of the virus from a farm within Abu Dhabi emirate (farm 1) and from another emirate regarding farm 3. It has been concluded that two lineages (lineage III in 1986) and (lineage IV in 2009 and 2022) have been reported in UAE since 1986, but only lineage IV is currently circulating according to recently published data.
Many similarities in the F gene analysis were reported, and the clustering was more apparent in the N gene tree topology than in the F gene tree as we observed in our study. This is due to the previously proved fact that the N gene provides a better epidemiological picture of PPRV compared to the F gene classification [8,24,48,49]. However, given that PPRV tends to mutate, phylogenetic analysis based on both F gene and N gene likely provides a more informative characterization as previously described [50].

Conclusions
In conclusion, PPRV circulation in the UAE was significantly influenced by the age of susceptible animals under one year old and migration of unvaccinated animals. In turn, this emphasizes how crucial it is to restrict the movement of animals within the country or at international borders, boost immunization rates, and closely monitor the disease in both domestic and wild animals. Based on the phylogenetic analysis of the F gene and N gene, the PPRV strains detected in our study were classified within Asian lineage IV. The present updated information on the sequences of PPRV strains circulating in domestic and wild small ruminants in the United Arab Emirates satisfies GCES standards and advances efforts to carry out the UAE-PPR national control plan, which aims to eradicate the disease by 2025. Institutional Review Board Statement: This research was approved by the research ethics committee Abu Dhabi Agriculture and Food Safety Authority (ADAFSA) (approval number: ADAFSA-EA-07-2022), and the study was conducted following the guidelines stated for animal use. A written consent (which was included in the sample request form approved by the ADAFSA research ethics committee) was obtained for the use of samples and animals from the owner before inclusion in the study.
Informed Consent Statement: Informed Consent was obtained from the owners of the animals.

Data Availability Statement:
The partial N and F gene sequences generated in this study are available in the NCBI database under accession numbers mentioned in the manuscript.