Detection, molecular and genetic characterization of peste des petits ruminants virus (PPRV) from sheep, goats and gazelles in the Sudan

Abstract

The present study was designed to identify, isolate and characterize PPRV currently circulating in small ruminants “sheep and goats” and gazelles in the Sudan. A total of 359 whole blood and tissue samples of sheep and goats collected from ten different States of the Sudan were screened for the presence of PPRV antigen by an IC-ELISA assay. The results showed that PPRV antigen could be detected in 180/359 samples with an overall antigenic prevalence of 50.1%. On the species basis, higher antigenic-prevalence was obtained from goats samples (63.4%, 45/71) compared to sheep samples (46.9%, 135/288). During 2015 to 2018, many suspected PPR outbreaks were occurred among sheep and goats in many localities within different States of the Sudan. These PPR suspected outbreaks were recognized mostly by the appearance of the typical clinical signs of the disease in small ruminants and were associated with a higher morbidity and mortality rates. Of the total 276 antigen samples from sheep and goats collected from suspected PPR outbreaks, 160/276 samples were found positive by an IC-ELISA, with an overall antigenic prevalence of 58%. Considering the animal species, of the total 223 antigen samples collected from sheep, 123/216 (56.9%)whole blood samples and 4/7 (57.1%) lung were found positive for PPRV antigen. Of the total 53 antigen samples collected from goats, 31/50 (62%) whole blood samples and 2/3 (66.7%) lungs were found positive for PPRV antigen. Moreover, of the five States under study, the highest antigenic-prevalence was demonstrated in Western Kurdufan State (81.2%) while the least incidence was present in Elgedarif State (50%). To investigate the presence of PPRV in sheep and goats in slaughter houses, screening of the collected 83 lung samples from sheep (65) and goats (18) by an IC-ELISA revealed that 20/83 samples were positive with an overall antigenic prevalence of 24.1%. Considering the animal species, higher overall antigenic prevalence (66.7%, 12/18) was detected among goats compared to sheep (12.3%, 8/65). Within States under study, results indicated that the highest antigenic-prevalence was demonstrated in Northern State (80%) while no incidence (0%) was present in Kassala State. From the several attempts, PPRV isolation was successful only from two filed samples in Vero cells inoculated with 10% lung homogenate. These two PPRV isolates designated as “PPRV/tc/Sudan/Khartoum/Bahri/2015 isolate” and “PPRV/tc/Sudan/Gedarif/2015 isolate” were originated from goat lung from Bahri (Elfaki hashim), Khartoum State and from sheep lung from Elgedarif State, respectively. Typical PPRV cytopathic effect (cpe) consisting of cell rounding, detachment of the cells from the monolayer sheet, destruction of the monolayer sheet and syncytia formation appeared starting from day 15 post infection (p.i.) and completed at 27 d.p.i. Subsequently, PPRV isolates were identified using IC-ELISA and RT-PCR assays. To confirm the positive results obtained by the IC-ELISA assay, 32 samples selected from sheep (27) and goats (5) were further analyzed using PPRV N-gene based RT-PCR assay. All of the analyzed samples from sheep and goats yielded bands of around 351 bp corresponding to the partial N-gene. The 351 nt partial PPRV N-gene nucleotide sequences of nine sheep and goat samples, which were positive when analyzed by RT-PCR, indicated that the PPRV obtained were clustered genetically with Lineage IV. PPR viruses sequenced in this study designated as “PPRV/Sudan/Khartoum/2015; PPRV/Sudan/Gedarif/2015; PPRV/Sudan/Gezira/Kab-Elgidad/2016; PPRV/Sudan/River-Nile/Garie/2016; PPRV/Sudan/Northern/Dongola/2017; PPRV/Sudan/Red-Sea/Port-Sudan/2017; PPRV/Sudan/White-Nile/Kosti/2017; PPRV/Sudan/Western-Kurdufan/Abuzabad/2017; PPRV/Sudan/Northern-Kurdufan/El-Obeid/2017” were deposited in the NCBI GenBank database under accession numbers MK371448.1-MK371456.1, respectively. Subsequently, sequence comparison and phylogenetic analysis confirmed that the nine PPRV strains from sheep and goats in the Sudan belonged to the PPRV lineage IV genotype and shared the closest sequence identity with PPRV strains originating from North African countries. During 2016 and 2017, specimens were collected from six apparently healthy semi-captive and captive Dorcas gazelles “Gazella dorcas” from three areas in Khartoum State. In May 2017, many free-ranging Dorcas gazelles with suspected signs of PPR were reported in Dinder National Park. PPRV antigen and nucleic acid were detected in specimens from gazelles by an IC-ELISA and RT-PCR assays. One PPRV isolate designated as “PPRV/Gazelle/tc/Sudan/DNP2017” was recovered in Vero cells, from a lung sample of one gazelle from Dinder National Park outbreak. The partial PPRV N-gene nucleotide sequences of three gazelle samples, which were positive when analyzed by RT-PCR,was determined. The N-gene (351 nt) partial nucleotide sequences of three PPRV designated as “PPRV/Gazelle/Sudan/Khartoum2016-1” and “PPRV/Gazelle/Sudan/Khartoum2016-2” were originated from two whole blood samples from Soba East and Elazhari, Khartoum State, respectively, and “PPRV/Gazelle/Sudan/DNP2017” was originated from one lung sample from a gazelle with suspected PPR from Dinder National Park were deposited in the NCBI GenBank database under accession numbers MG992016.1-MG992018.1, respectively. Further, genetic characterization clustered all PPRV from gazelles into lineage IV genotype. Antibodies developed against PPRV nucleoprotein were detected in sera from surviving gazelles from Dinder National Park outbreak using a C-ELISA assay. Based on the apparent clinical signs, higher fatalities and laboratory investigations the involvement of PPRV was confirmed. The present study demonstrates that gazelles are a potential wild small ruminant host for PPRV and may influence the epidemiology of PPR in the Sudan. This is the first report describes PPRV infection among gazelles in the Sudan and in Africa.