Epidemiological study of peste des petits ruminants in goats and sheep in South Kivu, Democratic Republic Of Congo

Author: Bwihangane, Birindwa

Supervisors: George C. G.; Lilly C. B. and Nicholas S.

Peste des petits ruminants (PPR) is an acute, contagious transboundary disease of sheep, goats and some wild (Gazelles, Springbuck, Impala) animals caused by Peste des petits ruminants virus (PPRV). The disease was first officially reported in Democratic Republic of Congo (D.R. Congo) with devastating losses since 2008. Since then PPR has spread to almost all pastoral counties in Eastern of D.R.Congo where goats and sheep are playing major role in farmer‟s livelihood. The control of PPRV in D.R. Congo have been limited due to lack of epidemiological information, including spatial prevalence distribution, genetic nature of the circulating virus, host-associated risk factors and socio-economic effects. Several pathogens including capripoxvirus (CaPV), Pasteurella multocida (PM), Peste des petits ruminants virus (PPRV), Mycoplasma capricolum and capripneumoniae (Mccp) are responsible for major respiratory syndromes of goats and sheep in D.R. Congo. The dual or multiple infections caused by several pathogens increase the morbidity and mortality rates within animals in flocks or between animals individually. Peste des petits ruminants (PPR) reduces the activation of the immune response to capripox in goats and sheep in a mixed infection resulting in increase of the mortality and morbidly rates and causing economic loss to farmers. The lumpy skin disease virus (LSDV), goatpox virus (GTPV) and sheeppox virus (SPPV) are species of Capripox genus. The objectives of this research were to assess the status, prevalence, coinfection with capripox, associated risk factors and socio-economic impact of Peste des petits ruminants in sheep and goats and investigate the historical demographic dynamics of goats from PPRV outbreaks through mitochondrial DNA D-loop control region in South Kivu. For prevalence of PPRV specific antibodies using competitive enzyme-linked immunosorbent assay (cELISA), 320 serum samples from both unvaccinated and asymptomatic goats and/or sheep were collected randomly from four different teritories of South Kivu, while associated infection determinants and socio-economic impacts of the disease were assessed by participatory interview through a structured questionnaire and farmer groups . The ArcGIS software was used to draw all PPR sero-prevalence maps. The molecular epidemiology of PPR virus (PPRV) targeting the conserved PPRV nucleoprotein (NP), fusion (F) and hemagglutinin (H) proteins was determined by Reverse-transcriptase Polymerase Chain Reaction (RT-PCR) using NP, F and H specific primers. A total of 150 samples including tissues, swabs and whole blood were collected from goats with PPR clinical signs, for virus characterization. Further, due to diagnostic constraints the same samples were used to check for coinfection with Capripox virus (CaPV) targets the P32, RPO30 and GPCR genes using conventional One-step PCR. Both positive PPRV and CaPV samples were sequenced using Sanger method for further phylogenetic analysis using both CLC Genomics work bench version 10 and Molecular Evolutionary Genetics Analysis (MEGA) version 6 software. Genomic DNA of 111 indigenous goats from both peste-des-petits ruminants virus susceptible and non-susceptible varieties was extracted and the mitochondrial control regions (D-loop region) were amplified and sequenced with specific primers. Additional 22 goat mtDNA Hypervariable I region reference sequences (HV1: 481bp) belonging to the six known haplogroups/lineages were also downloaded from GenBank and included in the r analysis. Results of PPRV sero-status and associated risk factors in South Kivu showed an overall PPRV sero-prevalence of 45.3% (n=320) from which 53.4% (n=240) was found in goats and 21.3 % (n=80) in sheep. A multilevel nominal logistic model using JMP-Pro software and Stata 11 indicated that the likelihood of goats and sheep being infected with PPRV increased significantly when the following occurred (p<0.05): animals shared water sources (p=0.003628), herd size increased (p=< 2.2e-16), animal age increased (p=2.630e-07), exotic breeds were used (p=0.005), animals were raised with goat flocks (p=0.0177310), animals were reared in communal grazing systems (p=0.0001004), different goat species were raised together (p=0.0073387), and when there was exchange of animals between farms (p=<2e-16). The overall molecular prevalence with RT-PCR was 64.7% (97/150). The molecular characterization revealed clustering of the PPRV within lineage III. Significant substitutions in the nucleoprotein (NP) gene sequences were observed. Live animals demonstrated the following clinical signs: serous to mucopurulent nasal and ocular discharge, fever, diarrhea, lacrimation, matting of eye lids, cutaneous nodules; one may also see self-resolving lips and muzzle lesions of the involved animals. Other lesions seen on dead animals, on post -mortem examination were: congestion of lungs haemorrhages in large intestine and liver. Samples taken for laboratory diagnosis included: oculo-nasal swabs, tissues, whole blood and serum. The results showed mixed infection between PPR and Capripox which emphasized on the importance of molecular techniques in differentiating the two diseases. Out of 150 tested animals, 64.7% (97/150) were PPRV positive, 52.7% (79/150) were Capripox positive and 38.7% (58/150) were both PPRV and CaPV positive. The pair-wise comparison of P32 gene of CaPV and F gene of PPRV showed 99.75% of identity percentage among CaPV sequences, 96.95% among PPRV sequences and 47.91% of nucleotide percentage identity between CaPV and PPRV. For the goat‟s mtDNA analysis, a total of 120 segregating sites, 56 haplotypes and 124 mutations were found in a 1,220-bp sequence. The mean haplotype diversity and nucleotide diversity were 0.971±0.007 and 0.01068±0.00206, respectively with the overall number of nucleotide differences of 10.731. The phylogenetic analysis showed that all goat sequences were clustered into two haplogroups A and B, of which haplogroup A was the commonest. The global analysis of molecular variance (AMOVA), indicated that 83.22% of the total genetic variation of studied animals was described by genetic dissimilarities between individuals (pv= 0.32658), 11.18% among groups (pv=0.0000*) and only 5.60% of the variation was attributed to genetic changes between goat populations (pv=0.00286*). Briefly, the high seroprevalence in unvaccinated animals indicates that PPRV is circulating in South Kivu. Several risk factors are associated with PPRV sero-status including spatial and farm management. Peste des petits ruminants virus (PPRV) obtained from Eastern D.R. Congo clustered genetically with PPRV strains of Lineage III from East Africa, including Tanzania, Uganda and Kenya. This study informs the transboundary importance of this disease in the region. The lumpy skin disease virus, which is one of the species of capripox genus; found in co-infection with PPRV in South Kivu is a lineage which is circulating and has a genetic relationship between its P32 gene of Capripox virus (CaPV) and the PPRV fusion gene. There are high levels of intrapopulation diversity in Mwenga-Shabunda, Fizi and Kalehe goats and the weak phylogeographic structuring, suggesting the existence of solid gene flow between goat populations probably due to extensive trans-border goat‟s movement in the past from countries that have reported previous outbreaks of the PPR disease. South Kivu pastoralist community recognised PPR among the economic disease affecting the goats and sheep and it has the potential of disrupting cultural set up and local economy with the daily economic losses due to morbidity rate associated with suspected PPR of 30.2 USD for sheep and 37.05 USD for goats and approximatively 10.26 USD for sheep and 120.72 USD for goats due to mortality rate. It is thus recommended that protective effective measures should be applied in South Kivu including animal control movement within and outside the country in order to control the spread of PPR from the infected regions to non-infected areas. Vaccines that protect animals for both PPR and capripox are recommended for a better protection. Some goat breeds resistant to PPR can be selected and used in breeding programs.