Research Project Proposal

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Stanley Underwood
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1 Research Project Proposal Applicant s full names AISSATOU DIDDI 1. Title of the proposed research project EPIDEMIOLOGY OF PESTE DES PETITS RUMINANTS IN NORTHERN PART OF CAMEROON 2. Research area Improved control of priority livestock and fish diseases including African swine fever (ASF); Contagious Bovine and Caprine Pleuropneumonia (CBPP and CCPP); Peste des Petits Ruminants (PPR); Rift Valley Fever (RVF); East Coast Fever (ECF); Capripox Virus diseases of ruminants; 3. Short summary (abstract) of the research project Peste des petits ruminants (PPR) is a serious disease that hampers cross-border trade in many countries in particular Cameroon. Our country has about Eight million, six hundred and Fifty five ( ) small ruminants that provide income to a large part of the rural population. The potential of animal production remains low valued and one of the main limiting factors is the presence of PPR. The socioeconomic impact of PPR results in heavy losses of production and its impact on market access of small livestock, affecting all players in the sector. At present, the epidemiological situation of the disease is poorly evaluated in Cameroon and very few molecular studies have been conducted on the lineages circulating. Faced with this whole issue and to ensure proper monitoring and implementation of appropriate control methods in Cameroon, it is necessary to make a precise study of the epidemiological situation of the disease. Page 1

2 The main objective of this study is to make a detail study of the epidemiological situation in the northern areas and to propose appropriate control measures. Specifically, the objectives of this study are to: determine the seroprevalence and identify the risk factors of PPR in the study area, isolate and identify the PPR virus by PCR and Cell Culture, characterize PPRV by molecular biology methods. The results of work will guide the actions of prevention and control in the most risky areas. This study could serve as support for decision making within the framework of national epidemiological surveillance network. 4. Statement of the problem, background & justification In Cameroon, the livestock sector is dominated by ruminants. Small ruminants play an important role in the area of animal production and the overall economy. According to FAO estimates in 2013, the herd of small ruminants is Eight million, six hundred and Fifty five ( ) (FAOSTAT 2015). Small ruminants are raised throughout of the territory of Cameroon, however, with varying development in different regions. Northern areas alone account for nearly 3/4 of the national herd population (FAO et al, 2003). Development of small ruminants in the country is under the influence of several constraints: the rearing facilities are not very adequate; they remain dominated by the extensive method (FAO et al, 2003). The potential of animal production remains low valued and one of the main limiting factors is the presence of infectious animal diseases such as high impact Peste des Petits Ruminants (PPR). PPR is a highly contagious disease of sheep and goats caused by a Morbillivirus closely related to rinderpest virus (Anderson et al., 1990; Couacy - Hymann et al., 1995). It is considered one of the most destructive livestock diseases in Africa, the near and Middle East and Asia (Taylor, 1984; Shaila et al., 1989; Lefevre and Diallo, 1990; Abu- Elzein et al., 1990; Nanda et al., 1996; Govindarajan et al., 1997). Page 2

3 Peste des petits ruminants virus (PPRV) is genetically grouped into four distinct lineages (I, II, III and IV) based on the analysis of the partial gene fragment F (fusion protein) or the N gene (nucleoprotein) of the virus (Dundon et al., 2014). The four lineages are present in Africa and currently the lineage II, III and IV are actively circulating (Banyard et al., 2010; Dundon et al., 2014; Libeau et al, 2014). The lineage IV which once seemed circulating in Asia (Shaila et al., 1996; Dhar et al., 2002; Banyard et al., 2010) has recently been detected in some African countries such as Cameroon, Central African Republic (Banyard et al., 2010), Sudan and Morocco (Kwiatek et al., 2011), Egypt (Sharawi and Abd al-rahim., 2009), Algeria (De Nardi et al., 2012) and Uganda (Luka et al., 2012). PPR is endemic in many countries in Africa and especially in Cameroon. It collectively disrupts trade security and prevents poor farmers to have access to potential lucrative markets for animals and animal products. Production losses resulting from high mortality up to 100% and high morbidity up to 90 % among susceptible animal (Lefevre and Diallo, 1990). Regarding control methods, vaccine against PPR is available in Cameroon and its efficacy was demonstrated by study carry out Awa et al., in 2002 but despite this, the vaccination coverage rate is still low and no national program exists. Although recognized as causing significant economic losses in farms of small ruminants, the epidemiological situation is still poorly evaluated in Cameroon and very few molecular studies have been conducted on the lineages circulating so it is necessary to make a precise study of the epidemiological situation of the disease. 5. Research question and hypothesis PPR was selected as the next target disease to be eradicated after rinderpest (Albina et al., 2013). It is also a model for the study of transboundary diseases because its dissemination is linked to regional movements of live animals. In order to understand the epidemiological situation and be able to proper monitor and implement of appropriate control methods in Cameroon, these questions needs to be examined: what is the actual prevalence rate in the northern regions of Page 3

4 Cameroon? What are the actual lineages circulating in Cameroon? What are the risks factors influencing the circulation of PPR? Hypothesis: PPR is endemic in Cameroon and there are more than one lineage circulations and their propagation is influenced by animal movements and networks of different actors. 6. Research objectives Specifically, the objectives of this study are to: - Determine the seroprevalence and identify the risk factors of PPR in the study area - Isolate and identify the PPR virus by PCR and Cell Culture - Characterize PPRV by molecular biology methods 7. Research plan 1. Determine the seroprevalence and identify the risk factors of PPR in the study area The field work will be carried out every two months in the selected sites for blood sampling. Sampling will be by puncturing the jugular vein of ovine and caprine animals aged at least six months and not vaccinated. Each time, farms will be identified; species and sex of the animal will be noted. The biological material (blood) collected in dry sterile tubes of 10 ml (type Venoject ND) will be processed on the field and in the laboratory to obtain the serum. Technique (ELISA), Enzyme Linked Immunosorbant Assay enables the detection of antibodies against the PPR virus. To this end, we will use the competition ELISA kit for the detection of anti- NP antibodies of the PPR in the serum. It is a universally recognized method as simple, fast and reliable. It has a high sensitivity and high specificity. Page 4

5 Alongside sampling, investigations will be carried out in each farm for identify risks factors. A structured questionnaire will be used to collect additional information about breeding system, movement of animals among others. A data processing model will be used to evaluate influence of variables and derive the risk factors. 2. Isolate and identify the PPRV by PCR and Cell Culture The detection and virus isolation will be done on swabs buccal products, nasal and ocular. These biological products will be collected along with the blood. Furthermore, in case outbreak during our studies, the animals presenting the PPR characteristic symptoms will be purchased and autopsied for organ collection: mesenteric and bronchial lymph nodes, spleen, lungs, intestinal mucosa, liver and kidneys. These organs will be transported in a sterile normal saline solution under cold chain and will be stored in a freezer -20 C until use. In the laboratory, the detection of PPRV will be done by RT-PCR on the N genes using NP3a * Forward primers 5'TCT AAA TCG CTC CGG CAC AGA CTG 3 ' from 1232 to 1255 / * NP4 Reverse 5' CCT CCT CCT CCT GGT CCA TCT GAA 3 ' from 1585 to 1560 (351 bp fragment) according to the protocol described by Couacy - Hymann et al; The F genes, the detection will be from 5 'primers ATC TTA GTG ACA AAG CTC GTA GAG G 3' gene F / 5 ' GAG GAG ACT TTT GTG ACC TAC AAG C 3' from 1124 to 1148 F gene (fragment of 372 bp) using the protocol described by Forsyth and Barett in The products of the RT-PCR on N and F genes will subsequently be sequenced, analyzed and phylogenetic classification of the isolated virus will be carried out. The PPRV isolation is done on Vero cells (African green monkey kidney cells) and other cell lines available 3. Characterize PPRV by molecular biology methods Viral genome will sequenced using Sangar sequencing methods. Sequences obtained from the PCR products will be aligned with those of PPRV present in the Page 5

6 National Center of the data bank for Biotechnology Information (NCBI). Multiple alignments of N and F gene will be performed using appropriate software package. Phylogenetic analysis based on the gene of the F and N protein is carried out using the criterion of the neighborhood based on the principle of parsimony (Saitou et al, 1987). Dissimilarities and distances between sequences are first determined with appropriate software. Construction of the phylogenetic tree will be based on the "unweighted neighbor-joining method" proposed by Gascuel in Phylogenetic trees are finally generated by to see relatives virus circulating with other PPRV. 8. Biological materials Biologicals materials such ocular and nasal swabs, mesenteric and bronchial lymph nodes, spleen, lungs, intestinal mucosa, liver and kidneys will be collected and used for this study. These samples will be screened using preliminary test in LANAVET. Positive samples will be shipped to BecA-ILRI Hub for further analysis. These samples are infectious to animal. 9. Expected outputs of the research The results of this study will provide more precise knowledge about the serological status of small ruminants in this study area. Furthermore, knowledge about the prevalence/incidence/circulating lineages of PPR distribution in space and/or time will provide valuable information to better understand the epidemiology of the disease. To this end, particular attention will be paid to the implications of these results in the field of active surveillance to support and guide the choice of the National Network of Epidemiological surveillance for the establishment of a possible National Control Program. 10. Tentative project timetable and start date Page 6

7 Activity Start Week Finish week 1 Isolation of PPRV of small ruminants samples on 1 8 Cell Culture May-June Identification of PPRV of small ruminants 1 5 samples by PCR and purification of PCR products July Characterization of PPRV by molecular biology 2 5 methods August Data Analysis September Report writing October Skills for the proposed research project List the research skills you currently possess that you will apply to the proposed research project List the research skills that you would like to gain during the proposed research project at the BecA-ILRI Hub - Serological analysis - Virus detection by RT-PCR - Cell Culture isolation - Molecular characterization 12. Potential impact of the research outputs In return to our home institute we will train other staff on knowledge gained and improve the technical platform of LANAVET in epidemiology. Particular attention will be paid to the implications of these results in the field of active surveillance to support and guide the choice of the National Network of Page 7

8 Epidemiological surveillance for the establishment of a possible National Control Program this in order to reduce the socioeconomic impacts of the disease. The results of our research will be widely disseminated through progress reports and in the national and international scientific community through communication in national seminars (consultation workshops, scientific seminars, meetings national or regional in scope). We will participate in international scientific conferences (WHO, FAO, IAEA) but we will valorize our results through scientific articles for publication in international journals with referees. This work should also contribute to building local research capacities and collaborations between teams from neighboring countries (Cameroon, Chad, Central Africa Republic, and Nigeria). 13. The proposed project linkage to my own research interests or a program of research at our institute LANAVET mission is the study of animal diseases, monitoring and control of epizootic diseases by diagnosis and vaccine production. My role in our institute constitute the following: vaccine production in particular viral vaccines production, (cell culture and maintenance, cell counting, virus culture and harvesting, virus and stem cell bank management), animal disease diagnosis by using molecular biology techniques (conventional PCR, RT -PCR), serological diagnosis. The research proposal closely tides with my research program since we do produce PPR vaccines and also carry out diagnosis of this disease. The project will enhance my skills on production of PPR vaccine and diagnosis taking to consideration circulating lineages. 14. The benefits of an ABCF Research Fellowship to my career as a researcher, and how it will build capacity for our research Programme, department or institution ABCF fellowship program will be an asset to my scientific career as it will on to strengthen my academic and professional skills, improve the technical platform of Page 8

9 LANAVET in epidemiology, and finally to write publications. Indeed, this research will aim to consolidate my skills in terms of molecular diagnostics and cell culture isolation. Restitution sessions will be organized to strengthen the capacity of LANAVET staff. 15. References cited in the proposal Anderson, E.C., Hassan, A., Barrett, T., Anderson, J., Observation on the pathogenicity for sheep and goats and the transmissibility of the strain of virus isolated during the rinderpest outbreak in Sri Lanka in Veterinary Microbiology 21, Abu-Elzein, E.M.E., Hassanien, M.M., Al-Afaleq, A.I., Abdehadi, M.A., Honsawai, F.M.J., Isolation of peste des petits ruminants from goats in Saudi Arabia. Veterinary Record 127, 309. Albina, E., Kwiatek, O., Minet, C., Lancelot, R., Servan de Almeida, R., and Libeau, G., Peste des Petits Ruminants, the next eradicated animal disease? Vet. Microbiol. 26, Awa, D.N., Ngagnou, A., Tefiang, E., Yaya, D. and Njoya, A., Post vaccination and colostral peste des petits ruminants antibody dynamics in research flocks of Kirdi goats and Foulbe sheep of north Cameroon. Prev Vet Med 55, Banyard, A.C., Parida, S., Batten, C., Oura, C., Kwiatek, O., and Libeau, G., Global distribution of peste des petits ruminants virus and prospects for improved diagnosis and control. J. Gen. Virol. 91, Couacy-Hymann, E., Bidjeh, K., Angba, A., Domenech, J., Diallo, A., Goats vaccinated with the attenuated peste des petits ruminants virus are protected against rinderpest disease. Research in Veterinary Science 59, Couacy Hymann, E., Roger, F., Hurard, C., Guillou, J.P., Libeau, G., Diallo, A., Rapid and sensitive detection of Peste des petits ruminants virus by Page 9

10 a polymerase chain reaction assay. J. Virol. Methods. 100: De Nardi, M., Lamin Saleh, S.M., Batten, C., Oura, C., Di Nardo, A., Rossi, D., First Evidence of peste des petits ruminants (PPR) virus circulation in algeria (sahrawi territories): outbreak investigation and virus lineage identification. Transbound Emerg Dis. 59: Dundon, W.G., Adombi, C., Waqas, A., Otsyina, H.R., Arthur, C.T., Silber, R., Loitsch, A., and Diallo, A., Full genome sequence of a peste des petits ruminants virus (PPRV) from Ghana. Virus Genes 49, Food and Agriculture Organization of United Nation (FAO)., World Association of Animal Production (WAAP)., Ministère de l Elevage, Pêches et industries Animales (MINEPIA)., Institut de Recherche Agricole pour le Développemen (IRAD)., Rapport national sur les ressources zoogénétiques des animaux d élevage du Cameroun. 109p Food and Agriculture Organization of United Nation (FAO). FAOSAT division de la statistique., 2015 Forsyth M.A., Barrett T., Evaluation of polymerase chain reaction for the detection and characterisation of rinderpest and peste des petits ruminants viruses for epidemiological studies. Virus Res. 39: Gascuel O BIONJ., 1997 an improved version of the NJ algorithm based on a simple model of sequence data. Mol Biol Evol. 14: Govindarajan, R., Koteesswaran, A., Venugopalan, A.T., Shyam, G., Shaquna, S., Shaila, M.S., Ramachandran, S., Isolation of peste des petits ruminants from an outbreak in Indian Buffalo Bubalus bubalis. Veterinary Record 141, Kwiatek O., Ali Y.H., Saeed I.K., Khalafalla A.I., Mohamed O.I., Obeida A.A., Abdelrahman M.B., Osman H.M., Asian lineage of peste des petits ruminants virus. Africa Emerg Infect Dis. 17(7): Page 10

11 Lefevre, P.C., and Diallo, A., Peste des petits ruminants virus. Rev Sci Tech Off Int Epiz, 9: Libeau, G., Diallo, A., and Parida, S., Evolutionary genetics underlying the spread of peste des petits ruminants virus. Anim. Front. 4, Luka, P.D., Erume, J., Mwiine, F.N., Ayebazibwe, C., Molecular characterization of peste des petits ruminants virus from Karamoja region of Uganda ( ) Arc Virol. 157: Nanda, Y.P., Chatterjee, A., Purohit, A.K., Diallo, A., Inui, K., Sharma, R.N., Libeau, G., Thevasagayam, J.A., Brünning, A., Kitching, R.P., Anderson, J., Barrett, T., Taylor, W.P., The isolation of peste des petits ruminants virus from northern India. Veterinary Microbiology 51, Saitou, N., Nei, M., The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol., 4: Shaila, M.S., Purushothaman, V., Bhavasar, D., Venugopal, K., Venkatesan, R.A., Peste des petits ruminants of sheep in India. Veterinary Record 125, 602. Sharawi, S.S.A., Abd-El-Rahim, I.H.A., Nucleotide sequencing and phylogenetic analysis of fusion (F) epitope for Egyptian peste des petits ruminants virus (PPRV) predicting unique criteria stated as Egypt Inter J Virol. 7(4): Taylor, W.P., The distribution and epidemiology of peste-des-petitsruminants. Preventive in Veterinary Medecine 2, Page 11

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