1. GFRA SCIENTIFIC MEETING 20-22 October 2015, Hanoi, vietnam
Serological Surveillance and Molecular Epidemiology of Foot-and-Mouth Disease in North Region of Cameroon.
Dickmu S1., Abdoulkadiri S.1,Hamet M.1, Babi D.1, Koulagna A.1,, Bravo de Rueda C.2, Bakkali K.L.3 Garabed R.4, El-Yuguda A.5, Rodriguez L.2 and Baba S.S.5
1. Laboratoire National Vétérinaire, LANAVET, Garoua, Cameroon
2. Foreign Animal Disease Research Unit, United States Department of Agriculture,
Agricultural Research Service, Plum Island Animal Disease Centre, Greenport, NY,USA
3. Agence Nationale de Securité Sanitaire de l'Alimentation, de l'Environnement et du travail, France
4. Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, OH, USA
5. Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Maiduguri, Nigeria.

2. Introduction (1) FMD is enzootic in Cameroon.
Serological evidence shown the circulation of all the seven serotypes in Adamawa region of Cameroon (Tanya, 1985),
Bronsvoort et al. (2003) and Ludi et al. (2014) reported the circulation of only three serotypes (SAT2, O and A).
No currently control program for FMD in Cameroon
There is the need for a thorough understanding of the
local epidemiology of the disease in order to design
and develop appropriate control measures.

3. Introduction (2)
There is paucity of information on the epidemiological situation of FMD in the North region of Cameroon
In order to understand the epidemiological pattern and ecology of FMDV in this part of the country, this project was aimed at carrying out a serological and molecular surveillance in order to obtain suitable data on the epidemiology of FMD in the North region of Cameroon.

4. Aims and Objectives of the Study
To determine the prevalence of FMD using serological and nucleic acid assay procedures on samples collected from cattle in the North region of Cameroon
To establish the molecular epidemiological pattern of FMDV infection identified in the study area
To identify the basic epidemiological factors influencing the circulation of FMDV in the study area

5. Methodology (1)
A targeted longitudinal study of sedentary cattle population in the North region was carried out over a period of six months.
A total of 1,117 samples comprising of 877 sera and 240 esophageal/ pharyngeal fluid were randomly collected from selected cattle in randomly selected six veterinary centres from the 53 Veterinary centres in the North region.

7. Methodology (2) Clinical cases Monitoring Sample collection
Clinical cases of FMD were sought for everywhere in the region. The affected farms were visited and appropriate samples collected. In a herd where more than twenty animals were clinically sick at least twenty animals were purposely selected and sampled.
If less than twenty animals were clinically sick, all the sick animals were sampled.
Sample collection
Oesophagal and phargngeal fluid were collected using probang cups.
Serum samples
Epithelial tissues from ruptured vesicular
The samples were conditioned in appropriate viral transport medium and shipped to LANAVET, for testing

8. Methodology (3) Laboratory Analysis Serum samples
NSP ELISA (PrioCHECK FMDV NS)
Serotyping: solid phase blocking ELISA (SPBE)
Sent to Plum Island Animal Disease Centre, USA for confirmation and serotyping.
Tissues:
Antigen detection - ELISA kit (IZSLER) for serotypes O, A, SAT1, SAT2

9. Methodology (4) Laboratory Analysis: Nucleic acid analysis
Positive antigen ELISA epithelium samples further tested RT-PCR
The esophageal/pharyngeal fluid (probang samples) tested using RT-PCR.
RT-PCR positive samples were sent to National Agency for Sanitary Food security (ANSES), France for genomic sequencing
Phylogeny: VP1 based phylogeny using MEGA 5.1

10. Determination of Risk Factors (Administering Questionnaires)
Methodology (5)
Determination of Risk Factors (Administering Questionnaires)
Sedentary animals
Two types of structured questionnaires were prepared and administered. One designed to collect information at herd level and one for individual animals sampled.
- Herd level: GPS coordinates, information on management system, contact with wildlife, gathering at watery point, new introduction of animal, total number of animals in herd among others were collected.
- Individual animal level, age, sex, breed, source of animal, colour coat among others were obtained and recorded. A total of 466 questionnaires were administered at individual animal level.
Monitoring of clinical cases
A structured questionnaire was prepared and administered to herds showing clinical signs of FMD. The GPS coordinates of the locality was recorded. Information relating to transhumance, contacts with wildlife, gathering at water point, management systems were obtained and recorded.

11. Statistical Analysis SPSS 16.0
Data were presented in mean value with standard deviations (X±SD) and compared by a Chi-square test. Statistically significant variables were determined at P≤0.05.

12. Results (1)
Spatio-temporal distribution of FMDV NSP-ELISA antibody among cattle herds in North region of Cameroon
First Sampling
Second Sampling
Location
Total No. tested
Total No. (%)
Positive
Period of sampling
Total No. (%) positive
Selifa 45
38 (84.4)
November. 2012 40
37 (92.5)
May, 2013
Mayo-Oulo
100
100 (100)
January. 2013 99
99 (100)
Lougguere
94 (94)
November.2012 56
50 (89.3)
March –April, 2013
Djalingo 30
30 (100) 27
26 (96.3)
Dembo 97
76 (78.4) 93
74 (79.6)
April, 2013
Hamakoussou 94
64 (68.1)
November 2012
January.2013 96
72 (75.0)
Total
466
(86.3)
411
358 (87.1)

13. Analysis of NSP seropositivity
Coat colour - not significant
Breed – significant p=0.006*
Contact with wildlife – not significant
Gender – not significant
Period since last outbreak- significant p=0.02
Age (<1 yr) – significant p=0.017
Chi square test

14. Results (2)
Distribution of FMDV NSP-ELISA antibodies positive serum samples into different serotypes (A and O) based on structural protein ELISA (SP-ELISA)
Location
Serotype A
Serotype O
Total No. tested
Total No. (%) positive
Mayo Oulo 15
10(66.7)
13(86.7)
Djalingo 18
12(66.7)
11(61.1)
Dembo
7(46.7)
Hamakoussou 22
16(72.7)
19(86.4)
Total 70
45(64.3)
53(75.7)

15. Results (3)
Results of analyses of epithelial tissues from infected cattle for presence of FMDV antigen
Location
Total samples
tested
No. (%) positive
Distribution of antigens to serotypes
No. of outbreaks reported
No. (%) outbreak confirmed
Dembo 10
3(30)*
SAT2 (2),SAT1 (1),O (1) 4
4(100)
Djalingo 22
12(54.6)a.b
SAT2(8), A(4),
SAT1(2)
4 (100)
Pitoa (Zibou) 3
1(33.3)
SAT2(1) 1
1 (100)
Velé 2
2 (100)
SAT2(2)
Total 37
18(48.7) 11
11(100)
*One animal had coinfection with the 3 serotypes (SAT1, SAT2 and O)
aOne animal had coinfection with the 3 serotypes (SAT1, SAT2 and A)
bOne animal had coinfection with 2 serotypes (SAT1 and A)

16. Second sampling (4-5 months later)
Results (4)
Results of PCR on probang samples obtained from NSP-ELISA positive cattle at different periods for presence of FMDV RNA
Location
First sampling
Second sampling (4-5 months later)
Total No. Tested
No. (%) positive
Total No. tested
Selifa 20
3(15)
1(5)
Mayo Oulo
4(20)
Lougguéré
2(10)
Djalingo
Dembo
5(25)
Hamakoussou
Total
120
21(17.5)
19(15.8)

17. No. (%) positive for Viral RNA No. (%) Outbreaks confirmed
Results (5)
Results of analyses by PCR of epithelial tissues samples obtained from infected cattle for presence of FMDV RNA
Location
Total samples tested
No. (%) positive for Viral RNA
No. Outbreaks reported
No. (%) Outbreaks confirmed
Dembo 10
10(100) 04
04 (100)
Djalingo 22
13 (59.1)
04(100)
Pitoa (Zibou) 03
03 (100) 02
02(100)
Velé
02 (100)
Total 37
28 (75.6) 12
12 (100)

18. Results (6)
Phylogenetic analysis of sequence data obtained from amplicons Cam ZIBOU 09, Cam VELE 03, Cam VELE 05.
Software: MEGA 5.1
Analysis
Analysis Phylogeny Reconstruction
Scope All Selected Taxa
Statistical Method Neighbor-joining
Phylogeny Test
Test of Phylogeny Bootstrap method
No. of Bootstrap Replications
Substitution Model
Substitutions Type Nucleotide
Model/Method Kimura 2-parameter model
Substitutions to Include d: Transitions + Transversions
Rates and Patterns
Rates among Sites Uniform rates
Pattern among Lineages Same (Homogeneous)
Data Subset to Use
Gaps/Missing Data Treatment Pairwise deletion
Codons Included st+2nd+3rd+Non-Coding
No. of Sites : 648
No Of Bootstrap Reps = 1000
Only bootstrap values of 70% and above are shown

19. Conclusion
From the results of this study, It could be concluded that FMD is endemic and has high prevalence among the cattle population in the North region of Cameroon.
The most prevalent FMDV serotypes are the SAT1, SAT2, A and O; occurring in single or in double and triple co-infections.
Phylogenetic analysis of the SAT2, the most common serotype, showed that the serotype is homologous to Libyan 2012 isolate and clustered with the Nigeria and Sudan 2007 isolates.
The basic epidemiological factors influencing the circulation of FMDV in the study area are age, breed and period of last outbreak.

20. Acknowledgements ARS USDA (Off shore project),
The Ohio State University Research Foundation
GFRA
ANSES
DTRA, USA
MINEPIA, Cameroon
LANAVET, Cameroon.

21. THANK YOU FOR YOUR ATTENTION