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Serological Surveillance and Molecular Epidemiology of Foot-and-Mouth Disease in

North Region of Cameroon.

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.

Introduction (1)

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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.

There is the need for a thorough understanding of the

local epidemiology of the disease in order to design

and develop appropriate control measures.

Introduction (2)

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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.

Aims and Objectives of the Study

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Methodology (1)

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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.

Methodology (2)

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Clinical cases Monitoring 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

Methodology (3)

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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

Laboratory AnalysisLaboratory Analysis

Methodology (4)

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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

Laboratory Analysis: Nucleic acid analysis Laboratory Analysis: Nucleic acid analysis

Methodology (5)

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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.

Statistical Analysis

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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.

Results (1)

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First Sampling Second Sampling

Location Total

No.

tested

Total No.

(%)

Positive

Period of

sampling

Total No.

tested

Total No.

(%)

positive

Period of

sampling

Selifa 45 38 (84.4) November. 2012 40 37 (92.5) May, 2013

Mayo-Oulo 100 100 (100) January. 2013 99 99 (100) May, 2013

Lougguere 100 94 (94) November.2012

January. 2013

56 50 (89.3) March. 2013 –

April, 2013

Djalingo 30 30 (100) November. 2012 27 26 (96.3) May, 2013

Dembo 97 76 (78.4) November. 2012 93 74 (79.6) April, 2013

Hamakoussou 94 64 (68.1) November 2012

January.2013

96 72 (75.0) April, 2013

Total 466 402 (86.3) 411 358 (87.1)

Spatio-temporal distribution of FMDV NSP-ELISA antibody among cattle herds in North region of Cameroon

Spatio-temporal distribution of FMDV NSP-ELISA antibody among cattle herds in North region of Cameroon

Analysis of NSP seropositivity

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Chi square test

• 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

Results (2)

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Distribution of FMDV NSP-ELISA antibodies positive serum samples into different serotypes (A and O) based on structural

protein ELISA (SP-ELISA)

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

Total No.

tested

Total No. (%)

positive

Mayo Oulo 15 10(66.7) 15 13(86.7)

Djalingo 18 12(66.7) 18 11(61.1)

Dembo 15 7(46.7) 15 10(66.7)

Hamakoussou 22 16(72.7) 22 19(86.4)

Total 70 45(64.3) 70 53(75.7)

Results (3)

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Results of analyses of epithelial tissues from infected cattle for presence of FMDV antigen

Results of analyses of epithelial tissues from infected cattle for presence of FMDV antigen

Location Total samplestested

No. (%) positive

Distribution of antigens to serotypes

No. of outbreak

s 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 4 (100)

Pitoa (Zibou) 3 1(33.3) SAT2(1) 1 1 (100)

Velé 2 2 (100) SAT2(2) 2 2 (100)

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)

Results (4)

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Results of PCR on probang samples obtained from NSP-ELISA positive cattle at different periods for presence of FMDV RNA

Results of PCR on probang samples obtained from NSP-ELISA positive cattle at different periods for presence of FMDV RNA

Location First samplingSecond sampling (4-5 months

later)Total No. Tested No. (%) positive Total No. tested No. (%) positive

Selifa 20 3(15)20 1(5)

Mayo Oulo 20 3(15)20 4(20)

Lougguéré 20 3(15)20 2(10)

Djalingo 20 3(15)20 4(20)

Dembo 20 5(25)20 4(20)

Hamakoussou 20 4(20)20 4(20)

Total 120 21(17.5)120 19(15.8)

Results (5)

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Results of analyses by PCR of epithelial tissues samples obtained from infected cattle for presence of FMDV RNAResults 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 04(100)

Pitoa (Zibou) 03 03 (100) 02 02(100)

Velé 02 02 (100) 02 02(100)

Total 37 28 (75.6) 12 12 (100)

Results (6)

Phylogenetic analysis of sequence data obtained from amplicons Cam ZIBOU 09,

Cam VELE 03, Cam VELE 05.

Phylogenetic analysis of sequence data obtained from amplicons Cam ZIBOU 09,

Cam VELE 03, Cam VELE 05.

AY343933.1 ERI/1/98

AY343934.1 ERI/4/98

AF367126.1 ERI/12/98

HM211082.1 CAR/P12/2000

AF367135.1 SAU/6/00

JX570631.1 LIB/1/2003

JX570632.1 LIB/7/2003

JX570615.1 CAR/1/2005

JX570616.1 CAR/8/2005

Cam ZIBOU 09

JX570636.1 NIG/2/2007

GU566071.1 SUD/1/2007

Cam VELE 05

JX570633.1 LIB/39/2012

JX570634.1 LIB/40/2012

Cam VELE 03 partiel

JX570635.1 LIB/41/2012

JX570622.1 EGY/9/2012

JX570624.1 EGY/11/2012

JX570618.1 EGY/3/2012

JX570625.1 EGY/13/2012

JX013980.1 EGY/23/2012

JX014256.1 PAT/1/2012

JX013978.1 EGY/7/2012

JX013979.1 EGY/26/2012

JX570619.1 EGY/4/2012

JX570620.1 EGY/5/2012

JX570617.1 EGY/2/2012

FJ461346.1 Murchison Falls National Park 2002

AY343939.1 SUD/6/77

AY442014.1 SUD/9/77

AY343938.1 ETH/2/91

AF367134.1 RWA/1/00

DQ009737.1 ZAI/01/74

AJ251473.1 KEN/3/57

AY343941.1 KEN/2/84

AY343963.1 UGA/51/75

AF367100.1 ZAI/1/82

AY343969.1 UGA/19/98

AF367115.1 KNP/32/92

AY593847.1 RHO/1/48

AF367127.1 NAM/286/98

AF367128.1 NAM/292/98

GU194491.1 ZIM/01/88

JQ639296.1 ZIM/7/89100

100

100

100

99

90

99

99

80

81

77

100

100

90

100

100

100

100

94

70

96

100

100

0.05

Software: MEGA 5.1Analysis Analysis ---------------------------- Phylogeny Reconstruction Scope ------------------------------- All Selected Taxa Statistical Method ------------------ Neighbor-joiningPhylogeny Test Test of Phylogeny ------------------- Bootstrap method No. of Bootstrap Replications ------- 1000Substitution Model Substitutions Type ------------------ Nucleotide Model/Method ------------------------ Kimura 2-parameter model Substitutions to Include ------------ d: Transitions + TransversionsRates and Patterns Rates among Sites ------------------- Uniform rates Pattern among Lineages -------------- Same (Homogeneous)Data Subset to Use Gaps/Missing Data Treatment --------- Pairwise deletion Codons Included --------------------- 1st+2nd+3rd+Non-CodingNo. of Sites : 648No Of Bootstrap Reps = 1000Only bootstrap values of 70% and above are shown

Conclusion

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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.

Acknowledgements

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ARS USDA (Off shore project),

The Ohio State University Research Foundation

GFRA

ANSES

DTRA, USA

MINEPIA, Cameroon

LANAVET, Cameroon.

THANK YOU FOR YOUR ATTENTIONTHANK YOU FOR YOUR ATTENTION