M. HABIELA, M. A. GAFFAR, Y. A. RAOUF, Y. H. ALI

The Global control of FMD - Tools, ideas and ideals – Erice, Italy 14-17 October 2008

Appendix 73

EPIZOOTIOLOGICAL STUDY OF FOOT-AND-MOUTH DISEASE IN THE SUDAN [THE SITUATION AFTER TWO DECADES]

M. Habiela1*, M. A. Gaffar2, Y. A. Raouf1, Y. H. Ali 2

1

Unit of Foot-and-mouth disease - Central Veterinary Research Laboratories (CVRL) Animal Resources Researches Corporation (ARRC)-Sudan. 2Department of Virology - Central Veterinary Research Laboratories-(CVRL) Animal Resources Researches Corporation (ARRC) Sudan. * Corresponding author: -E-mail addresses: mhabiela979@hotmail.com (M. Habiela), Present address: Central veterinary research laboratories (CVRL) - Unit of FMD- Madani Highway- Khartoum, Box: 8067 Amarat- Khartoum- Sudan, Cell: 00249 922 846856

ABSTRACT

To update the information on the situation of foot-and-mouth disease (FMD) in the Sudan, a serosurvey and disease survey were conducted. Recently collected data on FMD in the Sudan showed that FMD is a major constrain to cattle production in the country, but causes no threat or mild disease in sheep and goats. Disease with obvious clinical signs was detected in cattle only, which was caused by serotype "O" and "SAT2". Disease seasonal occurrence in the cold dry season was observed and animal movement seems to play a major role in virus dissemination. A total number of 1069 sera were collected from cattle, sheep, goats, and camel; from seven states in the Sudan, for detection of antibodies to FMDV. Application of liquid phase blocking (LPB) ELISA, revealed that antibodies to four serotypes were present in ruminants; namely O, A, SAT1 and SAT2. No antibodies to FMD were detected in camels’ sera. The results differed from early reports regarding the prevalence of serotype specific antibodies. In cattle antibodies to type "A" (78.13%) surpassed that of type "O"(69.39%) and antibodies to type "SAT2"(44%) surpassed that to type "SAT1"(20.2%). This work elucidates the current epidemiology of FMD in some parts of the Sudan.

1. INTRODUCTION

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of cloven-footed animals. Infection of FMD was reported in cattle, sheep, goats, swine, and antelopes, as well as more than 70 wild animal species (Shahan, 1960). FMD virus belongs to the Family Picornaviridae, Genus Aphthovirus. It has seven distinct serotypes O, A, C, South African Territories 1-3 (SAT-1, SAT-2, & SAT-3) and Asia-1 (Murphy et al. 1999). In most of sub-Saharan Africa, serotypes O, A, SAT-1 and SAT-2 are predominant (Rweyemamu et al., 2001). In the Sudan FMD is endemic and FMD outbreaks occur annually, the first record of the disease in the Sudan was in 1903 (Eisa and Rweyemamu, 1977), and four FMD serotypes out of the seven were reported in the country. These are O, A, SAT-1 and SAT-2 (Abu Elzein, 1983). Serotype O was firstly isolated then Serotype SAT1 before1952, Serotype A in 1957, and lastly Serotype SAT-2 in 1977, (Abu Elzein and Crowther 1979). Antibodies to these four FMDV serotypes were detected in cattle, sheep and goats sera, but their prevalence rate was quite different between species (Abu Elzein, 1987). camels' sera has been screened by the agar gel immunodiffusion test (AGID) for the presence of antibodies against FMD virus infection associated (VIA) antigen and proved to be negative (Abu Elzein et al., 1984). Since 1987 no study on FMD in the Sudan was carried out and type and subtype situation needs to be updated. However, recently Efforts were regenerated; a serosurvey was conducted in Khartoum state (Raouf et al., 2008), moreover, samples of suspected FMD outbreaks were sent more regularly to the World Reference Laboratory (WRL) at Pirbright in the UK (Anon, 2007). Detailed survey of FMD in different animal species in the Sudan is insufficient. In this study, an attempt was made to update and evaluate the FMD situation in some states of the Sudan, questionnaire and interviews with herdsmen and veterinarians, beside conduction of a serosurvey and a disease survey were carried out.

The Global control of FMD- Tools, ideas and ideals – Erice, Italy 14-17 October 2008 2. MATERIALS AND METHODS

2.1 Questionnaire and serosurveyThe survey was conducted from 2006 to 2008. A simple standardized questionnaire was formed and used for collection of information from the herdsmen emphasizing on the data on host and environment. Data was collected on blood-sampled animal (Species, age, sex, and breed) beside history of FMD in the herd. A total of 1069 sera were randomly collected from Cattle (469 sera), Sheep (319sera), Goats (88sera) and dromedary Camels (193sera) from seven states in the Sudan; namely, Gezira (Wad madani), Northern Kordofan (ElObied), Southern Kordofan (ElDeling), White Nile(Rabak), Gadarif (Gedarif), River Nile (Atbarah) and El-Shemalyah (Dongola) ( Fig. 1). These localities were selected according to the disease history and their density in animals’ population. 2.2 FMDV antibody Detection by LPB ELISA The LPBE Kits were provided by the Arab Organiztion for Agricultural development and the Federal Ministry of Animal Resources and Fisheries. They were obtained from the Institute for Animal Health (IAH), Pirbright Laboratory, UK. Screening assay for detection of FMD antibodies were carried out according to the instructions of the manufacturer after optimization of antigen dose for the test (Raouf et al., unpublished data). 2.3 Collection of FMD virus samples 2.3.1 Collection of Oesopharyngeal (OP) fluid (Probang samples) Samples were collected from suspected FMD- carrier animals using a probang cup, according to the method of Hedger (1968), added to equal volume of transport media ( 0.08 M phosphate buffer pH 7.2-7.6) with antibiotics (Kitching and Donaldson, 1987), Samples were transported in ice box to the Laboratory, and then stored at -70° C. 2.3.2 Collection of epithelial samples The epithelium samples were collected during the course of field outbreaks as described by Kitching and Donaldson (1987). Mouth lesions were taken from infected animals, put in transport media (0.04 M phosphate buffer pH 7.2.-7.6) with 50% glycerol and antibiotics, kept on ice, transported to the laboratory and stored at -30 to -5°C. 2.4 FMD virus isolation and serotyping Nineteen Probang samples and two swabs were collected from previously suspected FMD-infected cows. Eight epithelial samples that were collected from three out breaks, one in Gezira State and two outbreaks in White Nile State ( Table 4) . Some of the probang samples were packaged, as described by Kitching and Donaldson, (1987) and were submitted to the FAO-OIE World Reference Laboratory (WRL) for FMD at Pirbright, UK. Virus isolation was carried out in bovine thyroid (BTY) cell culture (Snowdon, 1966) and primary bovine kidney (PBK) cell culture (Patty et al., 1962) at CVRL. Virus isolates were serotyped using FMD antigen detection ELISA that obtained from the Institute for Animal Health (IAH), Pirbright Laboratory, UK. The ELISA procedures used were similar to those described by Roeder and Smith (1987) and Ferris and Dawson (1988).

3. RESULTS

3.1 Questionnaire and interviews From 50 distributed questionnaires, 23 were returned, eleven questionnaires from Gezira stats, four from White Nile state, three from each North Kordofan and Gedarif states, and one from each South Kordofan and El-Shymaliyah States. The collected data showed that FMD, which is locally known as (Abu Lisan = Tongue disease), is well-known to herdsmen and they are well-awarded of the disease, its clinical signs, seasonality, duration and transmission. The practiced- husbandry systems in the investigated herds, were either extensive or semiintensive with free animal movement. The questionnaire data showed that FMD clinical signs were only observed in cattle; and causes mild or no clinical signs in small ruminants specially that were intermingling with cattle. It was predominantly encountered in the cold dry season (November to March). The morbidity rate may approach 100%; especially in cross-bred cattle; while the mortality rate was low and only occurred in young animals. Losses were largely due to the death of newborn and suckling calves, loss of weight and milk production, decrease of draft power and infertility.

The Global control of FMD - Tools, ideas and ideals – Erice, Italy 14-17 October 2008 The data revealed that the current applied control policy is restriction of animal movement and quarantine. To reduce the effects of the disease, herdsmen apply powder of the ground seeds of the Acacia nilotica in the drinking water of infected animals to cure the mouth ulcers. These seeds are known to contain a high concentration of tannic acid. They also use glycerin and antibiotics for protection of infected animals from secondary bacterial infection.

= Cattle. = Sheep. = Goats. = Camel. = main location of sampling. Figure 1: Map of the Sudan showing the area of the Study and locations of sampled livestock between 2006- 2008

3.2 Serology The overall percent of positive sera for FMDV in the four tested animal species were 79.24 % in cattle, 22.95 % in sheep, 28.57 % in goats and no positive serum was observed in the tested camels’ sera (Table 1). Antibodies to the four FMDV serotypes used in the study, were observed in the animal sera from all the investigated states; with the highest prevalence in cattle, (Table 2).The results obtained by LPB ELISA showed that serotype A (78.1 %) was the most prevalent in cattle followed by serotype O (69.4 %), SAT2 (44%) and SAT1 (20.2%). In sheep, serotype O (27.5 %) was the most prevalent followed by SAT-2 (9.1%), A (8.7 %), and SAT-1 (5.1%). In goats, serotype O (27.5%) was the most prevalent followed by A (15.9%), SAT-1 (8.5 %) and SAT-2 (2.4 %) (Table 2; Figure 2). Table 1: Overall positive sera for FMDV antibodies per species

Animal species Positive sera Positive % Cattle 374 a / 472b 79.24% Sheep 70 / 305 22.95% Goat 28 / 98 28.57% Camel 0 / 176 0

a = positive sera b= tested sample Table 2: The percentage of positivity of the total sera for each of the four FMDV serotypes using LPBE

The Global control of FMD- Tools, ideas and ideals – Erice, Italy 14-17 October 2008

Serotype Species Positive sera

*Cattle

*Sheep *Goats *Camel 297 a /428 b 52

/189 22 /80 0 /193 O Positive % 69.39

27.51 27.5 0

A Positive sera 275 a /352 b

8 /92 11 /69 0 /193 Positive % 78.13

8.7 15.94 0

SAT1 Positive sera Positive %

60 a /297 b 20.2

5 /98 4 /47 0 /193 5.1 8.51 0

a = Number of positive sera, b= Number of tested sample. *= some of the serum samples were tested for more than one serotype.

Figure 2: Overall results of screened sera of different animal species for FMDV antibodies using LPBE 2006-2008 SAT2 Positive sera Positive %

176 a

/400 b 16

/178 2 /84 0 /193 44

8.99 2.38 0

3.3 FMD virus isolation and serotyping From the nineteen probang samples and two swabs that were inoculated in BTY cell culture, seventeen samples caused cytopathogenic effects (CPE) and progressive changes in cell culture within 24 -72 hours following inoculation. Eighteen cell culture harvests were re-inoculated in bovine kidney (BK) cell culture; ten samples produced CPE in BK cell culture (Table 3). A 10% suspension of collected epithelium samples from infected cows during FMD outbreaks were inoculated in BK cell culture and a progressive CPE started within two to three hours following inoculation. After 24 hours the monolayer was completely destroyed. (Table 3) 3.4 Identification of virus isolates by Antigen Detection ELISA All probang samples derived cell culture material that were tested by the antigen detection ELISA showed optical density less than 0.1 and were considered negative (Table 5). Probang samples dispatched to the FAO-OIE World Reference Laboratory (WRL) for FMD at Pirbright, United Kingdom (UK) also proved to be negative (Table 4). Epithelium samples from Gezira state (Al-Kiraiba) were positive for serotype SAT-2, whereas, one epithelium sample from the White Nile state (Jabal Biyout) was positive for serotype O. Epithelium samples from Alkonoz and Omshatain were negative for FMDV (Table 5).

The Global control of FMD - Tools, ideas and ideals – Erice, Italy 14-17 October 2008

Figure 4a: Gezira state results Figure 4b: North Kordofan state results

Figure 4c: White Nile State results Figure 4d: South Kordofan State results

Figure 4e: River Nile State results Figure 4f: Gedarif State results Figure 4: Results of screened sera of different animal species for FMDV antibodies using LPBE in some states in the Sudan between 2006- 2008:

The Global control of FMD- Tools, ideas and ideals – Erice, Italy 14-17 October 2008

Table 3: Results of inoculation of specimens sampled from cattle in different cell cultures

Type of specimen Number of samples passaged in cell culture Number of samples caused CPE in cell culture

Probang 21a 19

Swabs 2 a 2

Epithelium 7 b 7 a = Passaged in BTY cells and twice in BK cells b = At least two passages in BK cells only Table 4: Pirbright Laboratory- UK results of detection and serotyping of FMDV specimens sampled from cattle in Sudan-2007

Sample identity Description of sample WRL Reference code

Serotyping results by cell culture and ELISA

PCR results

SUD 7 -NK 1 SUD 7 -NK 2 SUD 7- NK 3 SUD 7 -NK 4 SUD 7 -NK 5 SUD8 -WN 1 SUD8- WN 2 SUD8- WN 3 SUD8 -WN 4 SUD8- WN 5 SUD8 -WN 6 SUD8- WN 7 SUD 9 -GAD 1 SUD 9 -GAD 2 SUD 9 -GAD 3 SUD10 DONG 1 SUD10- DONG 2 SUD 11GEZ-MD 19N SUD 11- GEZ-MD 21T SUD12 GEZ-MS 1 SUD12- GEZ-MS 2 PROBANG PROBANG PROBANG PROBANG PROBANG PROBANG PROBANG PROBANG PROBANG PROBANG PROBANG PROBANG PROBANG PROBANG PROBANG FLUID PROBANG FLUID FLUID PROBANG PROBANG SUD 18/2007 SUD 19/2007 SUD 20/2007 SUD 21/2007 SUD 22/2007 SUD 8/2007 SUD 9/2007 SUD 10/2007 SUD 11/2007 SUD 12/2007 SUD 13/2007 SUD 14/2007 SUD 15/2007 SUD 16/2007 SUD 17/2007 SUD 6/2007 SUD 7/2007 SUD 4/2007 SUD 5/2007 SUD 2/2007 SUD 3/2007 NVD NVD NVD NVD NVD NVD NVD NVD NVD NVD NVD NVD NVD NVD NVD NVD NVD NVD NVD NVD NVD NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG

The Global control of FMD - Tools, ideas and ideals – Erice, Italy 14-17 October 2008

Table 5: Results of epithelium and probang samples tested by Antigen detection ELISA

Sample ID GAD-3 GAD-2 DONG-2 NK-3 NK-4 GZ-MD19N GZ-MD21T WN-3 WN-7 AlKiraiba-2 AlKiraiba -3 AlKiraiba -4 Kuwait 133 AlKonoz calf Omshatain State Original sample

Gedarif Gedarif ElShymalyiah North Kordofan North Kordofan Gezira Gezira White Nile White Nile Gezira Gezira Gezira White Nile White Nile White Nile White Nile Probang Probang Probang Probang Probang Swab Swan Probang Probang Epithelium Epithelium Epithelium Epithelium Epithelium Epithelium Epithelium description of tested sample by Ag ELISA Cell culture harvest Cell culture harvest Cell culture harvest Cell culture harvest Cell culture harvest Cell culture harvest Cell culture harvest Cell culture harvest Cell culture harvest Cell culture harvest epithelium suspension Cell culture harvest Cell culture harvest Cell culture harvest Cell culture harvest Cell culture harvest Result

Negative Negative Negative Negative Negative Negative Negative Negative Negative SAT2 positive SAT2 positive SAT2 positive Negative Type O positive Negative Negative

4. DISCUSSION

In studying the current status of FMD in the Sudan, it is obvious that FMD is still endemic in the country. It occurs mostly, in the cold dry season. The extensive livestock husbandry systems adopted in the Sudan seems to favor the conditions for spread of FMD virus. Cattle reared under nomadic conditions in the Sudan use their feet to wander around for grazing which may extend for many kilometers and use the tongue for prehension of grass while eating, but when these functions are impaired by FMD lesions in feet and mouth they will be recumbent and mostly suffer from starvation (Abu Elzein, personal communication, 2008). These observations add a further dimension to the economic significance of FMD in the Sudan and clearly enfeeble the notion in enzootic areas that FMD is not particularly a serious disease and its relevance is only to international trade. Doel (2003) observed that FMD might have devastating effects on animals and herdsmen regardless of animal population. In the present study beside Gezira, North Kordofan and River Nile States that were previously screened by Abu Elzein et al. (1987), another four states were included in the recent study viz. South Kordofan, White Nile, Gedarif and El-Shymalyah states. After 20 years from the last study on FMD in the Sudan, and without applying any vaccination or eradication programmes, the prevalence of antibodies to FMDV serotypes has changed both in magnitude and order. In cattle antibodies to serotype "A" surpassed that of serotype "O" and antibodies to serotype "SAT2" surpassed that to serotype "SAT1". In this work in cattle, antibodies to serotype "A" and "O" showed a prevalence rate of 78.1% and 69.4% compared to 18% and 75.6% respectively in the previous study (Abu Elzein et al, 1987). Antibodies to serotype"SAT2" and "SAT1" showed a prevalence rate of 44% and 20.2% compared to 0.2% and 6.4% respectively in the previous study (Abu Elzein et al, 1987). These results coincided with the results of the recent serosurveilance in cattle species in Khartoum state (Raouf; 2007 report of FMD UNIT). In sheep and goats, the prevalence rate was also changed, serotype O was found to be the most prevalent serotype in sheep and goats (27.5% each) instead of serotype A in sheep and SAT1 in goats in previous reports (Abu Elzein et al, 1987), moreover, antibodies to SAT2 were detected for the first time in sheep and goat sera from investigated states 8.99% and 2.38 % respectively. Similar result was observed in recently surveyed sheep and goats' sera in Khartoum state (Habiela et al., unpublished data). Similar to previously published data ( Abu Elzein et al, 1987), detected antibodies to FMDV serotypes in sheep and goats sera revealed much lower prevalence rates than that detected in

The Global control of FMD- Tools, ideas and ideals – Erice, Italy 14-17 October 2008

5. ACKNOWLEDGEMENTS:

We thank Prof. M. Tibin, Director General of ARRC for his support, Ministry of animal resources and fisheries, and all colleagues who helped us in this study. We indebted to the staff of both FMD unit and Department of Virology at CVRL, to Dr. K. Sumption- EUFMD- Rome and Pirbright staff for their

cattle sera. This could be due to the fact that some of the screened flocks of sheep and goats graze without intermingling with cattle, as in some parts of North Kordofan, River Nile and Gedarif states.

Figure 3: Prevalence antibodies to FMDV in different animal species sera screened by Abu Elzein et al. in 1987.

The high prevalence rates of FMDV serotypes "A", "O" and "SAT2" antibodies detected in cattle species were consistent with the isolation of serotype "O" and "SAT2" in this work and with the recent reports of FMDV isolates of serotype O, A and SAT-2 from the Sudan by Pirbright Laboratory, UK (Anon, 2007 ) . In the Sudan, camels frequently browse in contact with other ruminants under free range conditions and at watering points. Screening of camels’ sera by LPB ELISA, which was used for the first time in the Sudan, revealed that no antibodies to any of the four FMDV serotypes used in the present study were detected. This result is in agreement with the findings of Abu Elzein et al (1984) who reported that Sudanese camels were seronegative to FMDV antibodies. Moreover, these findings are consistent with recent reported showed that dromedary camels are not susceptible to FMDV and do not show detectable serological response against it even under experimental conditions (Wernery and Kaaden 2004; Larska et al., 2008). Theoritically, FMDV carrier animals might harbor infectious virus in their esopharyngeal region (Sutmoller et al, 2003; Alexandersen et al, 2002), then the risk of transmitting the virus to other susceptible animals cannot be ruled out. Collection of OP samples from previously infected animals revealed that no FMDV was recovered or detected. In spite of that 81% of probang samples caused CPE in BTY cell culture and 56% of re-inoculated probang samples in BK caused CPE. This could be due to the known difficulty of isolation FMDV from such samples (Alexandersen et al., 2002; Abu Elzein, personal communication, 2008), or that, the examined animals were actually free from FMDV. SAT2 serotype being the most recently serotype introduced in the Sudan in seventies (Abu Elzein, 1979), this serotype is often associated with outbreaks in sub-Saharan Africa (Bastos et al. 2003). It showed wide spread as it could be deduced from our results of serosurvey and disease survey. Serotype SAT2 antibodies were detected in all the investigated states, and these virus serotype was isolated from one outbreak during the course of this study. In conclusion, Sudan is a vast country with different ecosystems and massive diverse animals' species. Our results indicated that FMD was detected in all the seven investigated states. The prevalence picture of the virus serotypes has changed. For instance, the SAT-2 serotype has spread and was involved in most of the recent FMD outbreaks in the country; this coincided with the results that obtained from Khartoum state survey (Raouf et al., 2008; Habiela et al., unpublished data). Further extensive serosurveillance, disease monitoring and phylogenetical analysis of the recent FMDV isolates are required so as to evaluate the real situation of FMD in the country.

The Global control of FMD - Tools, ideas and ideals – Erice, Italy 14-17 October 2008 assistance and to Prof. E. Abu Elzein and Dr. M. Badawi for reading the manuscript. This paper is published by permission of the Ministry of Animal Resources and Fisheries, Republic of the Sudan.

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