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Isolation and Identification of Foot and Mouth Disease Virus from Clinically
Infected Cattle in Ada Veterinary Clinic

Article · January 2015

DOI: 10.5829/idosi.aejsr.2015.10.6.101125

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American-Eurasian Journal of Scientific Research 10 (6): 368-374, 2015
ISSN 1818-6785
© IDOSI Publications, 2015
DOI: 10.5829/idosi.aejsr.2015.10.6.101125

Isolation and Identification of Foot and Mouth Disease Virus from

Clinically Infected Cattle in Ada Veterinary Clinic
1
Hassen Belay and 2Yimer Muktar

National Veterinary Institutes, Bishftue, Ethiopia

1

2
Haramaya University, College of Veterinary Medicine, DiraDwa, Ethiopia, P.O. Box 138

Abstract: Foot and mouth Disease (FMD) is a highly contagious and economically important disease of cloven-
hoofed animals, predominantly for cattle, sheep and pigs. This study was designed to isolate and identify the
serotypes of FMD virus from clinically suspected cattle in Ada veterinary clinic. Animals were clinically
examined and tissue and serum samples were collected from each of FMD suspected cases. Out of 38 submitted
samples, FMD virus was isolated from 36 (94.7%) of the samples on cell culture. Serotype identification was
made using virus neutralization test (VNT) of the viral isolates and complement fixation test (CFT) of sera of
positive cattle. The result from both tests Virus Neutralization Test (VNT) and Complement Fixation Test (CFT)
showed that 24 (66.7%) of the animals were positive for serotype O while the remaining 12 (33.3%) were infected
with South African territories type 2 (SAT2). Therefore, taking the most contagious nature of the disease, its
economic impact and the non-existence of specific treatment after infection into account; practicing preventive
strategies is the only means recommended to eradicate the disease from Ethiopia and to make the country to
benefit from its huge livestock industry.

Key words: SAT2 FMD Serotypes DebreZeit Virus Isolation Foot-And-Mouth Disease

INTRODUCTION Of the domesticated species, cattle, pigs, sheep,

goats and buffalo are susceptible to FMD while many
Foot and mouth disease (FMD) is the most species of cloven-hoofed wildlife, such as deer, antelope
contagious viral disease of cloven-hoofed animals. The and wild pigs may become infected [7]. [8]. Transmission
disease is characterized by fever and vesicular eruptions is generally affected by contact between infected and
on the feet, buccal mucosa and, in females, the mammary susceptible animals to the excretions and secretions of
gland [1, 2]. FMD is caused by a virus of the genus acutely infected animals [2].
Aphthovirus, in the family picornaviridae. There are seven FMD severely constrains international trade in
immunologically district serotypes; O, A, C, South African animals and animal products[9]. FMD has considerable
Territories (SAT) 1, SAT2, SAT3 and Asia1. FMD viruses economic consequence and can be attributed to both
are small RNA viruses that are enclosed with a non- direct and indirect costs. The direct effects of the disease
enveloped protein shell (Capsid) [3]. The presence of are loss of milk production, loss of draught power,
seven serotypes and multiple subtypes and variants has retardation of growth, abortion in pregnant animals, death
added to the difficulty of laboratory diagnosis and control in calves and lambs while the indirect losses are attributed
of FMD. The rise of new variants is inevitably caused by to the disruption in trade of animals and derivative
continued circulation of the virus in the field and quasi- products [10]. The disease causes the greatest production
species nature of RNA genome [5]. RNA viruses in losses in cattle and pigs and in particular in intensive
general and FMD virus in particular have very high dairy and pig production systems [11].
mutation rates, in the range of 10 3 to 10 5 per nucleotide Today, many countries have either eliminated FMD
site per genome replication, due to the lack of error by compulsory slaughter of infected animals or have
correction mechanisms during RNA replication [6]. reduced its incidence greatly by extensive vaccination

Corresponding Author: Yimer Muktar, Haramaya University, College of Veterinary Medicine,

DiraDwa, Ethiopia, PO. Box 138. Tel: +251 927847172. E-mail: yimermktr21@gmail.com.
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 Am-Euras. J. Sci. Res., 10 (6): 368-374, 2015

programs [8]. FMD is still a major global animal health Addis Ababa. It lies 9° N latitude and 4° E longitude at an
problem, but its geographic distribution has been altitude of 1850 meter above sea level central highlands of
shrinking in recent years as control and elimination Ethiopia. The area has an annual rainfall of 866 mm of
programs have been established in more and more which 84% is in the long rainy season from June to
countries [8]. Western and central Europe, North America, September. The dry season extends from October to
parts of South America, South Africa, Australia and some February. The mean annual maximum and minimum
island regions of Asia are currently recognized as being temperatures are 26°C and 4°C, respectively, with mean
free of FMD. However, the disease is still widespread in relative humidity of 61.3% [17].
many countries of Africa, Asia and the Middle East [9]. Cattle admitted in Ada veterinary clinic were
The epidemiology of FMD in sub-Saharan Africa is registered and all case histories were collected and
probably more complicated than in any other region of the detailed physical clinical examinations were carried out
world. Not only have six of the seven serotypes occurred according to standard procedures [2].
in Africa (Only Asia 1 has never been recorded), but also The cattle presented in the clinic were local breeds
marked regional differences in the distribution and which were brought from different peasant associations
prevalence of serotypes and intratypic variants occur [12, (PAs) around DebreZeit and they are managed in
13]. intensive production systems. However, all the cattle were
FMD was first recorded in Ethiopia in 1957 when not vaccinated against FMD. The owners complained that
serotypes O and C were found [14, 15]. The recent study the cattle showed rapid drop in feed and water
conducted by Ayelet et al. [16] on FMD samples consumption and they had lesions in their mouth and on
collected between 1981 to 2007 throughout the country the feet. Upon clinical examination of FMD suspected
from different species of animals showed that serotype O, animals; they have showed a varying degree of clinical
A, C, SAT1 and SAT 2 were recorded. Serotype O was the signs. Most of the animals were febrile with vesicular
dominant serotype identified with the degree of 73.3% and lesions on their feet, buccal mucosa and tongue with
widely distributed throughout the country, while the rate drooling of saliva. Generally, the disease was more severe
for serotype A was 19.5%, C 1.4%, SAT 2 was 4.1% and in young than in adult animals and milking cows showed
SAT 1 was 1.8 % with limited distribution. a drop in milk production. Animals showing such
Such a complex epidemiological situation of FMD in symptoms were tentatively diagnosed for Foot and mouth
Ethiopia due to the circulation of multi-serotypes of the disease (FMD) and thirty eight animals showing clinical
virus and involvement of four different host-species as signs of FMD were selected for the study.
well as additional factors such as the presence of high
numbers of wildlife (Especially African buffalo), rooming Collection and Transportation of Samples: Both epithelial
free cross borders between the neighboring countries and tissue and serum samples were collected from each of the
lack of control of animal movements urges the need of thirty eight FMD suspected cattle.Epithelial samples were
having appropriate research, diagnostic and controlling collected from unruptured vesicles in the buccal mucosa
programs of the disease. Vaccination against one and tongue of the cattle. The area over the vesicles was
serotype of FMD virus does not cross-protect against washed with phosphate-buffered saline (PBS) to remove
other serotypes and may also fail to protect fully or at all gross contamination such as feed. Using sterile scissors
against other subtypes of the same serotype [2]. The and forceps, a piece of epithelial sample at least 2cm x 2cm
objective of this study was therefore aimed: to isolate and (Postage stamp size) was collected without any attached
identify FMD virus serotypes causing disease outbreaks subcutaneous fat or muscle. The samples were then
around DebreZeit so as to design appropriate prevention placed in sterile screw capped test tubes containing
measures. transport medium composed of equal amounts of glycerol
and 0.04 M phosphate buffer, pH 7.2–7.6, with added
Study Area, Clinical Case Description and Clinical antibiotics (Penicillin [1000 International Units (IU)],
Examination: The study was conducted in Ada veterinary neomycin sulphate [100 IU], polymyxin B sulphate [50 IU],
clinic and National Veterinary Institute (NVI) over a period mycostatin [100 IU]). The samples were then labeled with
of November 2010 to May 2011. The clinic is located in the tag of the animals and dates of collection, kept in an
DebreZeit town near Genesis farms while NVI is also ice box, transported and submitted to NVI virology
located in DebreZeit in Kebele 15 next to AAUSVM. laboratory. Sampling was done following procedures
DebreZeit is located 45kms south east of the capital city, described in OIE [2].

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 Am-Euras. J. Sci. Res., 10 (6): 368-374, 2015

In addition, blood was collected from the jugular inactivated at 56°C for 30 minutes before testing. The
vein of each of FMD suspected cattle with a plain control standard serum was 21-day convalescent serum
vacationer tube, labeled and it was allowed to clot. The (Usually pig). Hank’s balanced salt solution with yeast
serum was then harvested in a cryovial and labelled. The lactalbuminhydrolysate and antibiotics was used as a
cryovials were packed with a plastic container and kept in medium. VNT was performed according to procedures
an ice box for transportation to NVI immunology described in OIE [2]. Positive wells (where the virus has
laboratory. been neutralized and the cells remain intact) were seen to
contain blue-stained cells sheets; the negative wells
Sample Storage and Processing: Standard sample (Where virus has not been neutralised) were empty. Titres
storage and processing for FMD test was employed (OIE, were expressed as the final dilution of antigen present in
2009). Epithelial samples were stored at -20°C until virus the serum/virus mixture at the 50% end-point. A titre of
isolation. The samples were taken from the transport 1/45 or more of the final antigen dilution in the
medium, blotted dry on absorbent paper to reduce the serum/virus mixture was regarded as positive. Titres of
glycerol content, which is toxic for cell cultures and 1/16 to 1/32 were considered to be doubtful and further
weighed. A suspension was prepared by grinding the antigen samples were requested for testing. Animals were
samples in sterile sand in a sterile pestle and mortar with considered to be positive if the second sample has a titre
a small volume of tissue culture medium (pH = 7.2) and of 1/16 or greater. A titre of 1/8 or less is considered to be
antibiotics under a class II safety cabinet. Further medium negative.
was added until a final volume of five times that of the
epithelial sample has been added, giving a 20% Complement Fixation Test (CFT): Serum samples were
suspension. This was clarified on a bench top centrifuge tested by CFT at National Veterinary Institute (NVI),
at 2000g for 10 minutes. Once clarified, such suspensions DebreZeit Ethiopia, according to the protocol described
of field samples suspected to contain FMD virus were in OIE manual [2].Sedimentation of hemolytic system
harvested and used to inoculate into cell cultures and the (SRBC) was taken as a positive result while full hemolysis
serum samples also stored at -20°C until they were tested was observed in negative results.
for the presence of specific antibodies by the complement
fixation test. Data Analysis: Data collected during the study period
were stored in the Microsoft Excel spread sheet
Analysis of Samples programand analyzed using STATA 17.and descriptive
Virus isolation on BHK-21 Cell Cultures: Established statistics and frequency was used.
cell layer of baby hamster kidney (BHK-21) cells were
inoculated with 0.2 ml of each of the clarified suspension RESULTS AND DISCUSSION
of epithelial tissue samples. The cell cultures were
examined for Cytopathic effects (CPE) for 48 hours within Virus Isolation: Of the total 38 clinical samples examined,
6 hrs of interval. Cytopathic effect (CPE) was observed cytopathic effect (CPE) was observed in 36 (94.7%)
after 48 hours (Or even less) in positive cases. If no CPE samples in BHK-21 cell cultures for FMD virus, but the
was detected, the cells were frozen and thawed, used to other 2 (6.3%) didn't show any CPE by blind passage 5
inoculate fresh cultures and examined for CPE for another times (Table 1). The CPE was characterized by fast
48 hours before the samples were declared to be negative. destruction of the cell monolayers and infected cells were
The infected BHK-21 monolayer cells were subjected to round and formed singly. Complete destruction of the cell
three freeze-thaw cycles to release the viral particles from sheet was mostly seen within 48 hours of inoculation
the cells. The viral suspension was clarified from the cell (Figures 2-5). The control cells remained intact after 48
debris by centrifugation at 800 × g for 10 min and stored hours of incubation.
at -70°C for virus neutralization test (VNT).
Virus neutralization (VNT) and Complement Fixation
Virus Neutralization Test (VNT): The quantitative VN Test (CFT): FMD virus serotype identification was
microtest for FMD antibody was performed with BHK-21 conducted by testing the first passages of those samples
cells in flat-bottomed cell-culture grade microtitre plates. that showed CPE using virus neutralization test and sera
Stock virus was grown in cell monolayers and stored at of those animals were prepared for complement fixation
–20°C after the addition of 50% glycerol. The sera were test.

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 Am-Euras. J. Sci. Res., 10 (6): 368-374, 2015

Table 1: Summary of cell culture results of suspected cattle in Ada veterinary clinic
Number of inoculated Number (%) of samples showing Number of samples that didn’t
Type of sample Animal species flask/sample CPE upto the 1st passage show CPE after 5th passage
Epithelial tissue cattle 1 36 (94.7%) 2 (5.3%)

Table 2: Results of virus neutralization and complement fixation tests

Number (%) of Serotypes identified from the samples examined
---------------------------------------------------------------------------------
Type of lab test Type of samples No of samples examined SAT2 O
VNT Isolated virus 36 12 (33.3%) 24 (66.7%)
CFT Serum 36 12 (33.3%) 24 (66.7%)

A. at the time of inoculation B. 48 hrs after inoculation

Fig. 1: BHK-21 cell uninoculated controls

Fig. 2: Cytopathic effect observed after 12 hrs of incubation

Fig. 3: Cytopathic effect observed after 18 hrs of incubation

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 Am-Euras. J. Sci. Res., 10 (6): 368-374, 2015

Fig. 4: Cytopathic effect observed after 24 hrs of incubation

Fig. 5: Cytopathic effect observed after 48 hrs of incubation

A. Antibodies virus isolates neutralized by specific antigen B. Virus isolates showing CPE on microplate that
was not neutralized by antibodies
Fig. 6: Results of virus neutralization test

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 Am-Euras. J. Sci. Res., 10 (6): 368-374, 2015

Out of 38 samples collected from clinically FMD ovine and caprine samples collected from the outbreak
suspected cattle, 36 (94.7%) were confirmed to be positive areas. However, serotype C was not detected since 1983
VNT and CFT. Samples from the remaining two cattle were [11].
found to be negative for FMD virus and circulating
antibodies. Serotype identification of FMD virus of CONCLUSION
positive animals was performed by virus neutralization
test (VNT) of the isolated virus and complement fixation The occurrence of 5 serotypes and multiple variants
test of their sera. Results from both tests have shown that of FMD virus in the country, its economic impact and the
24 (66.6%) of the positive cattle were infected with highly contagious nature of the disease urges to
serotype O while the remaining 12 (33.3%) were infected implement appropriate eradication of FMD from Ethiopia
with South African Territories type 2 (SAT2) serotype of so as to enable the country to benefit from its huge
FMD virus (Table 2). livestock potential. In this study, O and SAT2 serotypes
Based on the clinical examination and laboratory of FMD virus were detected on samples collected from
analysis performed, it can be concluded that 36 of the clinically presented and FMD suspected cattle in Ada
cases out of 38 under investigation were identified as veterinary clinic. This doesn’t mean that other serotypes
FMD. 66.7% of the cases under investigation were found of the virus are not occurring in and around DebreZeit
to be caused by serotype O while the remaining 33.3% since the study was conducted on those animals which
were caught by serotype SAT2 of FMD virus. The result came into the clinic within a period of six months only.
of the current study is fairly agreed with Ayelet et al. [11] Furthermore, it was not possible to identify the subtype
reviewed that, serotype O was identified in 1979 by variants due to the absence of FMD-specific primers to
WRLFMD on samples collected from DebreZeit in 1977. perform molecular techniques.
However, no published data is available on the Accordingly, the following recommendations are
occurrence of SAT2 in and around DebreZeit. Therefore, forwarded:
this study implied that other serotypes of FMDV are
emerging and this could lead to the difficulty of Efforts should be made to make information available
eradicating the disease from the area as well as the on the antigenic and geneticcharacteristics of
country at large. isolates collected in all regions of the country.
Since the first report of FMD, Serotypes A and SAT2 Ppolyvalent vaccines must be produced by the NVI
were not identified until 1969 and 1989, respectively [15]. including serotypes O, SAT2 and A which were
During 1988 to 1991 samples from 16 FMD outbreaks in found to be the most prevalent serotypes in recent
Ethiopia were examined at the National Veterinary years.
Institute (NVI), Ethiopia and at the Food and Agriculture A better coordination between regional and central
Organization World Reference Laboratory for Foot-and- laboratories must be made to improve the
Mouth Disease (WRLFMD), UK. Typing of the virus surveillance of FMD in the country.
responsible was possible in 13 of these outbreaks A more comprehensive system of vaccine matching
representing 10 separate disease events; eight of these should be made in order to select the subtypes of
were caused by serotype O and two by serotype SAT2. In FMDV serotypes with the best ability of cross-
contrast to earlier studies serotypes A and C were not protection as vaccinal strains.
detected [18]. Antibodies to SAT 2 were also detected in Further detailed Epidemiological and molecular study
1971 from cattle in North Omo, Southwestern Ethiopia [14, should be initiated to select the FMDV serotype
18]. From records of outbreak investigations conducted circulating in the county.
by NVI between 1982 to 2000, serotype O, A and SAT 2
FMD viruses were also identified [19]. The records of ACKNOWLEDGEMENTS
ministry of agriculture and rural development (MOARD)
from 2000 to 2006 indicated that FMD outbreak occurred We would like to forward our appreciation for staff
every year with the highest in 2001 with 88 outbreaks. members of National Veterinary Institute for their
FMDV is still active in many parts of Ethiopia; however, cooperation and allowance of the laboratory and all animal
only limited efforts were made to characterize FMD virus owners to allow sampling of their herd’s for the study to
isolates. During 1981 to 2007, a total of 5 serotypes (O, A, accomplish this work and Ada veterinary clinic the staffs
C, SAT 1 and SAT 2) were identified in bovine, swine, also acknowledged for their cooperation.

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 Am-Euras. J. Sci. Res., 10 (6): 368-374, 2015

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