DETECTION OF ORTHOPOXVIRUS ANTIBODIES IN SERA OF SOME DOMESTIC SPECIES IN THE SUDAN
BY Siham Tag Elsir Karam Alla Alkidir
(B.V.M.2002, University of Khartoum)
Athesis submitted to the University of Khartoum in
fulfillment of requirement for the degree of Master of Veterinary Medicine by Research
Department of Microbiology Faculty of Veterinary Medicine
University of Khartoum
(June 2008)
DEDICATION
To my mother, father
Husband, daughters,
Sisters, brothers
And friends
With deepest love
Acknowledgments
Praise to Allah who gave me the ability and health to accomplish,
this research work.
My sincere thanks and gratitude to my supervisor Prof.
Abdelmalik Ibrahim Khalafalla for his guidance, support and
encouragement throughout this work.
I am also indebted to Dr.Tag Elsir Mohamed, Central Veterinary
Research Laboratory in Soba for his help and provision of some essential
materials. My deep thanks to my colleague Dr. Afraa Tagelsir for her help
and support. I am extremely grateful to the staff of the Department of
Microbiology, Faculty of Veterinary Medicine, University of Khartoum,
for providing facilities to conduct this work, special thanks to Mr.Sharani
Omer for his technical assistance during the laboratory work, And to Miss.
Mawahib Awad for her assistance during this study. My gratitude is also
extended to all staff of the Department of Preventive Medicine, namely Dr.
Kitma Hassan Elmalek, Dr. Abdelwahid Saeed for their support, and Mr.
Hussein Abdelraheem and Miss. Zainab Altayeb for their assistance. Also I
want to thank Prof. Ahmed Gameel, Department of pathology, for his Help
in histopathology.
TABLE OF CONTENT
DEDECATION .................................................................................... i
ACKNOWLEDGMENT ..................................................................... ii
TABLE OF CONTENT ..................................................................... iv
LIST OF FIGUERS.......................................................................... viii
LIST OF TABLES ............................................................................. ix
LIST OF ABBREVIATIONS ............................................................. x
ARABIC ABESTRACT .................................................................... xi
ENGLISH ABESTRACT ................................................................. xii
INTRODUCTION............................................................................... 1
CHAPTERI: LITERATURE REVIEW .............................................. 3
1.1 Diseases caused by viruses of the genus orthopoxvirus ................ 3
1.2 Camel pox ..................................................................................... 3
1.2.1 Etiology of CP ............................................................................ 3
1.2.2 Importance of the disease ........................................................... 4
1.2.3 Epidemiology of CP ................................................................... 4
1.2.4 Clinical symptoms ...................................................................... 6
1.2.5 Diagnosis of CP.......................................................................... 7
1.3 Cowpox ......................................................................................... 8
1.3.1 Cause of cow pox ....................................................................... 8
1.3.2 Occurrence ................................................................................. 8
1.3.3 Transmission .............................................................................. 8
1.3.4 Clinical & Pathologic Features................................................... 9
1.3.5 Diagnosis.................................................................................... 9
1.3.6 Prevention ................................................................................ 10
1.3.7 Public health significance......................................................... 10
1.4 Poxviridae ................................................................................... 10
1.5 Taxonomic structure of the family .............................................. 10
1.6 Orthopoxvirus ............................................................................ 11
1.7 Classification............................................................................... 12
1.8 Morphology................................................................................. 13
1.9 Physiochemical and physical properties...................................... 14
1.9. 1 Nucleic acid............................................................................. 14
1.9.2 Lipids ....................................................................................... 14
1.10 Diagnosis of Orthopoxvirus ...................................................... 14
1.11 Vaccinia virus............................................................................ 16
1.12 Basic biology............................................................................. 16
1.13 Host resistance........................................................................... 17
1.14 Origin ........................................................................................ 18
1.15 Use as a vaccine ........................................................................ 18
1.16 History....................................................................................... 19
CHAPTER II: MATERIALS AND METHODS .............................. 20
2.1 Virus strain .................................................................................. 20
2.2 Hyper immune serum .................................................................. 20
2.3 Serum samples ............................................................................ 20
2.4 Preparation and sterilization of glassware ................................... 20
2.5 Preparation of solution and cell culture media ............................ 21
2.5.1 Phosphate buffer saline (PBS) and phosphate diluent (PD) ..... 21
2.5.2 Glasgow Minimum Essential Medium (GMEM) ..................... 21
2.5.3 Tryptose phosphate broth (TPB) .............................................. 21
2.5.4 Lactalbumin hydrolysate .......................................................... 21
2.5.5 1%Yeast extract solution.......................................................... 21
2.5.6 7.5%Sodium bicarbonate solution ............................................ 21
2.5.7 Thioglycolate medium.............................................................. 22
2.5.8 Trypsin-Versene solution ......................................................... 22
2.5.9 Penicillin-Streptomycin solution .............................................. 22
2.5.10 Fungizon solution................................................................... 22
2.6 Embryonated Chicken Eggs ........................................................ 22
2.6.1 Chorioallantoic Membrane Inoculation.................................... 23
2.6.2 Harvest of Chorioallantoic Membrane ..................................... 23
2.7 Pock lesion histopathology.......................................................... 24
2.8 Preparation of Cell Culture.......................................................... 24
2.9 Virus propagation........................................................................ 25
2.10 Virus titration ............................................................................ 25
2.11 Agar Gel preparation ................................................................. 25
2.12 Agar gel immunodiffusion test .................................................. 25
2.12.1 Preparation of agar gel ........................................................... 25
2.12.2 Standardization of AGID test ................................................. 25
2.12.3 Examination of the sera for antibodies against Orthopox viruses
using AGID test……………… …. ………………………………...26
CHAPTER III: RESULT .................................................................. 27
3.1Growth of Vaccinia virus on the chorioallantoic membrane ........ 27
3.2 Histopathology of the CAM0 ...................................................... 27
3.3 Growth of vaccinia virus in Cell Culture..................................... 27
3.4 Sero-prevalence of orthopox virus in Sheep, cattle and camel
serum samples ............................................................................. 27
CHAPTER IV: DISCUSSION.......................................................... 35
CONCLUSIONS............................................................................... 40
REFERENCES.................................................................................. 41
APPENDICES................................................................................... 52
LIST OF FIGURES
Figure 1 Pock lesions produced by Vaccinia virus on CAM of
embryonated eggs................................................................................ .31
Figure 2 CAM section showing swollen of the cells with pale cytoplasm
and some inclusions bodies……………………………………………32
Figure 3 CAM section showing foci of epithelial thickening…….…...33
Figure 4 Vero cell culture infected with Vaccinia Virus showing
rounding and plaque formation ………………………………………34
Figure 5 Precipitation line in AGID test for detection of antibodies
against orthopox viruses……….………………………………..…….35
LIST OF TABLES
Table 1 Testing sera collected from cattle, sheep and camels For antibodies against orthopox virus antibodies by AGID……………………………………………………………………29 Table 2 Testing sera collected from different areas in Khartoum for antibodies against orthopoxvirus antibodies by AGID……………………………………………………………………30
ABBREVIATIONS
AGID: Agar gel immuno diffusion test.
CAM: Chorioallantoic membrane.
CPE: Cytopathic effect.
CPV: Camel pox virus.
DNA: Deoxyribonucleic acid.
DDW: Deionized distilled water.
ELISA: Enzyme-linked immunosorbent assay.
GMEM: Glasgow minimum essential medium.
H&E: Haematoxillin and Eosin.
OPV: Orthopoxvirus.
PBS: Phosphate buffer saline.
PC R: Polymerase chain reaction.
PDB: Phosphate diluent buffer.
TPB: Tryptose phosphate broth.
VV: Vaccinia virus.
ملخص االطروحه
هدفت هذه الدراسه لمعرفة بعض الخصائص الحيويه لفيروس الفاآسينيا في جنين
آما هدفت ايضا لدراسة وبائية فيروس الجدري الحقيقي .البيض النامي والزرع الخلوي
.في االبقار والضان والجمال في السودان آأول دراسه من هذا النوع ) اورثوبوآس(
من فيروس الفاآسينيا وتم اآثارها في الغشاء عترهاستعملت في هذه الدراسه
واضحه تاخذ اللون االبيض المعتم فاعطت افات المشيمي اللقانقي لجنين البيض النامي
ايضا اخذت عينات من هذه االفات واجرى عليها اختبار . ملم0.2-0.1ويتراوح حجمها بين
انتفاخ بعض الخاليا ذات سيتوبالزم , واوضحت وجود تكاثر بؤري للخاليا االنسجه المريضه
.وجود اجسام اشتماليه قاعديه وارتشاح خاليا وحيده االنويه,شاحب
واعطت اثرا مرضيا خلويا يتمثل ) آلي القرد االفريقي( فيرو ايضا تم اآثار الفيروس في خاليا
.قنينة الزرع الخلوي الخاليا عن سطح لطعات وانفكاك في استدارة الخاليا وتكوين
استخدم في هذه الدراسه اختبار االنتشار المناعي في الجل لتحديد وجود اجسام مضاده لفيروسات
من السوداناالورثوبوآس في عينات مصل جمعت من ابقار وضان وجمال من مناطق مختلفه
. ل قياسىآمص_ الفاآسينيا حقنت بفيروس _ واستخدم مصل ممنع من ارانب
اوضحت الدراسه وجود اجسام مضاده لفيروسات االورثوبوآس بنسب متفاوته في هذه
وآانت في االبل77.9% وفي الضان 35.7% حيث آانت النسبه في االبقار الحيوانات
بصوره ) اوثوبوآس( وذلك يعني انتشار االجسام المضاده لفيروس الجدري الحقيقي 53.5%
.والضان والجمال في السودان واسعه في االبقار
Abstract
This study aimed to know the biological properties of Vaccinia
virus in the Chorioallantoic membrane of the chick embryo and cell
culture. Also to study the epidemiology of orthopox virus in sheep, cattle
and camels in the Sudan as the first study from this type.
Vaccinia virus strain is used in this study for propagation in
the Chorioallantoic Membrance (CAM) of embryonated eggs. Lesions
were seen as small pock ranging from 0.1-0.2 mm in diameter, round,
opaque and white in colour. The histopathology of these lesions showed
foci of epithelial thickening, swollen cell with pale cytoplasm,
eosinophilic inclusion bodies and infiltration of mononuclear cells.
When propagated in Vero cells, this virus gave clear Cytopathic
effect characterized by rounding of the cells, plaque formation, syncytia
and detachment of cells from flask surface.
Agar Gel Immuno Diffusion Test (AGID) was used in this study
to detect orthopoxviruses antibodies in serum samples collected from
cows, sheep and camels from different areas of Sudan.
The study revealed that orthopoxvirus antibodies are present in
these animals with varying range. The seroprevalence was 35.7% in
cattle, 77.9% in sheep and 53.5 % in camel. That means antibodies
against orthopox viruses are widely distributed in sheep, cattle and camel
in the Sudan.
INTRODUCTION
The orthopoxvirus genus encompasses eight members of the
Poxviridae family of viruses, Orthopoxviruses is characterized by large
brick-shaped virus particles, containing a double-stranded DNA genome of
approximately 200,000 bp, from which vaccinia virus (VV) is the prototypic
virus. Vaccinia virus shares with its closely related virus cowpox virus the
capacity to infect a wide range of hosts, among them humans, cows, rodents
and zoo animals (Moss, 1996).
While humans are the only natural host for variola virus, both vaccinia
virus and cowpox virus have a much broader host spectrum, and the natural
reservoir for cowpox virus is most likely the rodent (Marennikov et al.,
1977).
Orthopoxviruses were pathogenic for animals and man like cowpox
and monkeypox virus (Baxby, 1988). Cowpox viruses are of interest as they
have been identified more frequently during the last three decades (Bennett
et al., 1989). Several outbreaks of severe generalized poxvirus infection in a
variety of zoo animals have been shown to be caused by these agents
(Marennikov et al., 1977). Since cowpox virus has a wide host range,
sporadic cases occur in many other mammals. In particular infection of
domestic cats is an increasingly recognized condition and several reports
documented the transmission of virus from cats to man (Eis-Hubinger et al.,
1990, Egberink et al., 1986, Czerny et al., 1991). From another member of
the genus Orthopoxvirus, camelpox virus, caused severe generalized
infections especially in young camels were reported. However, transmission
to man has not been described so far (Jezek et al., 1983).
The ability of vaccinia virus (VV), the virus used for immunization
against smallpox and the best-studied laboratory model for poxvirus biology
and immunity, to infect almost any cell line in culture has resulted in
descriptions of its broad cellular tropism and presumed widespread
expression of a virus binding receptor (Moss, 2001 and Stuart, 2004).
However, certain biological properties of vaccinia virus are not studied yet.
In this work we studied the histopathology of pock lesion made by vaccinia
virus in the chorioallantoic membrance and beside that the infection by
orthopoxviruses in cattle, sheep and camel as measured by seroconversion in
the Sudan is not determined.
Objectives of the study:
1- To determine biological properties of Vaccinia virus in particular the
growth characteristic in Chorioallantoic membrane (CAM) of embryonated
egg and cell culture.
2- To determine the prevalence of antibodies against orthopoxvirus by Agar
Gel Immunodiffusion Test (AGID) test in cattle, sheep and camel in the
Sudan.
CHAPTER 1
LETERATURE REVIEW
1.1 Diseases caused by viruses of the genus orthopoxvirus
The genus orthopoxvirus within the family Poxviridae consists of
Several species causing diseases in a wide range of animal species and in
humans (Fenner, 1996). Member of the genus Orthopoxvirus such as
camelpox, cowpox and monckeypox are pathogenic for animals and Man
(Baxby, 1988, Fenner et al., 1989).
1. 2 Camel pox
Camel pox (CP) is a systemic disease. Atypical pox exanthema
appears over the entire body and on the head; in particular Camel pox causes
a severe generalized disease in camel, with extensive skin lesion (Murphy,
Gibbs, Marian, Horzinek and Studdert, 1999). Munz, et al., (1990) described
CP as a species-specific highly contagious disease of camel characterized by
pox lesion that causes a severe out break in young animals with mortality
rate up to 10%.
1.2.1 Etiology of CP
Camel pox is caused by a virus which has been classified under the
orthopoxvirus genus of family Poxviridae (Mahnel, 1974, Meyer,
Osterrieder and Pfeffer, 1993 and Wernery and Kaaden, 2002).
The virus has the characteristic and properties of a true poxvirus and
is closely related to the vaccinia-variola group (Mahnel and Bartenbach,
1973). Andrews, Pereira and Wildey (1978) considered the virus to be a
member of the orthopoxvirus group with resemblance to the smallpox virus.
1.2.2 Importance of the disease
Camels are important live stock resource adapted to hot and cold
environment. They have been utilized by man for meat, milk, wool, hides
and transportation. Sudan is the second most density camel populated
country in the world after Somalia (Schwarz and Dioli, 1992).
The mortality in infected herds range from 25% to 100% in young animals,
and from 5% to25% in older animals (Ramyar and Hessami, 1972; Kriz,
1982). The presence of CP in Sudan was first reported in 1953 (Anon,
1954). However, identification of its causative agent has not been made yet
(Shommein and Osman, 1987).
1.2.3 Epidemiology of CP
Camels may become infected with poxvirus through small abrasions
of the skin, by aerosol infection of respiratory tract or by mechanical
transmission through biting arthropods. Several scientists have reported an
increase in CP outbreaks during wet season (Munz, 1992; Wernery, Meyer
and Pfeffer, 1997 and Wernery, Kaaden and Ali, 1997) when the diseases
become more severe. During the dry season, it usually causes a milder
course (Pfahler and Munz, 1989).
Since the CPV has been isolated from the camel tick Hyalomma
dromedarii, it is generally believed that a larger arthropod population builds
up during rainy seasons, forcing a greater virus pressure and virus doses onto
the camel populations (Wernery and Kaaden, 2002). Camels aging 2-4 years
often develop the localized form, with lesion on skin and mucous
membranes of lips and nose. Young camels up to one year old and female
camels in the final month of pregnancy are affected mainly by the
generalized form (Khalafalla and Mohamed, 1998). In two principal camel-
rearing areas of Kenya, the disease was found in Turkana where outbreaks
were detected in two herds of young animals, while in Samburu, outbreaks
were found in two herds of adult animals, as well as in two herds of young
camels. In all cases, there was 100% morbidity in the affected herds. When
the young camels were involved, the main lesions were confind to the
mouth, nose and muzzle as distinct pustular lesions. In adult animals, there
was also extensive odema of the head and neck (Gitao, 1997).
According to Munz (1992) the high prevalence of antibodies against
CPV in camel sera and the occurrence of clinical outbreak in Kenya,
Somalia and Sudan indicate that the disease may be enzootic or sometimes
epizootic in these countries. In four areas of the Sudan Kalafalla, Mohamed
and Agab (1998) found that the prevalence of seropositive animals was
higher in adults more than 4years old (87%) than in calves less than 1 years
old (40%) and than in young animals of 1-4 years old (75%) and the
prevalence rates were higher in female camels (76.5%) than in males
(66.7%).
1.2.4 Clinical symptoms
The incubation period ranges from 9-13 days, pustules develop on the
nostrils and eyelids as well as on the oral and nasal mucosa in mild cases,
presenting with generalized clinical signs such as fever, lassitude, diarrhea,
and anorexia; the eruptions are distributed over the entire body ( Werney and
Kaaden, 2002). Abortion of female camels has been reported (Brisovich and
Orekhov, 1966; Buchnev and Sadykou, 1969; Munz, 1992). Mortality can
reach 28% in generalized forms of the disease (Jezek, Kriz and Rothbauer,
1983). Secondary bacterial and mycotic infections can complicate the course
of the disease (Werney and Kaaden, 2002).
Pox-lesions were also observed in the trachea and lungs of young
dromedaries (Werney and Kaaden, 1995; Kinne, Cooper and Werney, 1998).
Classical lesions in the skin start as erythematous macules, which develop
into papules and vesicles. Vesicles develop into pustules with depressed
centers and raised erythematous borders the so called pock. After the
pustules have ruptured, they become covered by crusts. Healing of pustules
might take 4-6 weeks with or without scars (Werney and Kaaden, 2002).
Associated lymph nodes are often swollen (Munz, 1992). Mammary glands,
genitalia and anal areas are also frequently affected (Kriz, 1982) Lesions
also develop on the mucous membranes of the oral cavity resulting in
difficulty in eating and consequent loss of condition (Munz, 1992).
1.2.5 Diagnosis of CP
Preliminary diagnosis of CP is based on clinical epizootiological and
pathological findings (Buchnev et al, 1987). Confirmatory diagnosis may be
accomplished by electron microscopic detection of orthopoxvirus particles
in pox lesions, cultivation of the virus in tissue culture cells or in the
chorioallantoic membrane (CAM) of embryonated chicken eggs as well as
by serological tests (Munz, 1992).
The systematization and laboratory differentiation is of great
importance in demarcating the orthopoxvirus from the Para poxvirus, as both
viruses can be found in the same camel (Wernery and Kaaden, 1995). Newer
diagnostic methods include the ELISA technique with monoclonal
antibodies, DNA restriction enzyme analysis (Munz et al, 1992) and a dot
blot assay digoxgenin-labeled DNA probes (Meyer et al, 1993). Czerny,
Meyer and Mahnel, 1989; Johann and Czerny, 1993 and Pfeffer, Wernery,
Kaaden and Meyer, 1998) have described various laboratory methods for the
diagnosis of CP. This include electron microscopy, ELISA, immuno-
histochemistry and polymerase chain reaction (PCR). CPV-antigen detection
by immuno-histochemistry is a new method for the diagnosis of CP, which
can easily be performed in laboratories not possessing an electron
microscope. In addition to the diagnosis, immuno-histochemistry is of
particular interest for histopathologists because it facilitates visualization of
the morphological changes induced by the poxvirus (Wernery and Kaaden,
2002). Kalafalla and Mohamed (1998) identified CPV using virus
neutralization, agar gel diffusion, immunofluorecent tests and
histopathological pictures of the skin lesions. Ropp, Jin, Knight, Massung
and Esposito, (1995) developed a PCR strategy to differentiate between
orthopoxvirus species including CPV. They have successfully used this
strategy to identify virus DNA in clinical material, infected cell culture and
CAMs. Meyer, Pfeffer and Rziha, (1994) established PCR and restriction
enzyme protocols for detection and differentiation of species of the genus
orthopoxvirus.
1.3 Cowpox
1.3.1 Cause of cow pox disease
Cowpox is caused by the cowpox virus, a member of the
orthopoxvirus genus of the family poxviridae, which also includes smallpox
and vaccinia.
Cowpox virus is complex double-stranded DNA virus that have
the Potential capacity of encoding more than 200 gene products along
their ~200 kb linear genomes. Their replication cycles occur entirely
within the cytoplasmic compartment of infected host cells (Moss, 1996).
1.3.2 Occurrence
Additional hosts of cowpox virus are human beings and various
animals, including large zoo cats, domestic cats, anteaters, and rodents. The
latter are considered the natural reservoir hosts (Marennikov et al., 1974).
1.3.3 Transmission
Milkers and milking machines are the main means of spread of the
virus. Insects may also serve as mechanical vectors for the virus.
1.3.4 Clinical & Pathologic Features
Cowpox virus produces what is usually a benign infection of the
udder and teats. Papules are first seen, followed by vesicles, which rupture
leading to scab formation. Scabs drop off in about two weeks. Decrease in
milk production result from the soreness of affected teats and also from
secondary bacterial infection, which may complicate the disease and
contribute to development of mastitis (Carter et al., 2005).
1.3.5 Diagnosis
Clinical specimens: Vesicular fluid, scabs, and scrapings from lesions.
It is difficult clinically to distinguish cowpox from pseudo cowpox and other
infections of the teats. Diagnosis is most easily confirmed by the
examination of distilled water lysates of lesion material by electron
microscopy. Orthopoxviruses are "brick-shaped" as opposed to the virions of
pseudo cowpox (a Para poxvirus), which are ovoid in appearance.
Cowpox virus can be grown in cell cultures of bovine and human
origin, and on the chorioallantoic (CAM) membrane of chicken embryos.
The latter method of cultivation also provides a means to differentiate
cowpox virus from pseudo cowpox virus, which does not grow on the CA M
membrane. Vaccinia virus produces smaller pocks on the CAM membrane
than does cow pox. (Carter et al., 2005).
1.3.6 Prevention
Vaccination is not practiced. Prevention is best accomplished by
sound milking practices. Milkers and milking machines can spread the virus.
1.3.7 Public Health Significance
Milkers may contract the infection from cows. The human infection
usually involves a single, benign lesion on the hand or face. Serious
systemic disease has been reported in immunosuppressed individuals.
(Carter et al., 2005)
1.4 Poxviridae
Poxviruses are large double-stranded DNA viruses with genomes
ranging from 130 to 380 kbp (Moss, 2001). Pox viruses are complex viruses
that replicate in the cytoplasm and encode many enzymes and
immunodulatory protein (Moss, 1996). The serological relationship between
several members of the poxvirus group were investigated by a variety of
techniques, including complement fixation ,gel diffusion and ring
precipitation,heamaglutinin inhibition pock and plague, neutralization, and
staining with fluorescent-coupled antibody.(Gwendo et al.,1961) .
1.5 Taxonomic Structure of the Family
Subfamily 00.058.1. Chordopoxvirinae
Genera
00.058.1.01.Orthopoxvirus
00.058.1.02.Parapoxvirus
00.058.1.03.Avipoxvirus
00.058.1.04.Capripoxvirus
00.058.1.05.Leporipoxvirus
00.058.1.06.Suipoxvirus
00.058.1.07.Molluscipoxvirus
00.076.0.01. Trichovirus.
By ICTV –International Committee on Taxonomy of Viruses (2006).
1.6 Orthopoxvirus
Orthopoxviruses are DNA viruses and can be identified by EM. They
are large and brick shaped virions of 220-450 nm long by 140-260 nm wide,
presenting an irregularly structured surface pattern .The orthopoxvirus
genome prototype comprises a160-220 kbp linear duplex DNA with a
variable- sized invated terminal repetition (ITR) that contains distally ,
ahypovariable array of tandemly repeated sequences adjacent to covalently
closed is omerized “ hairpin’’ ends (Hollowczak, 1982, Wittek, 1982,
McFadden and Pales, 1982, Pales and Pogo, 1981, Baroudy et al., 1982a,b
,1983). Previous comparisons of genome endonuclease cleavage
electrophores patterns and maps of different orthopoxvirus indicated that
there was considerable middle region DNA conservation within the genus
and that species ,strains and variant specific differences were represented
mainly as variations in terminal region (conserved and unconserved)
nucleotide sequences and DNA lengths (Wittek et al ., 1977, Muller et al.,
1978,Espoito et al., 1978, 1981b, Mackett and Archard ,1979, Archard and
Machett, 1979, Dumbell and Archard,1980., Moyer et al., 1980b,
Schumperli et al., 1980). Orthopoxviruses exhibit extensive serologic cross-
reactivity and nucleic acid homology (Woodroofe and Fenner 1962, Baxby,
1975, 1977, Esposito et al, Moss, 1978, Mackett and Archard, 1979).
Biological and serological techniques have been used to show that the
orthopox viruses are closely related (Andrewes and Pereira, 1978). Members
of the orthopoxvirus genus share common antigens, although they differ
biologically from one to another.
1.7 Classification
Orthopoxvirus classified by International Committee of Taxonomy of
Viruses (ICTV) in the subfamily Chordoviridae of the poxviridae family.
Orthopoxvirus
Virus classification
Group: Group I (dsDNA)
Family: Poxviridae
Genus: Orthopoxvirus
Species:
Buffalopoxvirus
Camelpoxvirus
Cowpoxvirus
Ectromeliavirus
Monkeypoxvirus
Rabbitpoxvirus
Raccoonpoxvirus
Sealpoxvirus
Skunkpoxvirus
Taterapoxvirus
UasinGishudiseasevirus
Vacciniavirus
Variolavirus
Volepox
1.8 Morphology
Virions consist of an envelope, a surface membrane, a core, and
lateral bodies, or a surface membrane, a core, and lateral bodies. Virions
have a buoyant density in CsCl of 1.23-1.27 g cm-3.During their life cycle,
virions produce extra cellular particles and produce intracellular particles;
can occur in two phenotypes; may be enveloped during their extra cellular
phase. The infection is initiated by extra cellular virions. Virus may be
sequestered within inclusion bodies that are not occluded and typically
contain one nucleocapsid. Virus capsid is enveloped and virions mature
naturally by budding through the membrane of the host cell. Virions are
generally brick-shaped, or pleomorphic and measure 200 nm in diameter;
250-300 nm in length; 250 nm in height displaying tubular units. The core is
biconcave with two lateral bodies nested between the core membranes, or
between the surface membranes.
1.9 Physicochemical and Physical Properties
1.9.1 Nucleic Acid
The genome is not segmented and contains a single molecule of linear
double-stranded DNA. The complete genome is 185000 nucleotides long.
The genome has a guanine + cytosine content of 36 %. The genome
sequence has termini with cross-linked hairpin ends (i.e. single-stranded
loopes thus forming one continuous polynucleotide chain). The genome has
terminally redundant sequences. The terminally redundant sequences have
reiterated inverted terminal sequences which are tandemly repeated. The
genome sequence is repeated at both ends. Double-stranded DNA is
covalently. Double-stranded DNA is linked at both ends.
1.9.2 Lipids
Lipids are present and located in the envelope. Virions are composed
of 4% lipids by weight. The composition of viral lipids and host cell
membranes are similar. The lipids are host derived and synthesized de novo
(during the early phase of virus replication) and are derived from plasma
membranes. Viral membranes include glycolipids. (ICTV management)
1.10 Diagnosis of Orthopoxvirus
Biologic and antigenic properties are often useful for identifying and
differentiating orthopoxviruses (OPV). However, polymerase chain reaction
(PCR) amplification, with either restriction cleavage or sequencing of
amplicons, has been gaining credibility as a more rapid, specific, sensitive,
and often cost-saving technique for research and diagnostic laboratories
( Meyer et al., 2004).
Other diagnostic methods have included differentiation by viral
protein-profile following separation by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and by various serologic assays ( Arita et
al., 1977, Obijeski et al., 1973). All of these methods required virus isolation
and propagation, and clear-cut differentiation was often difficult. Therefore,
a test that can directly detect OPV in clinical specimens is desirable. More
recently, PCR methods have been developed which eliminate the need to
propagate virus (Knight et al 1995, Meyer et al., 1994, Meyer et al., 1997,
Ropp et al, 1995). But they were not suitable for strain differentiation. OPV
strain differentiation is important for several practical applications including
epidemiological investigations. OPV PCR tests were developed based on the
determination of complete DNA sequences for VAC (Goebel et al., 1990,
Johnson et al, 1993) and VAR (Massung et al., 1993) and the availability of
partial genomic sequences from other OPVs. These sequence data have
shown that a large central genomic region is highly conserved among OPV
isolates, which explains the significant degree of cross-reactivity in various
tests (Fenner, F.1996, Fenner et al., 1988). However, the data also revealed
that genes within the terminal regions have both conserved and variable
segments, and several of these genes have been associated with host range,
tissue tropism, and/or virulence of different OPV species and strains (Goebel
et al., 1990, Massung et al., 1993, Massung et al., 1996). Thus, the species-
specific genes make these terminal variable regions ideal targets for use in
PCR-based diagnostics.
1.11 Vaccinia
Vaccinia virus (VACV or VV) is a large, complex enveloped virus
belonging to the poxvirus family ( Ryan et al (2004). It has a linear double-
stranded DNA genome, which is approximately 190 kbp in length and
encodes for approximately 250 genes. The dimensions of the virion are
roughly 360 × 270 × 250 nm. Vaccinia virus is well-known for its role as a
vaccine that eradicated the smallpox disease, making it the first human
disease to be successfully eradicated by mankind. This endeavour was
carried out by the World Health Organization under the Smallpox
Eradication Program. Post eradication of smallpox, scientists have been
studying vaccinia virus to use as a tool for delivering genes into biological
tissues (gene therapy and genetic engineering). Moreover, due to recent
concerns about smallpox resurfacing as a possible agent for bioterrorism,
scientists have renewed their interests in studying vaccinia virus. .(Smith et
al., 2002).
1.12 Basic biology
Vaccinia virus is unique amongst all DNA viruses because it
replicates only in the cytoplasm of the host cell outside of the nucleus.
(Tolonen et al., 2001). Therefore, the large genome is required for encoding
various enzymes and proteins involved in viral DNA replication and gene
transcription. During its replication cycle, VV produces several infectious
forms which differ in their outer membranes: intracellular mature virion
(IMV), the intracellular enveloped virion (IEV), the cell-associated
enveloped virion (CEV) and the extra cellular enveloped virion
(EEV)(Smith et al., 2002). Although the issue remains contentious, the
prevailing view is that the IMV consists of a single lipoprotein membrane,
while the CEV and EEV are both surrounded by two membrane layers and
the IEV has three envelopes. The IMV is the most abundant infectious form
and is thought to be responsible for spread between hosts. On the other hand,
the CEV is believed to play a role in cell-to-cell spread and the EEV is
thought to be important for long range dissemination within the host
organism (Smith and Law, 2004).
Maps and sequences of vaccinia (WR) virus and cow pox (Brighton)
virus donid DNA Tandem array regions have revealed as similar core
sequence constituling the repeated units in these species (Wittek and Moss,
1980, Baroudy et al., 1982a, b, 1983, Pick up etal, 1982). Sequence analysis
of vaccinia virus DNA tips (telomeres) showed two equirmolor isomeric in
averted complementary (Flip-Flop) forms of hair pin loops (Baroudy et al.,
1982a, 1983, pick up et al., 1983)
1.13 Host resistance
Vaccinia contains within its genome several proteins that give the
virus resistance to interferon. K3L is a protein with homology towards the
protein eIF-2alpha. K3L protein inhibits the action of PKR, an activator of
interferon. E3L is another protein encoded by vaccinia. E3L also inhibits
PKR activation; and is also able to bind to double stranded RNA. (Davies
MV, 1993)
1.14 Origin
Vaccinia virus is closely related to the virus that causes cowpox.
Historically the two were often considered to be one and the same
(Huygelen C, 1996). The precise origin of vaccinia virus is unknown due to
the lack of record-tracking as the virus was repeatedly cultivated and
passaged in research laboratories for many decades (Henderson DA, Moss
B, 1999). The most common notion is that vaccinia virus, cowpox virus and
variola virus, the causative agent for smallpox, were all derived from a
common ancestral virus. There is also speculation that vaccinia virus was
originally isolated from horses (Huygelen C, 1996).
1.15 Use as a vaccine
Avaccinia virus infection is very mild and is typically asymptomatic
in healthy individuals, but it may cause a mild rash and fever. Immune
response generated from a vaccinia virus infection protects the person
against a lethal smallpox infection. For this reason, vaccinia virus was, and
is still being used as a live-virus vaccine against smallpox. Unlike vaccines
that use weakened forms of the virus being vaccinated against, the vaccinia
virus vaccine cannot cause smallpox because it does not contain the
smallpox virus. However, certain complications and/or vaccine adverse
effects occasionally arise. The chance of this happening is significantly
increased in people who are immunocompromised. Approximately one in
one million individuals will develop a fatal response to the vaccination.
Currently, the vaccine is only administered to health care workers or
research personnel who have a high risk of contracting vaccinia virus, and to
the military personnel of the United States of America. Due to the present
threat of smallpox-related bioterrorism, there is a possibility the vaccine may
have to be widely administered again in the future. Therefore, scientists are
currently developing novel vaccine strategies against smallpox which are
safer and much faster to deploy during a bioterrorism event.(from
Wikipedia).
1.16 History
The original vaccine for smallpox, and the origin of the idea of
vaccination, was cowpox, reported on by Edward Jenner in 1798 (Henderson
DA, Moss B, 1999). The Latin term used for cowpox was variolae
vaccinaeX, essentially a direct translation of "cow-related pox". That term
lent its name to the whole idea of vaccination. When it was realized that the
virus used in smallpox vaccination was not, or was no longer, the same as
the cowpox virus, the name 'vaccinia' stayed with the vaccine-related virus.
Chapter II
Materials and Methods
2.1 Virus strain
Vaccinia virus (v.v) (Elstree strain) was obtained from the virus stock
of the Virology Laboratory, Department of Microbiology, Faculty of
Veterinary Medicine, University of Khartoum.
2.2 Hyper immune serum preparations
This was prepared in rabbits using Vaccinia virus. Four rabbits with
the same age inoculated with vaccinia virus. Then we collect the serum after
10 days, 20 days and 30 days.
2.3 Serum samples
305 serum samples were collected randomly from cattle and sheep
from Khartoum state, and 101 samples were collected from camels from
Gadarif and Tambool.
2.4 Preparation and Sterilization of Glassware
Flasks, beakers, bijou, and volumetric bottles, measuring cylinders,
tissue culture bottles, tubes and other glassware were rinsed in running tap
water, brushed with soap and then rinsed several times in tap and distilled
water (D.W).The clean dry glassware were sterilized in the hot –air oven at
160c° for 1hr.Volumetric glass pipettes were soaked overnight in potassium
dichromate, then, they were washed several times in (D.W).
2.5 Preparation of Solution and cell culture Media (see appendix 1).
2.5.1 Preparation:
Solutions a, b and c were autoclaved separately at 121 °c for 10
minutes and left to cool. To prepare working solution of PBS (Ix) solutions a
and b were mixed. Solution c was then added and the final volume was
brought to 2 liters with sterile DDW. To prepare PD (Ix) solution a was
made up to 2 liters with sterile DDW.
2.5.2 Glasgow Minimum Essential Medium (GMEM)
A- Stock Solution (5x)
The entire content (125.7g) of one bottle of powdered media (Sigma)
was dissolved in 2 liter of DDW. The solution was immediately filtered
through a Millipore filter (o.22 u) under positive pressure, tested for sterility
using thioglycolate media and store at 4c°.
B- Outgrowth and Maintenance Media (see appendix 2)
2.5.3 Tryptose Phosphate Broth (TPB)
Three grams of TPB powder were dissolved in 100 ml DDW,
sterilized by autoclaving at 121c° for 10 min and stored at 4c°.
2.5.4 Lactalbumin hydrolysate
Five grams of lactalbumin powder were dissolved in 100 ml of DDW,
sterilized by autoclaving at 121c for 10 min and stored at 4c°.
2.5.5 1% Yeast extracts solution
One gram yeast extract powder was dissolved in 100ml of DDW,
autoclaved at 121c for 10 min .and stored at 4c°.
2.5.6 7. 5% Sodium bicarbonate solution (NaHCO3)
Seven and half grams of NaHCO3 were dissolved in 100ml DDW and
autoclaved at 121c° for 10 min and stored at 4c°.
2.5.7 Thioglycolate medium
Twenty nine and half grams of thioglyconate medium were dissolved
in 100ml of DDW, dispensed in bijou bottles and autoclaved at 121c° for 10
min and stored at 4c°.
2.5.8 Trypsin – Versene solution
Trypsin (2.5 % solution ) was sterilized by filtration (Millipore filter
0.22ul ) versene (5% solution ) sterilized by autoclaving, then 6 ml of trypsin
were added to 4 ml of versene and the mixed solution completed to 100 ml
with sterile phosphate diluent (1x)
2-5-9 Penicillin – Streptomycin Solution
One gram of streptomycin powder and 2 million units of penicillin
were dissolved in 10 ml of sterile DDW so that 1 ml of the prepared solution
contained 100 mg streptomycin and 20.000 units of penicillin. The solution
was kept at -20c°.
2.5.10 fungizon solution
The content of one vial of fungizon (Amphotericin B 50 mg) was
dissolved in 10 ml of sterile DDW and kept at 4c°.
2.6 Embryonated Chicken Eggs
Embryonated chicken eggs were obtained from the poultry unit of
Virology Reseach Laboratory (VRL) Dept. of Microbiology, Faculty of
Veterinary Medicine, University of Khartoum, Shambat.The eggs were
cleaned, disinfected and incubated at 37c° in a humidity range of 60 -65%.
Embryonated chicken eggs were used for production of pocks on the
chorioallantoic membrane (CAM) at the age of 10- 12 days. Embryonated
eggs were candled in dark room to check for embryo viability.
2.6.1 Chorioallantoic Membrane Inoculation
A cross was made in an area over the air sac and another one on the
egg side using a pencil. Eggs were then swabbed with 70% alcohol and a pore
was made at the crosses and drilling was made just deep enough to penetrate
the shell membrane to ensure that each chorioallantoic membrane (CAM)
drops to from a false air sac. To drop the membrane, a rubber bulb was used.
The bulb was placed over the hole in the air cell end of the egg and slowly
aspirated from the cell by releasing pressure on the deflated bulb. Holes were
drilled to the proper depth, to create a false air sac to from in the area of the
second hole. Eggs were inoculated by chorioallantoic membrane (CAM) route
with sterile disposable I ml syringes. Using just the tip of the needle, 0.1-0 2
ml of inoculum was injected from the syringe. The pores were sealed with
melted paraffin wax. Eggs were incubated at 37c° for 5 days with daily
candling to check for embryo death.
Embryos that died during 24 hours of inoculation were considered as
nonspecific and discarded and those that survived thereafter were killed by
chilling at 4c°.
2.6.2 Harvest of Chorioallantoic Membrane
Eggs were removed from the refrigerator, disinfected with 70 %
alcohol and the shell over air sac was removed using sterile forceps. The
embryo and yolk were extracted with forceps. Care was taken not to disturb
the CAM, and the area of inoculation was examined for lesion before
removal from the shell, the CAM was then detached from the shell with
sterile forceps, stripped of excess fluid with another forceps and placed in a
sterile Petri dish, then examination of the pock lesion was performed. CAM
samples showing pock lesion were collected and homogenized using sterile
mortars and pestles with aid of sterile sand. Samples were centrifuged at
1000 rpm for 10 min. Supernatant fluids were collected into sterile bottles
and treated with antibiotics and then stored in sterile bijou bottles at -20c°.
2.7 Pock lesion histopathology
Pock lesion were collected and fixated in 10% neutral formalin for 2
days. First the tissues were cutting into small square pieces about one cubic
cm. Section were put in 60% alcohol for ½ an hour, 70% alcohol during the
day (4_5hrs), 90% alcohol overnight, 100% alcohol 6 hrs (2 hrs each),
chloroform overnight, and melted paraffin wax 3-4 hrs and quickly cooled.
Section were cutted with aid of rotary microtome ,and transferred in a warm
water path containing little amount of gelatin powder , and left to float then
fixed to the slide glass and then incubated for 30 minutes at 60 c° to dry.
We used zylene to remove wax from the section for 10 minutes, and used
100% alcohol to remove Zylene for 10 minutes, and then the section were
rehydrated by rinsing in 90% alcohol for 5 min., 70% alcohol for 5 min and
D.W for 5 min. The section were then stained with Heamatoxyline and
Eosin (H&E) and covered with a cover glass, then dried overnight at room
temperature and examined microscopically.
2.8. Preparation of Cell Culture
A flask containing confluent monolayer culture of Vero cell was
obtained from the Central Veterinary Research Laboratory (Soba), the
growth medium was removed and the cells were briefly washed with PD.
0.5ml of warm trpsin – versene solution was added and the flask incubated
at 37c° until cells flew freely when the flask was tilted. Few drops of bovine
serum were added to stop the action of trypsin and versene .The cells were
diluted in GMEM growth medium and sub cultured in appropriate plastic
tissue culture flasks and incubated at 37c°.
2.9 Virus Propagation
A 24 wells plate containing semi–confluent monolayer was
inoculated with 50 ul volume of inoculum (every 4 wells inoculated with
one sample). The inoculated plate was kept at 37c° for 60 minutes
adsorption time. Incula were then removed and monolayers washed twice
with PD and refed with maintenance medium. The plate and a set of control
wells were examined daily with an inverted microscope and, when
cytopathic effect (CPE) involved 70 % or more of the sheet cover, the whole
cultures were harvested after three repeated cycle of freezing and thawing.
The harvested cell lysate was used as inculum to infect new plastic tissue
culture flasks.
2.10 Agar gel Preparation (see appendix 3).
2.11 Agar gel immunodiffusion test (AGID)
2.11.1 Preparation of agar gel
The agar and other chemical powders were dissolved in it’s specific
buffer , heated in a microwave for 2 minutes, 9 ml of the agar gel was
poured in the Petri dish to give a thickness about 3mm , using a template and
a cutter, a rosette six peripheral wells and a central well were cut in the agar.
Then plugs were carefully removed.
2.11.2 Standardization of AGID test
All the agars that mentioned in the material were prepared and used in
AGID test to detect precipitin lines when a vaccinia virus antiserum was
added to the central well and the vaccinia virus antigen mixed with sodium
deoxycholate were added to the peripheral wells .The plates were incubated
in a humid chamber at 35_37c .The plates were examined daily for
precipitation bands. (Fig: 5)
2.11.3 Examination of the sera for antibodies against orthopoxviruses
using AGID test
Purified agar1 was used to test the sera since it was the best agar tried
.The antigen which was mixed with sodium deoxycholate was added to the
central wells, while the sera were placed in the peripheral wells. Then plates
were incubated at 37c in humidity chamber, Then result were obtained and
reported.
Chapter 111
Results
3.1 Growth of vaccinia virus on the Chorioallantoic Membrane
Vaccinia virus was inoculated onto the CAM of 12 embryonated eggs.
The results showed the production of small pock lesions, round, opaque-
white in color and approximately 0.1-0.2 mm in diameter and without
hemorrhagic or necrotic center (Fig: 1)
3.2 Histopathology of the Chorioallantoic Membrane (CAM)
A number of 2 CAM were collected from inoculated eggs for
histopathology. CAM section showed foci of epithelial thickening .The cells
appear swollen with pale cytoplasm and indistinct cell boundaries. Many
cells showed degenerative changes. Some cell exhibited small round
eosinophilic inclusion bodies in the cytoplasm. Increase in connective
tissues cells at the site of epithelial proliferation showed mononuclear cell
infiltration (lymphocytes, monocytes) (Fig: 2 and 3).
3.3 Growth of vaccinia virus in Cell Culture
Vaccinia virus replicated in Vero cell and produced 100% Cytopathic
effect (CPE) three days post inoculation which was characterized by
rounding, plaque formation and destruction of the cell sheet (Fig: 4).
3.4 Sero-prevalence of orthopox virus in Sheep , Cattle and camel
Serum Samples
Four hundred and sex sera were examined by AGID to investigate
orthopox virus antibodies. The Test showed that 222 samples were positive
(54.7%) and 184 samples were negative (45.3%) (Table1).
The result showed that 109 (77.9%) out of 140 sheep sera were positive, 59 (35.7 %) out of 165 cattle sera were positive and 54 (53.5%) out of 101
camel sera were positive (Table 1).
Table 2 showed that 97 (92.3 %) out of 105 sheep sera collected from Bahry
were positive, 12(34.3%) out of 35 sheep sera collected from Omdorman
were positive, and showed that 14 (28 %) out of 50 cattle sera collected from
Bahry were positive, 45(39.1%) out of 115 cattle sera collected from
Omdorman were positive.
In camel showed that 37 (61.6 %) out of 60 camel sera collected from
Gadarif were positive, 17(41.5%) out of 41 camel sera collected from
Tambool were positive.
Table 1: Testing sera collected from cattle, sheep and camels for antibodies against orthopox virus antibodies by AGID
Species
Positive (%)
Negative (%)
Total
Cattle
59(35.7%)
106(64.2%)
165
Sheep 109(77.9 %) 31(22.1 %) 140
Camel 54(53.5%) 47(46.5%) 101
Total 222(54.7%) 184(45.3%) 406
Table 2: Testing sera collected from different areas in Khartoum for antibodies against orthopox virus antibodies by AGID
Species areas No. samples
+ve (%) -ve (%)
cattle Bahary Omdorman
50 115
14 (28%) 45 (39.1%)
36 (72%) 70 (60.9%)
sheep Bahary Omdorman
105 35
97 (92.3%) 12 (34.3%)
8 (7.6%) 23(65.7%)
camel Gadarif Tambool
60 41
37 (61.6%) 17 (41.5%)
23 (38.4%) 24 (58.5%)
Fig 1: Pock lesions produced by Vaccinia virus on CAM of embryonated eggs
Small pock lesions, round, opaque-white in color and approximately 0.1-0.2 mm in diameter and without hemorrhagic or necrotic center
Fig 2: CAM section showed swollen of the cells with pale cytoplasm and some inclusions bodies
The cells appear swollen with pale cytoplasm and indistinct cell boundaries. Many cells showed degenerative changes
Fig 3: CAM section showed foci of epithelial thickening
CAM section showed foci of epithelial thickening .
Fig 4: Vero cell culture infected with Vaccinia Virus showing rounding and plaque formation
Cytopathic effect (CPE) three days post inoculation which was characterized by rounding, plaque formation and destruction of the cell sheet
Fig 5: Precipitation line in AGID test between Vaccinia virus antigen (control well) and cows sera.
35
Chapter IV
DISSCUSION
Biological methods like growth on the Chorioallantoic
Membrance of embryonated eggs (CAM) and cell cultures were used to
determine the biological properties of Vaccinia virus (VV) and diagnosis.
In this study, VV was able to grow on CAM and produced
small pock lesions, which were round, opaque, white in colour and with a
diameter of about 0.1-0.2mm.The results in this study confirm the
observations of Marennikova et al (1974) who report that when Vaccinia
Virus was propagated on CAM at 370c, monomorphic punctuated, rather
dense white pock lesions small in size were seen.
When Vaccinia virus was grown in Vero cells it produced
clear Cytopathic effect (CPE) consisting of rounding of cells, plaque
formation, syncytia and detachment from the glass in 3 days after
inoculation. These observations agree with Maria-Lucia (2002) who
observed that the CPE in Vero cell culture by VV consist of small
syncytia, or multinucleated giant cells, result from fusion of cell
membranes bearing viral glycoproteins. Also there are inclusion bodies,
which are seen as eosinophilic areas of altered staining in the cytoplasm.
36
We also determined here the histopathology of the pock lesions to
show the VV effect on the CAM. CAM sections showed foci of epithelial
thickening, swelling of the cells, eosinophilic inclusions bodies and
infiltration of mononuclear cells. This result is similar to that of Davies
(1975) in histopathological section of the CAM of chick embryos
inoculated with camelpox virus which gave cellular inclusions and the
eosinophilic intra-cytoplasmic inclusions and Feulgen positive reaction
could be shown in the cells in the pocks. Khanna et al (1996) made
sections through pock lesions formed by camel pox and showed
proliferation of CAM cells and a few intracytoplasmic eosinophilic
inclusions. This is expected since camel pox virus is also a member of
the orthopoxvirus of the family Poxviridae. On the other hand the section
of pock lesions which formed by fowlpox were hyper plastic and
exhibited large easinophillic intra cytoplasmic inclusion bodies (Nyaga
1979). Gaffar et al (1980) described the inclusions bodies in pock lesions
of CAM section formed by Fowl pox virus as eosinophilic bodies in the
cytoplasm. The inclusions bodies were rounded; oval or irregular in
shape, often had granular appearance, and lacked discrete boundaries.
Inclusions bodies frequently appeared occupy the entire cytoplasm
37
except from a thin margin, pushing the nuclei to one side. Some
inclusions bodies appeared vacuolated, containing remnants of the outer
layer. With haematoxylin-shorr S3 stain, these inclusions bodies were
distinguished very clearly, taking the brilliant- red stain.
Various serological tests have been used for demonstration of
antibodies against poxviruses. These include gel diffusion test (Tantawi,
1974, Gwendolyn et al, 1962), neutralization test (Al Falluji et al, 1979;
Davies et al, 1985; Hafez et al, 1992), plaque reduction test (Nguyen and
Richard, 1985) and ELISA test (Munz et al 1986).
In this study, we used AGID test to detect orthopoxviruses in
sera of some domestic animal species in the Sudan. This test is
commonly used for identify and classification of the orthopox virus
(Joseph et al, 1977), and previously used for survey on antibody against
Parapoxvirus among cattle in Japan (Hiroshi et al, 2000). We have
examined 406 sera, 305 sera collected from sheep and cattle from
Khartoum state and 101 sera collected from camel from Gadarif and
Tambool. Our finding showed that 240 (59%) samples out of 406
samples collected from sheep, cattle and camel were positive. 186 sera
out of 305 samples collected from sheep and cattle were positive
38
(55.1%), 109 (77.9%) samples were positive out of 140 samples collected
from sheep, 59 (35.7%) samples were positive out of 165 samples
collected from cattle and 54(53.5%) samples were positive out of 101
sera collected from camel.
These findings demonstrate for the first time a serological
evidence for orthopoxvirus infection in sheep and cattle. Cattle are
known to be infected by cow pox virus which is also a member of the
orthopoxvirus genus. This disease is not yet reported in the Sudan
probably due to its benign course and difficulty in clinically distinguish
cowpox from pseudo cowpox of the genus parapox and other infections
of the teats. Thus our study points out for the existence of this disease.
Cowpox is benign infection of the udder and teats that causes no
mortality in cattle.
Orthopox viruses are not pathogenic in sheep, but seroconversion
in sheep to orthopox virus could be due to exposure of the sampled sheep
to camel pox or cowpox virus. The production system in most areas of
the Sudan is characterized by raising different species of domestic
animals including sheep, goat, cattle and camels. According to Tantawi
39
(1974) sheep experimentally infected with camel pox virus developed
high serum antibodies but they remain susceptible to sheep pox.
Our study showed that 53.5% of camel sera collected from eastern
and central Sudan were positive for camel pox virus antibodies. Camel
pox was already reported in the Sudan (Shommein and Osman, 1987,
Khalafalla 1998). The disease is the most contagious viral disease in
camels characterized by localized and generalized pox lesions that cause
severe out break in young animals with mortality rates of up to 10%.
Khalafalla et al (1998) detected antibodies against camel pox virus
in sera collected from 4 different region of the Sudan using ELISA. The
result showed 72.5% seropositivity. Our findings support the finding of
Khalafalla et al (1998) on the seroprevalence of camel pox virus
antibodies in eastern and central Sudan.
Further studies are needed to determine the economical impact of
orthopox virus infection in the Sudan particularly in cattle and sheep.
40
CONCLUSIONS
From this study, it can be concluded that Vaccinia Virus when
grown on the chorioallantoic membrance of embryonated eggs and Vero
cell gives clear pock lesions and Cytopathic effect (CPE) that can
differentiate this virus from related viruses. Additionally histopathology
of the CAM can give characteristic lesions.
Antibodies against Orthopoxvirus are widely distributed in sheep,
cattle and camel in the Sudan.
41
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Appendices
Appendix 1: Phosphate buffer saline (PBS) and phosphate diluent (PD)
A-Ingredients:
Solution (a)
NaCl 16.0g
KCl 0.4g
Na2 HPO4 2.3g
KH2PO4 0.4g
DDW 1500.0 ml
Solution (b)
MgCI .6H2O 0.426g
DDW 200.0 ml
Solution (c)
CaCI2 2H2O 0.264g
DDW 200.0ml
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Appendix 2: Outgrowth and Maintenance Media Outgrowth and maintenance media were prepared according to Ali
(1971) as shown below:
Medium (liter)
Stock solutions Outgrowth Maintenance
GMEM 200 ml 200 ml
Lactalbumin hydroysate 25 ml 25 ml
Yeast extract (1%) 25 ml 25 ml
Sodium bicarbonate (7.5%) 7.5 ml 10 ml
Penicillin – streptomycin solution 1 ml 1 ml
Fungizon (5000ug / ml) 1ml -
DDW To IL To IL
Tryptose Phosphate broth (TPB) 50 ml 50 ml
Bovine serum 100 ml 20 ml
53
Appendix 3: Purified agar I (Procedure I)
Purified agar 1.60 g
Nacl 8.00 g
Phenol 0.5 ml
Distilled water 100 ml
Purified agar II (Procedure 2)
Purified agar 1.4. g
Nacl 8.00 g
Phenol 0.5 ml
Distilled water 100 ml
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