DETECTION of ORTHOPOXVIRUS ANTIBODIES in SERA of SOME DOMESTIC SPECIES in the SUDAN by Siham Tag Elsir Karam Alla Alkidir

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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 اﻻﻃﺮوﺣﻪ

هﺪﻓﺖ هﺬﻩ اﻟﺪراﺳﻪ ﻟﻤﻌﺮﻓﺔ ﺑﻌﺾ اﻟﺨﺼﺎﺋﺺ اﻟﺤﻴﻮﻳﻪ ﻟﻔﻴﺮوس اﻟﻔﺎآﺴﻴﻨﻴﺎ ﻓﻲ ﺟﻨﻴﻦ اﻟﺒﻴﺾ اﻟﻨﺎﻣﻲ واﻟﺰرع اﻟﺨﻠﻮي .آﻤﺎ هﺪﻓﺖ اﻳﻀﺎ ﻟﺪراﺳﺔ وﺑﺎﺋﻴﺔ ﻓﻴﺮوس اﻟﺠﺪري اﻟﺤﻘﻴﻘﻲ (اورﺛﻮﺑﻮآﺲ) ﻓﻲ اﻻﺑﻘﺎر واﻟﻀﺎن واﻟﺠﻤﺎل ﻓﻲ اﻟﺴﻮدان آﺄول دراﺳﻪ ﻣﻦ هﺬا اﻟﻨﻮع . اﺳﺘﻌﻤﻠﺖ ﻓﻲ هﺬﻩ اﻟﺪراﺳﻪ ﻋﺘﺮﻩ ﻣﻦ ﻓﻴﺮوس اﻟﻔﺎآﺴﻴﻨﻴﺎ وﺗﻢ اآﺜﺎرهﺎ ﻓﻲ اﻟﻐﺸﺎء اﻟﻤﺸﻴﻤﻲ اﻟﻠﻘﺎﻧﻘﻲ ﻟﺠﻨﻴﻦ اﻟﺒﻴﺾ اﻟﻨﺎﻣﻲ ﻓﺎﻋﻄﺖ اﻓﺎت واﺿﺤﻪ ﺗﺎﺧﺬ اﻟﻠﻮن اﻻﺑﻴﺾ اﻟﻤﻌﺘﻢ وﻳﺘﺮاوح ﺣﺠﻤﻬﺎ ﺑﻴﻦ0.1-0.2 ﻣﻠﻢ . اﻳﻀﺎ اﺧﺬت ﻋﻴﻨﺎت ﻣﻦ هﺬﻩ اﻻﻓﺎت واﺟﺮى ﻋﻠﻴﻬﺎ اﺧﺘﺒﺎر اﻻﻧﺴﺠﻪ اﻟﻤﺮﻳﻀﻪ واوﺿﺤﺖ وﺟﻮد ﺗﻜﺎﺛﺮ ﺑﺆري ﻟﻠﺨﻼﻳﺎ , اﻧﺘﻔﺎخ ﺑﻌﺾ اﻟﺨﻼﻳﺎ ذات ﺳﻴﺘﻮﺑﻼزم ﺷﺎﺣﺐ,وﺟﻮد اﺟﺴﺎم اﺷﺘﻤﺎﻟﻴﻪ ﻗﺎﻋﺪﻳﻪ وارﺗﺸﺎح ﺧﻼﻳﺎ وﺣﻴﺪﻩ اﻻﻧﻮﻳﻪ. اﻳﻀﺎ ﺗﻢ اآﺜﺎر اﻟﻔﻴﺮوس ﻓﻲ ﺧﻼﻳﺎ ﻓﻴﺮو ( آﻠﻲ اﻟﻘﺮد اﻻﻓﺮﻳﻘﻲ) واﻋﻄﺖ اﺛﺮا ﻣﺮﺿﻴﺎ ﺧﻠﻮﻳﺎ ﻳﺘﻤﺜﻞ ﻓﻲ اﺳﺘﺪارة اﻟﺨﻼﻳﺎ وﺗﻜﻮﻳﻦ ﻟﻄﻌﺎت واﻧﻔﻜﺎك اﻟﺨﻼﻳﺎ ﻋﻦ ﺳﻄﺢ ﻗﻨﻴﻨﺔ اﻟﺰرع اﻟﺨﻠﻮي . اﺳﺘﺨﺪم ﻓﻲ هﺬﻩ اﻟﺪراﺳﻪ اﺧﺘﺒﺎر اﻻﻧﺘﺸﺎر اﻟﻤﻨﺎﻋﻲ ﻓﻲ اﻟﺠﻞ ﻟﺘﺤﺪﻳﺪ وﺟﻮد اﺟﺴﺎم ﻣﻀﺎدﻩ ﻟﻔﻴﺮوﺳﺎت اﻻورﺛﻮﺑﻮآﺲ ﻓﻲ ﻋﻴﻨﺎت ﻣﺼﻞ ﺟﻤﻌﺖ ﻣﻦ اﺑﻘﺎر وﺿﺎن وﺟﻤﺎل ﻣﻦ ﻣﻨﺎﻃﻖ ﻣﺨﺘﻠﻔﻪ ﻣﻦ اﻟﺴﻮدان واﺳﺘﺨﺪم ﻣﺼﻞ ﻣﻤﻨﻊ ﻣﻦ اراﻧﺐ_ ﺣﻘﻨﺖ ﺑﻔﻴﺮوس اﻟﻔﺎآﺴﻴﻨﻴﺎ _ آﻤﺼﻞ ﻗﻴﺎﺳﻰ . اوﺿﺤﺖ اﻟﺪراﺳﻪ وﺟﻮد اﺟﺴﺎم ﻣﻀﺎدﻩ ﻟﻔﻴﺮوﺳﺎت اﻻورﺛﻮﺑﻮآﺲ ﺑﻨﺴﺐ ﻣﺘﻔﺎوﺗﻪ ﻓﻲ هﺬﻩ اﻟﺤﻴﻮاﻧﺎت ﺣﻴﺚ آﺎﻧﺖ اﻟﻨﺴﺒﻪ ﻓﻲ اﻻﺑﻘﺎر % 35.7 وﻓﻲ اﻟﻀﺎن %77.9 وآﺎﻧﺖ ﻓﻲ اﻻﺑﻞ %53.5 وذﻟﻚ ﻳﻌﻨﻲ اﻧﺘﺸﺎر اﻻﺟﺴﺎم اﻟﻤﻀﺎدﻩ ﻟﻔﻴﺮوس اﻟﺠﺪري اﻟﺤﻘﻴﻘﻲ (اوﺛﻮﺑﻮآﺲ) ﺑﺼﻮرﻩ واﺳﻌﻪ ﻓﻲ اﻻﺑﻘﺎر واﻟﻀﺎن واﻟﺠﻤﺎل ﻓﻲ اﻟﺴﻮدان .

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. +ve (%) -ve (%) samples cattle Bahary 50 14 (28%) 36 (72%)

Omdorman 115 45 (39.1%) 70 (60.9%)

sheep Bahary 105 97 (92.3%) 8 (7.6%)

Omdorman 35 12 (34.3%) 23(65.7%)

camel Gadarif 60 37 (61.6%) 23 (38.4%)

Tambool 41 17 (41.5%) 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.

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.

35 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

36 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

37 (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

38 (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.

39 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.

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50 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

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

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