PREVELANCE OF PESTE DES PETITS IN

THE BLUE NILE STATE

By Raja Eltahir Haj Omer B.V.Sc. (1996) University of Khartoum

Supervisor

Professor Abdel Rahim El Sayed Karrar

co-Supervisor

Dr. Yahia Hassan Ali

A thesis submitted to the University of Khartoum in partial fulfillment of the requirements for the Degree of Master of Veterinary Medicine (M.V.M)

Department of Medicine, Toxicology and Pharmacology Faculty of Veterinary Medicine University of Khartoum

2011 ﺑﺴــﻢ اﷲ اﻟﺮﺣـﻤـﻦ اﻟﺮﺣـﻴﻢ

ﻗﺎل ﺗﻌﺎﻟﻰ: (ﺳـﺒﺤـﺎن اﻟﺬي ﺳـﺨﺮ ﻟﻨﺎ هﺬا وﻣﺎ آـﻨﺎ ﻟﻪ ﻣـﻘﺮﻧﲔ)

ﺻﺪق اﷲ اﻟﻌﻈﻴﻢ

ﺳﻮرة اﻟﺰﺧﺮف - ﺁﻳﺔ (13)

LIST OF CONTENTS

Page

Dedication…………………………………………………………… ix

Acknowledgements…………………………………………………. iix

English Summary…………………………………………………….. x

Arabic summary ……………………………………………………. xi

List of Contents……………………………………………………… i

List of Tables ……………………………………………………….. vi

List of Figures………………………………………………………. vii

Introduction…………………………………………………………….. 1

iii

Page

CHAPTER ONE: LITERATURE REVIEW

1.1. Definition………………………………………………………………. 4

1.2. Etiology………………………………………………………………… 5

1.2.1. Virus structure…………………………………………………………... 5

1.2.2. Relationship between the PPR virus and virus………………. 6

1.3. Host Range and species variation………………………………………… 6

1.4. Historical Background……………………………………………………. 7

1.5. Epidemiology of PPR…………………………………………………… 9

1.5.1 Geographical Distribution…………………………………………….. 9

1.5.2. …………………………………………………………….. 10

1.5.3. Lineages of PPRV……………………………………………………… 11

1.5.4. Morbidity and Mortality……………………………………………… 12

1.6 Pathology………………………………………………………………… 13

1.6.1 Gross lesions…………………………………………………………… 13

1.6.2 Microscopic lesions (histopathology)………………………………… 14

1.7. Clinical Signs…………………………………………………...... 14

1.7.1. Per acute Syndrome………………………………………………… 15 page

1.7.2. Acute Syndrome…………………………………………………….. 15

1.7.3. Sub acute Syndrome………………………………………………… 17

1.8. Resistance and immunity……………………………………………… 17

1.8.1 Innate and passive immunity………………………………………… 17

1.8.2 Active immunity…………………………………………………….. 17

1.8.3 ………………………………………………………….. 18

1.9. Diagnosis of PPR…………………………………………………….. 19

1.9. 1. Laboratory Diagnosis……………………………………………… 19

1.9.1.1 Serological tests…………………………………………………… 20

1.9.2. Differential Diagnosis………………………………………...... 22

1.9.3. Control of PPR……………………………………………………… 26

CHAPTER TWO: MATERIALS AND METHODS page

2. Material and Methods………………………………………………….. 29

2.1. Area of the Study ……………………………………………………… 29

2.2. Data collection………………………………………………………. 31

2.2.1. Collection of samples……………………………………………… 33

2.2.2. Material for samples collection…………………………………… 33

2.2.3. Sampling Procedure………………………………………………. 34 Page

2.3. Competitive ELISA System for detection of specific to PPR virus…………………………………….... 35

2.3.1. Materials………………………………………………………….. 35

2.3.2. Reagents………………………………………………………….. 36

2.3.3. Protocol for competitive ELISA………………………...... 37

2.4. Immunocapture ELISA for detection of antigen………………….. 41

2.4.1. Materials………………………………………………………… 41

2.4.2. Protocol of Immunocapture ELISA…………………………….. 41

CHAPTER THREE: RESULT

Page

3.1 Epidemiology of PPR in Blue Nile state………………………… 43

3.2. The prevalence of PPR in ruminants in Blue Nile State……...... 44

3.3 The PPR Sero-prevalence in different species……………………. 44

3.4. The PPR sero-prevalence in different locations…………………. 44

3.5 Prevalence of PPR Antigen in the different species……………… 45

CHAPTER FOUR:

Page

DISCUSSION………………………………………………………… 58

Conclusions and Recommendation ……………………………………. 61

References………………………………………………………………. 63

LIST OF TABLES

Page

Table (1) The Number of the Serum samples collected from different

species of animals at Blue Nile State during(2009-2010)……………….. 32

Table (2) Number of Animals vaccinated against PPR in Blue Nile State during(2009 -2010)………………………………………………………. 45

Table (3) Seroprevalence of PPR in the different localities

in Blue Nile state, Sudan detected using cELISA during 2010…………. 46

Table (4) Result of cELISA for the detection of PPR antibodies

in the different species in Blue Nile state (2010)………………………… 47

Table (5) Determination of PPR Antibodies using cELISA in the different locations in Blue Nile state, Sudan (2010)………………………………… 48

Table (6) Seroprevalence of PPR determined using cELISA

in different species and localities in Blue Nile state during (2010)…...... 49

LIST OF FIGURES

Page

Figure (1) Geographic distribution of PPRV lineages

(Dhar et al., 2002)…………………………………………...... 10

Figure (2) Area of the Study ………………………………………………. 30

Figure (3) ELISA Plate showing

positive and negative serum samples……………………………………. 40

Figure (4) ELISA Plate showing Protocol

of Immunocapture ELISA………………………………………………. 42

Figure (5) Seroprevalence of PPR in the different localities in Blue Nile state during (2010)…………………………….. 50

Figure (6) PPR antibodies detected using cELISA

in the different species in Blue Nile state (2010)………………………. 51

Figure (7) Seroprevalence of PPR determined using cELISA in the different locations in Blue Nile State, Sudan during (2010)……………………… 52

Figure (8) Prevalence of PPR Antigen in the different species…………………………………………………… 53

Page

Figure 9 Erosive stomatitis involving: the inside of the lower

and adjacent gum ……………………………………………………………….. 54

Figure10:Serous nasal discharge becoming mucopurulent and resulting,

at times, in a profuse……………………………………………………………… 55

Figure11:Catarrhal exudate which crusts over and

occludes the nostrils……………………………………………………………….. 56

Figure 12:Severe non-haemorrhagic diarrhea, Congestion of conjunctiva, crusting on the medial canthus and sometimes profuse

catarrhal ……………………………………………………..... 57

DEDICATION

I dedicate this work to:

The soul of my father My dearest mother My great husband Ammar My lovely sons Ahmed Aymen Awab

My dearest Brothers and Sisters

With my best wishes

ACKNOWLEDGEMENT

First of all my thanks to Allah for giving me health and strength to complete this study. I owe my sincere Gratitude and thanks to my supervisor Professor. Abedel Rahim El Sayed Karrar for his guidance, advice, attention, kindness and unlimited help. My kind regards and thanks to my co- supervisor Dr. Yahia Hassan Ali, Dr. Intisar Kamil Saeed, Department of Virology especially Dr.Mahasin Elnour the Central Veterinary Research Laboratory (CVRL) Soba, for offering me space and facilities for work, and for performing ELISA. My thanks to IAEA Research contract for funding. The help and financial support of Ministry of Animal Resource and Fishers is very much appreciated. I extend my affable thanks to Animal health and Epizootic Disease Control General Directorate for his unreserved help. I would like to express my thanks to the staff members of the Blue Nile research laboratory (Abed Allah, Awad, Alawy) for giving a valuable support in sample collection. My full thanks to my colleague Elzain Basher for his helping in analysis of data. I am grateful to my mother, sisters, brothers, and sons with thankfulness, for their encouragement, patience and greatest help. Lastly I am really falling short of words to express my gratitude to my dearest husband Dr. Ammar Ismaiel for his moral support and blessing me with more than that I asked for. Prevalence of Peste des Petits Ruminants Virus In Blue Nile State A Thesis Submitted in partial fulfillment of the requirements of University of Khartoum for the Degree of Master of Veterinary Medicine Raja Eltahir Haj Omer B.V.Sc. (1996) Summary This study was carried out to investigate the epidemiology of peste des petits ruminants (PPR) in the Blue Nile State, Sudan, during 2009-2010, through collection of epidemiological data and antigen and detection.

Two methods were adopted to achieve this aim ; namely, collection of data from Veterinary services records and serological examination of , , camels and , using competitive enzyme- linked immunosorbent assay (c-ELISA) for PPR antibody detection and immunocapture ELISA (ic-ELISA) for(PPR antigen detection54. Out of 1037 serum samples collected from sheep , goats, camels and cattle in six Localities and tested for PPR virus antibodies using c-ELISA, 577 (55.64%) were positive. Among these, antibodies against PPR virus were detected in 83 samples(92.22%) from Baw Locality, 84 (63.16%) from Damazin locality, (84.38%) from Geissan Locality, 80(54.42%) from Rosirais Locality, 178 (43.84%) from Tadamon Locality and 98 (49.75%) from slaughterhouse. Throughout animal species, the highest antibodies titter of PPR was found to be in 206 samples(79.54%) of goats followed by sheep272(54.62%),cattle81 (40.50%) and camels 18 (22.50%).

Out of 303 lung samples collected from sheep, goats, camels and cattle in slaughterhouse and tested for PPR antigen, using ic-ELISA only six sheep samples were positive.

The results showed that PPR is an endemic disease in the Blue Nile State.

ﻣﻌﺪل اﻧﺘﺸﺎر ﻓﻴﺮوس ﻣﺮض ﻃﺎﻋﻮن اﻟﻤﺠﺘﺮات اﻟﺼﻐﻴﺮة ﻓﻰ وﻻﻳﺔ اﻟﻨﻴﻞ اﻻزرق رﺳﺎﻟﺔ ﻟﻨﻴﻞ درﺟﺔ اﻟﻤﺎﺟﺴﺘﻴﺮ ﻓﻲ اﻟﻄﺐ اﻟﺒﻴﻄﺮي ﺟﺎﻣﻌﺔ اﻟﺨﺮﻃﻮم ﺑﻮاﺳﻄﺔ رﺟﺎء اﻟﻄﺎهﺮ ﺣﺎج ﻋﻤﺮ ﺑﻜﻼرﻳﻮس اﻟﻌﻠﻮم اﻟﺒﻴﻄﺮﻳﺔ ﺟﺎﻣﻌﺔ اﻟﺨﺮﻃﻮم (1996) اﻟﻤــﺴﺘﺨـﻠـﺺ ﺃﺠﺭﻴﺕ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﻟﻠﺘﺤﻘﻕ ﻤﻥ ﻭﺒﺎﺌﻴﺔ ﻤﺭﺽ ﻁﺎﻋﻭﻥ ﺍﻟﻤﺠﺘﺭﺍﺕ ﺍﻟﺼﻐﻴﺭﺓ ﻓﻲ ﻭﻻﻴﺔ ﺍﻟﻨﻴل ﺍﻻﺯﺭﻕ ﻓﻲ ﺍﻟﻔﺘﺭﺓ ﻤﻥ 2009- 2011ﻡ ﻤﻥ ﺨﻼل ﺠﻤﻊ ﺒﻴﺎﻨﺎﺕ ﻋﻥ ﻭﺒﺎﺌﻴﺔ ﺍﻟﻤﺭﺽ ﻓﻲ ﺍﻟﻭﻻﻴﺔ ﻭﻤﺩﻯ ﺇﻨﺘﺸﺎﺭ ﺍﻷﺠﺴﺎﻡ ﺍﻟﻤﻀﺎﺩﺓ ﻭﺍﻷﻨﺘﻴﺠﻴﻨﺎﺕ ﻓﻲ ﺍﻟﺤﻴﻭﺍﻨﺎﺕ ﺍﻟﻤﺼﺎﺒﺔ. أﺳﺘﺨﺪﻣﺖ ﻃﺮﻳﻘﺘﺎن ﻹﻧﺠﺎز هﺬﻩ اﻟﺪراﺳﺔ هﻤﺎ: ﺠﻤﻌﺕ ﺒﻴﺎﻨﺎﺕ ﻤﻥ ﺴﺠﻼﺕ ﺍﻟﺨﺩﻤﺎﺕ ﺍﻟﺒﻴﻁﺭﻴﺔ ﻭﻓﺤﺹ ﺴﻴﺭﻭﻟﻭﺠﻲ ﻟﻌﻴﻨﺎﺕ ﻤﻥ ﺃﻨﺴﺠﺔ ﺍﻟﻀﺄﻥ ﻭ ﺍﻟﻤﺎﻋﺯ ﻭﺍﻹﺒل ﻭﺍﻷﺒﻘﺎﺭ ﻤﺴﺘﺨﺩﻤﻴﻥ ﻓﻲ ﺫﻟﻙ ﺇﺨﺘﺒﺎﺭ ﺍﻻﻟﻴﺯﺍ( ﻟﻠﻜﺸﻑ ﻋﻥ ﺍﻻﺠﺴﺎﻡ ﺍﻟﻤﻀﺎﺩﺓ) ﻭﺇﺨﺘﺒﺎﺭ ﺍﻻﻟﻴﺯﺍ ﺍﻟﻤﺘﻁﻭﺭﺓ ﻟﻠﻜﺸﻑ ﻋﻥ ﺍﻷﻨﺘﺠﻴﻨﺎﺕ . ﻓﺤﺼﺕ1037 ﻋﻴﻨﺔ ﺴﻴﺭﻡ ﻤﻥ ﺍﻟﻀﺄﻥ ﻭﺍﻟﻤﺎﻋﺯ ﻭﺍﻹﺒل ﻭﺍﻷﺒﻘﺎﺭ ﻓﻲ 6 ﻤﺤﻠﻴﺎﺕ ﻤﻥ ﺍﻟﻭﻻﻴﺔ ﻟﻠﻜﺸﻑ ﻋﻥ ﻭﺠﻭﺩ ﺍﻷﺠﺴﺎﻡ ﺍﻟﻤﻀﺎﺩﺓ ﻟﻠﻔﻴﺭﻭﺱ ﺒﻭﺍﺴﻁﺔ ﺇﺨﺘﺒﺎﺭ ﺍﻻﻟﻴﺯﺍ ﻭﺠﺩ ﺃﻥ 577 ﻋﻴﻨﺔ ﻤﻭﺠﺒﺔ ﺍﻱ ﺤﻭﺍﻟﻲ 55.64% ﻤﻭﺯﻋﺔ ﻜﻤﺎ ﻴﺄﺘﻲ: 83 ﻋﻴﻨﺔ ﻤﻥ ﻤﺤﻠﻴﺔ ﺒﺎﻭ (92.22%) ﻭ84 ﻤﻥ ﻤﺤﻠﻴﺔ ﺍﻟﺩﻤﺎﺯﻴﻥ (63.16%) ﻭ 54 ﻤﻥ ﻤﺤﻠﻴﺔ ﻗﻴﺴﺎﻥ (84.38%) ﻭ80 ﻤﻥ ﻤﺤﻠﻴﺔ ﺍﻟﺭﻭﺼﻴﺭﺹ (54.42%) ﻭ178 ﻤﻥ ﻤﺤﻠﻴﺔ ﺍﻟﺘﻀﺎﻤﻥ(43.84%) ﻭ98 ﻤﻥ ﺍﻟﺴﻠﺨﺎﻨﺔ (%49.75) . ﻭﺠﺩﺕ ﺍﻋﻠﻰ ﻨﺴﺒﺔ ﺍﺠﺴﺎﻡ ﻤﻀﺎﺩﺓ ﻟﻤﺭﺽ ﻁﺎﻋﻭﻥ ﺍﻟﻤﺠﺘﺭﺍﺕ ﺍﻟﺼﻐﻴﺭﺓ ﻓﻲ ﺍﻟﻤﺎﻋﺯ206 ﻋﻴﻨﺔ (%79.54) ﺗﻠﻴﻬﺎ اﻟﻀﺄن 272(54.62%) ﺛﻢ اﻻﺑﻘﺎر81(40.50%) واﺧﻴﺮا اﻻﺑﻞ (%22.50)18 ﺠﻤﻌﺕ 303 ﻋﻴﻨﺔ ﻤﻥ ﺃﻨﺴﺠﺔ ﺍﻟﺭﺌﺔ ﻤﻥ ﺍﻟﻀﺄﻥ ﻭﺍﻟﻤﺎﻋﺯ ﻭﺍﻹﺒل ﻭﺍﻷﺒﻘﺎﺭ ﻓﻲ ﺍﻟﺴﻠﺨﺎﻨﺔ ﻭﻓﺤﺼﺕ ﻟﻠﻜﺸﻑ ﻋﻥ ﻭﺠﻭﺩ ﺍﻷﻨﺘﻴﺠﻴﻨﺎﻨﺕ ﺒﻭﺍﺴﻁﺔ ﺇﺨﺘﺒﺎﺭ ﺍﻻﻟﻴﺯﺍ ﺍﻟﻤﺘﻁﻭﺭﺓ ﻭﺠﺩ ﻓﻘﻁ ﺃﻥ 6 ﻋﻴﻨﺎﺕ ﻤﻭﺠﺒﺔ ﻟﻔﻴﺭﻭﺱ ﻁﺎﻋﻭﻥ ﺍﻟﻤﺠﺘﺭﺍﺕ ﺍﻟﺼﻐﻴﺭﺓ. ﺩﻟﺕ ﻨﺘﺎﺌﺞ ﺍﻟﺒﺤﺙ ﻋﻠﻰ ﻭﺠﻭﺩ ﻤﺭﺽ ﻁﺎﻋﻭﻥ ﺍﻟﻤﺠﺘﺭﺍﺕ ﺍﻟﺼﻐﻴﺭﺓ ﺒﺼﻭﺭﺓ ﻭﺍﺴﻌﺔ ﻓﻲ ﻭﻻﻴﺔ ﺍﻟﻨﻴل ﺍﻻﺯﺭﻕ. Introduction

Peste des petits ruminants (PPR) is an acute or sub acute viral disease of goats and sheep characterized by fever, erosive stomatitis, conjunctivitis, gastroenteritis, and . Goats are usually more severely affected than sheep. (Appel et al, 1981).It is caused by a paramyxovirus of the genus.. PPRvirus is a single stranded RNA virus. It is enveloped, helical, and non- segmented. Its size ranges between 100-300nm, possessing six structural proteins

(Diallo, 1990). The disease usually appears in the acute form, with an incubation period of 4 to 5 days followed by a sudden rise in body temperature to 104-106° F

(40-41° C) (Hamdy et al, 1976).

PPR is not very contagious and transmission requires close contact. Ocular, nasal, and oral secretions and feces are the sources of virus. Contact infection occurs mainly through inhalation of aerosols produced by sneezing and coughing.

Fomites such as bedding may also contribute to the onset of an outbreak. Infected animals may transmit the disease during the incubation period. (Lefevre and

Diallo, 1990 ).

Poor nutritional status, stress of movement, and concurrent parasitic and bacterial infections enhance the severity of the clinical signs.

1

PPR occurs in most African countries situated in a wide belt between the

Sahara and Equator, the (Arabian Peninsula, Israel, Syria, Iraq,

Jordan), and the (Elhag and Taylor, 1983.).

The first outbreak of the disease in sheep and goats in Sudan was in three areas in south Gedarif (Eastern Sudan) in 1971 (El Hag, 1973). The disease was then reported in two caprine outbreaks in Central Sudan (Sinnar area) during 1971-

1972 and in Mieliq in 1972; in sheep in Western Sudan (Rasheed, 1992); in sheep and goats in Central Sudan (Hassan et al., 1994); in sheep and goats in Khartoum

State (Zeidan, 1994 and El Amin and Hassan, 1998). PPR was detected and isolated from different parts of Sudan.

PPR is a disease that is classified in the OIE list A diseases.

PPR may have passed unrecognized for years in some countries because it is frequently confused with other diseases that cause respiratory problems and mortality of small ruminants. PPR is an important disease in its own right, but it is also very important that it is rather difficult to be differentiated from Rinderpest

(RP).Clinical RP is rare in goats and sheep in Africa. In India, these species are quite often involved in RP outbreaks. Clinically, RP and PPR are similar, but the former should be the prime suspect if the disease involves both cattle and small ruminants. (Appel et al., 1981).

2

Continuous outbreaks of PPR were reported at Blue Nile State; however no study has been conducted to investigate the epidemiology of the disease in the

State.

This study is to investigate the epidemiology of PPR in Blue Nile State through collection of epidemiological data related to antigen and antibody detection.

The main objectives of this research work are:

To investigate the epidemiology of PPR in Blue Nile State throwgh:

1. The Seroprevallance of PPRV in Blue Nile State( PPR antibodies will be tested in collected sera using competitive ELISA (cELISA))

2. PPR antigen in diseased as well as clinically healthy animals (PPR antigens will be examined in collected tissue samples using immunocapture ELISA (icELIS).)

3

CHAPTER ONE

LITERATURE REVIEW

1.1. Definition:

Peste des Petits Ruminants was the French name of a similar disease in sheep and goats first described in in 1942. Both diseases were shown to be very close to each other (Rowland et al., 1971). Many authors prefer the appellation of "Ovine Rinderpest". But official institutions like FAO and OIE use the French name "Peste des Petits Ruminants", "Peste Des Petits Ruminants",

"Peste-des-Petits-Ruminants" or "Peste-des-petits-ruminants", even in

English.Peste des petits ruminants (PPR) is a highly contagious and infectious viral disease of domestic and wild small ruminants. It is an acute or subacute viral disease of goats and sheep characterized by fever, erosive stomatitis, conjunctivitis, gastroenteritis, and pneumonia. Goats are usually more severely affected than sheep. ( Appel et al., 1981).

The disease spreads from a region to another by sick animals. As the virus is early inactivated outside the body, indirect contamination is generally limited .In an affected flock, even in pest-free regions, the disease do not progress very rapidly, although there is close contact between animals. New clinical cases may be observed daily for a one-month period. (Mahin, 2008)

4

1.2. Etiology:

Peste des petits ruminants are caused by a paramyxovirus of the

Morbillivirus genus. Other members of the genus include rinderpest virus (RPV), virus (MV), canine distemper virus (CDV), and phocid distemper virus

(PDV) of sea mammals (seals). For many years, PPR virus was considered a variant of RPV, specifically adapted for goats and sheep that had lost its virulence for cattle. It is now known that the two are distinct though closely related antigenically.

1.2.1. Virus structure:

PPRvirus is a single stranded RNA virus. It is enveloped, helical, and non- segmented. Its size-ranges between 100-300nm, possessing six structural proteins

(Diallo, 1990) .The virus is sensitive to ether and chloroform. The half-life of hydrated virus held at 37°C is about 2 hours. At 50°C infectivity is destroyed within 30 minutes. PPR virus is also sensitive to lipid solvents and low pH. The virus present in lymph nodes is protected from pH changes after death of the animal .It was found in the lymph nodes of carcasses held for 8 days at 4°C

(Lefevre and Diallo, 1990).

PPR virus is composed of ribonucleoprotein core that carries the viral genome. The core constitutes the soluble antigen and is surrounded by lipoprotein envelope, which carries the viral antigen. PPR virus may appear as spheres or short

5 to long filaments and often includes irregular or dumbbell-shaped form (Avis,

FAO and OIE, 1992).

1.2.2. Relationship between the PPR virus and rinderpest virus:

PPRvirus is closely-related to rinderpest virus and predominantly affects sheep and goats. Although PPR virus has been shown experimentally to infect cattle, the disease is sub clinical and transmission between small ruminants and cattle in the field has been considered unlikely (Dardiri et al., 1977).

In the past differential serodiagnosis of rinderpest and PPR could only be accomplished by cross-neutralization tests. The recently-developed monoclonal antibody-based competitive ELISA (C-ELISA) for the detection of antibodies against rinderpest and PPR virus have allowed rapid, simple, differential serodiagnosis of the two diseases (Anderson et al., 1991). This has allowed serological surveys on the prevalence of antibodies against the two viruses in sheep, goats and cattle to give a better understanding of the interaction of these two infections under field condition.

1.3. Host Range and species variation:

PPR is primarily a disease of sheep and goats. There have been several reports of PPR occurring in other species. Clinically, PPR is a severe fast spreading disease among sheep and goats. Large ruminants like cattle, buffaloes and camels can become infected. But there is no evidence of clinical disease associated with

6 their infection. Some wild ruminants are also affected including Laristan sheep and

Dorcas gazelles (Mahin, 2008). The American white-tailed deer (Odocoileus virginianus) has been infected experimentally (Saliki and Wohlsein, 2008). Cattle, buffaloes, camels, and pigs are also susceptible to infection but do not exhibit clinical signs and are unable to transmit the disease to other animals

(EMPRES,1999). PPR is not infectious to humans.

1.4. Historical Background:

PPR was first discovered in Cote d, Ivoire in 1942 (Gargadenne and

Lalanne, 1942). For many years, it was considered as the mutant of rinderpest virus that was specifically pathogenic for goats and sheep which has lost its virulence in cattle. This concept has been changed and PPR virus identity has been proposed and accepted on the basis of lack of significant cross neutralization between the two viruses (Majiyagbe et al., 1984).

In recent years the disease has been seen in the Near East and the Arabian

Peninsula, in countries including the Islamic Republic of Iran, Iraq, Israel, Jordan,

Kuwait, Lebanon, Oman, Saudi Arabia, the United Arab Emirates and Yemen, and there is serological evidence from the Syrian Arab Republic and Turkey

.Outbreaks of PPR are now known to be common in India, , Bangladesh,

Pakistan and Afghanistan (Taylor, 1984).

7

It is still not clear whether the apparent geographical spread of the disease in the last 50 years is real or whether it reflects increased awareness, wider availability of diagnostic tools or even a change in the nature of the virus. It seems most likely that a combination of factors is responsible for the present knowledge of its range and it is known that confusion of PPR with pneumonic and other pneumonic diseases of small ruminants have delayed its recognition in some countries.(EMPRES,1999).

The first outbreak of the disease in sheep and goats in Sudan was in three areas in south Gedarif (Eastern Sudan) in 1971 (Ali, 1973). The disease was then reported in two caprine outbreaks in Central Sudan (Sinnar area) during 1971-

1972 and in Mieliq in 1972; in sheep and goats in Central Sudan (Hassan et al.,

1994); in sheep and goats in Khartoum State (El Amin and Hassan, 1998).PPR was detected and isolated from different parts of Sudan [Gezira State, White Nile State

(Central), Khartoum State, North Kordofan State (Western) and River Nile State

(Northern)] during 2000- 2002 (Intisar, 2002). A serological survey of PPR in

Sudan during 2002-2005 using cELISA revealed positive results in 70% of ovine sera collected from Kordofan state, 52.5% of ovine sera and 34.7 % of caprine sera collected from Darfur State (Intisar et al., 2007)).

8

1.5. Epidemiology of PPR:

1.5.1 Geographical Distribution:

PPR is known to be present in a broad belt of sub-Saharan Africa, Arabia, the Middle East and Southern Asia. Major outbreaks in Turkey and India in recent years have indicated a marked rise in the global incidence of PPR (Nanda et al.,

1996; Ozkul et al., 2002; Shaila et al., 1996). The virus was isolated in

(Taylor and Abegunde, 1979), Sudan (ElHag and Taylor, 1984), Saudia Arabia

(Abu Elzein et al., 1990), India (Shaila et al., 1989, Nanda et al., 1996) and Turkey

(Ozkul et al., 2002). Serological evidences were detected in Syria, Niger and

Jordan, while the virus presence was confirmed with cDNA probe in Ethiopia

(Roeder et al., 1994) and Eritrea (Sumption et al., 1998), respectively. Genetic relationship between PPR viruses isolated from different geographical regions was studied by sequence comparison of the F-protein gene Four lineages were revealed

(Shaila et al., 1996, Dhar et al., 2002) (Fig. 1).

9

Fig. (1) Geographic distribution of PPRV lineages (Dhar et al., 2002)

1.5.2. Transmission:

For PPR to spread, close contact between infected and susceptible animals is needed (Ozkul et al., 2002). There are several means of transmission between animals (Saliki 1998):

10

• Inhalation of aerosols produced by sneezing and coughing of infected animals the

dry, cold season (OIE 2002).

• Direct contact with ocular, nasal, or oral secretions

• Direct contact with feces

• Fomites such as bedding, water, and feed troughs

• No carrier state is known to exist

1.5.3. Lineages of PPRV:

Genetic characterization of PPR virus strains has allowed them to be

organized into four groups; three from Africa and one from Asia .One of the

African groups of PPRV is also found in Asia. The epidemiological significance of

these groupings is less clear at present than that of rinderpest virus groupings

(Roeder and Obi, 1999).

Lineage (I) have been found in and include: Senegalese strain,

Nigeria 75/1, 75/2, 75/3, 76/1 and Burkina Faso/99. Lineages (II) found also in

West Africa and include the isolates: Guinea Bissau/91 and Ivory Coast/89.

Lineage (III) includes viruses which have been isolated from and Asia:

Sudan/72, Oman/83, India/TN/92, Ethiopia/96 and Yemen/01. Lineage (IV) found

only in Asia and includes viruses whose origins are in the Middle East, Saudi

Arabia and : 15 Indian isolates (India/UP/94, India/MH/94,……),

Bangladesh/ 93 and Bangladesh/00, Nepal/95, Turkey/96 and Turkey/00, Israel/94,

11

Pakistan/94 and Pakistan/ 98, Saudi Arabia/ 94, Iran/94, Iraq/00a and Iraq/00 and

Kuwait/99 (Dhar et al. ,2002 ).

1.5.4. Morbidity and Mortality:

The incidence of PPR in an enzootic area may be similar to that of Rinderpest

(RP) in that a low rate of infection exists continuously. When the susceptible population builds up, periodic epizootics (outbreaks) occur, that receive more attention than usual. Such epizootics may be characterized by almost 100 percent mortality among affected goats and sheep populations. (Lefevre and Diallo, 1990).

The prognosis of acute PPR is usually poor. The severity of the disease and its outcome in the individual is correlated with the extent of mouth lesions.

Prognosis is good in cases where the lesions resolve within 2 to 3 days. It is poor when extensive necrosis and secondary bacterial infections result in an unpleasant, fetid odor from the animal's breath. Respiratory involvement is also a poor prognostic sign. A morbidity rate of 80-90 percent and a case fatality rate of 50-80 percent are not uncommon — particularly in goats (Saliki Wholsein 2008).

Young animals (4 to 8 months) have more severe disease, and morbidity and mortality are higher. Both field and laboratory observations indicate that PPR is less severe in sheep than in goats. Nevertheless, field outbreaks have been reported in the humid zones of West Africa in which no distinction could be made between the mortality rates in sheep and in goats. Poor nutritional status, stress due to long

12 distance movement, and concurrent parasitic and bacterial infections enhance the severity of clinical signs (Taylor, 1984, Saliki 1998)

1.6 Pathology:

There are several external lesions like erosive stomatitis involving: the inside of the lower lips and adjacent gum, Serous nasal discharge becoming mucopurulent and resulting, at times, in a profuse catarrhal exudate which crusts over and occludes the nostrils (Figure 9)

Severe non-haemorrhagic diarrhea, Congestion of conjunctiva, crusting on the medial canthus and sometimes profuse catarrhal conjunctivitis(Figures10)

Also the disease have :

1.6.1 Gross lesions:

Postmortem findings in acute cases include a dehydrated carcass with faecal soiling; necrotic lesions in the mouth and nose; congestion of the ileocaecal valve; linear engorgement and blackening (zebra striping) of folds of the caecum, proximal colon and ; enlarged spleen; and oedema of lymph nodes, especially the mesenteric lymph nodes. The , reticulum and omasum rarely show lesions. Unlike in rinderpest, primary bronchopneumonia is a common finding that is specific for the virus and is diagnostically important (Brown et al.,

1991). Pleuritis and hydrothorax may be found.

13

1.6.2 Microscopic lesions (histopathology):

Distinct changes similar to many morbillivirus infections are seen histologically, including multinucleated giant cells, especially in the lungs, and eosinophilic intranuclear and/or intracytoplasmic inclusion bodies

(AUSVETPLAN, 2009).

1.7. Clinical Signs:

PPR usually appears in the acute form, with an incubation period of 4 to 5 days followed by a sudden rise in body temperature to 104-106 F0 (40-41 C0)

(Taylor, 1984).

The disease is characterized by sudden onset of depression, fever, discharges from the eyes and nose, sores in the mouth, disturbed breathing and , foul smelling diarrhea and death (Roeder and Obi, 1999).

As it progresses, there is severe dehydration, emaciation, and dyspnea followed by hypothermia, and death usually occurs after a course of 5 to 10 days.

Bronchopneumonia, evidenced by coughing, is a common feature in the later stages of PPR. Pregnant animals may abort (EMPRES, 1999).

PPR is not very contagious and transmission requires close contact. Ocular, nasal, and oral secretions and feces are the sources of virus. Contact infection occurs mainly through inhalation of aerosols produced by sneezing and coughing.

Fomites such as bedding may also contribute to the onset of an outbreak. Infected

14 animals may transmit the disease during the incubation period. (Lefevre and

Diallo, 1990).

The disease has per acute, acute and sub acute syndromes (OIE, 2002) :

1.7.1. Peracute Syndrome

It is frequent in goats. Cases are found dead without previous symptoms.

They die with a serous, foamy or hemorrhagic discharge coming out of the nose(DEFRA,2005).

1.7.2. Acute Syndrome:

Is the most common form. Animals are recumbent, sometimes in self- auscultation position. Body temperature is high (40.5 -41 °C) in the beginning of the onset in acute cases. The most typical signs are seen in the digestive tract.

When entering an affected flock, one sees many animals with hind limbs stained by sticky feces. Some sheep have an arched back and show pain to defecate.

Tenesmus may be noticed when taking rectal temperature. Fluid feces are olive green to brown. Examination of the mouth shows ulceration of the buccal mucosae, especially on the inner face of the lips, and neighboring gum. There can be periodontitis. There is serous nasal exudates and conjunctivitis (Saliki

2008).fig(9)(10)

15

The sudden high fever remaining high for 5-8 days; either return to normal before recovery or drop below normal before death. Serous nasal discharge, becoming mucopurulent; can crust over and occlude nostrils fig(11).

Purulent ocular discharge with congested conjunctiva; can encrust, cementing eyelids together. Bronchopneumonia, necrosis ,ulceration of mucous membrane and inflammation of leading to sever non haemorrhagic diarrhea may occur (Salki, 1998; Empres, 1999; Defra, 2001; Dhar et al., 2002;

OIE, 2002; Ozkul et al., 2002).fig(12)

Respiratory distress, including dyspnea and sneezing in an attempt to clear nose.

Excessive salivation but not to the point of drooling. Anorexia. Severe dehydration and emaciation followed by hypothermia. Death usually occurs after 5-10 days.

Abortion in pregnant animals may occur. Mortality rate can reach 100%.

Beside coughing, there is an intensive labial dermatitis with scab formation. In its acute form it is characterized by high fever, discharges from the eyes and nose, sores in the mouth, lesions of the mucous membranes, laboured breathing, and diarrhea (FAO, 2008).

1.7.3. Subacute Syndrome:

It develops over 10- 15 days, and is characterized by

Pneumonia and inconsistent symptoms (OIE, 2002).

16

1.8. Resistance and immunity:

Susceptible sheep and goats of all ages and breeds can be infected with PPR virus and develop the acute disease. In countries that are free from the disease, the introduction of PPR into the totally susceptible population is likely to produce high morbidity and mortality, and the disease is likely to spread rapidly. However, there is always the possibility of mild disease as a result of lower susceptibility of individual animals or groups (AUSVETPLAN, 2009).

1.8.1 Innate and passive immunity:

Variable breeds of goats sometimes show varying degrees of resistance to

PPR. Maternal immunity provides protection for 3–4 months (AUSVETPLAN,

2009).

1.8.2 Active immunity:

Infection with PPR provides lifelong immunity in recovered animals

(AUSVETPLAN, 2009).

1.8.3 Vaccination:

Until recently, the most practical vaccination against PPR made was the use of tissue culture rinderpest (Roeder and Obi, 1999). The tissue culture rinderpest vaccine at a dose of 102.5 TCID50 protects goats for at least 12 months against PPR. In the past, the rinderpest vaccine has been used. However, this

17 practice is being phased out to avoid confusion during retrospective serologic studies (OIE, 2002).

The use of rinderpest vaccine to protect small ruminants against PPR is now contraindicated because its use produces antibodies to rinderpest which compromise serosurveillance for rinderpest, and

thereby the Global Rinderpest Eradication Program (GREP) (Roeder and Obi,

1999). A homologous PPR vaccine is now available and gives strong immunity

(OIE, 2002). Recently, a homologous PPR vaccine has been developed, the can protect small ruminants against PPR for at least three years (Roeder and Obi, 1999).

An effective live vaccine is currently in use, it was attenuated by serial passage of the Nigeria 75/1 strain of PPRV in Vero cells (Diallo et al., 1989). This vaccine is thermolabile, it is necessary to maintain it in an effective cold chain condition , which is difficult to achieve in many of the endemic countries. Thus a more heat – stable vaccine would be beneficial for use in countries with hot climates. There are also genetically engineered recombinant vaccines undergoing limited field trials

(OIE, 2002).

Goats were protected against a lethal challenge of PPRV following vaccination with a recombinant capripox virus containing either the fusion(F)gene of RPV or the haemagglutinin (H) gene of RPV. The H gene recombinant produced high titres

18 of neutralizing antibody to RPV in the vaccinated goats, where as the F gene recombinant failed to stimulate detectable levels of neutralizing antibody.

1.9. Diagnosis of PPR:

A presumptive diagnosis is based on clinical, pathological, and epidemiological findings and may be confirmed by viral isolation and identification. The specimens required are unclotted blood, lymph nodes, tonsils, spleen, and whole lung. Detection of viral antigens by complement fixation or agar-gel precipitin tests does not differentiate the disease from rinderpest.

Detection of virus-neutralizing antibodies with a rising titer in surviving animals is diagnostic. PPR must be differentiated from other acute gastrointestinal infections

(eg rinderpest), respiratory infections (eg, contagious caprine pleuropneumonia), and such other diseases as contagious ecthyma, heartwater, coccidiosis, and mineral poisoning. (Majiyagbe,K.A., Nawathe ,D.R. and AbegudeA.(1984)).

1.9. 1. Laboratory Diagnosis:

Includes serological tests for detection of specific antigens or the development of antibodies.

1.9.1.1 Serological tests: a) Antibody detection methods:

The C-ELISA is considered suitable for large scale testing due to its simplicity and availability of the recombinant antigen (Libeau et al., 1995).

19

The antibody is the major factor determining the sensitivity and specificity of an assay. The structure of antibodies is discussed more thoroughly in "The Use of

Antibodies in Immunoassays". Briefly, it is the three dimensional configuration of the antigen-binding site found in the F(ab) portion of the antibody that controls the strength and specificity of the interaction with antigen. The stronger the interaction, the lower the concentration of antigen that can be detected (other factors being equal). A competing factor is the specificity of binding or the cross- reactivity of the antibody to serum proteins other than the target antigen.

Depending on whether the antibodies being used are polyclonal or monoclonal, cross-reactivity will be caused by different forces.

In either case driving the assay to the limit of sensitivity may result in cross- reactivity, and one is faced with the conflicting needs of sensitivity versus specificity.

ELISAs can be qualitative or quantitative but they all need highly specific and sensitive antibodies.

At first glance, the choices in ELISA formats may be overwhelming, but don’t despair.

There are a wide variety of ELISA formats available that vary depending on the sensitivity required and whether one is trying to detect an antigen or the antibody response to it.

20

C-ELISA, sensitivity is 99.4 % and specificity is 94.5% (OIE, 2000).

A competitive ELISA based on a PPRV monoclonal antibodies specific for

haemagglutinin (H) protein (Anderson et al, 1991; Saliki et al, 1993; Singh et al,

2004) or nucleoprotein (N) ( Libeau et al , 1995) was developed for detection of

antibodies to PPRV in serum samples of sheep and goats. This test may be a useful

tool for a standardized and accurate determination of the immune status of animals

because of its superior sensitivity to conventional tests (Libeau et al., 1995).

b) Antigen detection methods:

These methods have been used to ensure the appearance of the disease by

detecting of the virus antigens.

Virus antigens can also be detected by immunocapture ELISA (ICE) which

is rapid and sensitive, and differentiates between PPR and rinderpest (FAO,1999).

The IC-ELISA allows a rapid differential identification of PPR or RP viruses, and

this is of great importance as the two diseases have a similar geographical

distribution and may affect the same animal species (Diallo, 2000; Diallo, 2004).

1.9.2. Differential Diagnosis:

The differential diagnoses of PPR include:

• Rinderpest (although many reports of ‘rinderpest’ among small ruminants may

have been PPR).

• Bluetongue.

21

• Contagious ecthyma.

• Foot and mouth disease.

• Heartwater.

• Coccidiosis and mineral poisoning.

The respiratory signs can resemble contagious caprine Pleuropneumonia

(CCPP) or pasteurellosis; Pasteurellosis can also be a secondary complication of

PPR (Saliki 2008, EMPRES 1999).

PPR is frequently confused with other diseases that present fever and grossly

similar clinical signs, especially when it is newly introduced. When carrying out an

investigation, examination of the way the disease behaves in the herd or flock is as

important as the findings on a single or sheep. The most frequent sources

of confusion are:

Mouth lesions:

Could be a symptom of: rinderpest, foot-and-mouth disease,bluetongue or

contagious ecthyma ( or “sore mouth”).

Difficult breathing:

Could be a symptom of: pneumonic pasteurellosis or contagious caprine

pleuropneumonia (CCPP).

Diarrhoea:

Could be a symptom of: coccidiosis or gastro-intestinal helminth infestations.

22

Pneumonia is usually a very obvious presenting sign in PPR so, without doubt, pneumonic pasteurellosis and CCPP have caused the most difficulty in differential diagnosis.

Pneumonic pasteurellosis is a purely respiratory disease of sheep and goats caused by the bacterium Pasteurella haemolytica. Dark red/purple areas, firm to the touch, are evident mainly in the anterior and cardiac lobes of the lung . There are no oral lesions nor is there diarrhoea. The numbers of affected and dead animals are usually lower than for PPR except under exceptional conditions of stress and crowding such as can occur when large numbers of sheep are assembled for trade.

The main problem of differentiation arises when oral lesions and diarrhoea are either absent or not very obvious in PPR, as is sometimes the case.

Using appropriate culture media, Pasteurella haemolytica bacteria are easily isolated in pure and profuse culture from pneumonic lungs of sheep, even from the lungs of PPR-affected animals.

Contagious caprine pleuropneumonia (CCPP) is a disease of goats (sheep are not affected) caused by a Mycoplasma sp. Like PPR, it is characterized by fever, difficult/abnormal breathing and coughing, but mouth lesions and diarrhoea are not present in CCPP. At post mortem examination, the lung lesions in CCPP are more diffuse and a fibrinous fluid is found in the chest cavity.

23

Fibrin deposits cover the lungs and are frequently connected to the chest wall by fibrinous strands . In PPR high-risk areas it is advisable to rule out PPR by laboratory testing of, at least, serum samples from convalescent flocks, even if

CCPP is suspected.

Rinderpest disease in small ruminants has been described primarily in Asia.

Generally, this disease occurs in small ruminants only when they are in contact with affected cattle or buffaloes, so it is important during investigations to examine all species. Confirmation requires the resources of a specialist laboratory . The samples required for laboratory confirmation of both rinderpest and PPR are identical. As the Global Rinderpest Eradication Programme (GREP) progresses, it becomes increasingly important that PPR and rinderpest be differentiated because, at this stage of the programme, any outbreak of rinderpest anywhere represents an international emergency.

Foot-and-mouth disease (FMD) is more commonly seen in sheep than goats.

The most important distinguishing features of FMD,other than the appearance of the lesions, are the absence of breathing problems and diarrhoea, and the presence of lameness (often marked). Sudden death of very young lambs without other signs often occurs. The oral lesions when present are often very small and difficult to see; the mouth does not exude such a foul odour as in PPR.

24

Bluetongue, like PPR, is characterized by fever, discharges and oral lesions .

However, it differs from PPR in: the presence of oedema of the head region; bluish discoloration of the oral cavity, the coronary band of the hooves and the less hairy parts of the body; and lameness. Bluetongue virus infection is endemic throughout the regions of the world affected by PPR. Clinical disease is, however, not generally experienced in indigenous breeds in these countries, being mainly restricted to exotic introduced animals. The presence of antibody to bluetongue viruses in single samples does not confirm a provisional diagnosis of bluetongue.

Contagious ecthyma (orf, “sore mouth”, contagious pustular dermatitis) is often confused with PPR because of the nodules and thick scabs sometimes seen on the lips in the late stages of PPR. Confusion is especially likely to arise in severe cases of orf where lesions extend into the mouth and nose. In uncomplicated orf, there is usually no oral necrosis, diarrhoea or pneumonia.

1.9.3. Control of PPR:

State and federal authorities should be notified when PPR is suspected.

Eradication is recommended when the disease appears in previously PPR-free countries; rinderpest eradication methods are useful. There is no specific treatment; however, treatment for bacterial and parasitic complications decreases mortality in affected flocks or herds. An attenuated vaccine has been prepared in embryonic caprine kidney cell culture; it affords protection from natural disease for ~1 yr.

25

Rinderpest cell culture vaccine also has been used successfully for immunization against PPR ((Majiyagbeet al., 1984).PPR usually spreads by direct contact, and it is usually introduced into a herd by an infected animal. Asymptomatically infected animals can shed the virus, and one report suggests that virus shedding may be possible for up to 12 weeks or longer in recovered animals. Quarantine and testing decrease the risk of infection. There is no specific treatment against the disease.

Control of PPR in non infected countries may be achieved using classical measures such as restriction of importation of sheep and goats from affected areas, quarantine, slaughter and proper disposal of carcasses and contact fomites and decontamination of affected premises in case of introduction. Control of PPR outbreaks can also rely on movement control (quarantine) combined with the use of focused ("ring") vaccination and prophylactic immunization in high-risk populations. Immunization of small ruminants with lymph node and spleen materials containing virulent virus inactivated with 1.5-5% chloroform was tried and the animals were immune to subsequent challenge 18 months latter (Braide,

1981). Until recently, the most practical vaccination against PPR was based on the use of tissue culture adapted rinderpest vaccine. Vaccination of animals with RP attenuated virus has been practiced for a long time. The tissue culture rinderpest vaccine (TCRV) at a dose of 102.5 TCID50 protected goats against PPR for 12 months and the animals were not able to transmit the infection following challenge

26 with PPR virus (Taylor, 1979), although the antigen was detected in lachrymal swabs from vaccinated animals after challenge with virulent virus (Gibbs et al.,

1979). However, it was reported previously that considerable residues of virulence were detected after 32, 42, even 65 serial passages in embryonic lamb kidney cells

(Taylor, 1979). This vaccine was successfully used to control PPR in some countries in west Africa (Bourdin, 1973) and is widely used in many African countries (Lefèvre and Diallo, 1990). It has been withheld from being used because of its interference with the Pan-African Rinderpest Campaign (PARC), since it is impossible to determine if seropositive small ruminants have been vaccinated or naturally infected with RPV. Sera from animals vaccinated with RP vaccine contain substantial level of RP antibodies with little or no cross neutralising antibodies to PPR but after challenge with PPR, neutralizing antibodies to PPR increase sharply. RP thermostable vaccine was developed for 36 protection of goats against PPR (Stem, 1993). Homologous PPR vaccine attenuated after 63 passages in vero cell (Diallo et al., 1989) was used and produced a solid immunity for 3 years (Diallo et al., 1995). The PPRV homologous vaccine was found to be safe under field conditions even for pregnant animals and it induced immunity in

98% of the vaccinated animals (Diallo et al., 1995). The PPRV vaccine has been tried for protection of cattle against RP and it was found very effective (Couacy-

Hymann et al., 1995)

27

CHAPTER TWO

2. Material and Methods:

2.1. Area of the Study :

Blue Nile State is one of the major animal producing states in Sudan. The animal population in the State is estimated as 2.977.600 head of cattle, 3.965.000 head of sheep, 222.600 head of goats, 130.000 head of camels, 15.000 head of donkeys and few numbers of horses and mules. This livestock is mainly owned by migratory tribes (Fellata groups, Kenanna and Ruffaa). The State is divided into 5 provinces. Ad-Damazin, Al-Roseres, Al-Kurmuk, Geissan and Baw. Fig. (2). The annual rainfall average is between 700 and 1200 mm, the majority of which falls between July to November, the average relative humidity during the hot- dry season is less than 50% and increase to 80% in the rainy season. The mean maximal temperatures ranges from 310c to420c in dry hot season (Feb. to Apr.) and the mean minimal temperature rang from170c to 210c in rainy season (Jun. to Sep.)

(Anon,2009).

28

Fig.(2) the localities (Provinces) of the Blue Nile State:

29

2.2. Data collection:

The Serosurviellance covered Blue Nile State. The study has been conducted to investigate the epidemiology of the disease in the State to determine the incidence of the PPR disease in the sheep, goats, bovine and camels in the different loc99alities of the State.

The selection criteria of this state included animals, population, species, vaccination, disease status and seasonal movement of animals within the State or between the States. Data about the occurrence of PPR in the State and adopted vaccination programmes during 5 years (2005-2010) were collected. Table (2)

Collected data was statistically analyzed by (SPSS) soft programme.

30

Table (1):

The Number of the Serum samples collected from different species of animals at Blue Nile State during(2009-2010):

Locality Camel Cattle Goat Sheep Toal

Baw 90 90

Damazin 69 35 29 133

Geissan 24 40 64

Rosirais 80 45 22 147

Tadamon 131 135 240 406

Slaughterhouse 30 167 197

Total 80 200 359 498 1037

31

2.2.1. Collection of samples:

A total of 1037 serum samples were randomly collected from sheep, goats, cattle and camels from different localities and different sites of Blue Nile State during (2009 to 2010 ). Age, sex, location and the history of vaccination against

PPR of sampled animals were recorded.

The puncture area of the jugular vein was cleaned by 70% ethanol. A plain glass vaccutainer with a tube –holder and two way needle was used. Then 5 ml of blood was withdrawn. The vaccutainer tubes were labeled indicating location, age and sex of the animal, put on a rack away from direct sun light, and transferred to the laboratory. The vacutainers were kept overnight at room temperature then centrifuged for 5 minutes at 1500 r.p.m. Each serum sample was decanted in eppendorf tube, labeled indicating location, species, age and sex of the animal then stored at -20ºC until used.

Three Hundreds and three (303) lung tissue samples were collected from

Aldamazien slaughter house to detect the PPR antigen by Immunocapture ELISA technique.

The samples was prepared to the test of immunocapture ELISA as follows:

• A little of sample include the lesion was taken

• Chopping the samples and keep it in normal saline

32

• Send the samples to the central laboratory in Suba with ice box

• Centrifuge and take the sedppmentation for the test

2.2.2. Material for samples collection:

1. Sterile 10 ml vaccutainer

2. Needles and needle holders

3. Pasteur pipettes

4. eppendorf tube

5. Sterile 10 ml disposable syringe

6. Racks

7. Ice boxes

8. Forceps

9. Sesser's

10. Gloves

11. Bottles for tissues collection

2.2.3. Sampling Procedure:

For serum collection: There are two sampling techniques which were carried

out:

a)Sampling by using vaccutainer:

With the needle fixed with the needle holder about 5ml of blood from the

jugular vein of the sampled animal were drawn into the vaccutainer tube as the

33 result of negative pressure .The tube was then placed in a rack and blood was left to clot at room-temperature for about 2-3 hours or incubated overnight at room temperature .The tube was then centrifuged to get clear serum. With sterile Pasteur pipette the serum was then transferred to a sterile Bijou bottle and the bottle is labeled. b)Sampling by using disposable syring:

The syring was used is 10 ml to collect 5ml blood from the jugular vein of tested animal. The syring was then put in the rack overnight at room temperature.

The clear serum was separated at the top of the tube .By pressing the bills of the syring carefully, the serum was drawn into the Bijou bottle, then the bottles were labeled and kept in deep-freezer.

2.3. Competitive ELISA System for detection of specific antibodies to PPR virus:

A competitive ELISA Kit supplied by CIRAD, Montpellier, France was used in this test.

2.3.1. Materials:

1. ELISA microtitre plates (NUNC immuno-1- Maxisorp) with 96 wells for solid phase.

2. Multi channel pipettes with 8 tips

3. Single channel pipettes

34

4. New pipette tips

5. Trough

6. Washing bottle with a single delivery nozzle attachment

7. Small volume bottles (Mc Cartney bottles)

8. Absorbent towel

9. Incubator with an orbital shaker

10. ELISA reader (Spectrophotometric reading)

2.3.2. Reagents:

1. PPR antigen

2. Strong PPR positive serum

3. Weak PPR positive serum

4. PPR negative serum

5. PPR monoclonal antibodies

6. Washing buffer prepared at the laboratory 1:5 phosphate buffered

saline(PBS)(200ml PBS+800ml distilled water).The PBS was prepared by

dissolving one tablet(sodium chloride 8g, potassium chloride 0.20g , sodium

hydrogen phosphaste 1.15g , potassium dihydrogen orthophosphate 0.20g in

a litter of distilled water.

7. The Blocking Buffer:

35

50 ml of PBS 0.01M+50 u1 of tween 20+150u1 of negative serum

for1-3 plates.100ml of PBS 0.01M+100 u1 of tween 20+300 u1 of

negative serum for 4-8 plates.

The conjugate solution- Rabbit anti-mouse IgG linked to enzyme Horse

Radish peroxidase (HRPO), in blocking buffer1:1000.

8. Substrate chromogen Solution:

The optimum substrate for (HRPO) enzyme is orthophenylene

diamine (OPD), prepared in the form of tablets .One tablet was

dissolved in 75 ml distilled water .The solution was prepared from

(OPD) and hydrogen peroxide at a dilution of 1:250

9. Stopping solution:

One molar sulphuric acid (1M H2SO4) was added to stop further conjugate- chromogen reaction.

2.3.3. Protocol for competitive ELISA:

The test was carried out according to the manufacturer instructions as follows:

A working dilution1:100 of PPR antigen was prepared in phosphate buffered saline (PBS)

50 micro-liters of diluted antigen were dispensed into each of the 96 wells.

The sides of micro-plates were tapped to ensure even distribution of the antigen dilution over the bottom of each well.

36

The micro-plates were then covered and incubated at 37°C for one hour with continuous shaking or overnight at 40c without shaking.

The micro-plates were washed after that, using washing buffer 3 times.

Washing was done by, inverting the micro-plate using an abrupt downward hand motion, the contents of all wells were discarded and the wells were filled with washing buffer. After the 3rd wash the micro-plate was inverted onto absorbent towel and slapped several times.

40 micro-liters of recently prepared blocking buffer were dispensed in all wells of micro-plates.

60 micro-liters of blocking buffer were added to the conjugate control (A1,

A2) wells.

10 micro-liters of blocking buffer were added to monoclonal control wells

(F1, F2, G1, and G2)

10 micro-liters of strong positive control serum were added to the wells

(B1, B2, C1, and C2).

10 micro-liters of weak positive control serum were added to the wells (D1,

D2, E1, and E2).

10 micro-liters of negative control serum were added to the wells (H1, H2).

According to the plate layout, 10 micro-liters of test sera were added to the appropriate wells to get a final dilution of 1:5.

37

50 micro-liters of immediately prepared working dilution1:100 of the monoclonal antibody in blocking buffer were added to all wells of micro-plates, except the conjugate control wells (A1, A2) as in figure(3)

The plates were then covered and incubated at 37°C for one hour with continuous shaking.

Then, the plates were washed 3 times using the same procedure of the first wash.

50 micro-liters 1:1000 conjugate working solution were added to all 96 wells of micro-plates .The sides of micro-plates were tapped to insure that the conjugate solution is evenly distributed over the bottom of each well.

The plates were then covered and incubate at 37°C for 1hr

A third washing phase using the same previous procedure was done.

50 micro-liters of substrate/chromogen solution (4 micro-liters of H2 O2

(substrate) were added to each 1 ml of OPD) were added to each well of the test micro-plates.

50 micro-liters of substrate/chromogen solution were also added to one column of a clean micro-plate and this was used as blank.

The plates were incubated at room temperature in the dark for 10 minutes without shaking.

After colour develop that, 50 micro-liters of stop solution (1M sulphuric acid) were immediately added to all wells of the test micro-plates and to the wells of blank.

38

The micro-plate was then read with ELISA reader using the 492 nm filter.

The reset samples were tested again and they had been added to either positive or negative values.

The positive serum samples were indicated by colourless wells while negative samples were indicated by yellow colour wells.

Figure (3)

The plate layout for the (PPR) protocol for competitive ELISA:

1 2 3 4 5 6 7 8 9 10 11 12

A Cc Cc 1 5 37

B C++ C++ 1 5 37

C C++ C++ 2 6

D C+ C+ 2 6

E C+ C+ 3

F MAb MAb 3

G MAb MAb 4 40

H C- C- 4 40

39

2.4. Immunocapture ELISA for detection of antigen:

2.4.1.Materials:

As described in 2.3.1

2.4.2.Protocol of Immunocapture ELISA:

The test was carried out according to the manufacturer instructions as follows:

The plate was coated with 100 µl of capture antibody diluted 1:400 in 1XPBS

(1PBS+9DDW) incubated overnight at 40c without shaking.

The plate was washed three times with washing buffer (washing buffer: 20ml of

PBS+980DDW+500 µl of Tw20) .

50 µl of control antigens (PPR and RP) were added in columns 1,2 and 7,8 then 50

µl of BB was added in wells A1. A2. B1. B2 and A 7. A8 B7. B8 as in the

Figure(4).

50 µl of tested antigen was added in duplicate horizontally in columns 3-6, then

25 µl of detecting antibodies diluted1:300 in blocking buffer

(1xPBS+0.05%Tw20+0.5% negative serum e.g. 1ml of PBS +9ml of DDW+5 µl of Tw20+50 µl of negative serum) was added to each well ( PPR in1-6 RP IN 7-

12).

Then 25 µl of conjugate diluted1:100 in BB were added to all wells, the plate was incubated for 45 minutes in orbital shaker, the contents were discarded then the plate was washed three times and blotted to dry.

40

100 µl of substrate( OPD+H2O2 )were added to each well, the plate was then incubated in darkness without shaking for 10 minutes.

The reaction was stopped by adding 50 µl of stop solution( sulfuric acid )to each well, then the Plate was read using ELISA reader using the 492nm filter, with blank (OPD+H2 O2+H2SO4).

Figure (4)

The protocol of immunocapture ELISA for PPR:

1 2 3 4 5 6 7 8 9 10 11 12

A Cc Cc 1 1 Cc Cc 1 1 Cc Cc 2 2 Cc Cc 2 2 B PPR PPR 3 3 PPR PPR 3 3

C Ag Ag Ag Ag PPR PPR PPR PPR D

Ag Ag Ag Ag E RP RP RP RP

F Ag Ag Ag Ag

G RP RP RP RP

H Ag Ag Ag Ag N N N N

Ag Ag Ag Ag

N N 16 16 N N 16 16

Ag Ag Ag Ag

41

Chapter three

RESULT

3.1 Epidemiology of PPR in Blue Nile state:

The relatively good infrastructure available for veterinary services in the state, including manpower, transportation and the vaccine, may help in the control of the diseases spread, but in spite of that there was high prevalence of PPR disease as shown by the results of the serum samples.

This study investigated the epidemiology of PPR in Blue Nile state through collection of epidemiological data antigen and antibody detection. Continuous outbreaks of PPR were reported at Blue Nile state, there were many sing of respiratory disease, diarrhea and many animals aborted. The owners vaccinated their animals in the rainy seasons the samples of serum were collected before the . Table (2) .

The seasonal movement of animals had significant influence on spreading of the disease in different localities .

Migratory flocks of nomads have been thought to be a source of infection of

PPR in new areas. Environmental stress, particularly hot and humid climate contributes for the precipitation of the disease (Wous, 1995).

42

3.2. The prevalence of PPR in ruminants in Blue Nile Localaties:

Using cELISA in total of 1037 tested sera, variable seroprevalance of PPR was noticed in the state. Highest seroprevalance in Baw (92.22%) then Geissan

(84.38%) ,Damazin(63.16%),Rosirais (54.42%),Tadamon(43.84%) and

Slaughterhouse 49.75%) .

The difference in the sero-prevalence in the different localities was found to be statistically significant (P-value<.01) when the data was screened with Pearson chi-square .fig(5).

.3.3 The PPR Sero-prevalence in different species:

PPR antibodies were detected in different species.The highest sero- prevalence of PPR was found to be in Goats(79.54%) followed by sheep(54.62%),cattle (40.50%) and camels (22.50%).

The difference in the sero-prevalence in the different species was found to be statistically significant (P-value<.01) when the data was screened with Pearson chi- square, details are shown in Table(4) and Figure (6).

3.4. The PPR sero-prevalence in different locations in Blue NileState:

Within Blue Nile state PPR seroprevalance ranged between 31%-100%.

The highest sero-prevalence was found to be in Ofoud (100%), four locations were found to have above 90% seroprevalance (Azaza, Wadabook, Baw and

43

Slaughterhouse), while the lowest sero-prevalence was recorded in Agade

(31.77%), details are shown in Table(5) and Figure (7) .

3.5 Prevalence of PPR Antigen in the different species:

The prevalence of PPR antigen in samples collected from slaughterhouse during a period of three month in(2010) 303 lung and analyzed with immunocapture technique for PPR antigen and the result showed that six sample from sheep samples were found positive(4.37%). Figure(8) .

Table ( 2 ) Number of Animals vaccinated against PPR in Blue Nile State during(2009 -2010):

Years Vaccination Outbreaks areas

2005 41235 -

2006 145529 -

2007 257800 -

2008 6000 Baw

2009 6500 Tadamon

2010 9000 Tadamoun

44

Table3.Seroprevalence of PPR in the different localities in Blue Nile state,

Sudan detected using cELISA during 2010 :

Locality Sample Positive Sero-prevalence

collected

Baw 90 83 92.22%

Damazin 133 84 63.16%

Geissan 64 54 84.38%

Rosirais 147 80 54.42%

Tadamon 406 178 43.84%

Slaughterhouse 197 98 49.75%

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Table4. Result of cELISA for the detection of PPR antibodies in the different species in Blue Nile state (2010) :

Species Sample collected Positive

Camel 80 18

Cattle 200 81

Goat 259 206

Sheep 498 272

46

Table5. Determination of PPR Antibodies using cELISA in the different locations in Blue Nile state, Sudan (2010)

Location Sample Collected Positive

Aazaza 22 21

Agade 192 61

Albangadeed 19 10

Aldeesa 69 33

Baw 90 83

Boot 166 71

different places 40 34

Geissan 109 43

N-eldmazeen 24 20

Ofoud 16 16

Salha 197 98

Slaughter house 48 46

Wadabook 48 46

Total 1037 577

47

Table(6).Seroprevalence of PPR determined using cELISA in different species

and localities in Blue Nile state during (2010)

Locality goat sheep cattle camel total

Tested % tve Tested % tve Tested % tve Tested % tve Test % tve

ed

Baw 90 92.20 - - - - - 90 92.20%

Damazin 35 74.29 29 13.79 69 47.83 133 74.43%

Giessan 24 83.33 40 85.00 64 84.37%

Rosirais 45 91.11 22 95.45 80 22.50 147 54.42%

Damazin 30 43.33 167 50.90 197 49.74% slaughterhouse

Tadamon 35 65.71 240 44.58 131 36.64 406 43.84%

48

FIGURE 5.Seroprevalence of PPR in the different localities in Blue Nile state during (2010)

Sero-prevalence of PPR in different Localities

100.00% 92.22% 84.38% 90.00%

80.00% 63.16% 70.00% 54.42% 60.00% 43.84%

50.00%

40.00%

30.00%

20.00%

10.00%

0.00% Baw Damazin Geissan Rosirais Tadamon Slaug

49

FIGURE 6. PPR antibodies detected using cELISA in the different species in Blue Nile state (2010)

Sero-prevalence of PPR in different species

79.54%

80.00%

70.00% 54.62

60.00% 40.50%

50.00%

40.00% 22.50%

30.00%

20.00%

10.00%

0.00% Camel Cattle Goat Sheep

50

FIGURE 7. Seroprevalence of PPR determined using cELISA in the different locations in Blue Nile state , Sudan during (2010)

9999999999999999999999999999

Peste Des Petits Sero-positivity in different locations in Blue Nile

100.00% 100.00% 95.45% 92.22% 90.00% 85.00% 83.33% 80.00%

70.00%

60.00% 52.63% 49.75% 50.00% 47.83% 42.71% 39.45% 40.00% 31.77% 30.00%

20.00%

10.00%

0.00% Aazaza Agade Albangadeed Aldeesa Baw Boot different places Geissan N-eldmazeen Ofoud Salha

51

Figure 8. Prevalence of PPR Antigen in the different species

Prevalence of PPR in lung samples collected from slaughterhouses

4.37% 5% 4% 4% 3% 3% 2% 2% 1%

1% 0% 0% 0% 0% Goats Sheep Cattle Camels

52

Figure( 9):

Erosive stomatitis involving: the inside of the lower lips and adjacent gum

53

Fig(10):

Serous nasal discha9rge becoming mucopurulent and resulting, at times, in a

profuse

54

Fig(11):

Catarrhal exudate which crusts over and occludes the nostrils

55

Fig(12):

Severe non-haemorrhagic diarrhea, Congestion of conjunctiva, crusting on the medial canthus and sometimes profuse catarrhal conjunctivitis

56

Chapter four

DISCUSSION

Peste des petits ruminates is a viral disease caused by a paramyxovirus of the

Morbillivirus genus and poses a serious threat to the development of small ruminants’ production. It was observed for the first time in Eastern Sudan in

February 1971 at the Ethiopian border not far from the Dinder National Park. The disease spread in January 1972 to the west and to the north, reaching Khartoum and Gazira State in the centre of the country. Another outbreak was recorded in

1974. Sudan remained free from the disease until 1989 when it was reported again in the central area of the country between Rufaa and Wad Rawa. The disease spread to Khartoum State and to the Northern Dafur in Western Sudan in 1990(El

Hag, 1995). PPR virus was isolated from different parts of Sudan [Gezira State,

White Nile State, Khartoum state, North Kordofan State and River Nile State during 2000- 2002 (Intisar 2002). A serological survey of PPR in Sudan during

2002-2005 using cELISA discovered positive results in 70% of ovine sera collected from Kordofan state, 52.5% of ovine sera and 34.7 % of caprine sera collected from Darfur State (Intisar 2007).

. The results of this study showed that PPR is widely spread in the Blue Nile State and its prevalence seems to be increasing in most areas where ruminants are raised.

57

The general detected seroprevalence of PPR was 55.64% which is higher than that earlier reported(50%) in Sudan (Haroun et al. 2002; Intisar et al. 2007).This is probably due to the termination of rinderpest vaccination campaigns in the country during which sheep and goats were vaccinated with rinderpest vaccine.In the present study the highest sero-prevalence of PPR was found to be in Goats

(79.54%) followed by sheep (54.62), cattle (40.50%) and camel (22.50%). Sheep was noticed to have higher percentage of seropositivity than goats (67.7%, 55.6%), similar finding was reported by Intisar et al (2007). However in this study much higher seroprevalence was detected in goats (79.54%) similar finding was reported by Intisar et al. (2010).) in the previous report (55.6%). The higher prevalence of

PPR in sheep than in goats has been formerly recognized which is mostly due to the high mortality observed in goats so lower percentages of survivor's sexists

(Khan et al. 2008). The observed antibodies to PPR in cattle sera may be due to contacts with PPR infected small ruminants or due to Vaccination with PPR vaccine. Although Camels are comparatively hardy animal species they are also susceptible to many infectious diseases. Serological surveys have demonstrated that camels are susceptible to the infection with PPR virus (Haroun et al., 2002),

Khalafalla et al., (2010) reported an outbreak of (PPR) in camels in the Eastern

Sudan.

58

The result of the assessment of sera samples from different localities by C-ELISA proved that about 55.64% were positive .High prevalence of PPR was detected in the samples from Bow locality followed by Geissan, Damazin, Rosirais and

Tadamon. The prevalence rates obtained by this study appear high, and this proportion considered high.

The detection of a high prevalence of antibodies against PPRvirus in sera collected from field samples of sheep, goats, cattle and camel, explained the exposure of these animals to the field virus. The wide spread of the disease all over the state was also indicated. This result agreed with the previous reports( Nussieba, 2005;

Nussieba et al., 2009)who reported that PPR is widespread in Sudan, and high prevalence of PPR antibodies detected by C-ELISA was in Blue Nile State, hence this prevalent of the disease and high prevalence in the State may be attributed to the animal movement between bordering States and countries. Variations in seroprevalance could be due to differences in sample size, age, prevailing manage mental practices, humidity and season (Radostits et al., 2007).

PPR antigen detection was carried out in this study using IcELISA; the overall positive samples were 6 out of 303 (1.98%). Nussieba (2005) reported the detection and isolation of PPR antigen in sheep and goat tissue specimens collected from different areas in Sudan. 519 sera of sheep and goats were tested for PPR antibodies using cELISA, collected from River Nile State (North), and Darfur

59

(West) with 50.7% positives. Khalafalla et al (2005) reported a new emerging respiratory disease of camels in Eastern Sudan during Sep. 2004; PPR antigen was detected using IcELISA and PCR, PPR was supposed to be the main causative agent of that outbreak.

Nussieba et al (2008) using haemagglutination test detected PPR antigen in 92.5% of 40 tissue samples of sheep in the Sudan; this great difference could be due to the collection of samples during an active outbreak and reported in the present work is probably due to that most of samples collected from healthy slaughter ruminants from slaughterhouse.

Conclusions and Recommendations

Conclusion:

In most countries where the disease is endemic, PPR is the main infectious killer disease of small ruminants, and therefore the most important threat to the livelihoods of poor farmers. Because of its high mortality rate, PPR affects food security directly by reducing the availability of meat and milk for family consumption and of funds for purchasing other commodities and foods, for which prices are increasing. The overall consequence of PPR outbreaks will be increased vulnerabilities for communities that are already facing such challenges as agro climatic changes and civil unrest. PPR prevention and control should be considered a public good.

60

In conclusion, according to the result of the present study PPR is widely spread in

the Blue Nile State and its prevalence seems to be increasing in most areas where

ruminants are raised.

Continuous outbreak of PPR and the result obtained in this study, concluded that

PPR is endemic in the Blue Nile State.

Recommendations:

Based on the results of the study, and depending on the information about the

transmission of the disease. The following recommendation should be considered:

• Increase the awareness of animal owners of the seriousness of the disease and

educate them that the disease is untreatable.

• Strict quarantine measures should be carried out around the infected areas.

• Mass vaccination against PPR to cover all the state and Should keep vaccinating

the animals continuously

61

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