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DETECTION OF ANTIBODIES AGAINST Mycoplasma capricolum subsp. capripneumoniae IN SERA COLLECTED FROM GOATS IN KHARTOUM STATE
By Gasim Mohammed Abuidris Elmustafa (B.Sc.V., University of Khartoum, 1999)
Supervisor Dr. Ahmed Zaki Saad Ahmed Zaki
A thesis submitted to the University of Khartoum in partial fulfillment of the requirements for M.Sc. Degree in Microbiology By courses and complementary research
Department of Microbiology Faculty of Veterinary Medicine University of Khartoum
September 2007
Preface
This study was carried out at the Department of Microbiology, Faculty of Veterinary Science, University of Khartoum under the supervision of Dr. Ahmed Zaki Saad
i
Dedication
To my beloved father and mother To my beloved family To my closed friends.
With love….
ii ACKNOWLEDGEMENT
I am indebted to “Allah” who granted me everything including the mind, health and patience to accomplish this work.
I would like to express my deepest appreciation and sincere gratitude to my supervisor Dr. Ahmed Zaki Saad for his guidance, valuable advices, suggestions and kind supervision throughout the study period.
A lot of thanks are expressed to all members of Microbiology laboratory; Faculty of Veterinary
Medicine; University of Khartoum for their valuable help.
iii Least but not last, my deep gratefulness are due to my colleagues Ali Elkarrar and Mohammed
Elfadil for their help, patience, and encouragement throughout the period of the study.
iv Abstract
The main goal of this research was to study the prevalence of contagious caprine pleuropneumonia (CCPP) in Khartoum State.
A total of 160 serum samples were collected from different areas in the State and investigated for antibodies (Abs) against
Mycoplasma capricolum subsp. capripneumoniae (Mccp) using agar gel immunodiffusion (AGID) and indirect haemagglutination (IHA) tests.
Sonicated SDS-treated soluble antigen was the most suitable antigen preparation for AGID compared to whole cell or cell debris
SDS-treated antigens.
60% of samples were found positive with AGID whereas
63.75% of samples were found positive with IHA respectively. All
IHA positive sera with antibody titre ≥ 1/8 were found positive with
AGID.
Regardless of sex and age all animals are equally affected.
Foreign goat breeds were more susceptible to infection than local ones.
v It was concluded from the study that CCPP was widely distributed in Khartoum State and further work should be done to isolate the causative agent.
vi ﻤﻠﺨﺹ ﺍﻷﻁﺭﻭﺤﺔ
ﻫﺩﻑ ﻫﺫﺍ ﺍﻟﺒﺤﺙ ﺇﻟﻲ ﺩﺭﺍﺴﺔ ﻤﺩﻱ ﺍﻨﺘﺸﺎ ﺭ ﻤﺭﺽ ﺫﺍ ﺕ ﺍﻟﺭﺌـﺔ ﻭﺍﻟﺠﻨـﺏ
ﺍﻟﺴﺎﺭﻱ ﻓﻲ ﺍﻟﻤﺎﻋﺯ ﻓﻲ ﻭﻻﻴﺔ ﺍﻟﺨﺭﻁﻭﻡ.
ﺘﻡ ﺠﻤﻊ 160 ﻋﻴﻨﺔ ﻤﺼﻠﻴﺔ ﻤﻥ ﺍﻟﻤﺎﻋﺯ ﻤﻥ ﻤﺨﺘﻠﻑ ﺍﻟﻤﻨﺎﻁﻕ ﺒﺎﻟﻭﻻﻴﺔ ﻭﺘﻡ
ﻓﺤﺼﻬﺎ ﻤﻌﻤﻠﻴﺎ ﻟﻠﻜﺸﻑ ﻋـﻥ ﻭﺠـﻭﺩ ﺍﻷﺠـﺴﺎﻡ ﺍﻟﻤـﻀﺎﺩﺓ ﻟـﺴﺒﺏ ﺍﻟﻤـﺭﺽ capricolum sub sp capripneumoniae ) (Mycoplasma ﺒﻭﺍﺴﻁﺔ ﺍﺨﺘﺒﺎﺭﻱ ﺍﻻﻨﺘﺸﺎﺭ ﺍﻟﻤﻨﺎﻋﻲ ﺍﻟﻤ ﺯﺩﻭﺝ ﻓﻲ ﺃﺠﺎﺭ ﺍﻟﺠل ﻭ ﺍﺨﺘﺒﺎﺭ ﺍﻟـﺘﻼﺯﻥ
ﺍﻟﻐﻴﺭ ﻤﺒﺎﺸﺭ.
ﻟﻘﺩ ﻭﺠﺩ ﺇﻥ ﺍﻟﻤﺴﺘﻀﺩ ﺍﻟﺫﻱ ﺘـﻡ ﺘﻜـﺴﻴﺭﻩ ﺒﻭﺍﺴـﻁﺔ ﺍل (Sonicator)
ﻭﻋﻭﻟﺞ ﺒﻭﺍﺴﻁﺔ (Sodium dodecyl suphate)ﻫﻭ ﺍﻷﻨﺴﺏ ﻻﺨﺘﺒﺎﺭ ﺍﻻﻨﺘﺸﺎﺭ
ﺍﻟﻤﻨﺎﻋﻲ ﺍﻟﻤﺯﺩﻭﺝ ﻓﻲ ﺃﺠﺎﺭ ﺍﻟﺠل ﻋﻨﺩ ﻤﻘﺎﺭﻨﺘﻪ ﺒﻤﺴﺘﺤﻀﺭﺍﺕ ﻤﺴﺘﻀﺩﻴﺔ ﺍﺨﺭﻱ.
60% ﻤﻥ ﺍﻟﻌﻴﻨﺎﺕ ﻗﺩ ﻭﺠﺩﺕ ﻋﻨﺩ ﺍﺨﺘﺒﺎﺭﻫﺎ ﺒﻭﺍﺴﻁﺔ ﺍﺨﺘﺒـﺎﺭ ﺍﻻﻨﺘـﺸﺎﺭ
ﺍﻟﻤﻨﺎﻋﻲ ﺍﻟﻤﺯﺩﻭﺝ ﻓﻲ ﺃﺠﺎﺭ ﺍﻟﺠل ﺒﻴﻨﻤﺎ 63.75% ﻗـﺩ ﻭﺠـﺩﺕ ﻤﻭﺠﺒـﺔ ﻋﻨـﺩ
ﺍﺨﺘﺒﺎﺭﻫﺎ ﺒﻭﺍﺴﻁﺔ ﺍﺨﺘﺒﺎﺭ ﺍﻟﺘﻼﺯﻥ ﺍﻟﻐﻴﺭ ﻤﺒﺎﺸﺭ . ﻭﺠﺩﺕ ﺃﻥ ﻜل ﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﺘـﻲ
ﺘﺤﺘﻭﻱ ﻋﻠﻲ ﺘﺭﻜﻴﺯ ﺠﺴﻡ ﻤﻀﺎﺩ ⅛ ﻓﺎﻜﺜﺭ ﻋﻨﺩ ﻓﺤﺼﻬﺎ ﺒﺎﺨﺘﺒﺎﺭ ﺍﻟـﺘﻼﺯﻥ ﺍﻟﻐﻴـﺭ
ﻤﺒﺎﺸﺭ ﺘﻜﻭﻥ ﻤﻭﺠﺒﺔ ﻋﻨﺩ ﺍﺨﺘﺒﺎ ﺭﻫﺎ ﺒﻭﺍﺴﻁﺔ ﺍﺨﺘﺒﺎﺭ ﺍﻻﻨﺘﺸﺎﺭ ﺍﻟﻤﻨﺎﻋﻲ ﺍﻟﻤـﺯﺩﻭﺝ
ﻓﻲ ﺃﺠﺎﺭ ﺍﻟﺠل .
vii ﺃﻭﻀﺤﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺃﻥ ﻜل ﺍﻟﺤﻴﻭﺍﻨﺎﺕ ﻤﻌﺭﻀﺔ ﻟﻺﺼﺎﺒﺔ ﺒﺼﺭﻑ ﺍﻟﻨﻅـﺭ
ﻋﻥ ﻋﻤﺭﻫﺎ ﻭﺠﻨﺴﻬﺎ ﻟﻜﻥ ﺍﺘﻀﺢ ﺃﻥ ﺍﻟﺴﻼﻻﺕ ﺍﻷﺠﻨﺒﻴﺔ ﺃﻜﺜﺭ ﻋﺭﻀﺔ ﻟﻺﺼـﺎﺒﺔ
ﻤﻥ ﺍﻟﻤﺤﻠﻴﺔ.
ﺨﻠﺼﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺇﻟﻲ ﺃﻥ ﺍﻟﻤﺭﺽ ﻤﻨﺘﺸﺭ ﻓﻲ ﻜل ﻭﻻﻴﺔ ﺍﻟﺨﺭﻁﻭﻡ ﻭﻴﻘﺘﺭﺡ
ﺇﺠﺭﺍﺀ ﻤﺯﻴﺩ ﻤﻥ ﺍﻟﺒﺤﺙ ﻟﻌﺯل ﻤﺴﺒﺏ ﺍﻟﻤﺭﺽ ﺒﺎﻟﻭﻻﻴﺔ.
viii
LIST OF CONTENTS Preface ……………………………………………………………… i Dedication ii ……………………………………………………………… Acknowledgment iii ………………………………………………………. English Abstract iv ………………………………………………………... Arabic abstract……………………………………………………….. v List of contents vii ………………………………………………………… List of tables xi …………………………………………………………… List of figures.. xii List of Plates xii …………………………………………………………… Introduction 1 ……………………………………………………………. Chapter One: Literature Review 3 1.1 Contagious caprine pleuropneumonia ………………... 3 1.1.1 Definition………………………………………………. 3 1.2 History…………………………………………………. 3 1.3 Causative agent……………………………………….... 3 1.4 8 Classification 1.5 Epidemiology …………………………………………. 8 1.5.1 Host range …………………………………………….. 8 1.5.1.1. Goats of Sudan ………………………………………... 9 1.5.1.2 Breeds …………………………………………………. 9 1.5.1.2.1 Sudanese Nubian goats ……………………………….. 9 1.5.1.2.2. Sudanese desert goats ………………………………… 9 1.5.1.2.3. The Nilotic goats 10 1.5.1.2.4. Saanen 10 1.5.1.2.5. Damascus 10 1.5.2. Transmission 12 1.5.3. Distribution 12` 1.6. Clinical signs 14 1.7s. Pathology 15
ix 17.1. Gross lesions 15 1.7.2. Histopathology 16 1.8. Molecular epidemiology 16 1.9. Diagnosis 17 1.9.1. Field Diagnosis 17 1.9.2. Specimens for Laboratory 18 1.9.3. Laboratory Diagnosis 18 1.9.4. Differential Diagnosis 20 1.10. Disease Prevention and Control 20 1.11. Control and Eradication 21 1.12. Immunology 22 1.13. Serology 22 1.13.1. Serological identification 23 1.13.1.1. Growth inhibition test (GIT) 23 1.13.1.2. Immunofluorescence tests (IF) 23 1.13.2. Serological tests 23 1.13.2.1 Immunodiffusion 23 1.13.2.2. Indirect (Passive) hem agglutination test (IHA) 24 1.13.2.3. Latex agglutination test (LAT) 25 1.13.2.3.1. Polysaccharide-specific antibody-detection (LAT) 25 1.13.2.3.2. polysaccharide-specific antigen-detection (LAT) 25 1.13.2.4. Complement fixation test (CFT) 26 1.13.2.5. Enzyme linked immunosorbent assay (ELISA) 26 1.13.2.6. Polyacrylamide gel electrophoresis (PAGE) and 27 immunobloting CHAPTER TWO: Materials and Methods 29 2.1 Samples ……………………………………………. 29
x 2.1.1. Collection of samples ………………………………… 29 2.1.2. Separation of serum 30 2.2. Mccp strains 30 2.2.1. Growth medium 30 2.2.1.1. Medium ingredients 30 2.2.1.1.1. Horse serum 30 2.2.1.1.2. Fresh yeast extract 31 2.2.1.1.3. Glucose solution 31 2.2.1.1.4. Penicillin solution 31 2.2.1.1.5. Thallium acetate 10% (w/v) 31 2.2.1.1.6. Sodium pyruvate 32 2.2.1.2. Modified Hayfilick’s medium 32 2..2.3. Cultivation of Mccp 33 2.2.4. Harvesting and washing of cells 33 2.2.5. Sonication of Mccp cells 33 2.2.6. SDS treatment 34 2.3. Serological test 34 2.3.1. Agar gel immundiffusion test (AGID) 34 2.3.1.1. Material 34 2.3.1.1.1. Antigens 34 2.3.1.1.2. Serum samples 34 2.3.1.1.3. Phosphate buffered saline (PBS) 35 2.3.1.1.4. Other substances 35 2.3.1.2. Method 35
xi 2.3.1.2.1 Preparation of the gel 35 2.3.1.2.2. Test procedure 36 2.3.2. Indirect haemagglutination test (IHA) 37 2.3.2.1. Materials 37 2.3.2.1.1. Antigen 37 2.3.2.1.2. Serum samples 37 2.3.2.1.3. Sheep red blood cells (SRBC) 37 2.3.2.1.4. Normal saline 37 2.3.2.1.5. Phosphate buffered saline (PBS) 37 2.3.2.1.6. Others substances 37 2.3.2.2. Method 38 2.3.2.2.1. Test procedure 38 CHAPTER THREE RESULTS 40 3.1 Selection of antigen for AGID 40 3.2 Detection of antibodies against Mccp 40 3.3 Distribution of CCP in Khartoum State 43 3.4. CCPP in different goat breeds 45 3.5. CCPP in different age groups of goats 47 3.6. CCPP in different sex of goats 49 CHAPTER FOUR DISCUSSION 52 Conclusion 55 Recommendations 55 REFERENCE 56
xii LIST OF TABLES
Table Page 1.1 Mycoplasmas, Including M. capricolum subsp. 6 capripneumoniae, Isolated from Small Ruminants with Respiratory Disease (Nicolas, 2002) 1.2 Major Biochemical Differences between Mycoplasmas 7 of Small Ruminants (Nicolas, 2002) 1.3 Distribution of CCPP (Nicolas, 2002) 14 2.1 Samples collected from different areas in Khartoum 29 State.
3.1 Comparison of results of the two tests. Result of IHA 41 was recorded as strong positive, weak positive and negative 3.2 Distribution of CCP different areas of Khartoum State 43
3.3 Antibodies against Mccp in different goat breeds in 45 Khartoum state:
3.4 47 CCPP in different age groups of goats 3.5 CCPP in different goat’s sex 49
LIST OF FIGURES
Figure Page 2.1 Loading of wells with different reagents in the AGID 36
xiii test 3.1 Distribution of CCPP in Khartoum State 44 3.2 Antibodies against Mccp in different breeds of goats in 46 Khartoum State 3.3 Antibodies against Mccp in different age groups of 48 goats 3.4 CCPP in different goat sexes in Khartoum state. 50
LIST OF PLATES
Plate Page 1.4.1.1. Goats of Sudan 11 3.1 Selection of antigen for AGID 42 3 AGID test for goat serum samples collected from 51 Khartoum State
xiv Introduction: Contagious caprine pleuropneumonia (CCPP) is a major disease of goats in Africa, Middle East and western Asia. It is both highly contagious and particularly virulent with up to 100% and 70% morbidity and mortality rates respectively in susceptible herds. The disease, which is caused by Mycoplasma capriculum subsp. capripnemoniae (Mccp), results in major economic losses to goats industry in nearly 40 countries; hence it is placed by the OIE in list B, which includes the important disease from economic standpoint. While clinical disease has so far been reported in 38 countries, only 11 countries have isolated the causative organism, principally because Mccp is difficult to culture. In the Sudan, the disease is known Abu nini and it was described in the Sudan Veterinary Report since 1902 (Anon, 1902). However, isolation attempts of the causative agent were unsuccessful till 1960s where Abdulla and Lindly (1967) achieved the primary isolation of a mycoplasma from a goat with pleuropneumonia, but they failed to maintain the isolate. The presence of the disease in the Sudan was officially declared in 1981, following the successful isolation of the type strain (F38) from a goat showing signs of Abu-nini in Khartoum State (Harbi et al .1981). Thereafter, diagnosis of the disease in the Sudan was based mainly on clinical signs and postmortem findings.Isolation of Mccp was then not reported in the Sudan till the beginning of the 21th century,when Abbass.M.Sawsan (2006) achieved the isolation of 7 Mccp strains from Darfur, Khartoum and Kassala States.Unfortunately 6 isolates were lost due to improper storage. Failure to isolate more strains in the Sudan in the last 22 years resulted in lack of data concerning the characteristics of the local
1 strains responsible for the disease; their origin and distribution in the country, and their immunogenic properties which would facilitate diagnosis of the disease. The current research represented a part of a detailed study funded by the Ministry of Higher Education aiming to study the prevalence of CCPP in the Sudan. Objectives: This study was carried out in Khartoum State with the following objectives: i.Detection of antibodies against Mccp in sera from goats in different parts of the State. ii.Study the role of breed, age and sex in susceptibility of goats to CCPP. iii.Compare the sensitivity and specificity of AGID &IHA in detection of antibodies against Mccp.
2 CHAPTER ONE LITERATURE REVIEW
1.1. Contagious Caprine Pleuropneumonia: 1.1.1. Definition: Contagious caprine pleuropneumonia (CCPP) is an acute highly contagious disease of goats caused by a mycoplasma and characterized by fever, coughing, severe respiratory distress, and high mortality. The principal lesion at necropsy is sero fibrinous pleuropneumonia.
1.2.History: Contagious caprine pleuropneumonia (CCPP) was first described in 1873 in Algeria by Thomas and known under the local name of "bou frida" (McMartin et al., 1980) because, in the majority of diseased goats, only one lung was affected. Its contagiousness was not initially recognized because the disease was endemic in most areas under examination, so climatic conditions were thought to be responsible for disease outbreaks. However, a major outbreak in South Africa in 1881 following the introduction of goats from Turkey led the colonial veterinary surgeon, Duncan Hutcheon, to conclude that CCPP was highly infectious and referred to as the classical CCPP.
1.3. Causative agent: Research into the control of CCPP was initially hampered by confusion over the exact cause of the disease. Two mycoplasmas, M. mycoides subsp. mycoides (LC) and M. mycoides subsp. capri, were for sometime implicated in the etiology of the disease because they
3 caused a pleuropneumonia in small ruminants that resembled CCPP. It was not until 1976 that a highly fastidious mycoplasma designated F38 but later named M. capricolum subsp. capripneumoniae, was isolated for the first time in vitro by MacOwan and Minette (1976). Once these workers had developed a suitable medium for the mycoplasma and carried out experimental infections, its role as the primary cause of classical CCPP was confirmed. However, in spite of this confirmation, respiratory diseases, caused by M. m. capri and M. m. mycoides (LC), are still referred to erroneously as CCPP particularly in the Middle East and India. A condition should only be termed as CCPP when the following criteria have been satisfied:
• Mccp is isolated or there is strong serological evidence of the mycoplasma
• Lesions are restricted to lung and pleura and consist of a pleuropneumonia
• The condition is highly infectious with high levels of morbidity and mortality rates.
• There is no enlargement of the interlobular septa of the lung. The taxonomic status of F38 has long been unclear and it was only in 1993 that it became a subspecies of M. capricolum, and classified as M. capricolum subsp. capripneumoniae (Mccp) (Leach Erno and MacOwan, 1993). Mccp in grouped in what is known as the Mycoplasma mycoides cluster. This cluster contains six important ruminant mycoplasmas including M. m. mycoides SC, M. m. mycoides LC and M. m. capri. Member of the cluster share immunological and biochemical properties. Accordingly, their close relationship can lead to problems for diagnosis. Table (1-1) and Table (1-2) summarize the
4 properties of Mccp some members of this cluster as well as other mycoplasmas capable of causing diseases in small ruminants.
5 Table (1.1): Mycoplasmas, Including M. capricolum subsp. capripneumoniae, Isolated from Small Ruminants with Respiratory Disease (Nicolas, 2002) Primary site of In vitro Mycoplasma Host Disease* Pathogenicity isolation (other) growth M.c.. Slow (5-7 Goat (Sheep) Lungs CCPP High capripneumoniae d) Goat (Sheep, Resp. tract (udder, Pp, M, A, M. m. mycoides LC Moderate Fast Cattle) joints) C Respiratory tract M. m. capri Goat (Sheep) Pp, A, C Moderate Fast (joints) Joints/Respiratory M. c. capricolum Goat, Sheep Pp, M, A High Fast tract (udder) M. ovipneumoniae Sheep, Goat Respiratory tract Pneumonia Low Moderate Sheep and M. conjunctivae Eyes KC Moderate Moderate Goats Sheep and M, A, KC, M. agalactiae Udder (joints, eyes) High Fast Goats P Goats M. putrefaciens Udder (joints) M, A Moderate Fast (Sheep) Low/non M. arginini Ubiquitous Respiratory tract None Fast pathogenic
*Pp = pleuropneumonia; P = pneumonia; M = mastitis; A = arthritis; C = conjunctivitis; KC = keratoconjunctivitis; CCPP = contagious caprine pleuropneumonia.
6 Table (1.2): Major Biochemical Differences between Mycoplasmas of Small Ruminants (Nicolas, 2002)
Film Glucose Arginine Phosphatase Casein Tetrazolium Reduction Mycoplasma and fermentation hydrolysis activity digestion aerobic anaerobic spots
M. + - - - + varies weak/+ capripneumoniae
M. mycoides LC + - - - + + +
M. m. capri + - - - + + +
M.c. capricolum + + + - + + +
M. + - - - - varies + ovipneumoniae
M. conjunctivae + - - - - - +
M. agalactiae - - + + - + +
M. putrefaciens + - + + - varies +
M. arginini - + - - - - +
+ Positive. - Negative.
7 1.4. Classification: Mycoplasma put under Class: Mollicutes Order: Mycoplasmatales. Family: Mycoplamataceae. Genus: Mycoplasma. Mycoplasma has no cell wall, but a three layered cytoplasmic membrane, and produced fried-egg appearance colonies by most species. They replicate by binary fission. The membrane of family Mycoplasmataceae requires sterol for growth. It has genome size of 5.0ҳ 10 Daltons and their NADH oxidase is localized in the cytoplasm (Freunt,1983).The genus Mycoplasma don’t hydrolyzed urea contrary to the other genus ureaplasma.
1.5. Epidemiology: 1.5.1. Host range: Contagious caprine pleuropneumonia is a disease of goats, and where the classical disease has been described; only goats were involved in spite of the presence of sheep and cattle. Sheep were believed to be refractory to disease although possibly acting as a reservoir of infection .However; there have been reports of sheep with respiratory signs from which Mccp was isolated and some of which were also seropositive. Additional studies are needed to clarify the susceptibility to sheep to infection and to understand their role in the epidemiology of CCPP by acting as carrier animals. M. mycoides capri, which was considered the cause of CCPP will result in a fatal disease in experimentally inoculated sheep and
8 can spread from goats to sheep. It is however, not recognized as a cause of natural disease in sheep.
1.5.1.1. Goats of Sudan: The goat population of the Sudan was estimated approximately to be about 11 millions (Yassin and Hussein, 1985). However, more recent data from the Animal Wealth Sector Performance Report, Federal Ministry of Animal Resources in 2000 reported 37 million heads of goats in the Sudan.
1.5.1.2. Breeds: At least six methods of classifying domestic goat are used and based on origin, utility, body size, ear shape and ear length exist (Payne, 1966). Devendra and Burns (1983) classified goat into three categories on the basis of body size. There are many goat breeds in the Sudan but the main breeds are the Nubian, Sudanese Desert and the Nilotic (Devendra, 1983).
1.5.1.2.1. Sudanese Nubian goats: The Nubian breed, also referred to as local Nubian in Egypt, has curved horns in both sexes and small ears which may rector dropping. The hair is long, and the color black or black and white (Devendra, et al., 1977).
1.5.1.2.2. Sudanese Desert goats: These goats belong to the arid area of northern Sudan. The desert goats have long legs and short fine gray coats which are
9 adaptation characteristics to arid regions. The breed is very prolific, producing a high proportion of twins, with production of 9 – 10 kids.
1.5.1.2.3. The Nilotic goats: A typical example is the type found in Southern Sudan. The male has short horns, and females have either very short horns or none at all. These goats show some similarities with the small east African goat.
1.5.1.2.4. Saanen: Saanen is a foreign breed originated in West Switzerland; they are white, pale cream or pale biscuit in color, with black spot on the nose, ears and udder. They have a short coat and are generally polled, and the ears are erect and point forward. They have the highest average milk yield of all breed of goats, and for this reason have been introduce into many countries.
1.5.1.2.5. Damascus: The Damascus is probably the most important in the Middle East, and has often claimed to be the most suitable dairy goat for the area. The breed is common in Lebanon, Syria and Cyprus. Hirsch (1932) described Damascus breeds as polled in both sexes, generally red or red and white in color, with a convex head profile and Long lap ears. Females weight about 55-60kg, and have a height at withers of about 73cm. It gives daily milk yield as 2-4 liters. Damascus goats are more prolific than the Saanen, with average of 1.7 kids per birth (Epstein and Hers, 1964).
10 A: Nubian B: Desert
C: Saanen D: Damascus
Plate (1): Goats of Sudan
11 1.5.2. Transmission: Contagious caprine pleuropneumonia is transmitted by direct contact through inhalation of infective aerosols. Outbreaks of the disease often occur after heavy rains (e.g., after the monsoons in India) and after cold spells. This is probably because recovered carrier animals start shedding the mycoplasmas. After the stress of sudden climatic change. It is believed that a long-term carrier state may exist.
1.5.3. Distribution: Contagious caprine pleuropneumonia is endemic in many countries in Africa Asia and the Middle East. Mccp has only been isolated in 13 countries because of the difficulty encountered in isolation (Table 1.3). In the Sudan, the disease is known as Abu Nini. The earliest reference to this disease was in the beginning of the last century in the annuals reports of the Sudan Veterinary Department (Anon, 1902). Goats with pleuropnemonia were first report in Kassala and later in Upper Nile, Blue Nile, Khartoum, Kordofan and Bahar El Gazal Provinces. Even as late as 1958 attempts to isolate the casual organism had been largely unsuccessful (Anon, 1958). Karib (1958) succeeded in setting up lung lesion by spraying diluted lung fluid from an infected animal into the nostrils of three goats. In Khartoum State El Nasri(1967);Abdulla et al (1980), Abdulla and Lindly (1967) achieved the primary isolation of a mycoplasma from a goat with pleuropneumonia. However, the maintenance of this isolate was always unsuccessful. They reported also attempts to cross infect cattle, goats and sheep with caprine and bovine strains of mycoplasma of local origin but it was considered necessary to repeat and confirm
12 their observations. Sudan is the second country which has established the role of the former mycoplasma sp. strain F38 as the causative agent of CCPP (Harbi et al., 1981; Abbas, 1986). The first successful isolation trail was carried out by Harbi et al. (1981), who recovered Mccp isolate from a goat with acute pleuropnemonia in Khartoum State and they designated that isolate as strain SPG1.Abbas.I.Sumaya (1986) was able to isolate four different species of mycolasma including M.capricolum subsp.capripneumoniae,M.mycoides subsp.mycoides (LC),M.ovipneumoniae and M.arginini (Abbas,1986). The second successful isolation of Mccp in the Sudan was reported by Abbas.M.Sawsan (2006). She isolated 5 strains of Mccp; three of them were recovered from Southern Darfur State including Nyala2 (N2) strain. The other two strains were isolated from Khartoum and Kassala States. The only reports of suspected CCPP in Europe date back to the 1920s when an outbreak occurred in Greece following the seizure of goats from Turkey. Conclaves (1982), however, reported a disease in goats in Portugal in 1980 which was very closely resembled classical CCPP but from which M. m. mycoides LC was isolated. In 1996, a suspected outbreak of pleuropneumonia clinically resembling CCPP was investigated in a goat herd in England containing some imported goats and which had suffered severe respiratory disease resulting in many deaths (Nicolas, 2002). CCPP was quickly ruled out by the newly developed polymerase chain reaction (PCR) tests and a combination of M. ovipneumoniae and Pasteurella (Mannheima) haemolytica were eventually identified as the causes of disease. There have been no reports of the isolation of Mccp on the American
13 continent although other members of cluster have been described there.
Table(1.3): Distribution of CCPP (Nicolas, 2002)
Confirmed by isolation Clinical disease reported or
of mycoplasma suspected
Algeria, Burkina Faso, Benin, Chad, Eritrea, Ethiopia, Cameroon, Central African Republic, Africa Kenya, Niger, Sudan, Djibouti, Egypt, Libya, Mali, Nigeria, Tunisia, Uganda Somali, Zaire
Nepal, Oman, United Afghanistan, Bangladesh, India, Iran, Asia Arab Emirates, Turkey, Iraq, Israel, Jordan, Kuwait, Lebanon, Yemen Pakistan, Saudi Arabia, Syria
1.6. Clinical signs: Goats of all ages and sex can be affected. The acute disease is more noticeable in naive populations in newly affected areas. The incubation period generally lasts on average 10 days but may vary between 2 and 28 days. The first signs are reluctance to walk and the onset of fever (typically 41ºC and occasionally 42ºC) although animals continue to feed and ruminate. Respiration accelerates and becomes painful with violent coughing episodes. Animals stand with
14 limbs abducted and neck extended .There is continuous salivation and noses become blocked with a mucopurulent discharge. In the terminal stages, the goats are unable to move and death follows quickly. In sub acute or chronic forms signs are milder with coughing usually noticeable only following exercise. A study to correlate clinical signs and early lesions showed that affected goats, killed up to a week after contact with affected animals, were free of lung lesions or clinical signs; between two and three weeks after contact, lung lesions were generally small and superficial characterized by hyperemia and edema with clinical signs being restricted to an infrequent cough; fever was first seen after nearly 4 weeks which correlated with lung consolidation, the area of which increased as the fever progressed (Wesonga et al.,1993). Morbidity can be 100 percent and mortality may be in the range of 70% to 100 % (Rurangirwa et al.1981). Gathering or increased confinement of animals facilitates the spread of the disease.
1.7. Pathology: 1.7.1. Gross lesions: The gross lesions in classical CCPP are confined to the thoracic cavity. Pea-sized yellowish nodules are seen in the lungs in early cases, whereas in more established cases there is marked congestion around the nodules. The lesions may be confined to one lung or involve both, and an entire lobe may become solidified. The pulmonary pleura become thickened, and there may be adhesions to the chest wall. Hutcheon emphasized that the lesions of CCPP do not resemble those of contagious bovine pleuropneumonia (CBPP) in that "no thickening of the interlobular tissue" occurs, a classical lesion of
15 CBPP. In sharp contrast, M. mycoides capri has been reported to cause lesions in a wide variety of organ systems and to produce lung lesions closely resembling those seen in CBPP (OIE, 1991). The generalized lesions described include encephalitis, meningitis, lymphadenitis, splenitis, genitourinary tract inflammations, and intestinal lesions, none of which are a feature of classical CCPP. The lung lesions, which resemble those seen in CBPP, are usually confined to one lung and reflect various stages of fibrinous pneumonia. Extensive pleuritis is usually present, and various stages of hepatization and marked dilation of interlobular septa is commonly seen .The cardiac and diaphragmatic lobes are the ones most commonly involved.
1.7.2. Histopathology: Histological examination of the lung tissues often reveals an acute serofibrinous to chronic fibrino-necrotic pleuropneumonia with infiltrates of serofibrinous fluid and inflammatory cells, mainly neutrophils, in the alveoli, bronchioles, interstitial septae and sub pleural connective tissue. Intralobular edema is more prominent but interlobular edema has also been reported. Peribronchial and perbronchiolar lymphoid hyperplasia with mononuclear cell infiltration are prominent in affected lung.
1.8. Molecular epidemiology: Unlike other members of the M. mycoides cluster, Mccp shows a surprising degree of heterogeneity particularly in the sequence of its two rRNA operons which both contain the genes for 5S, 16S and 23S rRNA .These polymorphisms, representing point mutations in either gene, can be used to subtype strains, some of which may have
16 epidemiological and, possibly, evolutionary significance. Sequencing the 16S rRNA genes of African strains identified two distinct lines, I and II, both of which were represented in Central, North and East Africa; isolates from the Middle East were of the type II only although they could be further divided (Pettersson, et al., 1998; Heldtander, et al., 2001). Sequencing the amplified products of a different gene(s), the H2 locus, (Lorenzon et al., 2002) divided strains into 4 major groups in which lineage A occurred exclusively in East Africa, B mostly in North Africa and the Middle East, C in Central Africa, and D, represented only by a single strain, was restricted to the United Arab Emirates. Subtyping with amplified fragment length polymorphism (AFLP) strongly supported the 16S rRNA sequence analysis by identifying two main lineages ,but all studies have been hampered by a lack of strains; clearly more strains are necessary to provide a better understanding of the epidemiology and evolution of CCPP (Kokotovic et al., 2000). On a more local level, 10 of 11 strains of M. c. capripneumoniae isolated from four different regions of Tanzania had very similar profiles with AFLP. These profiles were also indistinguishable from two Kenyan and one Ugandan strain indicating the close association between small ruminants in these three neighboring countries.
1.9. Diagnosis: 19.1. Field Diagnosis: A highly contagious disease occurring in goats and characterized by severe respiratory distress, high mortality, and postmortem lesions of fibrinous pleuropneumonia with pronounced hepatization and pleural adhesions warrants a field diagnosis of CCPP.
17 1.9.2. Specimens for Laboratory: From a dead animal that has had severe clinical disease, the best specimens to submit are affected lung, swabs of major bronchi, and tracheobronchial or mediastinal lymph nodes. All samples should be collected aseptically and if possible, placed in transport medium (heart infusion broth, 20 percent serum, 10 percent yeast extract, benzyl penicillin at 250 to 1000 IU/ ml). Samples should be kept cool and shipped on wet ice as soon as possible. If transport to the laboratory is delayed (more than a few days), samples may be frozen (Cipran; and Pijoan, 1978). Blood should be collected for serum.
1.9.3. Laboratory Diagnosis: Diagnosis must be confirmed by isolation of the agent Mccp. The causative agent, once isolated, can be identified by immunofluorescence or by growth or metabolic inhibition tests. Several serological tests can be used in the laboratory for the detection of antibodies to Mccp. These include complement fixation (CF), passive haemagglutination (PH), and enzyme-inked immunosorbent assay (ELISA). The latex agglutination test is a very convenient field test for detecting antibodies in whole blood or in serum (Rurangirwa, et al., 1987a). In spite of greatly improved media formulations, the isolation of Mccp remains one of the more difficult tasks for the mycoplasma diagnostic laboratory. A major problem is the transport of the samples over long distances to a specialist laboratory which often results in the inactivation of the fragile mycoplasma. The samples of choice are the pleural fluid which contains high numbers of mycoplasmas and sections of hepatized lung preferably at the interface of normal and
18 diseased tissue. The samples must be sent quickly in a cool condition but will become of little value if journey time is longer than two days. Sending frozen samples is recommended but not always practical. During the recent investigation of CCPP in Eritrea, excellent isolation rates of Mccp were achieved from lyophilized lung samples even though isolation was not carried out for several weeks after arrival (Nicolas. 2002). Choice of medium is critical and best results were obtained during the same investigation with a commercial medium (Mycoplasma experience medium). Other media which have been shown to support the growth of most strains of Mccp include H25P or FP medium supplemented with 2 g per liter of sodium pyruvate (Bölske et al., 1996). The development of PCR has greatly improved CCPP diagnosis as it is now possible to detect the mycoplasma quickly even in mixed cultures and directly from clinical material such as pleural fluid and lung. PCR based on the 16S RNA genes have been reported which enable the detection of all members of the M. mycoides cluster followed by specific identification of Mccp by restriction enzyme digestion. PCR has also been reported to be successful for detecting Mccp directly from pleural fluid dried on filter paper which can be sent, overseas if necessary, to a reference laboratory (Lorenzon et al., 2002). Serodiagnosis of CCPP, on the hand, is a relatively easy task thanks to a rapid, specific and relatively sensitive test developed initially in Kenya. The latex agglutination test (LAT) uses a carbohydrate extracted from Mccp linked to latex particles which agglutinate in the presence of specific antibodies in the blood of affected goat’s .The test, which takes minutes to complete, is more
19 sensitive than the complement fixation test and easier to perform than the competitive ELISA which should be used for confirmation (Thiaucourt et al., 1994). A LAT has also been described for circulating antigen and could provide earlier detection in affected animals before antibodies have appeared (March et al., 2000).
1.9.4. Differential Diagnosis: Clinically, CCPP may be confused with other pneumonic conditions such as pasteurellosis and peste des petits ruminants.
1.10. Disease Prevention and Control: Protection against CCPP was shown to be possible more than a century ago when Hutcheon subcutaneously inoculated goats with lung extract from affected animals (McMartin et al., 1980). Furthermore goats vaccinated with an attenuated broth culture of Mccp did not succumb to contact infection (MacOwan; and Minette, 1978). This clearly demonstrated control was possible. Since then a number of different preparations have been produced which are reported to produce solid immunity even after one year. These include a vaccine composed of sonicated antigens emulsified with incomplete Freund’s adjuvant and another in which lyophilized Mccp is inactivated with saponin immediately before immunization (Rurangirwa et al., 1987). The latter vaccine has been in use in Kenya for the last few years but has been modified so that the mycoplasma is inactivated with saponin for at least 12 hours at 4ºC (OIE., 2000). Kids older than 10 weeks of age are vaccinated to avoid interference by maternal antibody. In the Sudan, Zaki (2001) formulated a dead vaccine using the continuous culture technique and assessed that
20 vaccine in an immunization trail. The immune response of vaccinated animal was measured using indirect ELISA and growth inhibition test. Antibodies against Mccp were detected in sera of vaccinated animals up to 5 months post-vaccination when the experiment was discontinued. Growth inhibiting antibodies were detected in sera from goats that were received a vaccine containing 0.6 mg/ml. 21 immunogenic bands were detected using immunoblotting. Almost half of the bands ranged between 33-7.2 kDa In other countries where vaccination is not practiced, other control measures are used. Antibiotics such as the tetracycline, fluoroquinolones and macrolide family are generally effective clinically if used earlier enough (Onovarian et al., 1974; El Hassan et al., 1984). However, the complete elimination of the mycoplasma is rarely achieved and treated animals should always be considered as potential carriers. Movement restrictions and slaughtering of infected and contact animals is recommended for countries or regions that are newly infected.
1.11. Control and eradication: Sufficient regulatory restrictions should be maintained to prevent introduction of CCPP into apparently healthy animals. Serological testing of susceptible animals for importation is a recommended safeguard. Successful control of the spread of CCPP rests on removing susceptible animals from any possible contact with CCPP-infected animals, whether they are clinically affected or sub clinical carriers only. On-farm quarantine of suspicious and contact animals would be very advantageous in stemming the spread of the
21 disease. In an outbreak situation, testing, slaughter, and quarantine would be the methods of choice. 1.12. Immunology: Little is known of the immunology of CCPP despite a number of reported experimental infections. More recently March et al., (2002) monitored the humoral response of goats infected with a multipassaged Mccp strain 19/2 with several serological tests and PCR. While there was little evidence after infection of the infectious agent or clinical or pathological disease, apart from elevated temperatures and a transient cough in one goat, serological responses were detected by latex agglutination test and competitive ELISA. Immunodominant bands of 23, 40 and 44 kDa were seen by immunoblotting in all experimentally infected animals as well as in some sera from a natural outbreak of CCPP in Eritrea which additionally showed bands of 62, 70 and 108 kDa.
1.13. Serology: Serological tests are either used for identification of isolates (growth inhibition test and immunofluorescence) or detection of antibodies in goat’s sera. The role of the later grouping diagnosis of CCPP is limited due to the fact that animals die in the acute phase of the disease before they develop antibodies. There are a great number of various serological tests available for comparison of the mycoplasma. However, their value as diagnostic tools differs from test to another.
22 1.13.1. Serological identification: 1.13.1.1. Growth inhibition test (GIT): The GIT is the simplest and most specific but least sensitive of the test available. It depends on the direct inhibition of growth on solid media by specific hyper immune serum, and detects primarily surface antigens (Dighero et al., 1970). This test is highly recommended, together with immunofluorescent test (IF) for species of mycoplasma (Freundt et al., 1979; Subcommittee on the Taxonomy of Mollicutes, 1979).
1.13.1.2. Immunofluorescence tests (IF): It is one of the most reliable methods for the identification of mycoplasmas. It’s either direct (antigen and labeled IgG) or indirect (antigen, antibody and labeled IgG prepared as anti-species specific antibodies). The test detects mainly the outer membrane antigens (Bölske., 1995). It is simple, rapid, sensitive, yet economical in the use of antiserum.
1.13.2. Serological tests: 1.13.2.1. Immunodiffusion: Gel diffusion techniques for immunoprecipitation analysis have a history of about 70 years. In the thirties some bacteriologists made attempts to apply the diffusion principle for serological typing of various bacterial species but well-standardized techniques for immunodiffusion (ID) were not established until the middle of forties. In 1946 Oudin in France gave his first report on the principles and application of the simple diffusion technique in tubes (Oudin .,1946). Somewhat later double diffusion methods were introduced in 1947 in
23 Sweden by Ouchterlony (1948) and in1948 in England by Elek (1948) and Oakley and Fulthorpe in 1953. Since then the basic techniques have been further developed and several new ID methods based on the original principles have been reported. Agar gel immunodifution (AGID) test is very simple and inexpensive test that can be used for the detection of CCPP. It can be used in any laboratory and even in the field. The success of the test depends largely on the preparation of the potent specific hyper immunoserum. Result can be obtained in one day but the test isn’t sensitive enough .the results of the test are meaningful only if they are positive and the negative results don’t confound the provisional diagnosis. Standard CCPP hyper immunoserum is also effective in detecting CCPP antigens.
1.13.2.2. Indirect (Passive) haemagglutination test (IHA): This test has been used frequently in the detection of antibodies against various species of mycoplasma in man and animal. It was used with complement fixation for diagnosis of CCPP (Muthomi and Rurangirwa, 1983). The IHA was found to have a positive correlation (r: 0.89) with the complement fixation test in detection of antibody to strain F. 38. However, the test was described to be more sensitive and less specific compared to complement fixation. It detected antibodies to Mccp in sera from infected, treated or immunized goats. However, it did not detect a rise in antibody level in the first seven days after experimental infection. Antibodies are found in experimental CCPP infection 7-9 days after the onset of clinical signs, and peak titers between days 22 and 30 (Muthomi and Rurangirwa, 1983). Abbas et
24 al., (1978) used membrane antigens of Mmm LC type, Mmc, Mccp and Mcc strains in IHA test and applied it to goats in CCPP herds and healthy goats. Among animals with signs of CCPP, 72% had antibodies to the two subspecies of M. mycoids, but only 7% had antibodies to M. capripneumoniae. In Oman Sultanate, Jones and Wood (1988) used IHA in the field detect antibodies against caprine mycoplasma. The result revealed the wide spread of seropositivity to Mmc, whereas strain F38 reactors were largely confined to herds in which the F38 biotype had been shown by cultural means to occur.
1.13.2.3. Latex agglutination test (LAT): 1.13.2.3.1. Polysaccharide-specific antibody-detection (LAT): Rurangirwa and others (1987) showed that the polysaccharide produced by Mccp could by bound to untreated goat erythrocytes, and utilized for the detection of Mccp antibodies in natural and experimental cases of CCPP. The antibody-detection LAT is now routinely applied in Kenya (Office International des Epizooties, 1996) for quick screening of goat sera.
1.13.2.3.2. polysaccharide-specific antigen-detection (LAT): The antigen-detection LAT is a rapid detection test of CCPP, it has been developed recently (March et al., 2000). In this test, latex micro spheres (diameter, 8µm) were coated with anti-Mccp polyclonal immunoglobulin G (IgG) antiserum (anti-F38 biotype). The coated microspheres, when used in an agglutination test (LAT), detect Mccp in the serum of the goats with CCPP. The development of LAT which detect circulating Mccp antigen should allow the diagnosis of the
25 current infection; since antigens has been detected within 3to 9 days of the onset of clinical signs (Rurangirwa et al., 1995). So the use of antigen detection latex in conjunction with current antibody-detection LAT should allow effective field screening at all stages of infection.
1.13.2.4. Complement fixation test (CFT): Complement fixation test was used for the detection of CCPP infection (MacOwan, 1976; MacOwan and Minette, 1977). It has been found to be more specific, though less sensitive, than IHA test (Muthomi and Rurangirwa, 1983).Its main disadvantage is the high level of technical expertise require performing the test.
1.13.2.5. Enzyme linked immunosorbent assay (ELISA): Indirect ELISA was developed to screen goat sera at a single dilution of antibody to Mccp (WamWayi et al., 1989).This test is based on the fact that either the antigen or antibodies can be linked to an enzyme and bound to an insoluble phase (polystyrene or poly vinyl plates) while they retain immunological and enzymatic activity in spite of conjugation and linkage. The test was done using monoclonal antibodies (mAb) for the diagnosis of both CBPP (Abusugra et al., 1997; Goff and Thiaucourt, 1998) and CCPP (Rurangirwa et al., 1997; Thiaucourt et al., 1994). Blocking ELISA was found also highly specific for CCPP and it detected antibodies in sera for naturally infected or artificially immunized animals while remained negative with hyperimmune sera to related strains such as PG 50 of group 7 of leach (Thiaucourt et al., 1994).The test was more sensitive than CFT in detecting antibodies in goat serum.
26 Competitive enzyme linked immuosorbent assay (C-ELISA) was development by the OIE Collaborating Centre for the Diagnosis and Control of animal Diseases in Tropical Countries (Le Goff and Thiaucourt et al., 1998). Evaluation and comparison with CFT was carried out by Thiaucourt et al., (1998). The C-ELISA had equal sensitivity and greater specificity. The ELISA used a herd test and is easier to perform than CFT, but it performance characteristic has not yet been fully assessed. An extensive examination for at least one mycoplasma species indicate an excellent inter laboratory reproducibility (Mia et al., 1981) and high degree of correlation between cultural isolation and ELISA (Cassel et al., 1981; Davidson et al., 1981).
1.13.2.6. Polyacrylamide gel electrophoresis (PAGE) and immunobloting: Whole-cell protein analysis by sodium dodecyl sulphate- polyacrylamide gel electrophoresis [SDS-PAGE] and/ or immunoblotting is widely used to characterize bacteria, including mycoplasma isolates (Costas et al., 1987; Khan et al., 1987).The procedure is characterized by high specificity which allows the differentiation of distinct groups within a species. For CCPP, monoclonal antibody to Mccp, designated E8-18, was tested against different strains in the mycoplasma mycoides cluster with western blotting (Rurangirwa et al., 1997). E8-18 reacted with a 24-kDa antigen of Mccp but did not with any antigens of the other strains in the cluster. The identified antigen was found to be an integral protein on the surface of live Mccp. According to this finding, western blotting with monoclonal antibodies can be used in
27 differentiating Mccp from closely related and difficult-to-distinguish mycoplasma species.
28 CHAPTER TWO Materials and Methods
2.1. Samples: A total of 160 blood samples were collected from goats of different breed, age and sex. The samples were obtained from different areas in Khartoum state (Table 2.5) during the period between February and April 2006. Table (2.1): Samples collected from different areas in Khartoum State. Area Number of Time of collection samples East Nile 26 February 2006 El-Drushab 30 March 2006 Khartoum North Abu-Halima 22 March 2006 Shambat 10 April 2006 Omdurman Umbaba 50 March 2006 Khartoum Soba 22 March 2006
Goat breeds involved in the study included the Nubian (53 samples), the Desert (37 samples), the Saanen (50 samples) and the Damascus (20 samples).
2.1.1. Collection of samples: Blood samples were collected from the jugular vein. A piece of cotton soaked in alcohol was used to clean the injection area. The vein was raised and 5 ml of blood was taken using sterile syringe.
29 2.1.2. Separation of serum: Immediately after collection, the blood samples were held at room temperature for 1 hour, then at 4°C overnight. Carefully, the separated serum was transferred into sterile plastic container, which was labeled and centrifuged at 3000 rpm for 10 minutes. Then the clear serum was transferred into new plastic containers, labeled and stored frozen till subjected to serological investigation. Data including number of samples, area, age, sex and breed of goats was recorded and every sample was labeled.
2.2. Mccp strains: Mycoplasma capricolum subsp. capripneumoniae (Mccp) strain N2 was provided kindely by Dr. Sawsan M. Abbas, Central Veterinary Research Laboratory. It was isolated from Nyala, Southern Darfur State (Sawsan, 2006) and the identification was confirmed by CIRAD- EMVT, France.
2.2.1. Growth medium: 2.2.1.1. Medium ingredients: 2.2.1.1.1. Horse serum: Twenty percent trisodium citrate was prepared in blood collection bottle. The blood was collected aseptically from jugular vein of a healthy horse. It was put overnight on the bench. The plasma was separated and measured. Then 10% calcium chloride was added to separate fibrin and the serum was heated in water bath at 56°C for half an hour to destroy the complement. The serum was then sterilized by filtration through cellulose acetate filter (0.2µm pore size, Sartorius). Aliquoteus of 50 and 100 ml of serum was prepared aseptically and
30 put into the refrigerator at 4°C for short period or stored frozen for long period till used.
2.2.1.1.2. Fresh yeast extract: Two hundred and fifty grams of pure fresh yeast were added to one liter of distilled water, heated at 100°C for 30 minutes and then centrifuged at 6000 rpm for 10 minutes. The supernatant was then separated, sterilized by filtration through cellulose acetate filter (0.2 µm pore size, Sartorius) and stored frozen until used.
2.2.1.1.3. Glucose solution: Five grams of glucose were dissolved in 10 ml double distilled water, sterilized by autoclaving at 108°C for 5 minutes and then stored at 4°C until used.
2.2.1.1.4. Penicillin solution: It was prepared by dissolving Benzyl Penicilin Sodium powder (one million IU) in five ml sterile distilled water to give 200000 IU/ml and stored at 4°C.
2.2.1.1.5. Thallium acetate 10% (w/v): This solution was prepared by dissolving 10 g of thallium acetate in 100 ml of double distilled water, sterilized by autoclaving at 120 °C for 15 minutes then stored at 4 °C till used.
31 2.2.1.1.6. Sodium pyruvate: Five grams of sodium pyruvate (sigma) were dissolved in 10 ml double distilled water and sterilized by autoclaving at 108°C for 5 minutes and stored at 4°C until used within a day.
2.2.1.2. Modified Hayfilick’s medium: The medium was used as described by Thiauciurt et al. (1996) with one modification by addition of dehydrated commercial yeast extract. Part A: PPLO broth w/v crystal violet (Difco) 21.0 g Yeast extract (Difco) 5.0 g Distilled water 7000ml Part B Horse serum 200 ml Fresh yeast extract 100 ml Glucose (0.5 g/ml) 2.0 ml Sodium pyruvate (0.5 g/ml) 8.0 ml Benzyl penicillin 0.5 ml Thallium acetate (10%) 0.3 ml
Part A was dissolved by heating. The pH was adjusted to 7.6, and then the medium was sterilized by autoclaving at 121°C for 15 minutes. Part B was then added aseptically to part A at 50 °C. The medium was gently mixed and distributed in 5 ml amount into sterile test tubes or in 500 ml amounts into1-litre sterile conical flask. To check sterility, the medium was incubated at 37°C overnight, then stored at 4 °C and used within four weeks.
32 For solid media and after addition of part A, agar (Oxoid) was dissolved by heating, sterilized by autoclaving at 121 °C for 15 minutes and then added to broth medium at 50 °C to give final concentration of 1.5%. The medium was then poured aseptically in 15 ml amount into sterile Petri dishes.
2.2.3. Cultivation of Mccp: The lyophilized culture of Mccp, N2 was rehydrated with 1ml of medium and used to inoculate 2 tubes of 5ml broth medium. The culture was incubated aerobically at 37°C for 6 days. Growth of Mccp was detected as fine turbidity. Larger volume (1litre) of culture was obtained by using inoculums representing 1/10 off the inoculated medium.
2.2.4. Harvesting and washing of cells: Mccp were harvested from 1 liter of culture by centrifugation at 10000 rpm for half an hour at 4°C using Heraeus Suprafuge (10.9cm radius).Cells were twicely washed in PBS, harvested using the same centrifuge and then resuspended in 10 ml PBS and kept frozen.
2.2.5. Sonication of Mccp cells: Washed cells of Mccp were treated in an MSE (Measuring and Scientific Equipment, England) ultrasonic disintegrator at 10 microns for 15 minutes. Soluble antigen was then separated from cell debris by centrifugation at 10000 rpm at 4°C for half an hour. Both soluble antigen (supernatant) and cell debris (sediment part) were stored frozen till used.
33 2.2.6. SDS treatment: Whole antigen, soluble antigen and cell debris were subjected to sodium dodecyl sulphate treatment. Equal volumes of antigen suspension and SDS (Pharmacia Bioteck) solution (4%) were mixed and incubated at 37°C for 1 hour. The antigen solutions were stored frozen and used in serological tests.
2.3. Serological tests: 2.3.1. Agar gel immundiffusion test (AGID): The test was performed as described by Ouchterlony (1948). 2.3.1.1. Materials: 2.3.1.1.1. Antigens: • SDS-treated soluble antigen. • SDS-treated cell debris antigen. • SDS-treated whole cell antigen.
2.3.1.1.2. Serum samples: - Reference Lyophilized hyperimmune antisera against Mccp was provided kindly by Dr. Thiaucourt (CIRAD-EMVT, France). The hyperimmune antiserum was reconstituted using half ml of double distilled water, distributed in sterile 100 µl volumes into 1ml sterile containers and stored frozen. The hyperimmune antiserum was prediluted 1000 times before use in the test. - Goat sera A total of 160 goat serum samples were obtained from different parts in Khartoum state.
34 2.3.1.1.3. Phosphate buffered saline (PBS): Na Cl 8 g
Na2 H2PO4 1.15 g
KH2PO4 0, 2 g K Cl 0, 2 g D.D. W 1000 ml pH 7. 4 After constitution, the buffer was autoclaved at 121°C for 15 minutes.
2.3.1.1.4. Other substances: - Agrose (HAS) (SIGMA). - Different sterile microtitre pipette and tips. - Petri dishes.
2.3.1.2. Method: 2.3.1.2.1. Preparation of the gel: One gram of agarose powder was boiled in 100ml of 0.1 M PBS, 8g of sodium chloride (help in diffusion of Ag and Sera), and 0.5 ml of phenol (as a bacteriostatic agent). The solids were dissolved by heating in 100 ml PBS. The agarose solution was then poured in 12 ml amounts into sterile Petri dishes (5 cm in diameter). After solidification wells were then cut (Fig 1) in a standard pattern, consisting of 6 mm diameter wells, 3.2 mm apart, one central well surrounded by six, and all equidistant
35 2.3.1.2.2. Test procedure: The method of AGID test was carried out according to Ouchterlony (1948). The central well was kept for the antigen and the peripheral wells for the tested sera (Fig. 1). To select the most suitable antigen for AGID, the three antigens preparations were tested against the hyperimmune antisera and other known sera. Wells were filled with 30 µl of each reagent and plates were read after 48-72 hours following incubation in a humidified chamber at room temperature. Clear precipitin lines were photographed and recorded as positive result.
HS
Sera Sera
Ag
Sera Sera
Sera
Fig. (2.1): Loading of wells with different reagents in the AGID test. HS: Hyperimmune serum. Ag: Antigen.
36 2.3.2. Indirect haemagglutination test (IHA): 2.3.2.1. Materials: 2.3.2.1.1. Antigen: Sonicated soluble antigen of Mccp strain N2.
2.3.2.1.2. Serum samples: - Hyperimmune antisera against Mccp (CIRAD-EMVT, France). - Goat sera:. A total of 160 goat serum samples were obtained from different parts in Khartoum State.
2.6.2.1.4. Sheep red blood cells (SRBC): Fresh blood was collected from the jugular vein of a healthy sheep with sterile syringe and it was evacuated immediately in a container containing ½ ml sodium citrate as anticoagulant. The uncloted blood was centrifuged at 500 rpm for 10 minutes. The packed red blood cells (RBCS) were washed three times in sterile normal saline and used immediately in the test.
2.3.2.1.5. Normal saline: 8.5g of NaCl was dissoloved in 1000 ml of Distilled water and sterilized by autoclaving at 121°C for 15 minutes
2.3.2.1.6. Phosphate buffered saline (PBS): Prepared as described for (AGID).
2.3.2.1.7. Others substances: - Microtiter plates. - Different microtitre pipettes and sterile tips.
37 2.3.2.2. Method: This was originally performed using antigen-sensitized human type O red blood cells (RBCs) (Carter, 1955), but more recently sheep red blood cells (RBCs) have been used (Sawada, 1982). The soluble antigen extract described above was used to sensitize the red blood cells (RBCs) or absorbed on to the red blood cells (RBCs). This was done by adding 15volume of the antigen extract to the 1volume of the packed red blood cells (RBCs) and the mixture was incubated for one hour at 37°C with frequent shaking. The sensitied RBCs was separated from the mixture suspension by centrifugation as described before and washed 3 times in normal saline. The sensitized RBCs was used immediately in the test as 1% suspension in normal saline.
2.3.2.2.1. Test procedure: The test was carried out in U shaped microtiter plates and preformed as follows: 1. All wells were filled with 25 µl PBS. 2. One row was used for each serum sample and the first well of the row was filled with 25 µl of a serum sample without a predilution step. 3. The sample was then 2 fold serially diluted till the 10th well. The 11th & 12th wells were kept for positive and negative controls respectively. 4. All wells were then filled with 25 µl of sensitized RBCs (1% solution), shaken slightly and left at room temperature. 5. The test was read an hour after incubation, then readings were taken hourly for up 3 h. A course agglutination of the RBCs along the
38 side of the concave wells is taken as a positive reading, and the formation of a button at the centre of the wells as negative.
39 CHAPTER THREE RESULTS
3.1. Selection of antigen for AGID: Three antigen preparations of Mccp strain N2 were tested with AGID test using known sera and results were shown in Figure 2 (a, b, and c). All antigen preparations gave precipitation bands with hyperimmune antiserum and other positive samples. However, the SDS-treated soluble antigen was the best; it gave very clear thick precipitation bands. Accordingly all serum samples collected in this study were tested for antibodies against Mccp using SDS-treated soluble antigen.
3.2. Detection of antibodies against Mccp: A total of 160 goats sera were investigated using AGID and IHA. Ninety-six of samples (60%) were found positive with AGID (Figures ) while 102 samples (63.75%) were found positive with IHA (Figure ). The results of the two serological tests were identical in 110 samples (69% of samples); of these 74 samples were positive and 36 were negative. In general difference in the results between the two tests was detected in samples that were weak positive with IHA (Ab titre ≤1/8) (Table 3.1). With few exceptions, all IHA negative samples were also negative with AGID.
40 Table (3.1): Comparison of results between AGID and IHA.
Serological test Strong positive Weak positive (Ab. Titer 1/32) (Ab titre ≤ 1/8 ) negative
IHA 58 74 samples 28 samples samples (46.3%) (17.5%) (36.2%) AGID 64 Positive 74 22 samples samples samples (46.3%) (13.7%) (40%)
41
A: AGID using SDS-treated Soluble Ag
B: AGID using SDS-treated WholeAg
C : AGID using SDS-treated Cell debris Ag
Plate (2)
42 3.3. Distribution of CCPP in Khartoum State: The results are shown in table (3.2) and Figure (1). The highest percentage of the positive samples was detected in Shambat (Khartoum North) and Umbada (Omdurman).
Table (3.2): Distribution of CCPP in different areas of Khartoum State: No. of Negative Positive percentage of Area samples Positive AGID IHA AGID IHA AGID IHA Khartoum East Nile 26 13 12 13 14 50% 53.9% North: El- 30 18 13 12 17 40% 56.7% Droushab Abu- 22 10 9 12 13 54.6% 59.1% Halima Shambat 10 2 4 8 6 80% 72% Omdurman: Umbada 50 12 14 38 36 76% 60% Khartoum Soba 22 9 9 13 13 59.1% 59.1%
43
90%
80%
70%
60%
50%
Positive 40%
30%
20%
10%
0% East Nile El-Drushab Abu-Halima Shambat Umbada Soba- –Khartoum Khartoum Khartoum Khartoum Omdurman khartoum North North North North Area
Positive percentage (AGID) Positive percentage (IHA)
Figure (3.1):Figure Distribution (3.1): Distribution of ofCCPP CCPP in Khartoum in State Khartoum State
44 3.4. CCPP in different goat breeds: Results are shown in table (3.3) and figure (2). The results of the two testes were generally similar. The highest percentage of positive samples was detected in Damascus goats while the lowest percentage of positive samples was detected in the Nubian goats.
Table (3.3): Antibodies against Mccp in different goat breeds in Khartoum state: Positive No. of Negative Positive Breed percentage samples AGID IHA AGID IHA AGID IHA Desert 37 17 15 20 22 54.1% 59.5% Nubian 53 26 24 27 29 51% 62% Saanen 50 22 19 28 31 56% 62% Damascus 20 1 5 19 15 95% 75%
45 100%
90%
80%
70%
60%
50% Positive
40%
30%
20%
10%
0% Desert Nubian Saanen Damascus Breeds
Positive percentage (AGID) Positive percentage (IHA)
Figure (3.2) Antibodies against Mccp in different breeds of goats in Khartoum State Figure (3.2): Antibodies against Mccp in different breeds of goats in Khartoum State.
46 3.5. CCPP in different age groups of goats: Results are shown in table (3.3) and figure (3). In general the highest percentages of positive samples were detected in animals between 1-3 years of age compared to goats aged more than 3 years.
Table (3.4): CCPP in different age groups of goats: Positive Age No. of Negative Positive percentage (year) samples AGID IHA AGID IHA AGID IHA 1-2 57 21 21 36 36 63.2% 63.2% 2-3 45 15 18 30 27 66.7% 60% 3.4 51 25 24 26 27 51% 53% > 4 7 3 3 4 4 57.1% 57.15
47 80%
70%
60%
50%
40% Positive
30%
20%
10%
0% 1_2 2_3 3_4 >4 Age
Positive percentage (AGID) Positive percentage (IHA)
Figure (3.3) Antibodies against Mccp in different age groups of goats
48 3.6. CCPP in different sex of goats: Results are shown in table (3.6) and figure (4). The percentage of positive samples was generally similar in both sexes with AGID.
Table (3.5): CCPP in different goat’s sex: Positive No. of Negative Positive Breed percentage samples AGID IHA AGID IHA AGID IHA Female 152 61 58 91 94 60% 61.8% Male 8 3 2 5 6 62.5% 75%
49 80%
70%
60%
50%
40% Positive
30%
20%
10%
0% Female Male Sex
Positive percentage (AGID) Positive percentage (IHA)
Figure (3.4) CCPP in different goat sexes in Khartoum State
50
Plate (3): AGID test for goat serum samples collected from Khartoum state: Central well contained SDS treated sol. Ag of strain N2 Peripheral wells contained positive51 and negative serum l CHAPTER FOUR DISCUSSION
Contagious caprine pleuropneumonia is a serious disease of goats in Africa, Middle East and Asia. In the Sudan the disease was first reported in 1902 (Anon, 1902). However, successful isolation of the causative agent was only achieved in the early eighties of the last century (Harbi et al., 1981). The current study represent a part of a project, funded by the Ministry of the Higher Education, aimed to study the distribution of the disease in the country and to provide new Mccp isolates that will help in epidemiological studies and control programmes. Latex agglutination test was proved valuable in screening goat sera for antibodies against Mccp. In this study, we failed to prepare the reagent due to unavailability of latex beads. Accordingly, collected goat sera from different areas in Khatoum state were investigated for antibodies against Mccp using agar gel immunodiffusion test (AGID) and indirect haemagglutination test (IHA). It was reported that other serological tests can be used successfully for the diagnosis of CCPP. Sero-convension to the F38 strain in experimentally infected animals was observed by complement fixation and direct haemagglutination tests to start after 7-9 days,to peak between days 22nd to 30th, and to decline rapidly thereafter (Muthomi and Rurangirwa,1983). AGID test was used extensively for the immunological analysis of mycoplasma membranes (the main antigen) and to compare different mycoplasma species (Robinson et al.,1963; Kahane and Razin,1969;Armstrong and Ya,1970).
52 In one study, AGID and CFT were used to study the antigenic relationships among four human mycoplasmas. The antigen for AGID test was prepared by repeated freezing and thawing. The four mycoplasmas antigens were found antigenically distinct. However, evidence of shared antigenic components was obtained with the two tests. The AGID was found superior to CFT in distinguishing some other recent human mycoplasma species (Robinson et al., 1963). In the current study, it appeared that the method used for disrupting mycoplasma was important in preparing satisfactory antigen for gel diffusion. Sonicated mycoplasma cells were used first as an antigen in the test against known reference antisera to Mccp and no precipitation lines were produced. However, all antigenic preparations (whole cells, sonicated cells supernatant and cell depris) had a potential for producing lines of precipitate in agar gel immunodiffusion when treated with 2% SDS. Prominant bands were observed when sonicated SDS-treated supernatant of mycoplasma cells was used. SDS is an anionic detergent solublizes proteins by cleaving hydrogen bonds, unfolding polypeptide and prevention of protein aggregation (Westermeier, 1997). It was applied successfully for the solublization of mycoplasma membrane antigens for agar gel double diffusion test (Kahane and Razin, 1969). The authors found that most membrane antigens active in immunodiffusion tests were separated from crude hydrophobic protein fraction. These antigens showed no serological cross-reaction between M. laidlawi and M.gallisepticum with antisera prepared against these fractions. They concluded that membranes of diverse origin do not contain the same structural protein, hence immunodiffusion was specific.
53 In the current study,indirect haemagglutination test was used beside AGID. Both tests were simple, easy to perform and require minimum laboratory equipments, hence can be used routinely in small laboratories for diagnosis of the disease. The indirect haemagglutination test was found statistic but less specific than AGID. This result was similar to that obtained by Muthomi and Rurangirwa (1983) who compared the complement fixation test (CFT) and IHA test for the detection of antibodies in goat sera. He concluded that the IHT give non-specific reaction in non-diluted sera. Accordingly only antibody titres >1/8 should be regarded as positive. In this study all samples contained antibody titre >1/8 were found positive with AGID. However samples contained antibody titres less than that gave different readings with AGID.
The study revealed that CCPP was prevalent in all investigated areas in Khartoum state. Although the number of serum samples collected in this study was small, the result showed that Umbada (Omdurman) and Shambat (Khartoum north) were the most affected areas and the disease was most prevalent in foreign breeds.
54 Conclusions:
- Antibodies against Mccp were detected in different areas of Khartoum State using AGID and IHA. - IHA was more sensitive than AGID while the later gave precipitation lines with goat sera having titres ≤ 1:8. - Sonicated SDS-treated supernatant of Mccp cells was the most suitable soluble antigen for AGID.
Recommendations: - The accuracy of AGID &IHA should be evaluated using a test with more defined antigens e.g. polysaccarides in latex agglutination test. - The prevalence of CCPP in the Sudan should be determined in all states of the country. - Mccp isolates should be recovered for epidemiological studies and control programmes.
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