ADDIS ABABA UNIVERSITY FACULTY OF VETERINARY MEDICINE

SEROLOGICAL, CLINICAL AND PARTICIPATORY EPIDEMIOLOGICAL SURVEY OF CONTAGIOUS BOVINE PLEUROPNEUMONIA IN SOMALI REGION, ETHIOPIA

By GEDLU MEKONNEN GIZAW

JUNE, 2004 DEBRE ZEIT, ETHIOPIA

ADDIS ABABA UNIVERSITY FACULTY OF VETERINARY MEDICINE

SEROLOGICAL, CLINICAL AND PARTICIPATORY EPIDEMIOLOGICAL SURVEY OF CONTAGIOUS BOVINE PLEUROPNEUMONIA IN SOMALI REGION, ETHIOPIA

A thesis submitted to the Faculty of Veterinary Medicine, Addis Ababa University in partial fulfillment of the requirements for the Degree of Master of Science in Tropical Veterinary Epidemiology

BY

GEDLU MEKONNEN GIZAW

JUNE, 2004 DEBRE ZEIT, ETHIOPIA

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SEROLOGICAL, CLINICAL AND PARTICIPATORY EPIDEMIOLOGICAL SURVEY OF CONTAGIOUS BOVINE PLEUROPNEUMONIA IN SOMALI REGION, ETHIOPIA

BY: GEDLU MEKONNEN GIZAW Board of Examiners

1. Prof Ph. Dorchies

Signature

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2. Prof. Feseha Gebreab

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3. Dr. Wondwossen Abebe Gebreyes

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4. Dr. Giles Innocent

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5. Dr. Andy Catley

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6. Dr. David Barrett

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Academic Advisors

1. Dr. Ademe Zerihun

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2. Dr. Pascal Bonnet

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3. Dr. Laike Mariam Yigezu

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TABLE OF CONTENTS Page LIST OF TABLES ................................................................................................................. VI LIST OF FIGURES ..............................................................................................................VII LIST OF APPENDICES .................................................................................................... VIII LIST OF ABBREVIATIONS ............................................................................................... IX ACKNOWLEDGEMENTS ....................................................................................................X ABSTRACT............................................................................................................................ XI 1.

INTRODUCTION ............................................................................................................1

2.

LITERATURE REVIEW ................................................................................................4 2.1.

CONTAGIOUS BOVINE PLEUROPNEUMONIA .................................................................4

2.1.1. The disease..............................................................................................................4 2.1.2. Aetiology .................................................................................................................4 2.1.3. Epidemiology ..........................................................................................................6 2.1.4. Pathogenesis ...........................................................................................................7 2.1.5. Clinical signs ..........................................................................................................8 2.1.6. Necropsy findings .................................................................................................10 2.1.7. Diagnosis ..............................................................................................................11 2.1.7.1.

Cultural examination.....................................................................................11

2.1.7.2.

Biochemical test............................................................................................12

2.1.7.3.

Serological examination ...............................................................................12

2.1.8. Differential diagnosis ...........................................................................................14 2.1.9. Treatment ..............................................................................................................16 2.1.10.

Control and prevention.....................................................................................16

2.1.10.1. Control of cattle movement ..........................................................................17 2.1.10.2. Vaccination ...................................................................................................17 2.2.

THE EPIDEMIOLOGY OF CBPP IN ETHIOPIA ...............................................................18

2.2.1. CBPP control methods in Ethiopia.......................................................................22 3.

MATERIALS AND METHODS ...................................................................................24 3.1.

DESCRIPTION OF THE STUDY AREA ............................................................................24

3.2.

STUDY POPULATION ..................................................................................................24 iv

3.3.

STUDY DESIGN ..........................................................................................................25

3.4.

SAMPLING FRAME AND SAMPLE SIZE DETERMINATION .............................................25

3.5.

CLINICAL EXAMINATION AND SAMPLE COLLECTION .................................................26

3.5.1

Clinical examination.............................................................................................26

3.5.2

Serum sample collection .......................................................................................26

3.5.3

Sample collection for isolation .............................................................................26

3.5.4

Sample collection for histo-pathological examination .........................................27

3.5.5

Serological testing ................................................................................................27

3.5.5.1 3.6.

Competitive ELISA (cELISA)......................................................................27

PARTICIPATORY EPIDEMIOLOGY ................................................................................29

3.6.2. Methods.................................................................................................................29

4.

3.6.2.1.

Participatory Disease searches (PDS)...........................................................30

3.6.2.2.

Proportional piling ........................................................................................31

3.6.2.3.

Seasonal calendar..........................................................................................31

RESULTS ........................................................................................................................33 4.1.

PREVALENCE OF CONTAGIOUS BOVINE PLEUROPNEUMONIA (CBPP) USING C-ELISA 33

4.2.

OUTBREAK INVESTIGATION .......................................................................................36

4.2.1. Clinical and post-mortem findings .......................................................................36 4.2.2. Histopathology......................................................................................................40 4.2.3. Microbiological findings.......................................................................................40 4.3.

PARTICIPATORY EPIDEMIOLOGY ................................................................................41

4.3.1. Participatory Disease Searches (PDS).................................................................41 4.3.2. Proportional piling ...............................................................................................44 4.3.3. Seasonal calendar.................................................................................................45 5.

DISCUSSION ..................................................................................................................49

6.

CONCLUSION AND RECOMMENDATIONS..........................................................52

7.

REFERENCES................................................................................................................54

8.

CURRICULUM VITAE.................................................................................................75

9.

SIGNED DECLARATION SHEET..............................................................................80

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LIST OF TABLES Page

TABLE 1. CATTLE POPULATION AT RISK IN 4 CBPP AFFECTED AREAS OF ETHIOPIA ..................21 TABLE 2. CBPP OUTBREAK REPORTS TO THE FEDERAL MINISTRY OF AGRICULTURE DURING THE YEARS OF 1992 TO 2002.....................................................................................................22

TABLE 3. ESTIMATED SAMPLE SIZE ...........................................................................................25 TABLE 4. SERO-PREVALENCE OF CONTAGIOUS BOVINE PLEUROPNEUMONIA IN CATTLE IN SHINILLE AND JIJIGA ZONES (OCTOBER 2003 TO FEBRUARY 2004) ...................................33 TABLE 5. SERO-PREVALENCE OF CONTAGIOUS BOVINE PLEUROPNEUMONIA IN CATTLE IN DIFFERENT AGRO-ECOLOGICAL ZONES OF SHINILLE AND JIJIGA (OCTOBER 2003 TO

FEBRUARY 2004) ...............................................................................................................33 TABLE 6. SERO-PREVALENCE (ANIMAL LEVEL AND HERD LEVEL) OF CONTAGIOUS BOVINE PLEUROPNEUMONIA IN CATTLE IN DIFFERENT DISTRICTS OF JIJIGA AND SHINILLE ZONES (OCTOBER 2003 TO FEBRUARY 2004) ...............................................................................34 TABLE 7. SERO-PREVALENCE RESULT BASED ON CBPP STATUS ................................................34 TABLE 8. SERO-PREVALENCE RESULT OF CBPP BASED ON HUSBANDRY STATUS ......................35 TABLE 9. THE PREVALENCE OF CBPP IN DIFFERENT STUDY SITES IN SHINILLE AND JIJIGA ZONES BETWEEN OCTOBER 2003 AND FEBRUARY 2004................................................................35

TABLE 10. SUMMARY OF CLINICAL EXAMINATION DATA ..........................................................36 TABLE 11. SUMMARY OF POST-MORTEM FINDINGS ...................................................................39 TABLE 12. SUMMARIZED TABLE OF PROPORTIONAL PILING ON IMPORTANT LIVESTOCK DISEASES IN JIJIGA AND SHINILLE ZONES...........................................................................................45

TABLE 13. PROPORTIONAL PILING DATA FORMAT FOR CATTLE DISEASES ..................................68 TABLE 14. TABLE. SEASONAL CALENDAR OF CATTLE DISEASES IN SOMALI REGION .................69

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LIST OF FIGURES Page FIGURE 1. MAP SHOWING THE DIFFERENT CBPP ZONES IN ETHIOPIA .......................................23 FIGURE 2. YELLOWISH THORACIC FLUID COLLECTED FROM A SEVEN-YEAR OLD COW SUFFERING FROM CBPP.......................................................................................................................37

FIGURE 3. THORACIC CAVITY OF A SIX YEAR OLD COW SHOWING EXTENSIVE FLUID IN THE THORACIC CAVITY..............................................................................................................37

FIGURE 4. THE LUNG OF A SEVEN YEAR OLD COW SHOWING FIRM AND FLESHY LESIONS. (HEPATISATION). ................................................................................................................38 FIGURE 5. THE LUNG OF A FOUR-YEAR OLD COW SHOWING MARBLED LUNG COATED WITH FIBRIN ..........................................................................................................................................38 FIGURE 6. GROSS LESIONS OF THE KIDNEY OF A SEVEN YEAR OLD COW SHOWING WHITISH SPOTTED AREAS OF DEAD TISSUE (INFARCTS).....................................................................39

FIGURE 7. MYCOPLASMA MYCOIDES SUBSPECIES MYCOIDES COLONY WITH THE CLASSICAL APPEARANCE OF ‘FRIED-EGG’ WITH A DENSE CENTRE ........................................................40

FIGURE 8. MYCOPLASMA MYCOIDES SUBSPECIES MYCOIDES COLONY WITH THE CLASSICAL ‘FRIEDEGG’ APPEARANCE .............................................................................................................41

FIGURE 9. A LARGE NUMBER OF FLIES’ THROUGHOUT THE BODY OF A COW WITH CBPP .........42 FIGURE 10. THE FORELEGS SLIGHTLY SPREAD APART IN COW WITH CBPP ...............................43 FIGURE 11. DRUG USED BY PASTORALIST AGAINST CBPP.........................................................44 FIGURE 12. SUMMARIZED SEASONAL CALENDAR FOR LIVESTOCK DISEASES IN AFDEM DISTRICT. (SOMALI REGION IN ETHIOPIA). .........................................................................................46 FIGURE 13. SUMMARIZED SEASONAL CALENDAR FOR LIVESTOCK DISEASES IN DEMBEL DISTRICT.

(SOMALI REGION IN ETHIOPIA). .........................................................................47

FIGURE 14. SUMMARIZED SEASONAL CALENDAR FOR LIVESTOCK DISEASES IN MIESO DISTRICT. (SOMALI REGION IN ETHIOPIA). .........................................................................................48 FIGURE 15. MAP SHOWING THE STUDY AREA (SOMALI REGION) ...............................................65 FIGURE 16. MAP SHOWING THE STUDY SITES............................................................................66 FIGURE 17. MAP SHOWING THE STUDY AREAS ...........................................................................67 FIGURE 18. DISTRIBUTION OF SERA AND MAB ..........................................................................72

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LIST OF APPENDICES Page

APPENDIX 1. HUMAN AND LIVESTOCK POPULATION IN SRS .....................................................61 APPENDIX 2. SRS HOUSEHOLD SIZE & AVERAGE NUMBER OF PERSONS PER HOUSEHOLD .........61 APPENDIX 3. SRS LIVESTOCK POPULATION AND ITS TROPICAL LIVESTOCK UNIT (TLU)...........62 APPENDIX 4. CBPP SERO-PREVALENCE IN VARIOUS ZONES OF ETHIOPIA (1995 TO 1997) ........63 APPENDIX 5. PERCENTAGE INHIBITION WITH C-ELISA AS COMPARED TO HERD .......................64 APPENDIX 6. FIELD SURVEY DATA COLLECTION FORMAT ..........................................................68 APPENDIX 7. SERUM SAMPLE COLLECTION FORM ......................................................................70 APPENDIX 8. INSTRUCTIONS FOR USE OF C-ELISA ....................................................................71 APPENDIX 9. MAP SHOWING CBPP OUTBREAK REPORTED ZONES FROM 1996 TO AUGUST 2001 ..........................................................................................................................................74

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LIST OF ABBREVIATIONS

AGID

Agar Gel Immuno Diffusion

BQ

Black Quarter

CBPP

Contagious Bovine Pleuropneumonia

C-ELISA

Competitive Enzyme Linked Immunosorbent Assay

CFT

Complement Fixation Test

CIRAD/EMVT

Centre de coopération Internationale en recherche agronomique pour le développement/ Département élevage et médecine vétérinaire

CSA

Central Statistical Authority

DGIT

Disk Growth Inhibition Test

FAT

Fluorescent Antibody Test

FMD

Foot and Mouth Disease

G.C.

Gregorian calendar

GDP

Gross Domestic Product

HS

Hemorrhagic Septicemia

IFA

Indirect Fluorescent Antibody Test

LSD

Lumpy Skin Diseases

MAbs

Monoclonal Antibodies

MOA

Ministry of Agriculture

NAHRC

National Animal Health Research Centre

PA

Participatory Appraisal

PCR

Polymerase Chain Reaction

PPLO

Pleuropneumonia Like Organisms

SAST

Serum Agglutination Slide Test

SNNPR

Southern Nation, Nationalities and People Region

SRS

Somali Regional State

TBD

Tick Borne Diseases

TLU

Tropical Livestock Unit

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ACKNOWLEDGEMENTS

I would like to extend my best regards to my advisor Dr. Ademe Zerihun for his constructive advice, provision of literature and overall intellectual guidance and devotion of his time to correct the manuscripts. It is also my pleasure to thank my co-advisors Dr. Pascal Bonnet who supported me in data analysis, GIS and literature materials and Dr. Laikemariam Yigezu for his valuable support during laboratory works. Dr. Bayleyegn Molla is also very much acknowledged for his valuable contribution on encouragement and criticisms during my study. I am indebted to FranÇois Thiaucourt (CIRAD/EMVT) who supplied me with cELISA kit from France.

My sincere thanks go to Haraghei Catholic Secretariat of Dire Dawa office, and in particular to Ato Belihu Negesse and Ato Hilina Mikre, for the material and logistic support during my field work. I am very much grateful to Dr. Mohammed Sheik Said and Dr. Abdullahi Hussein of the Somali Regional State Livestock, Crop and National resource Bureaus for facilitating the field activities. Ato Eshetu Zewdie and Ato Terfe Feleke who are the Somali Regional State veterinary laboratory staff deserves my appreciation for being involved in sample collection during the difficult field activities of the research work.

Appreciations are due to the Sebeta National Veterinary Diagnostic Laboratory Institute that allowed me to use the laboratory facilities. I am well pleased to gratify Dr. Asegedech Sirak, Ato Berhanu Wolde Aregay Ato Melaku Sombo, W/o Huluagerish Adamu and W/o Leteberhan Yimesgen for their unreserved technical support on the laboratory procedures. Using this opportunity, I would like to extend my heartfelt thanks to Save the Children (UK) Jijiga and Addis Ababa offices for their cooperation in material support.

I would also like to acknowledge the intellectual contributions of the livestock keeper in Jijiga and Shinille zones of the Somali region.

It is my pleasure to acknowledge my families, Shimeles Mekonnen, Gizaw Mekonnen, Haregua Mekonnen, Askale Mekonnen, Mulu Mekonnen, Roman Yilma, Lema W/Aregay, Asrat Bagale, Elias Mulugeta, Yusuf Haji Adem and Shewangizaw Mersea who in the first place helped me to start and accomplish my study.

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ABSTRACT

During this study almost all combination of activities that leads Contagious Bovine Pleuropneumonia (CBPP) investigation to a conclusive decision has been carried out. Accordingly, an epidemiological survey on CBPP has been undertaken to assess the distribution and prevalence of CBPP and its related risk factors in the Shinille and Jijiga zones of the Somali region. This study were based on the diagnosis of clinical cases, seroepidemiological investigation of the herd, detection of typical gross and histopathological lesions, followed by microbiological analysis of the sampled organs, built around a participatory epidemiological surveillance approach in the low land of the Ethiopian Somali region.

In this study a total of 793 serum samples were tested from 56 herds, six districts and two zones (Jijiga and Shinille) of the Somali region. The overall CBPP herd sero-prevalence with at least one sero-positive animal was 17 (30.4%). The sero-prevalence based on agro-ecology shows that it is highly significant in lowland with 39.0% than in medium altitude with 6.7%. The participatory epidemiology result which was consistent with the conventional veterinary method of diagnosis was found out to be among the best approach in pastoral area to gather disease intelligence. The local names given to CBPP by the pastoralists refer to the organ affected and its symptom, such as Sombob means lung, and Ofta means difficulty in breathing. The major risk factors identified during this study were the type of husbandry system and associated cattle movement. The highest herd sero-prevalence was in pastoral area 36.0% followed by transhumance and sedentary, respectively. However, there is no significant sero-prevalence variation between transhumance and sedentary.

An extensive post-mortem examination was undertaken in seven animals showing clinical signs. In all these animals, only the right lung was affected and all had large amount of thoracic fluid which coagulated after the thoracic cavity was opened. In 5 out of 7 animals, the lung was attached to the thoracic wall.

The causative agent Mycoplasma mycoides subspecies mycoides was isolated by culturing lung tissue samples obtained from study area. After two weeks of culture, the Mycoplasma colony was observed as small colonies, 1 mm in diameter, with the classical ‘fried-egg’ appearance. This result gave conclusive evidence about the accuracy in the use of participatory epidemiology methods and conventional veterinary diagnostic methods.

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1.

INTRODUCTION

Ethiopia has more domesticated animals than any other country in Africa and agriculture is the mainstay of the Ethiopian economy. Agriculture accounts for about 45% of the total Gross Domestic Product and 85% of export earnings of the country. Official figures from the Central Statistics Authority give a national cattle population of 35.4 million heads (CSA, 1998). Livestock plays a critical role for the majority of the Ethiopian population in terms of income, savings, food security, employment, traction, fertilizer, and fuel. This sector is also important to the national economy, contributing 16 % of the total GDP, one-third of agricultural GDP, and eight percent of export earnings (MOA, 1997). An improvement in this sector, therefore, has the potential to contribute significantly to national income and to the welfare of the majority of rural families.

Given the large livestock population and distribution in the country and poor supply of veterinary services, infectious and parasitic diseases consequently cause death and debilitation to a significant number of animals. Animal health problems are generally exacerbated by drought, concentration of livestock at watering points and dry grazing grounds, combined with reduced resistance, intensifies the spread of communicable and parasitic diseases, which often cause higher losses than the forage or water shortages (The World Bank, 2001).

With the imminent eradication of rinderpest, Contagious Bovine Pleuropneumonia (CBPP) has become the most important cattle disease that hinders livestock development in Ethiopia (Fig. 1). This is mainly due to the stoppage of the consecutive yearly blanket vaccinations with combined rinderpest and CBPP since 1992/93, which had certainly contained the disease to a relatively low level during the past years by reducing the susceptible bovine population. With the deteriorating economic situation of many African countries, vaccination programs have been allowed to slip; drought conditions have resulted in great movements of livestock and the disease (CBPP) has spread rapidly to the east and south (Windsor et al, 1998).

Therefore, CBPP is now re-emerging as one of the most economically important diseases that impede livestock production in Ethiopia. It is a highly contagious disease with serious socioeconomic consequences at the farmer and national level hampering the export potential. Although export of live bovine from Ethiopia is minimal, it is thought to be a potential barrier by being an additional to other list A diseases existing in the country. Besides, the poorly understood pathogenesis, relatively ineffective vaccines or with adverse effects and poor 1

diagnostic assays, further exacerbates the impact of CBPP. The main problem in eradication is the frequent occurrence of sub-acute or asymptomatic infections and the persistence of chronic carriers after the clinical phase. Serological analysis is the most important diagnostic tool for the control of CBPP, but it is significantly hampered by the relatively low sensitivity and specificity of the methods (Abdo et al, 2000; March et al, 2003; Vilei et al, 2000; Monnerat et al, 1999).

Contagious Bovine Pleuropneumonia (CBPP) is both an epidemic and endemic disease of cattle that affects production through mortality and reduction in productivity. It also retards genetic improvement and limits working ability of cattle. The economic impacts of CBPP in a number of African countries, including Ethiopia were studied (Tambi and Maina, 2004). Accordingly, cattle in CBPP-infected areas (epidemic and endemic) are divided into three classes namely, calves and yearlings below 3 years, adult males and reproductive females. The losses due to CBPP (epidemic and endemic) are measured as the number of deaths that occur per class of animal, the quantity of beef lost for each class of animal, the quantity of milk lost from reproductive females and the loss in draft power from oxen. In Ethiopia, the average physical losses from CBPP in terms of cattle deaths are 25,115 heads (8,372 in endemic and 16,743 in epidemic), 1,852 and 13,396 metric tones of beef and milk, respectively. In terms of animal power, averages of 3,135,000 ox days are lost. Ethiopia experiences the largest number of cattle deaths, and reduction in cattle products under both endemic and epidemic conditions compared to the other African countries, due probably to its large cattle population (Tambi et al., 2004).

Although vaccination has been considered as a strategy for the control of CBPP in Ethiopia for the last 30 years, the disease still persists in several regions of the country, with its incidence increasing from year to year. This is, mainly due to lack of effective vaccine, irregular and low rate/coverage of vaccination, lack of livestock movement control, and absence of systematic disease surveillance and reliable data. For the time being, therefore, mass vaccination and where possible control of animal movement remains the most practical option in sub-Saharan Africa (Litamoi, 2000).

The vaccines are exclusively monovalent live attenuated freeze-dried products derived from broth culture of T1SR (streptomycin resistant variant) or T1/44 seed strains of Mycoplasma mycoides subspecies mycoides small colony (SC) that gives protection for 6 months up to one year. The other vaccines are V5, KH3J that gives protection for 2 and 6 months (Bamhare, 2

2000). The available major diagnostic assays are Serum Agglutination Slide Test (SAST), Complement Fixation Test (CFT) and Competitive Enzyme Linked Immunosorbent Assay (C-ELISA) which are used at herd level (Amanfu et al, 1998).

The control of CBPP can be achieved by restriction of animal movement and stamping out of infected and exposed animals along with attendant zoo-sanitary measures. This requires adequate financial, infrastructural, and human resources and adequate information system. However, under the current Ethiopian situation, these control strategies could not be implemented to control and eradicate the disease. Therefore, control measures through vaccination and restriction animal movement remain the most practical option in the country.

Therefore, to carry out an effective control of CBPP through strategic vaccination the prerequisites are a thorough understanding of the epidemiology of the disease in the country. Hence, from the outset the epidemiological assessment of CBPP should be implemented in order to envisage a rational plan for the control and eventual eradication of CBPP from Ethiopia.

Accordingly, the aims of this epidemiological survey on CBPP were to assess the distribution and prevalence of CBPP and its related risk factors, and to validate the livestock keepers’ perceptions and its seasonality of Contagious Bovine Pleuropneumonia in the Jijiga and Shinille zone of the Somali region. To fulfil the aforementioned aims, this study were based on the diagnosis of clinical cases, sero-epidemiological investigation of the herd, detection of typical gross and histo-pathological lesions, followed by microbiological analysis of the sampled organs, built around a participatory epidemiological surveillance approach in the low land of the Ethiopian Somali region.

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2.

LITERATURE REVIEW

2.1. Contagious Bovine Pleuropneumonia

2.1.1. The disease Contagious Bovine Pleuropneumonia (CBPP) is an acute, sub-acute or chronic respiratory disease of cattle caused by a Mycoplasma called Mycoplasma mycoides subspecies mycoides (bovine biotype) SC (small colony) (OIÈ, 2002). It is a serious threat and obstacle to livestock production and development in Sub-Saharan Africa, some Asian countries, and still occurring in some European countries (Windsor, 2000; Abdo et al, 2000; March et al, 2003;). Once introduced to a new area, initial losses are can be very high and its eradication is very difficult requiring major expenditure for control.

CBPP is an economically important and highly infectious septicaemia characterized by localization in the lungs and pleura (Radostits et al., 1994). It causes a respiratory disease that ranges from a persistent, sub-clinical infection to an acute, some times fatal disease. Anorexia, fever and respiratory signs, such as dyspnoea, polypnoea, cough and nasal discharges, are the main manifestation of CBPP. The main problems for control or eradication are the frequent occurrence of sub-acute or asymptomatic infections and the persistence of chronic carriers after the clinical phase (OIÈ, 2002). However, clinical signs are not always evident and could be confounded with other respiratory disease symptoms. Sub-acute or asymptomatic forms occur frequently and serve as a source for maintaining and spreading infection in the herd. Most infections are limited to the respiratory tract, although arthritis occurs in calves (Walker, 1999). Sequestra and chronic cases are possible but still remains debated the fact that these cases might be infectious in all cases and time (lungers as a new source of infection).

2.1.2. Aetiology

The Mycoplasmas (Mollicutes), formerly called PPLO (pleuropneumonia-like organisms), are non-sporulating, Gram-negative, non-motile bacteria, which do not posses a determined shape of the cell. The Mollicutes are members of the order Mycoplasmatales and class Mollicutes (soft skin) and they are the smallest of the free-living prokaryotes. Mollicutes is the correct

4

term to use when collectively referring to members in this order; however, the trivial name mycoplasma(s) is also used for this purpose (Walker, 1999).

There are no internal membrane structures and no cell wall external to the plasma membrane; however, many strains possess surface structures equivalent to a capsule. With the exception of Acholeplasmas, Mycoplasmas depend on a supply of intact cholesterol, which they incorporate into the membrane, creating sufficient osmotic stability for survival under normal physiological conditions. The Acholeplasma synthesize carotenol as a substitute for cholesterol, but will incorporate cholesterol if it is provided.

Their polymorphism is the consequence of the missing cell wall. Mycoplasmas are devoid of not only cell walls but also lack the genetic capacity to produce one, which also renders them completely resistant to ß-lactam and other cell-wall active drugs. Due to their small size (0.10.3 µm) and their polymorphism, they are able to pass through the usual bacteriological filters (0.1-0.3 µm). Cell shapes include spherical, pear shaped, spiral shaped, and filamentous forms. Cell sometimes appear as chains and beads, the result of asynchronized genomic replication and cell division. Mollicutes stain poorly with Gram stain method, although they are classified as gram negative. The preferred stains are; Giemsa, Castañeda, Dienes and methylene blue (Walker, 1999).

Members of the Mollicutes infect a wide range of animal species and human being. Infections range from sub-clinical to severely debilitating and sometimes fatal disease. Clinical manifestations include respiratory and uro-genital tract infections, arthritis, mastitis, and septicaemia. Most pathogenic species exhibit a high degree of host specificity. Mycoplasma mycoides subspecies mycoides is the cause of CBPP in cattle. Mycoplasma belongs to the order Mycoplasmatales and class Mollicutes (soft skin). Mycoplasmas are unique in microbiology because of their extremely small size and their growth on complex but cell-free media. Members of the M. mycoides group, M. capricolum group, and Leach’s group 7 form the so-called M. mycoides cluster, which consists of six Mycoplasma species, subspecies or groups of strains, originating from bovines and goats (OIÈ, 2000).

These six Mycoplasmas share serological and genetic characteristics, and this causes taxonomic and diagnostic problems. In natural conditions, Mycoplasma mycoides subspecies mycoides Small Colony type (MmSC) affects only the ruminants of the Bos genus (mainly bovine). Two types of Mm are recognized: large colony (LC) and small colony (SC). They 5

cannot be differentiated serologically but are different morphologically, culturally and in their pathogenecity, and can be distinguished through mouse protection tests.

Large colony types occur almost exclusively in goats, rarely in sheep while SC types cause CBPP in cattle (OIÈ, 2000). M. mycoides subspecies mycoides LC also cause mastitis, arthritis, and, occasionally, Contagious Caprine Pleuropneumonia and a fatal systemic disease in goats. They can be maintained readily in special culture media and in embryonated hens’ eggs.

2.1.3. Epidemiology

Mycoplasma mycoides subspecies mycoides SC type, the aetiological agent of contagious bovine pleuropneumonia (CBPP), can be grouped into two major, epidemiologically distinct, clusters. One cluster contains strains isolated from different European countries since 1980 and a second cluster contains African and Australian strains collected over the last 50 years (Vilei et al, 2000). Epidemiological and clinical observations indicate that the European outbreaks of CBPP are less virulent than the disease encountered in Africa. Furthermore, CBPP in Europe seems to be far more insidious, as it is usually chronic, and affected cattle show few distinctive clinical signs and rarely die (Vilei et al, 2000).

CBPP is still an endemic disease in Africa, Asia, Eastern Europe, and the Iberian Peninsula (Radostits et al., 1994). Because of the method of spread, outbreaks tend to be more extensive in housed animals and in those in transit by train or on foot and the incubation period can last from a few days up to several months (in occasional instance up to 6 months). In groups of susceptible cattle the morbidity approaches 90%, the case mortality may be as high as 50% and 25% of the infected ones remain as recovered carriers with or with out clinical.

Contagious bovine pleuropeumonia epidemiology is characterized by the following: (1) transmission by direct contact; (2) long incubation period; and (3) possibility of early excretion of mycoplasma (up to 20 days before apparition of clinical signs), during the course of the disease and after recovery in “lungers” (up to two years). These epidemiological features on the one hand, and the lack of a reliable screening test to pick up early carriers and lungers on the other hand, make it essential to control cattle movements in order to limit the spread of the disease.

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The epidemiology of CBPP in Africa is dominated by different factors. These are; cattle are the only species affected; there is no reservoir host in wild animals; transmission is through the direct contact of susceptible animal with clinical cases or chronic carriers and cattle movements play a very important role in the maintenance and extension of the disease (Bessin and Connor, 2000).

Contagious bovine pleuropeumonia CBPP is widespread in Africa and, according to the OIE and to various reports in 1995, the disease is present in 24 countries of tropical Africa including Ethiopia. CBPP is now emerging as the most important animal epidemic in subSaharan Africa where hitherto disease-free areas are being infested and the incidence is increasing within endemic zones at an alarming rate (Litamoi, 2000). It has proved extremely difficult to make a rational estimate of the cost of CBPP in the areas where it still exists for methods of animal husbandries are geared more to survival than to profit. Nevertheless, one can estimate losses from self-subsistence farming system by evaluating the losses at farm gate market price.

2.1.4. Pathogenesis

CBPP is typical example of multi-factorial diseases, where factors such as intercurrent infections, crowding, inclement climatic conditions, age, genetic constitution, and stress from transportation, handling, and experimentation are important determinants of the final outcome of infection (Rosendal, 1993). An essential part of the pathogenesis of the disease is thrombosis in the pulmonary vessels, probably prior to the development of pneumonic lesions. The mechanism of development of the thrombosis is not well understood, but is considered, at least in part, mediated through induction of cytokines (Rosendal, 1993). Contagious bovine pleuropeumonia is lobar variety of pneumonia in which the inter-lobular septa are dilated and prominent due to a great out pouring of plasma and fibrin in to them and it this dilated septa that give the “marbling” effect to the lung in these areas (Radostits et al., 1994).

Bronchitis, bronchiolitis, and alveolitis with predominantly neutrophils and mononuclear cellular response constitute the very early inflammation in Mycoplasma pneumonia. CBPP is characterized by substantial unilateral pulmonary necrosis, sometimes sequestration, and marked serosanguinous fluid accumulation in interstitia and pleura (FAO, 1997). Vasculitis appears to be an important component of the pathological changes in this disease, explaining 7

the marked exudation and pleurisy. Thrombosis can explain ischemic necrosis and infarcts of the lung. Death results from anoxia and presumably from toxaemia (Walker, 1999).

There are various substances produced by the Mollicutes, which are potentially important in disease pathogenesis. Peroxide and super-oxide production may be important in disruption of host cell integrity.

Urease produced by Ureaplasma species may be involved in injury to host tissue because of the production of ammonia by urea hydrolysis. Mycoplasma phospholipases are potentially important in pneumonia for they may reduce surface tension of the alveolar surfactants, thus resulting in atelectasis. A galactan polymer in M. mycoides ssp. mycoides has been shown to modulate the immune response and promote dissemination.

2.1.5. Clinical signs

There is considerable variation in severity of signs observed in cattle affected by CBPP, ranging from hyperacute through acute to chronic and sub-clinical forms (European Commission Health & Consumer Protection Directorate General, 2001) and summarized as follows.

(a)

Hyperacute forms

The clinical signs observed in the hyperacute form are much accelerated. Affected animals may die within a week exhibiting classical respiratory signs. In fatal cases, death occurs after a variable course of from several days to 3 weeks.

(b)

Acute forms

The early stages of CBPP are indistinguishable from any severe pneumonia with pleurisy. Animals show dullness, anorexia, irregular rumination with moderate fever and may show signs of respiratory disease. Coughing is usually persistent and is slight or dry. Sometimes fever goes up to 40 – 42 °C, and the animal prostrates with difficulty of movement. As the typical lung lesions develop, the signs become more pronounced with increased frequency of coughing and the animal becomes prostrate or stands with the back arched, head extended and

8

elbows abducted. While classical respiratory signs may be evident in calves, articular localization of the causative agent with attendant arthritis usually predominates.

(c)

Subacute forms

Signs may be limited to a slight cough only noticeable when the animal is exercised. Cattle that recover naturally are extremely weak and emaciated. Many infected animals develop chronic or milder forms of the disease, which may be either symptomless or associated with only a slight temporary rise in body temperature, and some loss of condition.

Recovered animals may be clinically normal but in some, an inactive sequestrum forms in the lung, with a necrotic centre of sufficient size to produce a toxaemia causing unthrift ness, a chronic cough, and mild respiratory distress on exercise.

The length of the incubation period depends upon the volume of the infective dose, the virulence of the strain, and the immune state of the animal and it can last from a few days up to several months (in occasional instance up to 6 months) (FAO, 1997). Depending on the resistance level of the animal, and the intensity of exposure, the disease takes an hyper-acute, acute to chronic, or the acute course is sometimes followed by a chronic stage which may last for 2 to 3 years (lunger) as a latent phase of the disease. The hyperacute form, involving up to 10 per cent of infected animals, may be observed at the onset of an outbreak; death is sudden and is often not accompanied by any other signs. The acute form is observed in approximately 20 per cent of the diseased animals. The course is 5 to 7 days (FAO, 1997). The earliest signs are a sudden onset of fever to 40o C or more and, in milking cows, a drop in milk yield, anorexia and cessation of rumination. There is severe depression and the animals stand apart or lag behind a traveling group and stop eating. The clinical symptoms start with the characteristics short, dry cough, which becomes more and more painful. Later, the cough usually becomes more severe; the animals shows signs of pain, standing with arched back and extension of the head and neck forwards and downwards, increased grunting respiration, salivation and nasal discharge. At this stage one could try to get sample of thoracic fluid from the chest by tapping before any fibrin is formed that would hamper the sampling (and auscultation of the lung is possible at this stage to identify formation of liquid).

9

2.1.6. Necropsy findings

The characteristic post mortem findings in CBPP are localized in the chest cavity except in young calves where inflammation of the limb joints (usually the carpal and tarsal joints), with increased fluid, is sometimes seen. There is thickening and inflammation of the pleura often with heavy deposits of fibrin and large amounts of clear, serous effusion containing shreds of fibrin. A most striking feature of the acute disease is the very large volume of yellow fluid (up to 30 liters) containing clots, which can accumulate in the chest and therefore causing extremely difficult breathing (FAO, 1997).

The lungs (almost always only one, the left) and pleura are affected and in most cases, only the diaphragmatic lobe is involved . Affected lobules show various stages of gray and red hepatization and the interlobular septa are greatly distended with serofibrinous exudatethe classical ‘marbled’ lung of this disease (Radostits et al., 1994). In acute forms, the yellowish fluid in the chest cavity may solidify and cover the lining of the chest and surface of the lung (the pleura) with a yellow or yellowish-grey fibrin coating resembling an omelet. Accumulation of fibrin on the pleura causes the lung and chest wall to stick together (adhesion). In the recovered and chronic form, fluid is rarely seen in the pleural cavity but adhesions between lung lobes and between lungs and the chest wall are commonly found. Infarcts, varying in size from about 10-300 mm, are frequently preset in the affected lung tissue, which are the result from thrombosis of inter- or intra-lobular arteries and lymph vessels. The infarcts subsequently become sequestered from the adjacent parenchyma by granulation tissue/fibrous capsulethe sequestra of carrier cases. The diameter of a sequestrum can vary from 2 to 25 cm and the capsule can be as much as 1 cm thick (FAO, 1997).

Lymph nodes in the chest may be enlarged and wet (edematous), with small necrotic foci and pinpoint hemorrhages, the difference between cortex and medulla may be indistinguishable. In the kidney cortex, white spots of dead tissue of variable size, called infarcts, can sometimes be seen. Because the lesions are so characteristic, slaughterhouse monitoring is a powerful tool to use in detecting introduction and spread of the disease although in the Ethiopian context slaughtering is achieved in almost all small butchery at backyard slaughtering.

10

2.1.7. Diagnosis

The diagnosis of CBPP is based on a history of contact with infected animals, clinical findings, immuno-diagnosis tests, necropsy findings and cultural examination. 2.1.7.1. Cultural examination

Samples to be taken from live animals are nasal swabs and/or broncho-alveolar washings or pleural fluid obtained by puncture. Samples to be taken at necropsy are lung lesions, lymph nodes, pleural fluid and synovial fluid from those animals with arthritis. The causal organisms can be isolated culturally from animals during the febrile phase or shortly post mortem from blood, pleural exudates (chest fluid) and/or affected lung tissue & lymph nodes.

Because of the Mycoplasmas’ fastidious nature, samples should be submitted to the laboratory as soon as possible after collection (Walker, 1999). During transportation, it is advisable to use a transport medium that will protect the Mycoplasma and prevent proliferation of other bacteria such as ampicilin and amoxyciline, which are not effective for Mycoplasma but works for contaminants.

Mycoplasma m. ssp. mycoides needs appropriate media to grow. In attempting isolation, 2-3 blind passages may be required. Most isolation attempts fail because the organism is labile, is often present in small quantities, and is demanding in its growth requirements. Therefore, the media should contain a basic medium (such as heart-infusion or peptone) yeast extract (preferably fresh), and horse serum (10%). Besides, several other components can be added, such as glucose, glycerol, DNA, and fatty acids, but the effects vary with the strains. To avoid growth of other bacteria, inhibitors, such as penicillin, colistin or thallium acetate, are necessary (OIÈ, 2000). For demonstration of proteolysis, growth is carried out on casein agar and/or coagulated serum agar.

Depending on careful use of the appropriate procedures and media the isolation and identification of the CBPP agent can be difficult and time consuming procedure. Whenever possible, classical bacteriology laboratories should set up a special section for handling Mycoplasmas only.

11

2.1.7.2. Biochemical test

Mycoplasma m. ssp. mycoides is sensitive to digitonin (like all members of the order Mycoplasmatales), does not produce ‘film and spots’, ferments glucose, reduces tetrazolium salts (aerobically or anaerobically), does not hydrolyse arginine, has no phosphatase activity, and has no or weak proteolytic properties (OIÈ, 2000). For routine field use, the immunological tests are sufficient, but where these give dubious results and in all cases of identification of first isolates, biochemical tests should be confirmed by a reference laboratory.

For this purpose, after two or three subcultures, antibiotics should be omitted from the medium to check if the isolate is a Mycoplasma or an L-form of a bacterium that will regain its original shape in the medium without inhibitors. Once this test is done and after cloning (at least three colonies should be selected), the organism can be identified using biochemical tests (OIÈ, 2000). The peroxidase-antiperoxidase (PAP) test is an excellent confirmatory test (FAO, 1997).

However, differentiation of MmmSC strains by serological and biochemical means has been difficult. This difficulty is caused by immunological cross-reactions and biochemical similarities of MmmSC strains with M. mycoides subsp. mycoides large-colony (MmmLC). Therefore, immunological test using growth inhibition test is used as a confirmatory test. The mechanisms by which antibody inhibits the growth of Mycoplasma organisms are not clearly understood. It has been proposed that growth-inhibiting antibodies are directed against exposed surface membrane proteins in which the cells are enclosed in a capsule of carbohydrate which is composed of galactan (Rurangirwa et al 2000)

2.1.7.3. Serological examination

To detect latently or chronically infected animals, almost all serological tests are suitable. Galactan, the major antigenic component of Mycoplasma m. ssp. mycoides can be found in all body fluids during and for some time after the acute stage of the disease. It has been found that not all Mycoplasma m. ssp. mycoides strains, which have been isolated so far, present immunological differences. Moreover, the level of antibodies against Mycoplasma m. ssp. mycoides that can be detected serologically does not necessarily also gives information about the level of protective antibodies being present (Seifert, 1996). 12

The presence of pathogens varies with the development of the lesions, and a negative result is not conclusive, particularly after treatment with antibiotic (due to sensitivity of culture). Direct examination of the exudates or smears is possible, but requires great skill. The causal organism, Mycoplasma mycoides, can be demonstrated in the fluid present in the chest and in diseased lung by culture, by biochemical tests, immunological tests (indirect fluorescent antibody (IFA) test, fluorescent antibody test (FAT), disk growth inhibition test (DGIT), agar gel immunodiffusion (AGID) test, the interfacial precipitation test, dot immunobinding on membrane filtration, and by a polymerase chain reaction (PCR). It is important to remember that Mannheimia [Pasteurella] species can be cultured frequently from any pneumonic lung (and even from normal lung), thus, their isolation does not indicate a diagnosis of pasteurellosis nor does it rule out the diagnosis of CBPP. At present, the most reliable test for detecting serum antibodies that is currently prescribed test for international trade by the OIE is the Complement Fixation (CFT) test which applies at herd level for interpretation. Great care is needed in collecting and storing sera to be used for this test which is complex to perform. False negative results can be found both early and late in the disease course. A competitive ELISA (C-ELISA) test has undergone evaluation and is possible to apply at animal level (for interpretation) and compared with CF test, the C-ELISA has equal sensitivity and greater specificity. The C-ELISA is an individual test but you can aggregate the results and therefore interpret it at herd, and it is easier to perform than the CF test but its performance characteristics have not yet been fully assessed (OIE, 2002; Amanfu et al, 1998). The rapid slide agglutination test with either whole blood or serum (better with serum) has been developed to detect specific agglutinins. Due to lack of sensitivity, the test detects only animals in the early stages (i.e. acute phases) of the disease and therefore, should be used only on a herd basis and applied on several animals of a given herd and to detect the presence of the disease (not to measure the quantity of disease). It is performed by mixing a drop of a suspension of killed and stained Mycoplasma mycoides organisms with a drop of serum or blood on a glass slide; in a positive result aggregates form within one minute. The slide agglutination test can give false positive results in uninfected animals, however, and also antibodies become undetectable by this test as the disease progresses. The specificity and sensitivity of the complement fixation test (CFT) in Botswana was assessed in 82 cattle affected by the disease and held in a double fenced quarantine camp. Using post mortem lesions as the gold standard and a 2 x 2 contingency table, the two tests were compared in terms of their sensitivity and specificity in detecting antibodies to MmmSC. 13

The CFT was found to be slightly more sensitive than the c-ELISA, and this could be related to the stage of the disease (Amanfu et al., 2000b).

2.1.8. Differential diagnosis

In carrying out a CBPP diagnosis, it is necessary to differentiate this disease from other diseases which may present similar clinical signs or lesions. The way the disease behaves in the herd is as important as the findings in a single animal when carrying out an investigation. The following diseases should be considered in differential diagnosis of CBPP (FAO, 1997): •

Rinderpest: The confusion with rinderpest results from the fever and discharges observed from the eyes, nose and mouth. However, the characteristic lesions of rinderpest those are essentially erosions in the mouth and throughout the digestive tract, together with the profuse, often bloody, diarrhoea in advanced cases, should enable easy differentiation from CBPP in which these are not seen. Lung lesions are seen in more chronic cases of rinderpest and these consist of red areas of collapse together with emphysema of lung lobules and the septa separating them. At this stage the erosive lesions of rinderpest may have healed.

•

Foot-and-mouth disease:

Salivation, lameness and fever are the cause of

confusion. •

Haemorrhagic septicaemia:

This is a very acute disease and most affected

animals die within 6 to 72 hours after the onset of clinical signs. Buffaloes are particularly susceptible. Oedema of the throat and neck to the brisket is often very pronounced. The lung lesions seen in animals that survive the longest can appear very similar to the marbling lesion of CBPP, there may be yellow fluid in the chest and the affected lung may adhere to the inside of the rib cage. Thus, in the individual case distinguishing between HS and CBPP can be difficult. •

Bacterial or viral bronchopneumonia:

Clinical signs may resemble closely those

of acute CBPP. Post-mortem examination shows usually both lungs to be affected, fibrinous exudates may be present but not to the same extent as in CBPP. While dark, solid areas of lung may be seen, these are usually restricted to the anterior lobes (not the diaphragmatic lobe as in CBPP) and marbled lungs are not often seen. 14

•

Theileriosis (East Coast Fever):

Coughing, nasal and ocular discharge and

diarrhoea are observed. Affected cattle show general enlargement of superficial lymph nodes and especially those of the head. The lungs contain much clear liquid which is also present in the chest cavity; the airways in the lung may be filled with white froth. Cigarette urn-like ulcers are seen in the abomasal folds. Neither pneumonia nor inflammations of the pleura are present. •

Ephemeral fever:

In most cases this is a self-limiting disease of short duration;

most affected cattle recover quickly, even those which are severely affected. The fever fluctuates with two or more peaks. Pneumonia is not a main feature of the disease but a secondary pneumonia can occur with lung oedema and emphysema in a small proportion of cases. •

Confusion with CBPP arises from the presence of fever, discharges from the eyes and dripping of saliva from the mouth, lameness and swollen joints (but in animals of all ages unlike CBPP).

•

Abscesses: They can be mistaken for sequestra. When cut open the contents of abscesses are seen to be offensive smelling, liquid purulent material, absent in sequestra. In abscesses a total destruction of the lung tissue occurs. Old thickly encapsulated hydatid cysts can also cause some confusion.

•

Tuberculosis: Tubercular nodules can superficially resemble sequestra but they are degenerative cheese-like lesions, sometimes calcified. The lung tissue is destroyed and the same lesions are also seen in lymph nodes in the chest. The capsule of the tubercular nodules is not well defined when compared to that of sequestra.

•

Farcy: The lung lesions of farcy differ from sequestra as they are filled with foul smelling purulent material (same as abscesses). Similar lymph node lesions are always present.

•

Actinobacillosis: The pulmonary lesions, when found, could be mistaken for sequestra.. Lesions are generalized and seldom present in lungs.

•

Echinococcal (hydatid) cysts: These cysts having a double wall and contain a clear liquid, often calcified when old. 15

•

Foreign body reticulum pericarditis: Mostly one animal is affected. The two diseases could be clinically misunderstood, but not epidemiologically and pathologically.

2.1.9. Treatment

Under practical field conditions, when the disease breaks out in a new area, treatment is not applicable and not recommended because of reasons of disease prevention (Seifert, 1996). Treatment is usually undertaken and indicated only in areas where the disease is endemic (Radostits et al., 1994), but in practice farmers are treating their animals when they have no other alternative. Although the Mycoplasmas are susceptible to a number of antibiotics in vitro, treatment failures are common (Walker, 1999).

Commonly used antibiotics include tetracyclines, tylosin, erythromycin, lincomycin, spectinomycin, and tilmicosin (Walker, 1999). Tylosin and spiramycin are effective in the control of excessive vaccination reactions and should be of value in the treatment of clinical cases (Radostits et al., 1994). Resistance to some of these antimicrobials has been noted. Animals that do not respond to treatment often become carriers. Penicillin is of little value, streptomycine has some curative effect.

2.1.10. Control and prevention

With Africa's burgeoning human population and decreasing agricultural output per capita, the resources needed to implement and maintain widespread animal disease control are becoming scarcer every year as these funds are stretched to help solve the many other problems facing the African agricultural industries. To make the most efficient use of the increasingly scarce resources, disease control programs must be tailored to the needs of particular communities and to high-priority cattle populations to ensure their efficacy, acceptance and sustainability and therefore economic evaluation should be generalized.

2.1.10.1. Control of cattle movement

Uncontrolled animal movements during transhumance, trade, and cattle theft have facilitated the spread of the disease throughout Tanzania. Although the quarantine and checkpoints have 16

been in place, weak legislation and a lack of means and resources to enforce control of livestock movements are making the situation worse (Msami et al., 2001).

The ideal method to control a trans-boundary disease like CBPP is the application of the stamping out policy of complete elimination of infected and exposed animals along with attendant zoo-sanitary measures. However, this presupposes that there are adequate financial, infrastructural, and human resources to execute the task. Nevertheless, the current reality in sub-Saharan Africa is that these pre-requisites are either lacking or inadequate. Moreover, no efficiency relies on economics only and there is no evidence on efficiency for efficacy effectiveness. In general, feasibility is debatable and hampers efficiency.

2.1.10.2. Vaccination

In most African countries, for the time being, mass vaccination (or restricted to target key areas) and where possible controls of animal movement remain the most practical option. To obtain the desired results an exercise aimed at controlling CBPP through mass vaccination should endeavour to achieve high immunization coverage using high quality vaccines, which should be administered, at short intervals especially during the initial stages (90 – 92% of the population).

Botswana is a typical example of African country in eradicating CBPP recently from the country. This was in February 1995 after an initial freedom from the disease of almost half a century, the last outbreak having been reported from Chobe district in 1939. Since the disease has been absent for so many years, laboratory diagnostic capability for effective diagnosis of the disease was limited to gross and histo-pathological diagnosis and cultural isolation of the aetiological agent of CBPP. Therefore, the government of Botswana and FAO carried out training of laboratory technicians and supervising officers on different serological techniques. Training of field staff in the clinical recognition of the disease and pathological differentiation from other types of bovine pneumopathies was done locally (Amanfu, 2000a).

With the veterinary department’s effort and assistance from FAO, the micro-method of Complement Fixation Test (CFT), which was OIE approved was established in the laboratory. In addition, the Serum Agglutination Slide Test (SAST) was likewise used as a pen-side test. Later, the Competitive Enzyme Linked Immuno-Sorbent Assay (C-ELISA) test and Polymerase Chain Reaction (PCR) were introduced as additional diagnostic test (Amanfu, 17

2000a). All these tools i.e. serology, isolation, clinical symptoms, pathology and epidemiology were used as effective means for decision making during the outbreak and for surveillance after the last suspected animal has been destroyed.

It must be pointed out that the commitment of government to get rid of CBPP and maintain freedom from the disease thereafter was responsible for the success that was achieved. It is also due to the role that cattle play in the socio-economic activities of the people of Botswana which accounts to 46 % of the GDP (Amanfu, 2000a). Generally, the major feature of Botswana success in CBPP eradication are; availability of equipment and reagents, veterinary staff and farmer training, quality assurance, proficiency testing and considerable efforts at achieving substantially.

2.2. The epidemiology of CBPP in Ethiopia

The origin of CBPP in Central, West and East Africa is obscure and it has been suggested that the infection was introduced by zebu cattle when they first migrated to the African continent. There is a suggestion that CBPP was introduced into East Africa from India by the army of field Marshal Napier when he invaded Ethiopia in 1867-1868 (Masiga et al., 1996), while Tulasne et al., (1996) have reported that the traditional practice of provoking "Willems reaction" was rediscovered by willemms in 1854. This indicates that CBPP had existed in Africa before 1854.

After Rinderpest has been brought under control, CBPP is considered to be among the most important cattle diseases and impediments to livestock development in Ethiopia, particularly in the lowlands. CBPP is one of the great plagues which continue to devastate cattle herds on which so many people are dependent in the lowlands. In the highlands, the consecutive yearly blanket vaccinations with combined Rinderpest and CBPP have certainly contained the disease to a relatively low level during the past years. But with the adoption of a strategy towards Rinderpest eradication, the vaccinations in the highlands have ceased since 1992/93. Generally, the irregularity and low rate of vaccinations since 1993 seem to contribute to the increased incidence of the disease and its further spread (MOA, 2003). The usual blanket coverage was around 50% and never reached the desired 80-100% level.

According to eleven years (1992 – 2002 G. C.) disease outbreak reports by Federal Ministry of Agriculture, several CBPP epidemics have been recorded from the south, south-west, west, 18

north-west and north-east regions of the country (Table 2). The passive disease outbreak reports from 1992-2002 shows 587 outbreaks, 16,806 cases, and 3,262 deaths. The highest record was in 1998 when 187 outbreaks with 5,652 cases and 1071 deaths were reported (MOA, 2002). However, this data cannot be used to determine, the level and geographic feature of the disease, determine the importance of the disease, set priorities for the use of resources for disease control activities, plan, implement and monitor diseases control program, or demonstrate disease status to trading activities. Due to the insidious nature of the disease, such official data do not necessarily convey the extent of the problem caused by CBPP in Ethiopia.

Recent studies conducted in Western Ethiopia (Regassa, 2001; Beyene, 1997), Northwest Ethiopia (Takele, 1998), Southern Ethiopia (Wondimu, 1996) and different regions of the country (NAHRC, 2000) revealed that CBPP is posing a major threat to cattle in many parts of the country thereby causing considerable economic losses through morbidity and mortality and warranting for serious attention (Afework, 2000).

The cattle population at risk of CBPP and livestock production systems in CBPP endemic and epidemic zones of Ethiopia is estimated to be a total of 13,325,700 heads of cattle. All of them are considered to be at risk of CBPP, of which 5,510,700 are in endemic zones and 7,815,000 are in epidemic zones (Table 1). Generally, based on the available information, the epidemiological situation of CBPP in various parts of Ethiopia can be summarized as follows (Afework, 2000): §

Western Ethiopia including Western Wollega and Assosa Zones (and possible a part of Gambella Region) are considered endemic and epidemic

§

Southern Ethiopia: (Southern Nation, Nationalities and People region, SNNPR). Borena Zone as a whole (Oromia Region), infected since long time, is an endemic area and characterized by pastoralism; South Omo, Konso Derashe and Amaro Zones (SNNP Region) are considered as endemic, with recent outbreaks in the neighbouring Zones such as Bench Maji and North Omo Zones

§

Gondar and Gojam areas have declared numerous outbreaks since 1993 and South Gondar and West Gojam are categorized as epiendemic areas. West Gojam zone comprises of Seven districts, namely Burie-Wonberma, Denbecha, Jabitenan, Dega-Damot, Quarit, Sekela and Achefer, of which the first two district are considered CBPP endemic and the last four districts are considered CBPP free. 19

§

The highlands of North Shewa were considered as CBPP free, however, LaikeMariam and Roger (1997) reported sero-prevalence rate of 54% using CFT.

§

Southern Tigray seems to be recently infected with sero-revealence rate of 50% reported in 1996 (Laike Mariam and Roger, 1997), and this can be categorized as epiendemic.

§

Agew Awi zone comprises of four districts, namey Dangela, Ankesha-guagusa, Gungua and Banja- shikudad, of which the first three are considered as CBPP endemic and the last is with sporadic occurrence. Here mixed crop-livestock production system is practiced and the dominant livestock species are cattle (see table 1)

§

North Eastern Ethiopia: Afar Region as a whole and Northern Somali Region may be considered as endemic, with recent outbreaks encroaching on the edge of endemic are in Southern Tigray, North Wello, North Shewa, East Shewa, (Amhara Region), and Arsi Zone (Oromia Region).

§

Eastern Ethiopia: In Somali Region except one zone, Shinille, which is considered to be CBPP epidemic zone, the status of the disease in all the other zones is unknown. Once introduced to a new area, initial losses in pastoral communities can be very high and its eradication is very difficult requiring major expenditure for control.

Table 1. Cattle population at risk in 4 CBPP affected areas of Ethiopia Area

Administrative Zones

Cattle

Livestock system

population Endemic Zones

Western Ethiopia

- Western Wellega (Oromia)

1 005 500

mixed crop-livestock

- Asosa (B. Gumuz)

84 200

mixed crop-livestock

Epidemic Zones

20

- Part of W. Wellega (Oromia)

272 700

mixed crop-livestock

1 188 000

mixed crop-livestock

470 000

mixed crop-livestock

768 000

Nomadic

- southern Tigray (Tigray)

450 000

mixed crop-livestock

- North Wello (Amhara)

620 000

mixed crop-livestock

- North Shoa (Amhara)

1 018 000

mixed crop-livestock

- Eastern Shoa (Oromia)

1 019 000

mixed crop-livestock

- Arsi (Oromia)

2 509 000

mixed crop-livestock

- Borena (Oromia)

1 419 000

Nomadic

- South Omo (SNNP)

413 000

mixed & nomadic

- Konso S.D. (SNNP)

70 000

mixed

crop-livestock

- Derashe S.D. (SNNP)

34 000

mixed

crop-livestock

- Amaro S.D. (SNNP)

59 000

mixed crop-livestock

- North Omo (SNNP)

1 715 000

mixed crop-livestock

- Maji (SNNP)

212 000

mixed & nomadic

Endemic zones

5 510 700

Epidemic zones

7 815 000

Endemic Zones Western - Western Gojam (Amhara)

North

- Awi (Amhara)

Ethiopia

Endemic Zones - Afar Zones (Afar) Eastern Epidemic Zones

North Ethiopia

Endemic Zones

Southern Ethiopia

Epidemic Zones

Total

Source: Cattle population in the Zones: CSA, Livestock, Poultry and beehives population, November 1998

Table 2. CBPP outbreak reports to the Federal Ministry of Agriculture during the years of 1992 to 2002

Year No.

of Cases

Death Slaughter PARa

Outbreaks

Morbidity Mortality Rate (%)

Rate (%) CFRb (%)

1992

1

4

1

0

1500

0.27

0.07

25.00

1993

3

484

127

8

72,655

0.67

0.17

26.24

21

1994

0

115

46

0

20800

0.55

0.22

40.00

1995

48

429

160

7

161645

0.27

0.10

37.30

1996

96

505

183

5

83484

0.60

0.22

36.24

1997

43

753

131

16

93895

0.80

0.14

17.40

1998

187

5652

1071

77

844833

0.67

0.13

18.95

1999

94

4025

596

740

534938

0.75

0.11

14.81

2000

56

1918

274

600

210,375

0.91

0.13

14.29

2001

27

1595

252

20

312,516

0.51

0.08

15.72

2002

32

1326

421

34

267,381

0.5

0.16

31.75

16806

3262 1507

2,604,022 0.6

0.13

19.4

Total 587

Source: MOA, Veterinary Services, Epidemiology Unit, 2000 a

PAR: Population At Risk; bCFR: Case Fatality Rate

2.2.1. CBPP control methods in Ethiopia

The major control method practiced in Ethiopia is Vaccination. The control of CBPP by vaccination has been carried out for the last 30 years in Ethiopia. Previously the consecutive yearly blanket vaccination with combined Rinderpest and CBPP vaccine was adopted as a strategy to control CBPP. It was this strategy that is believed to have contained the disease to a relatively low level until 1992/93. And this method was considered as a successful achievement in the control of CBPP. However with the adoption of a strategy towards Rinderpest eradication, the vaccinations in the highlands and most parts of the Somali region have ceased since 1992/93. Besides, the vaccination coverage was around 50% and did not reach the desired 80 – 100% level. Currently, CBPP control in Ethiopia was based on targeted and ring vaccination in the face of outbreaks (MOA, 1997).

22

Figure 1. Map showing the different CBPP zones in Ethiopia

23

3.

MATERIALS AND METHODS

3.1. Description of the Study Area The Somali region covers the rangeland of south-eastern parts of Ethiopia. The total area estimated is at about 382.000 square kilometres. It is bound to the north with Djibouti and Afar region, to the east and southeast with Somalia and to the west by Oromia and Harari regions (Fig 14, 15 and 16).

The climate is arid and semi-arid but, influenced by its proximity to the maritime influences of the Gulf of Aden and the Indian Ocean to the north and east and to the influence of high lands of Ethiopia lying North West of the region. Rainfall is bimodal with an average precipitation of less than 200 mm in the south east to some 600-700 mm in the north and west. Rainfalls not only vary dramatically in intensity and duration, spatial (geographical) distribution is also erratic. This has important consequences for the pastoral and farming communities (Woodroof, 1987). In areas bordering the highlands, there are four seasons locally known as Gu, Dayr, Kerenta and Jilal representing April to June, September to December, July to September and March to May, respectively.

The Somali Regional State livestock population in the 1999s estimate at 2.03 million camels, 3.75 million cattle and 17.6 million sheep & goats (Appendix1). The land use pattern closely related to both the climate and vegetation and, varies from rain fed farming in the sub- humid area to pure pastoralism in the more arid lowlands. Most of the rangelands are used for various forms of semi-nomadic pastoralist. Much of the rainfall agriculture is concentrated in the northern north-western and western sides of the region, although the incidence of cropping declines towards the south of the area. The veterinary service delivery is extremely poor.

3.2. Study Population

The target study population comprised cattle of all ages in the Somali Regional State. Two zones (Jijiga and Shinille) bordering the highlands of other regional states were included in the sample population. The status of CBPP in Jijiga and Shinille zones of the Somali region was unknown (Fig. 1). Furthermore, CBPP vaccination was carried out during the consecutive yearly blanket vaccination with combined Rinderpest and CBPP vaccine and stopped since 1992/93 in most parts of Ethiopia but in the study areas it has been stopped earlier than this. 24

3.3. Study Design

A Cross-sectional survey was carried out in two zones of the Somali Regional State namely, Jijiga and Shinille. Six districts were purposively selected based on their husbandry and CBPP status. Animals were examined with a combination of clinical, pathological, serological, microbiological methods. Participatory epidemiological techniques were applied to analyze community based approaches to CBPP and other diseases prevalence in the area. Geographical information system (GIS) was applied to label the spatial distribution of diseases from participatory epidemiological analysis and serological survey.

3.4. Sampling frame and Sample Size Determination

The sampling frame consisted of list of districts and associated cattle population in these areas. The population in each village could not be obtained except the list of the villages. The sampling methods were purposive sampling based on CBPP status namely, outbreak area, suspected area, and free area. Moreover, husbandry systems of pastoral, transhumance and sedentary were taken in to consideration to get a better picture of the areas.

Since the approximate prevalence of the disease in the region was not known, 20% prevalence was considered and more number of samples was taken. From each herd 20% of animals were selected using the random sampling procedure (Thrusfield 1995). Therefore, during this study the 20% prevalence with more number of samples were selected (Table 3).

Table 3. Estimated sample size Description

Number

Number of zones

2

Number of districts

6

Total number of sites

11

Percentage of samples /herd

20

Total number of herds

56

Total number of samples collected

793

25

The risk factors were identified based on the retrospective data from the local agriculture bureaus and semi-structured interview with the key informant groups who were selected from the communities. 3.5. Clinical Examination and Sample collection 3.5.1

Clinical examination

Clinical examinations of sick animals due to CBPP were done in order to cross-check and triangulate the perception of the livestock keeper with other participatory appraisal results. Twenty five animals which showed respiratory signs were clinically examined by the research team. The pastoralists identify these animals well. Clinical signs considered were anorexia, fever, dyspnoea, exercise intolerance, polypnoea, cough and nasal discharges and lagging behind the group. 3.5.2

Serum sample collection

Animals were restrained by owners and 10 ml of blood sample were collected from the jugular vein using vacutainer tubes. The samples were kept under the shade in a slant position for four hours and centrifuged. The sera sample were transferred to serum tubes and kept at 20 0C until brought to the laboratory for analysis. Corresponding to each sample, the age and sex of every animal and geo reference information were collected and registered on a separate case book. 3.5.3

Sample collection for isolation

The CBPP outbreak areas were identified using Participatory Disease Searches (PDS) methods. In the study areas where CBPP outbreak existed, the history of the disease in the herd and particular animals were asked. Among the clinical cases which showed respiratory signs regarded to be that of the disease, two animals with severe signs were purchased from the pastoralist. One of the animals was slaughtered for post-mortem on the field and the other animal was transported by truck to the National Veterinary Institute, Debre Zeit (Fig. 9 and 10). An extensive post-mortem examination was undertaken and the finding registered in a case book. Additional six clinical cases which were followed at the site were also investigated at post-mortem. These latter animals died natural death the examination was undertaken as soon as they died. Therefore, all in all post-mortem examinations were done on seven animals.

26

Samples for isolation were taken from the lung, kidney and mediastinal lymph nodes and thoracic fluid. The samples were immediately put in the freezer. For cultivation of the pathogen, the lung tissues were ground in medium with antibiotics and inoculated into media that contain inhibitors to prevent the growth of contaminating bacteria. As the growth of M. m. subsp. mycoides SC takes several days, desiccation of the solid medium was limited by closing the Petri dishes.

The Disk Growth Inhibition Test was used as a confirmatory diagnosis. It is based on the direct inhibition of the growth of the agent on a solid medium by a specific hyperimmune serum. It is a specific and simple test to perform, using the following interpretation: small inhibition zones (less than 2 mm wide), partial inhibition with ‘breakthrough colonies’, falsenegative and false-positive reactions (very rare).

3.5.4

Sample collection for histo-pathological examination

Samples for histopathology were also collected from the same tissue namely, lung, kidney and mediastinal lymph nodes (except thoracic fluid) from both the field samples and in Debre Zeit and kept in 10% buffered formalin.

3.5.5 3.5.5.1 A)

Serological testing Competitive ELISA (cELISA) Principles of Competitive ELISA test principle

1) The wells of the polystyrene micro-plate are coated with an MmmSC lysate. 2) Teat Serum samples are diluted and incubated at 370 C with the specific monoclonal antibody in a pre-plate. This mixture is then transferred into the MmmSC antigen coated micro-plate. Any antibody specific to MmmSC in the serum forms an MmmSC / bovine antibody immune-complex, which effectively masks the MmmSC sites. In this case the monoclonal antibody cannot bind to the corresponding epitope. 3) After washing, an anti-mouse-IgG antibody coupled peroxidase is incubated in the wells. In presence of specific MmmSC antibodies in the serum to analyse, the monoclonal antibody does not fix in the plate and the conjugate cannot bind in the wells. On the contrary, the conjugate can bind to the monoclonal antibody. 27

4) After washing, the enzyme substrate (TMB) was added to the conjugate, forming a blue compound becoming yellow after blocking. The intensity of the colour was an inverse measure of the proportion of anti-MmmSC antibodies in the serum sample to test.

The cut-off was calculated by using the results obtained from a monoclonal control (Cm) and a conjugate control (Cc). The positive and negative controls are delivered with the kit. They have to be added to each micro-plate, in order to validate the results.

B)

i.

Competitive ELISA test procedure

ELISA plates were coated with a lysed antigen solution in PBS, pH 7.4 (100 µl/well) and incubated overnight at 4°C.

ii.

The plates are washed once in PBS diluted 1/5 with 0.05% Tween 20.

iii.

Sera (diluted 1/10) and MAb diluted in PBS with 0.5% horse serum and 0.05% Tween 20 were left in contact with the antigen for 1 hour at 37°C under moderate agitation in a humid chamber.

iv.

The plates were washed twice and conjugate is added to all the wells (100 µl); the wells were then incubated for 1 hour at 37°C.

v.

The plates were washed three times and the substrate was added to all the wells (100 µl).

vi.

Reading was performed at 405 nm when the absorbance in the control MAb had reached 0.8-1.6.

28

3.6. Participatory Epidemiology 3.6.1

Participatory Appraisal

The development of PA was influenced by two main bodies of experience which can be summarised as negative and positive experiences. Negative experiences included decades of poor success in rural development in non-industrialised countries and the wide spread failure of aid programmes to improve the lives of poor people (Chambers, 1983, cited from Catley, 1999). The positive experiences which contributed towards PA development included lessons from a diverse group of disciplines and professional experiences. But the main influences were drawn from social sciences and novel systems approaches in agricultural and ecological researches such as, Freirian-style adult education in Latin America; the methods of agroecosystem analysis developed at the University of Chiang Mai in Thailand; the value of indigenous knowledge from applied social anthropology (Catley, 1999).

Participatory appraisal (PA) methods have been used by veterinarians in Africa since the late 1980s. The methods can be categorized as interviews, ranking and scoring method, and visualization methods (Catley et al, 2001). This study was designed to use participatory appraisal methods and conventional veterinary investigation methods to obtain information on CBPP in pastoral systems of Somali region. The information required using participatory appraisal methods were, CBPP status and its priority, risk factors (husbandry, movement), seasonality of CBPP, and time and source of infection.

3.6.2. Methods

The study was conducted in three groups of areas which were based on their CBPP status (Afdem, Mieso and Dembel) of Somali region (Fig. 14, 15 and 16). The field work was carried out over a period of 8-week (December 2003 up to February 2004). Afdem (latitude 09o 27’, longitude 040

o

59’) and Mieso are located at the North-western part of Somali

region, whereas Dembel lies at the North-eastern part of the region (latitude 09

o

48’,

longitude 042 o 35’). The methodology was based on a comparative assessment of diseases and an initial stage of community identification of 10 ‘important’ diseases.

29

The sites were selected based on the need to sample cattle for serum collection. In each site, informants included those people whose cattle were being sampled and other people who were present nearby and interested or invited to join the discussion by the community. The number of informant groups in Afdem, Mieso and Dembel were 10, 10 and 3 respectively. Group size varied from 5 to 10 individuals. The numbers of informant groups were based on the availability and willingness of the livestock herders.

Since the researcher had received participatory training at different times and practiced the method for the last 3 years, the time was used efficiently. First identification of the disease to be scored was done using semi-structured interview. This was done by asking the ten most important cattle diseases found in the area all the year round. During this step the local names of the disease that we were interested were identified. However, whenever some important priority diseases that were not included in the survey were mentioned by the community, it was taken as ‘controls’. This was because the local people will be mostly willing and interested to discuss if their priority disease was included.

3.6.2.1. Participatory Disease searches (PDS)

Participatory Disease searches (PDS) are probably the most powerful tool available for active surveillance and it is a sensitive and rapid intelligence-gathering activity for the detection and investigation of animal health events (Catley et al, 2002). In this study PDS was used with the objective of finding clinical cases of CBPP that enables to categorize the study areas based on CBPP status. Therefore, rather than using preset methods and designs such as those used in surveys, an inductive approach was used. With this approach, each information gathering exercise generates insights that guide or induce the next stage of the process.

Accordingly, secondary sources of information were sought that could indicate the spatial and temporal distribution of recent CBPP outbreak. The next step used was participatory methods in order to understand local perceptions of animal health situation in general and CBPP in particular. The methods used were semi-structured interviews (SSIs), focus group discussions and visualization methods such as mapping. In addition, participatory enquiries using of both open questions (non-directed questions using words such as ‘why’, ‘who’, ‘what’, ‘where’, ‘when’, ‘how’,) and probing questions (which may need to be directed) were used (Catley et al, 2002). 30

3.6.2.2. Proportional piling

The ten most important cattle diseases were selected by the informant groups and asked to rank them according to their impact using 100 stones against 10 diseases. The impact of CBPP that was defined by the communities was reduced milk production, treatment cost and mortality over a year period. The group was asked to discuss among themselves and reach a group decision regarding the placement of the stones. When placing of the stones against the diseases was complete, the group was requested to thoroughly check the scores and if they wanted, rearrange the scores until they were contented with the result. Most of the time the informants do not count the stones when assigning to the particular disease, rather they simply pile the stones in to piles and show visually the varying amount of the items being scored. The types of data obtained were non-parametric and therefore, median was used during statistical analysis.

3.6.2.3. Seasonal calendar

Temporal variations in disease occurrence are a common aspect of epidemiological investigation. Seasonal calendars are a useful method for understanding local perceptions of seasonal variations in disease incidence or population of ticks, biting flies or other factors. Seasonal calendar can also generate new hypothesis about associations between diseases, environmental factors, and interactions with wildlife and vectors (Catley et al, 2002).

To construct a seasonal calendar the researcher put four materials on the ground and told them that it represented the four seasons. The bigger stick with green leaf represented Kerenta (July-Sept), followed by the bigger dry stick Jilal (Oct-Feb), the smaller stick with green leaf Dirac (March-Apr), and the shorter dry stick Xagay (May-June). The informants were requested to explain the meaning of each symbol to know whether they have understood what it represented.

The informants were given 16 stones and asked to show the relative occurrence of each disease in each season. When placing of the stones for one disease against the season was complete, the group was requested to thoroughly check the scores and if they wanted, rearrange the scores until they were contented with the result. Based on similar scoring 31

method, more items were added one by one to the seasonal calendar and scored by the informant groups. These items were ten cattle diseases called Gubulo, Sogudud, Xaran, Burunbur, Malig, Dhigis, Abeb, Bogta, DhabarJabiye, and Cal. The local disease names were written on card boards and passed around the group before scoring to check that informants recognized the disease name they were going to score. A completed seasonal calendar comprised four seasons along the x-axis of the diagram and 10 diseases along the y-axis of the diagram. Following scoring, open and probing questions were used such as ‘why do you see Gubulo mainly in Kerenta and Dirac?’and ‘when do you come back to your village?’. These exercises were done repeatedly with all 23 groups of informants in all sites using the same types and number of diseases and seasons.

Agreement between informant groups was assessed using Kendall’s coefficient of concordance (W) (SPSS, 2002). This non-parametric test measures the association between sets of ranks assigned to objects by judges (or groups of judges) and computes a W value between 0 and 1. A high or significant W value (nearer to 1) means that the judges are ranking the objects using a similar standard. The test is particularly useful for determining inter-judge reliability (Seigel and Castellan , 1994, cited from Catley et al, 2002).

32

4.

RESULTS

4.1. Prevalence of Contagious bovine Pleuropneumonia (CBPP) using c-ELISA

During this study a total of 56 herds were surveyed from two zones and six districts of the Somali region. The prevalence of CBPP in both study zones is given in Table 4. There was an average sero-prevalence of 30.4% in 17 herds with at least one infected animal per herd (Appendix. 5). It was higher in Shinille zone (35.1%) than in Jijiga zone (21.1%). On individual animal basis, the prevalence was in their respective zones was 11.6% and 4.3% which were much lower than herd prevalence.

Table 4. Sero-prevalence of Contagious Bovine Pleuropneumonia in cattle in Shinille and Jijiga zones (October 2003 to February 2004) Zones

Tested serum

Shinille Jijiga Total

653 140 793

Number of positive 76 6 82

No. of herds tested 37 19 56

No of positive herds 13 4 17

% Positive 95 % CI animal herds level prevalence 35.14 21.05 30.36

(9.28 - 14.35) (1.59 - 9.09) (8.31 - 12.67)

The prevalence of CBPP based on agro-ecology of the study sites is given in Table 5. On herd level, the prevalence in the lowlands was higher (39%) than in medium altitudes (6.7%). The majority (81/82) of all cases were found in the lowlands.

Table 5. Sero-prevalence of Contagious Bovine Pleuropneumonia in cattle in different agroecological zones of Shinille and Jijiga (October 2003 to February 2004) Agro-ecology

Tested serum

No. of herds tested 41 15

No of positive herds 16 1

% positive (herd)

637 74

Number of positive animals 81 1

39.02 6.67

95 % CI animal level prevalence 9.06-13.83 0.03-7.21

Lowland Medium altitude Total

793

82

56

17

30.39

8.31-12.67

Table 6. shows the distribution of CBPP in different districts of Shinille and Jijiga zones of the Somali Region. The highest herd sero-prevalence was observed in Mieso (100%) followed by Qabribeyah (75%), and Afdem (71.4%) districts. However, result in relation to districts 33

shows Except Jijiga district which is medium altitude, the remaining districts are located in the lowland. The herds in Jijiga were sedentary while others except Dembel and Qabribeyah which are agro-pastoralists, the remaining districts are pastoral. Therefore, the sampled herds were smaller in size in Jijiga and become larger in agro-pastoral and pastoral areas.

Table 6. Sero-prevalence (animal level and herd level) of Contagious Bovine Pleuropneumonia in cattle in different districts of Jijiga and Shinille zones (October 2003 to February 2004) District

Total sample size

Afdem Dembel Jijiga Qabribeyah Mieso Shinille

130 134 75 65 46 343

No. of positive animals 67 1 1 5 4 4

No of positive herds 5 1 1 3 3 4

No. of herds tested 7 9 15 4 3 18

% positive herds 71.43 11.11 6.67 75.00 100.00 22.22

95 % CI of animal level prevalence 42.62-60.39 0.02-4.09 0.03-7.21 2.54-17.04 2.42-20.79 0.32-2.96

In areas where a clinically confirmed outbreak of CBPP was reported, prevalence was 71.4% compared to suspected areas (33.3%). Half of the sampled herds (28/56) included in both groups and most (75/82) of the animal that tested positive were found in CBPP outbreak areas and suspected sites. Table 7. Sero-prevalence result based on CBPP status CBPP status Outbreak Suspected Free

Total sample size 130 389 274

No. of positive animals 67 8 7

No. of herds tested 7 21 28

No of positive herds 5 7 5

% positive herds 71.43 33.33 17.86

95 % CI of animal level prevalence 42.62-60.39 0.89-4.01 1.03-5.19

With regard to the husbandry status, the highest prevalence (43.8%) was observed in transhumance areas than pastoralist (36%) or sedentary (6.7%) areas. Since the 95% confidence interval at individual animal level seropositivity did not overlap between the pastoral and the remaining husbandry systems, the difference was statistically significant at 5% level. On the other hand, the different between transhumance and sedentary was not significant.

34

Table 8. Sero-prevalence result of CBPP based on husbandry status Husbandry status

Total sample size Pastoral 473 Transhumance 245 Sedentary 75

No. of positive 71 10 1

No of positive herds 9 7 1

No. of herds tested 25 16 15

% positive herds 36.00 43.75 6.67

95 % CI of animal level prevalence 11.91 - 18.55 1.97 - 7.38 0.03 - 7.21

The prevalence in the study sites in the six districts investigated at individual animal and herd level are shown in Table 7. At both levels prevalence was high in Kaha study site found in Afdem district where an active outbreak was also observed. There was no evidence of infection in Dembel (Dembel district) and Gad (Shinille district), though they are bound to some sites which were CBPP seropositive.

Table 9. The prevalence of CBPP in different study sites in Shinille and Jijiga zones between October 2003 and February 2004 Sites

Total sample size

Afdem Dembel Hurso Jijiga Kaha Qabribeyah Qarenley Shinille Gad Harmukale Tuleytu

53 59 92 75 77 65 75 86 91 74 46 793

Number of positives 2 0 1 1 65 5 1 2 0 1 4 82

No. of herds tested 3 4 5 15 4 4 5 4 5 4 3 56

35

No of positive herds 1 0 1 1 4 3 1 2 0 1 3 17

% positive herds 33.33 0.00 20.00 6.67 100.00 75.00 20.00 50.00 0.00 25.00 100.00 30.36

95 % CI of animal level prevalence (0.46, 12.98) (0.03, 5.91) (0.03, 7.21) (74.36, 91.68) (2.54, 17.04) (0.03, 7.21) (0.28, 8.15) (0.03, 7.30) (2.42, 20.79) (8.31, 12.67)

4.2. Outbreak Investigation

4.2.1. Clinical and post-mortem findings

In the outbreak area, CBPP outbreak was observed in 4 herds, all in Afdem district sand more specifically in Kaha site. Twenty five animals which showed signs of respiratory distress were examined. The signs observed were fever (40

oC), fast, difficult or noisy breathing,

coughing, after exercise, forelegs spread apart (slight), flies on the body, lack of exercise tolerance, and lack of immediate sensitivity to fly bite and lag behind herds. However, classical signs of CBPP signs such as difficulty in breathing, standing with the head and neck extended and legs widely placed and turning out elbows were not observed (Table. 10).

An extensive post-mortem examination was undertaken in six animals showing clinical signs. In all these animals, only the right lung was affected and all had large amount of thoracic fluid (Fig. 5). Ten litres of the thoracic fluid was collected from one side of the cavity immediately after the animal was killed for post mortem examination. The fluid coagulated within two hours after exposed to the environment.

Table 10. Summary of clinical examination data

Case number

Clinical signs A B C D E F G H I Fever (40

J

K L M N O P Q R S T U V W X Y

o

C)

+ +

Fast, difficult or noisy breathing Coughing Coughing, after

+ + + + + + +

+ + + + + +

+ + + + + + + + + +

+ + + + + + +

+ + + + + + + + + + + + + +

exercise Head and neck extended Forelegs spread

+

apart (slight) Nasal or mouse discharge Flies on the body

+ + + + + + + + + + + + + + + + + + + + + + + + +

36

Figure 2.

Yellowish thoracic fluid collected from a seven-year old cow suffering from CBPP.

The owner sits in despair after losing this cow and expecting further loss from the disease.

In 4 out of 7 animals, (Table. 11) the lung was attached to the thoracic wall as shown in figure 3, 4 and 5 and a large amount of (10 litres) yellowish thoracic fluid were obtained (Fig. 2).

Figure 3. Thoracic cavity of a six year old cow showing extensive fluid in the thoracic cavity.

(Note the coagulated yellowish thoracic fluid which shows fibrin deposit.) 37

Figure 4. The lung of a seven year old cow showing firm and fleshy lesions. (hepatisation).

Figure 5. The lung of a four-year old cow showing marbled lung coated with fibrin

The lung shows reddish and light red colour.

38

The kidneys were involved (Table. 11) with pin point infarcts on the surface as it is shown on Fig. 6. When cut, the lesions covered the cortex part. The mediastinal lymph nodes were oedematous and swollen.

Figure 6. Gross lesions of the kidney of a seven year old cow showing whitish spotted areas of dead tissue (infarcts)

Table 11. Summary of post-mortem findings

Post mortem findings

Case number A

B

C

D

E

F

G

Yellowish thoracic fluid

+

+

+

+

+

+

+

One lung affected (right)

+

+

+

+

+

+

+

Lung covered with yellowish material

+

+

+

+

+

+

+

Lung adhered to the chest wall

+

+

Lung do not collapse and are solid

+

+

Marbling of lung

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Sequestra in the lung White spots on the kidney

+

+

39

+

4.2.2. Histopathology

Tissue samples from lung, kidney and mediastinal lymph nodes were fixed with 10 percent buffered formalin solution for histopathology. The microscopic appearance in the lung included widening of the interlobular septa. The lesion observed in the lymph node was accumulation of oedematous fluid and fibrin in the cortical and medullary sinuses.

4.2.3. Microbiological findings

Lung tissue sample was ground in medium with antibiotics and inoculated into PPLO media containing inhibitors of other contaminants or concurrent infectious agents. After two weeks culture, small colonies of 1mm in diameter with classical ‘fried-egg’ appearance of Mycoplasma (Fig. 7 and 8). Diagnosis was confirmed by immunological tests, using the growth inhibition test.

Figure 7. Mycoplasma mycoides subspecies mycoides colony with the classical appearance of ‘fried-egg’ with a dense centre

40

Figure 8. Mycoplasma mycoides subspecies mycoides colony with the classical ‘fried-egg’ appearance

4.3. Participatory Epidemiology

4.3.1. Participatory Disease Searches (PDS)

The secondary sources of information collected during PDS were information on CBPP outbreak, cattle movement, and types of intervention. The result had indicated the spatial and temporal distribution of recent CBPP outbreak. And in fact, this step was vital to be specific where to look the disease and minimize from two zones and six districts to one zone and one district. Besides, we came to know that no veterinary service interventions were carried out specifically to the outbreak areas, called Afdem district. The research team went to Afdem and participatory methods were used in order to understand local perceptions of animal health situation in general and CBPP in particular. The methods used were semi-structured interviews (SSIs), focus group discussions and visualization methods such as mapping. In addition, participatory enquiries using of both open questions (non-directed questions using words such as ‘why’, ‘who’, ‘what’, ‘where’, ‘when’, ‘how’,) and probing questions (which was directed to CBPP) were used.

41

We went to the district town called Afdem, and discussed with the local people for three days, where most of them heard the problem of CBPP as a rumour and told us to go and ask to a place called Selahdemeer. Then in Selahdemeer we have stayed for another two days and discussed with the communities the diseases’ (CBPP) whereabouts. Although we met livestock owner whose cattle were found at the place where now considered as out break area, they could not specify either the disease type or the exact location of the place. We have been told that they could not know day to day information because their livestock were found with the younger and stronger men who are going to distant areas which depend on the availability of pasture and water that can not be predicted. Finally, they recommended that we better go to Alijir which might had better information. Accordingly, we went further to Alijir and repeated the PDS methods with the communities.

Eventually, the local communities at Alijir told us that the herders with the livestock came to a place called Kaha because that place has got unexpected rain which they call it locally ‘Hayes’. Then we travelled further to kaha and met the livestock herder who showed us clinical cases of CBPP. Since the clinical cases we have been showed did not show ‘classical CBPP signs’, we agreed to purchase one sick animal for post-mortem (Fig. 9). Accordingly, we have done clinical and post-mortem examinations to probe the knowledge of the disease, issues and terminologies and found out that the pastoralist knew the clinical and post-mortem lesions accurately.

Figure 9. A large number of flies’ throughout the body of a cow with CBPP 42

During semi-structured interview open and probing question were used to gather intelligence about CBPP. The pastoralist identified animals that suffered from CBPP by observing the clinical signs animals show. These signs are accumulation of flies all over the body (Fig. 9) regardless of the distance from the tail or the head. Animals do not move these fly removal mechanisms. One animal which was brought to Debre Zeit for post-mortem examination was selected from the herd by the pastoralists themselves and the signs observed in this animal were not indicative of classical CBPP except pain on touch and slight elbow abduction (Fig. 10). The body temperature was also in the normal range. Even the experienced veterinarians at the National Veterinary Institute had doubts until the post-mortem findings showed lesions typical of CBPP. However, in addition to typical post-mortem findings, Mycoplasma mycoides subspecies mycoides colony with the classical ‘fried-egg’ appearance was isolated (Fig. 7 & 8).

Figure 10. The forelegs slightly spread apart in cow with CBPP Moreover, the methods of CBPP control were asked and found out to be using various types of antibiotic treatment. They responded that they use antibiotic treatment by CommunityBased Animal Health Workers. They have also mentioned there is no public or private veterinary service in their vicinity. In a place called Kaha one herder said that he had used 60 bottles of oxytetracycline within three months for treating his herd. The pastoralists unanimously agreed that the use of antibiotic treatment for CBPP cases was best and the only 43

option they have got. Since the veterinary service delivery was very poor in the area, they are even using tetracycline capsules to inject directly in the lung after they dissolved with water (Fig. 11). The local names mentioned for CBPP by the informant groups were; Gubulo, Ofta, and Sombob that refers to difficulty in breathing and lung which indicates that the community knows not only the sign and symptom of the disease but also the organ affected.

Figure 11. Drug used by pastoralist against CBPP. (Note: It will be diluted with water and directly injected on the lung) 4.3.2. Proportional piling

Table 10 shows the proportional piling results of 10 important diseases prevailing in the Somali Region. CBPP was ranked first in Afdem areas, second in Mieso area and ninth in Dembel areas. The first and second were CBPP outbreak and suspected areas, respectively. In the last district the disease is not considered as an important disease even as compared to gastrointestinal parasitism and coincided with the serological findings.

44

Table 12. Summarized table of proportional piling on important livestock diseases in Jijiga and Shinille zones. Types of diseases English name Local name CBPP TBD Anthrax LSD Trypanosomes Black leg FMD HS Paralysis Internal parasite

**

Gubulo Sogudud Xaran Burunbur Malig Dhigis Abeb Bogta DhabarJabiye

Districts with CBPP status Median Rank (Outbreak area)

Median Rank (Suspected area)

Median Rank (Free area)

25.5 (21.8-30.5) 10.5 (7.8-12.3) 9.5 (6.5-11.0) 9.0 (5.5-17.5) 7.5 (4.8-10.5) 7.0 (6.0-13.3) 6.5 (2.8-9.8) 6.5 (5.0-10.5) 6.0 (4.8-7.3)

28.5 (7.8-34.8) 7.5 (6.0-9.0) 5.0 (4.0-7.0) 8.5 (6.8-14.3) 13.0 (10.0-15.3) 8.5 (6.5-11.5) 6.0 (4.0-7.3) 5.0 (4.0-5.0) 12.5 (5.0-19.5)

0.0 (0.0-12.0) 14.0 (7.0-25.0) 11.0 (6.0-18.0) 5.0 (4.0-7.0) 8.0 (4.0-11.0) 11.0 (6.0-12.0) 9.0 (7.0-15.0) 10.0 (10.0-12.0) 13.0 (13.0-26.0)

7.0 (5.8-8.5) 10 0.50

8.0 (7.0-9.0) 3

Cal 4.0 (2.0-9.3) 10 N ** 0.39 Kendall's W

P< 0.05

Kendall’s W = Coefficient of concordance between groups

4.3.3. Seasonal calendar A summarized seasonal calendar for livestock diseases against four local seasons is shown in Figure 2, 3 and 4. This activity was carried out in the three districts namely Afdem, Mieso and Dembel considered as CBPP outbreak, suspected and free areas, respectively. In total 23 groups were included. These results were discussed with informants and in particular, findings were related to seasonal movements of cattle. The group explained that more cattle were present in the villages during the two rainy seasons, Kerenta (July-Sept) and Dirac (March-Apr). During these seasons cattle were returning back from where they had migrated and congregated in small areas that favour the transmission of CBPP.

However, during the long and short dry seasons, Jilal (Oct-Feb) and Xagay (May-June), cattle were moved out of the village and migrated to different areas in search of pasture and water which reduces the contact rate of animals. However, they had mentioned that the infection of CBPP was occurring during migration and contact with other animals. The consistency of agreement in describing different diseases and seasonality were observed and cross checked with other diseases. The result shows that informants had scored accurately disease that increase or decrease in its incidence as a result season such as, black leg, internal parasite and anthrax. 45

Seasons Kerenta Sept)

(July- Jilal (Oct-Feb)

Dirac Apr)

(March- Xagay (May-June)

Gubulo Contagious Bovine • • • Pleuropneumonia •••

•• ••

•• ••

••

(W=0.20) Sogudud Tick borne diseases

5.5 (3-8) •• •

3.5 (2-6) ••• •••

3.5 (2-6)

2.0 (1-3)

•• ••

•• •

(W=0.07)

3.0 (0-8)

6.0 (2-9)

Dhigis Blackleg

••• ••• ••• 9.1 (7-10)

3.5 (0-5) ••• ••

3.5 (0-5) ••• •••

0 (0-3) •• •

4.5 (2-6) •• •

6.0 (4-11)

3.0 (0-6) ••• ••• •••

3.0 (0-6)

7.4 (0-16)

1.0 (0-4) ••

4.5 (2-7) ••• •••

2.0 (0-5) ••• ••

6.0 (0-8) ••• ••

5.0 (4-7)

5.0 (3-8) ••• ••

(W=0.26) Cabeb FMD

••

(W=0.26) Dhabar jabiye Paralysis

2.0 (0-2)

(W=0.46) Kud Anthrax

0.0 (0-0)

(W=0.19) Burunbur LSD

1.0 (0-4) •

9.0 (7-12) ••• ••• •• 8.0 (2-10) •• ••

(W=0.07) Malig Trypanosomoses

1.0 (0-5) •• ••

3.5 (0-6) ••• •••

(W=0.13) Cuno-barar Hemorrhagic septicemia (W=0.3) Cal Internal parasite

3.5 (2-9)

2.5 (0-4)

••

6.0 (0-7) ••• •••

•••

4.5 (0-6) ••• •••

2.0 (0-7)

5.5 (3-8)

2.5 (0-4)

5.5 (4-9)

•• ••• •• 6.5 (6-9)

•••

•• ••

•••

2.5 (0-5)

4.0 (2-5)

2.5 (0-3)

(W=0.3)

•

•

•••

Figure 12. Summarized seasonal calendar for livestock diseases in Afdem district. (Somali region in Ethiopia). N = 10; W, Kendall’s coefficient of concordance between groups (p