Epidemiology of Brucellosis in Yaks in the
Tibet Autonomous Region of China
By
Jiangyong Zeng
BAgr (Vet Med), MAgr (Vet Med)
College of Veterinary Medicine
School of Veterinary and Life Sciences
Murdoch University
Western Australia
This thesis is presented for the degree of Doctor of Philosophy of
Murdoch University
2017 Declaration
I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution.
Jiangyong Zeng
ii
Dedication
I dedicate this Doctoral Thesis to the most precious people in my life, for their everlasting love, full understanding and strong support in my study:
My wife - Xu Wang
and
My daughter - Junhan Zeng
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Abstract
Brucellosis, caused by members of the genus Brucella, is a highly contagious production-limiting disease and one of the most important zoonosis in many countries of the world, including China. Prior to the study outlined in this thesis, few studies on the epidemiology of brucellosis in Tibet had been undertaken. Consequently, the aims of this study were to determine the epidemiological characteristics and economic impact of brucellosis in yaks in Tibet.
In a study examining historical data, significant differences were found in the spatial and temporal distribution of brucellosis in both livestock and humans (p<0.05). In the period from 2011 to 2013 there was a positive correlation between the seroprevalence in livestock and humans (r=0.93). Brucellosis was shown to be more common in the spring/summer seasons when parturition occurred.
A cross-sectional serological study of 1,523 randomly selected yaks belonging to 181 herders was conducted in Damxung, Maizhokunggar and Yadong counties. Sera were tested using a Rose Bengal Test (RBT) and a Competitive immune-enzymatic assay (C-
ELISA) and the results interpreted in parallel. The individual yak seroprevalence was
2.8% (95%CI: 2.0, 3.7) with a herd level seroprevalence of 18.2% (95%CI: 12.9, 24.6).
At the individual animal level, age and production system were significantly associated with seropositivity in a multivariable logistic regression model. The odds of Brucella infection were significantly higher in older yaks (3-5 years old, OR=4.51; 95%CI: 1.53,
19.29; >5 years old, OR=3.89; 95%CI: 1.23, 17.21) compared to younger yaks (<3 years old). The odds of seropositivity for yaks managed under an agro-pastoral production system was 2.9 (95%CI: 1.48, 5.86) times higher compared to those iv
managed under a pastoral production system. At the herd level an association between seropositivity and a history of herd-abortions was observed (OR=4.98, 95%CI: 1.48,
16.62). Surprisingly vaccination of calves in Pali township of Yadong county was not associated with a lower level of infection (p=0.49 and p=0.99 for individual and herd level data, respectively).
A total of 317 yak pastoralists were interviewed using a structured questionnaire to determine their knowledge, attitudes and practices (KAP) relating to brucellosis.
Although 60.6% of the respondents had heard of the disease, there was an overall low level of knowledge about the disease. Pastoralists did, however, adopt management/husbandry practices which would reduce transmission of the disease to humans and other animals. Multivariable logistic modelling showed that better knowledge was predicted by age (≥50 years old, OR=1.98; CI: 1.15, 3.46), production system practiced (pastoral, OR=10.57; CI: 5.47, 21.54), education level
(primary/secondary school, OR=2.19; CI: 1.22, 3.96) and number of persons in a household (≥6 persons, OR=2.77; CI: 1.59, 4.91). Difference in attitudes and practices were predicted by education level (primary/secondary school, OR=1.72; CI: 1.03, 2.88), number of persons in a household (≥6 persons, OR=2.80; CI: 1.68, 4.76) and production system practiced (pastoral, OR=2.43; CI: 1.38, 4.33).
An economic evaluation of brucellosis found that the disease could result in a loss of
US$ 3,126,256.68 (95%CI: US$2,006,644.50, US$4,559,176.80) in the total population of yaks over a six-year period in Damxung and Maizhokunggar counties and Pali township of Yadong county in Tibet, with an average loss per yak estimated at
US$ 1.42 (95%CI: 0.91, 2.07) annually. Through benefit-cost analysis, vaccination was v
found to be the most economically sound control method with a benefit-cost ratio (BCR) of 3.19 (95% CI: 2.17, 4.66) and a net present value (NPV) of US$313,354.87 (95%CI:
US$157,678.46, US$541,061.80). In a sensitivity analysis the NPV of the vaccination control program was shown to be most sensitive to the loss from an abortion. In contrast the price of yaks that were slaughtered had the largest influence on the NPV for the test- and-slaughter control program and the combination control program (vaccination and test-and-slaughter programs).
It is concluded that both public health education and implementation of a routine vaccination program are needed to effectively control brucellosis in yaks on the Tibetan plateau.
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Table of Contents
Title ...... i Declaration ...... ii Dedication ...... iii Abstract ...... iv Table of Contents ...... vii Publications arising from this research ...... xii Conference Presentations (Oral) ...... xii Poster Presentations ...... xiii List of Tables...... xiv List of Figures ...... xvii Acknowledgements ...... xviii List of Acronyms and Abbreviations ...... xxi CHAPTER ONE ...... 1 Introduction ...... 1 1.1 Introduction ...... 1 1.2 Background of Tibet ...... 3 1.2.1 Location and administration of Tibet ...... 3 1.2.2 Climate of Tibet ...... 4 1.2.3 Livestock production system ...... 5 1.2.4 Livestock population ...... 7 1.3 Prevention and control of animal disease ...... 8 1.3.1 Veterinary services in Tibet ...... 8 1.3.2 Regulations and Laws ...... 9 1.3.3 Prevention and control system ...... 10 1.3.4 Brucellosis control program ...... 12 1.4 Aims and Objectives of this study ...... 13 CHAPTER TWO ...... 14 Literature review ...... 14 2.1 Introduction ...... 14 2.2 Aetiology ...... 14 2.3 Clinical signs, Pathology and Pathogenesis ...... 16
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2.3.1 Clinical signs ...... 16 2.3.2 Pathology ...... 17 2.3.3 Pathogenesis ...... 18 2.4 Epidemiology ...... 19 2.4.1 Transmission ...... 19 2.4.2 Survival and resistance ...... 20 2.4.3 Epidemiology of brucellosis ...... 21 2.4.3.1 Epidemiology of animal brucellosis...... 21 2.4.3.2 Epidemiology of human brucellosis...... 22 2.4.3.3 Epidemiology of brucellosis in China ...... 24 2.5 Diagnosis ...... 25 2.5.1 Identification of the agent ...... 25 2.5.1.1 Staining ...... 25 2.5.1.2 Culture ...... 25 2.5.1.3 Identification ...... 26 2.5.1.4 Nucleic acid recognition methods ...... 26 2.5.2 Serological tests ...... 27 2.5.2.1 Common in-use serological tests ...... 27 2.5.2.2 Buffered Brucella antigen tests ...... 29 2.5.2.3 Reducing agents ...... 29 2.5.2.4 Milk ring test ...... 29 2.5.2.5 Complement fixation test (CFT) ...... 30 2.5.2.6 Indirect ELISA ...... 30 2.5.2.7 Competitive enzyme immunoassays (or blocking ELISA) ...... 31 2.5.2.8 Fluorescence polarization assay (FPA) ...... 32 2.6 Treatment, prevention and control of brucellosis ...... 32 2.6.1 Treatment ...... 33 2.6.2 Prevention ...... 34 2.6.2.1 Main vaccines used in the past ...... 34 2.6.2.1.1 B. abortus strain 19 and B. melitensis Rev 1 ...... 34 2.6.2.1.2 Other live vaccines used in the past ...... 36 2.6.2.2 Main current vaccines ...... 36 2.6.2.2.1 B. abortus RB51 ...... 36 viii
2.6.2.2.2 DNA vaccines ...... 37 2.6.3 Control ...... 37 CHAPTER THREE ...... 41 Retrospective study of the seroprevalence of brucellosis in yaks in Tibet ...... 41 3.1 Introduction ...... 41 3.2 Materials and methods ...... 42 3.2.1 Data ...... 42 3.2.2 Statistical analyses ...... 43 3.3 Results ...... 43 3.3.1 Early records before the mid-1980s ...... 43 3.3.2 Results of recent animal surveillance (2009 to 2015)...... 47 3.3.2.1 Spatial distribution ...... 47 3.3.2.2 Temporal distribution ...... 48 3.3.2.3 Species susceptibility ...... 49 3.3.3 Results of surveillance for brucellosis in humans (2011 – 2013) ...... 49 3.3.3.1 Spatial distribution in humans ...... 50 3.3.3.2 Temporal distribution of seroprevalence in humans ...... 51 3.3.4 Correlation between animal and human seroprevalence ...... 51 3.4 Discussion ...... 52 CHAPTER FOUR ...... 58 Seroprevalence of bovine brucellosis in domesticated yaks (Bos grunniens) ...... 58 4.1 Introduction ...... 58 4.2 Materials and methods ...... 59 4.2.1 Study design ...... 59 4.2.2 Study sites ...... 59 4.2.3 Sample size and sampling ...... 62 4.2.4 Serological Tests ...... 63 4.2.5 Questionnaire design and implementation ...... 65 4.2.6 Preliminary analyses ...... 65 4.3 Results ...... 66 4.3.1 Sampling profile ...... 66 4.3.2 Seroprevalence at the animal level ...... 66 4.3.3 Herd level seroprevalence results ...... 68 ix
4.3.4 Seroprevalence in yaks raised under different production systems ...... 70 4.3.5 Seroprevalence of vaccinated and unvaccinated yaks ...... 71 4.4 Discussion ...... 74 CHAPTER FIVE ...... 78 Risk factor analysis ...... 78 5.1 Introduction ...... 78 5.2 Materials and methods ...... 78 5.3 Results ...... 80 5.3.1 Univariable and multivariable logistic regression analyses of risk factors for seropositivity in individual yaks ...... 80 5.3.2 Analyses of the seroprevalence data at the herd level ...... 82 5.4 Discussion ...... 83 CHAPTER SIX ...... 88 A study on the knowledge, attitudes and practices of herders with respect to brucellosis in yaks ...... 88 6.1 Introduction ...... 88 6.2 Materials and methods ...... 90 6.2.1 Study area ...... 90 6.2.2 Questionnaire design ...... 90 6.2.3 Procedure ...... 94 6.2.4 Data management and analyses ...... 95 6.3 Results ...... 96 6.3.1 Demographic characteristics ...... 96 6.3.2 Knowledge ...... 97 6.3.3 Association of risk factors with respondent’s knowledge ...... 97 6.3.4 Attitudes and practices ...... 102 6.3.5 Association of risk factors with the respondent’s attitudes and practices .... 102 6.4 Discussion ...... 106 CHAPTER SEVEN ...... 114 Evaluation of the economic impact of brucellosis in domestic yaks of Tibet ...... 114 7.1 Introduction ...... 114 7.2 Materials and methods ...... 115 7.2.1 The study area and design ...... 115 x
7.2.2 Economic model ...... 116 7.2.3 Strategy to control the disease ...... 120 7.2.4 Sensitivity analysis ...... 123 7.3 Results ...... 124 7.4 Discussion ...... 131 CHAPTER EIGHT ...... 139 General discussion ...... 139 8.1 Seroprevalence ...... 139 8.2 Risk factors for infection ...... 142 8.3 Economic assessment ...... 145 8.4 Limitations of the present study ...... 147 8.5 Recommendations ...... 148 8.6 Future research ...... 152 8.7 Conclusions ...... 155 Appendices ...... 156 Appendix 1. Basic information collected while sampling...... 156 Appendix 2. Farmer questionnaire for risk factors, KAP and economic survey ...... 157 Appendix 3. Farmer questionnaire for economic survey ...... 166 References ...... 168
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Publications arising from this research
Zeng, J.Y., Duoji, C., Yuan, Z.J., Yuzhen, S., Fan, W.X., Tian, L.L., Cai, C., Robertson,
I.D. 2017. “Seroprevalence and risk factors for bovine brucellosis in domestic yaks (Bos grunniens) in Tibet, China.” Tropical Animal Health and Production. 49 (7) 1339-1344.
Zeng, J.Y., Douji, C., Yundan, D.Z., Pu, Q., Gongjue, C.W., Jiumei. D.J., Robertson,
I.D. “A study on the knowledge, attitudes and practices of Tibetan yak herders with respect to brucellosis” has been accepted on 20th, Dec, 2017. International Health，doi:
10.1093/inthealth/ihx076.
Conference Presentations (Oral)
Zeng, J.Y., Robertson, I.D. (2017) Seroprevalence and risk factors for bovine brucellosis in domestic yaks (Bos grunniens) in Tibet, China. 3rd International
Conference of Animal Health Surveillance, 30th April to 04th May, Rotorua, New
Zealand.
Zeng, J.Y., Cai, C., Robertson, I.D. (2016) Progress on “Epidemiology of brucellosis in yaks in the Tibet Autonomous Region of China”. ACIAR workshop, 19th to 23rd
September, Canberra, Australia.
Zeng,J.Y., Cai, C, Robertson, I.D. (2014) Introductory Seminar on “Epidemiology of brucellosis in yaks in the Tibet Autonomous Region of China”. 3rd May, School of
Veterinary and Life Sciences, Murdoch University, Perth, Western Australia
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Poster Presentations
Zeng, J.Y., Cai, C., Robertson, I.D. (2016) A study on knowledge, attitudes and practices of herders in the main yak reared communities of Tibet, China with respect to brucellosis. Annual Poster Day for year 2016. School of Veterinary and Life Sciences,
Murdoch University
Zeng, J.Y., Cai, C., Robertson, I.D. (2015) Seroprevalence and risk factors for Brucella in yaks in main yak raising areas of Tibet, China. Annual Poster Day for year 2015.
School of Veterinary and Life Sciences, Murdoch University
Zeng, J.Y., Cai, C., Robertson, I.D. (2014) Epidemiology of brucellosis in yaks in the
Tibet Autonomous Region of China. Annual Poster Day for year 2014. School of
Veterinary and Life Sciences, Murdoch University
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List of Tables
Table 3.1 Results of the brucellosis vaccination program in Tibet from 1979 to 1985 .. 45 Table 3.2 Comparison of the seroprevalence to brucellosis prior to and after implementing vaccination programs in yaks, cattle, sheep and goats from 1979 to 1985 ...... 45 Table 3.3 Comparison of seroprevalence to brucellosis in three species prior to and after vaccination from 1979 to 1983 ...... 46 Table 3.4 Seroprevalence of brucellosis in yaks, cattle, sheep, goats and pigs at the prefecture-level in Tibet (2009-2015) ...... 47 Table 3.5 Seroprevalence of animal brucellosis during the period of 2009 to 2015 ...... 48 Table 3.6 Seroprevalence in different seasons ...... 49 Table 3.7 Seroprevalence in different susceptible species groups (2009 – 2015) ...... 49 Table 3.8 Seroprevalence of brucellosis in humans at the prefecture-level in Tibet (2011-2013) ...... 50 Table 3.9 Seroprevalence of human brucellosis (2011 to 2013) ...... 51 Table 4.1 Summary of sampling for the cross-sectional study ...... 63 Table 4.2 Serological detection of antibodies against Brucella using the RBT and C- ELISA ...... 67 Table 4.3 Animal level apparent (test) prevalence of brucellosis in yaks in the three counties sampled in Tibet ...... 67 Table 4.4 Herd level apparent (test) seroprevalence of brucellosis in yaks in the three sampled counties ...... 68 Table 4.5 Herd and animal level apparent (test) seroprevalence of brucellosis in yaks reared under two production systems in Tibet ...... 72 Table 4.6 Comparison of seroprevalence in individual yaks and herds adopting different production systems in three townships containing herds adopting both production systems ...... 73 Table 4.7 Comparison of apparent (test) seroprevalence in individuals and herds from vaccinated and non-vaccinated yaks ...... 73 Table 5.1 Characteristics of the sampled yaks and herds in the two counties (n=1,283) 79
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Table 5.2 Univariable analysis of risk factors associated with brucellosis seropositivity among individual yaks in Tibet using the results of the Rose Bengal Test (RBT) and the competitive enzyme linked immunosorbent assay (C-ELISA) interpreted in parallel. .. 81 Table 5.3 Multivariable logistic regression analysis of risk factors associated with brucellosis seropositivity among individual yaks ...... 82 Table 5.4 Descriptive statistics and univariable associations between seropositivity (herd level) and potential management risk factors in yaks in Tibet (n=181) ...... 83 Table 6.1 Distribution of households in villages of the three counties surveyed for the knowledge, attitudes and practices study ...... 92 Table 6.2 Demographic characteristics of respondents in the surveyed areas of Tibet (n=317) ...... 96 Table 6.3 Descriptive analyses of the respondent’s knowledge for brucellosis categorised by gender and production system ...... 99 Table 6.4 Association of demographic and socio-demographic variables with categorised knowledge scores of the respondents ...... 101 Table 6.5 Multivariable logistic regression model of factors associated with the respondent’s knowledge ...... 101 Table 6.6 Bivariate analyses of respondents’ attitudes and practices of brucellosis as influenced by sex and production system ...... 104 Table 6.7 Association of demographic and socio-demographic variables with the categorised scores of the respondent’s attitudes and practices ...... 106 Table 6.8 Multivariable logistic regression model of factors associated with the level of the respondent’s attitudes and practices ...... 106 Table 7.1 Parameters used in the financial analysis ...... 118 Table 7.2 Description of the three control strategies ...... 120 Table 7.3 List of input variables used as @Risk functions ...... 124 Table 7.4 Estimated economic losses (US $ and 95%CI) of brucellosis in the study region (366,350 yaks) during the six-year study period ...... 127 Table 7.5 Estimated numbers of newly infected female yaks in the different control strategies (95% CI) over the six-year study period ...... 127 Table 7.6 Prevalence of brucellosis in each year for the different control strategies (%) ...... 128
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Table 7.7 Benefit-cost analysis (US$) for evaluating three control strategies for brucellosis in the total population of yaks over a six-year period (95%CI) in Damxung and Maizhokunggar counties and Pali township in Tibet ...... 128 Table 7.8 Result of sensitivity analysis using different protection rates under the two control strategies of vaccination and ...... 129
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List of Figures
Figure 1.1 Geographical location of Tibet ...... 4 Figure 1.2 Organisational structure of the veterinary services (animal health) in Tibet ... 9 Figure 3.1 Spatial distribution of animal brucellosis in Tibet (2009-2015) ...... 48 Figure 3.2 Spatial distribution of human brucellosis in Tibet (2011-2013) ...... 50 Figure 3.3 Annual seroprevalence of animal and human brucellosis in Tibet (2009-2015) ...... 52 Figure 4.1 Map showing the location of the selected counties in Tibet, China ...... 60 Figure 4.2 Map showing the distribution of brucellosis seropositive and seronegative herds sampled in Maizhokunggar County...... 69 Figure 4.3 Map showing the distribution of brucellosis seropositive and seronegative herds sampled in Damxung County ...... 69 Figure 4.4 Map showing the distribution of brucellosis seropositive and seronegative herds sampled in Yadong County ...... 70 Figure 4.5 Yaks grazing in fenced areas ...... 75 Figure 5.1 Picture showing yaks, sheep and goats co-grazing on grasslands in Tibet ... 87 Figure 6.1 Map of locations of three counties of Yadong (left), Damxung (middle) and Maizhokunggar (right) surveyed in Tibet with the number and overall percentage of households interviewed in the respective counties ...... 93 Figure 7.1 Regression coefficients of the sensitivity analysis for the vaccination control program ...... 130 Figure 7.2 Regression coefficients of the sensitivity analysis for the test-and-slaughter control program ...... 130 Figure 7.3 Regression coefficients of the sensitivity analysis for the combination control program ...... 131
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Acknowledgements
This thesis would not have been possible without the support, assistance and guidance of many people and institutions.
First and foremost, I would like to express my deepest appreciation and gratitude to my principal supervisor, Prof. Ian D Robertson, for patient guidance, enthusiasm, statistical advice, academic critiquing and timely feedback. It also is appreciated that he accepted and allowed me to attend his classes, which has brought me an understanding of the subject of epidemiology and has enabled me to obtain “fresh blood” for completing my expedition.
I wish to thank Emeritus Prof. Nick Costa for assisting me to come to Australia and thereby allowing me to study in the field.
I am greatly indebted to generous financial supports, in the form of fellowship and research, provided by the Australian Centre for International Agricultural Research
(ACIAR), Murdoch University and the Tibet Livestock Research Institute (TLRI).
I would like to thank my co-supervisor Dr. Mieghan Bruce for sharing her economic knowledge and for reviewing the economic chapter. I would also like to thank my other co-supervisor Dr. Chang Cai for her support in the initial stages of the research and for reviewing my poster presentations. I owe my gratitude to Dr. Karma Rinzin (Bhutan) for assisting and sharing his knowledge of statistics in the aspect of epidemiology and enlightenment in the use of R, as well as Dr. Ernest Mochankana (Botswana), Dr.
Abdirahman Salah (Somaliland), Dr. Petrus Malo Bulu (Indonesia), Mr. Chih-Hsien Lin xviii
(Taiwan), Mr. Harish Tiwari (India), Mr. Mohanad Alamura (Iraq), Mr. Ali Gaddan
(Iraq) and Mr. Arash Osmani (Afghanistan) for sharing their views, insights and inspirations during my study. Many thanks go to other colleagues: Dr. Sivapiragasam
Thaya (Malaysia), Dr. Elviva Lee (Singapore), Mrs. Sharie Aviso (Philippines), Mr.
Emad Aziz (Iraq), Mr Yin Li (China) and Mr. Hamidreza Sodagari (Iran) for their camaraderie.
My sincere appreciation goes to the staff of the TLRI, particularly Mr. Ciren Duoiji for contacting heads of the veterinary stations of the selected counties, organizing field investigation and collecting samples during the fieldwork, Prof. Qiumei Ji (Director
General of the Institute), Emeritus Prof. Sezhu (Former Vice Director of the Institute) and Prof. Dawa Yangla (Head of Tibetan Yak Centre) for their assistance in the field and in this project, and finally I would like to acknowledge my colleagues: Mr. Zhenjie
Yuan, Mrs. Silang Yuzhen and Mr. Wujin Cuomu for their assistance in the laboratory.
I am gratefully to the heads of the veterinary stations in the three sampled counties during the survey including Mr. Yundan Dunzhu of Maizhokunggar county, Mr.
Puqiong and Mr. Gongjie Caiwang of Damxung county, and Mr. Jiumei Dorjie of
Yadong county for their assistance and I would also like to thank all participating farmers and pastoralists for their cooperation.
Special thanks go to the technical assistance provided by some staff of the China
Animal Health and Epidemiology Centre such as Prof. Weixing Fan, Mrs. Lili Tian,
Mrs. Zhen Yang and Mr. Zhigang Jing for carrying out laboratory tests of the sera samples.
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Last but not least, I don’t know how to thank my family members: my wife Xu Wang, my daughter Junhan Zeng for their unrelenting love, support and encouragement, and my mother, my parents-in-law, my siblings and friends for their understanding throughout my study.
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List of Acronyms and Abbreviations
BCR Benefit-cost ratio
BPAT Buffered antigen plate agglutination test
BSE Bovine spongiform encephalopathy
C-ELISA Competitive ELISA
CFT Complement fixation test
CI Confidence interval
DTT Dithiothreitol
ELISA Enzyme Linked Immunosorbent Assay
FAO Food and Agriculture Organization of the United Nations
FMD Foot-and-mouth disease
FPA Fluorescence polarization assay
GDP Gross Domestic Product
HPAI Highly pathogenic avian influenza iELISA Indirect ELISA
KAP Knowledge, Attitudes and Practices
ME Mercaptoethanol
MOA Ministry of Agriculture
MRT Milk ring test
NPV Net present value
OIE World Organization for Animal Health
OR Odds Ratio
PPR Péste des petits ruminants
PRRS Porcine reproductive and respiratory syndrome
RBT Rose Bengal test xxi
SAT Standard tube agglutination test
TAAAS Tibet Academy of Agriculture and Animal Husbandry Science
TBAA Tibet Bureau of Agriculture and Animal Husbandry
TBV Tibet Bureau of Veterinarians
TCADC Tibet Centre for Animal Disease Control
TCDC Tibet Centre for Disease Control
TIAHS Tibet Institute of Animal Health Supervision
TIVDC Tibet Institute of Veterinary Drugs Control
TLRI Tibet Livestock Research Institute
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CHAPTER ONE
Introduction
1.1 Introduction
Livestock are a major resource for farming households throughout the world, playing a crucial role in producing food, generating income for both rural and urban populations, and providing fuel, transport and employment opportunities (Seré et al., 1996).
Livestock have been raised by Tibetan households for thousands of years with their products being an important source of foreign trade and playing a large role in the economic development of local communities (Goldstein and Beall, 1991; Zhang, 2013a).
Yaks (Bos grunniens) are a unique plateau species that have supplied products to
Tibetans for over 4,500 years (Gerald et al., 2003) and the livelihood of local communities is closely associated with the rearing of these animals. Diseases that affect the productivity of livestock have the potential to cause significant economic losses to both individual animal owners and the local economy as a whole, and may even jeopardise the viability of individual livestock industries (Seré et al., 1996). Yaks are susceptible to many diseases, of which brucellosis has been reported to be the most serious (Gerald et al., 2003).
Brucellosis, caused by the genus Brucella, is a zoonotic disease that is prevalent worldwide (Perry et al., 2002; Perry and Grace, 2009; WHO, 2009; International
Livestock Research Institute (ILRI), 2012). Livestock can be infected directly through contact with either infected animals or contaminated materials such as aborted foetuses, milk or fluid from hygromas (Geering et al., 1995; Romich, 2008). Human infection is
1
closely linked to infection in livestock and other animals. Due to traditional customs and adoption of different sanitary practices, the epidemiology of human brucellosis can vary dramatically between regions (Seleem et al., 2010). Also the disease is frequently misdiagnosed and under-reported in humans, particularly in developing countries, resulting in the impact of the disease frequently being overlooked and underestimated
(Paul et al., 1995).
Brucellosis is a significant disease of livestock in many countries, and its epidemiology has been studied throughout the world (McDermott and Arimi, 2002; Pappas et al.,
2006). In several countries the spread of animal brucellosis has been controlled with the use of vaccination, test-and-slaughter methods, pasteurisation of milk, improved surveillance and controlling the movement of cattle (Godfroid et al., 2011). Bovine brucellosis, caused by B. abortus, has been successfully eradicated from Australia,
Canada, Cyprus, Denmark, Finland, the Netherlands, New Zealand, Norway, Sweden and the United Kingdom (Seleem et al., 2010). However, in many developing countries the disease remains endemic due to a lack of resources allocated by both governments and farmers, and a lack of awareness about the risk factors associated with the disease
(McDermott and Arimi, 2002).
In China, brucellosis has had a significant impact on the livestock industry, resulting in the government listing it as a Class Ⅱ notifiable disease through the Law on Prevention and Control of Infectious Diseases, and it is considered an important disease by the
Implement Detailed Rules of the By-law on Disease Prevention and Control of
Livestock and Poultry (Deqiu et al., 2002). Prior to the 1980s, the incidence of both human and animal brucellosis was high in China. Subsequently its prevalence decreased 2
significantly after the government implemented a vaccination and slaughter campaign; however this reduction was only temporary as human infection again increased during the late 1990s (Deqiu et al., 2002; Zhong et al., 2013). In Tibet, the seroprevalence of the disease in livestock and humans was also high in the 1970s and early 1980s.
Through the implementation of vaccination and slaughter of affected animals the disease was reported to have been controlled (Anonymous, 1985); however the disease subsequently re-remerged in the 21st century (Zhandui. et al., 2008; Wu et al., 2011; Liu et al., 2014). There is no compulsory vaccination program against brucellosis in China, and for an eradication or control campaign to be effective it is critical to understand both the epidemiology of the disease and the current attitudes and practices of the local herders (Díaz, 2013).
1.2 Background of Tibet
1.2.1 Location and administration of Tibet
Tibet is located in the western region of China, north-east of the Himalaya mountain range on the Qinghai-Tibetan plateau. It neighbours the Chinese provinces of Xinjiang,
Qinghai, Sichuan and Yunnan, as well as the countries of India, Burma, Bhutan and
Nepal (Figure 1.1). The whole border, including both international and with other
Chinese provinces, runs for more than 4,000 km (Lan et al., 2015). The average altitude of Tibet is more than 4,000 meters above sea level, and the region is characterised by rugged terrain, unique ecology and abundant natural resources. The Tibet autonomous region, a provincial region in China, contains five prefectural cities (Lhasa, Shigatse,
Chamdo, Nyingchi and Shannan) and two prefectures of Nagqu and Ngari. There are 74 counties in the region which contain 900 townships and more than 7000 villages (Tashi et al., 2005). There are 3.12 million permanent residents in Tibet with 2.38 million 3
(76.3%) living in rural areas (Duoji and Yang, 2014). Almost all residents in rural areas are ethnic Tibetans. However, there have been large numbers of migrants entering Tibet over the past 40 years, with the majority of these migrants settling in the urban areas
(Goldstein et al., 2003; Fischer, 2006).
Figure 1.1 Geographical location of Tibet
1.2.2 Climate of Tibet
The northwest part of Tibet is generally dry and cold, while the southeast part is relatively warm and humid. The main climatic characteristics of the region are prolonged sunlight with high levels of radiation and low, but variable, temperatures.
However, in summer the temperature can reach above 25℃ (Gerald et al., 2003). The average annual temperature in most regions is below 5℃. Annual precipitation varies between regions, from more than 1,000 mm in the east to less than 100 mm in the west 4
(Zuo et al., 2009). Due to the large size of Tibet the temperature varies dramatically between the north and south. In the south, the average temperature is 8℃ (lowest: -16℃ and highest: 16℃). In contrast, the temperature in the north is extremely low, with an average below 0℃ (Lan et al., 2015). The mean surface temperature on the Tibetan
Plateau has increased by an average of 0.3°C per decade over the past five decades, which is approximately three times the global average (Wang et al., 2014). The increase in temperature in the central and western regions has been considerably higher than that of the eastern region (Yang et al., 2007). Researchers have further predicted that the
Tibetan plateau will warm at a faster rate than the global mean (Christensen et al., 2007).
Precipitation has also increased by approximately 3.4 mm per decade over the past 50 years (Wang et al., 2014).
1.2.3 Livestock production system
The total output from the agriculture, livestock and fishery industries amounts to just under 20% of the Gross Domestic Product (GDP) of Tibet (Anonymous, 2013). Tibet is one of the largest pastoral regions in China. The total area covers 1.2 million km2, including approximately 203 million acres of natural grassland, 18 million acres of forest and 556,000 acres of arable land (Ciren, 2006; Lan, 2013).
There are four livestock-production systems adopted in Tibet: the pastoral production system; the agro-pastoral production system; the crop-based livestock production system; and the agro-forestry–pastoral mixed production system (Tashi et al., 2002). In the pastoral production system, predominantly located in northern Tibet, livestock are usually grazed at higher altitudes in warmer seasons and then moved to graze in valleys during the colder seasons. For the crop-based production system in central Tibet, 5
livestock are fed mostly crop residues, such as straw, during winter and spring, and are grazed in the upper valley and mountain areas during summer. An integrated system is adopted during autumn. The agro-pastoral production system is an amalgamation of the former two systems. In this system, livestock primarily graze on rangelands and are occasionally supplemented with crop residues. The agro-pastoral-forestry mixed production system is a blend of livestock, crop and forestry production and is mainly adopted in south-eastern Tibet (Tashi et al., 2005).
The grazing of livestock is an important aspect of global food supply (Kellems and
Church, 2002), and the process has evolved in Tibet over several thousand years
(Goldstein and Beall, 1991; Goldstein, 1992; Miller, 1999a). However, in order to sustain the industry the industry must reduce the degradation of rangelands caused by overgrazing and burrowing by pikas (Ochotona spp.). Grazing lands are divided into seasonal pastures and pastures are allocated to, and fenced by, herders (Sheehy et al.,
2006; Lan, 2013). Herders generally prefer grazing yaks, sheep and goats together to maximize the use of rangeland resources while minimizing the risk of livestock losses from extreme weather events (Miller, 2005). In the crop based production system, summer and autumn are ideal seasons for crop growth. Cropping systems are characterised by frequent irrigation, heavy tillage, high seeding rates and the use of fertilizer. The predominant crops are spring barley and winter wheat, followed by canola (rapeseed), winter barley, and minor fodder and vegetable crops (Paltridge et al.,
2009).
6
1.2.4 Livestock population
There is a large variety of livestock grazed in Tibet including yaks, cattle, zo
(yak×cattle crosses), horses, donkeys, sheep, goats and pigs. In 2014, there were a total of 19.5 million livestock in the region, including 6.4 million large animals (cattle, yaks and horses), 12.7 million small ruminants (sheep and goats) and 0.4 million pigs (Duoji and Yang, 2014).
Livestock products have played an important role in the Tibetan economy for centuries and provide products for both export and local consumption and processing (Tudeng and Huang, 2004). The yak (Bos grunniens) is one of the most important animals for
Tibetan herders (Wang, 2002), and was originally domesticated over 4,000 years ago
(Zhang, 2013a). The yak is integrated into the local culture, social life and religion of herders and communities (Gerald et al., 2003). The number of yaks in Tibet has been estimated at 4.7 million, which represents over 30% of the total in China. China has a total of 14 million yaks, which makes up 95% of the world’s population (Gerald et al.,
2003; Ciren, 2006). There are 12 recognised domestic breeds of yak in China, three of which are widespread in Tibet: Pali yak in Yadong County, Jiali (“Alpine”) yak in Jiali
County and Sibu yak in Maizhokunggar County. The Tibetan term for yak is ‘Nuo,’ which means wealth, and it is commonly referred to as “the treasure of the plateau” or
“the boat of the plateau” due to its ability to adapt to highland pastures more efficiently than other livestock (Yang, 2002). Yaks are prevalent in seven prefectures of Tibet, and are primarily raised under the pastoral production system (Ji et al., 2003).
7
1.3 Prevention and control of animal disease
1.3.1 Veterinary services in Tibet
Veterinary services in Tibet are controlled by the local government. The veterinary system in Tibet is based on the standard Chinese administrative divisions: province, prefecture (prefectural city), county, township and village. At the provincial level, there is one administrative agency (Tibet Bureau of Agriculture and Animal Husbandry -
TBAA) and one academic agency (Tibet Academy of Agriculture and Animal
Husbandry Science - TAAAS). The TBAA is responsible for overall livestock developmental activities in the region. Within the bureau, there are three main administrative agencies: Tibet Bureau of Veterinarians (TBV); Tibet Institute of Animal
Health Supervision/Tibet Institute of Veterinary Drugs Control (TIAHS/TIVDC); and the Tibet Centre for Animal Disease Control (TCADC). The TBV is exclusively an administrative agency. The TIAHS/TIVDC is a mixed agency that is responsible for inspecting livestock for disease and evaluating livestock products. It is also responsible for administration and law enforcement related to animal disease prevention and the supervision of veterinary drugs. The TCADC is responsible for disease surveillance and control. The three agencies operate at both the prefecture and county level, and are responsible for animal disease control, inspection and quarantine, veterinary drug administration and residue control. There are veterinary stations in townships and villages, and a large number of veterinarians are also based in the villages. In addition, there is a veterinary technical support agency, the Tibet Livestock Research Institute
(TLRI), which is part of TAAAS, and is primarily responsible for research into animal disease. The structure of veterinary services in Tibet is summarised in Figure 1.2. The total number of staff in the veterinary services in Tibet, including administrators, academic staff and veterinarians, is approximately 9,680. 8
Tibet Autonomous Region
Tibet Bureau of Agriculture Tibet Academy of Agriculture and Animal Husbandry and Animal Science
Tibet Bureau of Tibet Institute of Tibet Centre for Tibet Livestock
Veterinarians Animal Health Animal Disease Research Supervision Control institute
Prefecture Bureau Prefecture Department Prefecture Centre of Veterinarians of Animal Health for Animal Supervision Disease Control
County Bureau of County Department of County Centre for Veterinarians Animal Health Animal Disease
Supervision Control Supervision
Veterinary Station in Township
Figure 1.2 Organisational structure of the veterinary services (animal health) in Tibet
1.3.2 Regulations and Laws
China has passed a number of veterinary laws and has established a formal system for disease regulation. The Law of the People’s Republic of China on Animal Disease
Prevention was enacted on the 3rd July 1997, and then amended and passed by the legislature on 30th August 2007. This law promotes the prevention, control and
9
eradication of animal disease in China. It contains explicit rules regarding the reporting, notification and release of animal disease information, regulations pertaining to the control and eradication of disease, inspection of animals and animal products, diagnosis and treatment of animals and the administration of animal disease preventive measures.
Based on this law, supporting regulations have been formulated for the prevention, reporting, surveillance, quarantine, control and eradication of animal diseases (e.g.
Administrative Measures for Animal Disease Reporting, Lists of Category A, B and C
Animal Diseases, List of Zoonoses, and Regulations on Emergency Response to Major
Animal Diseases).
1.3.3 Prevention and control system
In China comprehensive measures for disease control, including compulsory vaccination, epidemiological surveys, quarantine, disinfection, movement controls, culling infected animals and surveillance, have been adopted and have significantly reduced the extent of some animal diseases (Cai, 2014). On January 14th 2009, the
Ministry of Agriculture (MOA) issued the Circular of the National Animal Disease
Surveillance Plan and the Epidemiologic Investigation Plan for Major Animal Diseases.
Five days later, it issued the Circular of Vaccination against Major Animal Diseases
(Bureau of Veterinary, 2009). Surveillance points were set up at the provincial, prefecture, county, township and village levels. In Tibet, the government established six national monitoring stations of animal diseases and 16 border (international and domestic) surveillance stations for animal diseases (Lan, 2013).
In order to improve animal health and accelerate the development of the local animal industry, Tibetan authorities also implemented several policies regarding the prevention 10
and control of animal diseases. These included policies to increase vaccinations, disease surveillance, epidemiological surveys and quarantine. These efforts at the national and provincial levels were effective at eradicating some infectious diseases, including rinderpest in 1967, bovine contagious pleuropneumonia in 1995 and glanders in 1999
(Lan, 2013).
Based on the epidemiological features of some animal diseases, the Tibetan authorities have formulated their own regulations. Compulsory vaccination against five animal diseases, FMD (Foot and mouth disease), HPAI (Highly pathogenic avian influenza),
PPR (Peste des petits ruminants), swine fever and PRRS (Porcine reproductive and respiratory syndrome), has been mandated for livestock across Tibet. Each year, 100% of at-risk animals are required to be vaccinated against these diseases. Surveillance and epidemiological surveys are undertaken annually in Tibet, with more than 10 animal diseases being monitored (FMD, HP-PRRS, swine fever, Newcastle disease, brucellosis, bovine tuberculosis, rabies, bovine spongiform encephalopathy (BSE), bovine contagious pleuropneumonia, PPR, Africa Swine fever, glanders and scrapie).
The reporting system for animal diseases follows the administrative divisional structure.
Farmers initially report occurrences of a disease (infectious or non-infectious) within their herds to the county’s veterinary department(s). If only individual livestock are affected, local veterinarians are tasked with the treatment of the affected animal. If a major outbreak of a disease occurs (such as FMD, PPR or HPAI), it is first reported to the TBAA by the County and Prefecture veterinary departments, then to the MOA. The
TBAA initiates emergency responses and carries out slaughter of affected animals and disinfection of the relevant premises. Expenses are covered by the government (Lan, 11
2013). Simultaneously, samples of sera, fluid and tissue are collected and dispatched to the relevant national reference laboratories for pathogen identification.
1.3.4 Brucellosis control program
In the early 1990s a national brucellosis eradication program was implemented in China.
Twenty-one human brucellosis surveillance sites and 10 animal surveillance sites were built throughout China to monitor potential outbreaks (Jiang et al., 2012). The National
Brucellosis Intervention Pilot County Survey was established in 2005 in order to monitor and prevent both animal and human brucellosis (Liu et al., 2008).
The previous program of compulsory vaccinations of all livestock against brucellosis in
Tibet was halted in 1985 after the disease was deemed to be under control. According to the regulations of the national MOA, the disease is currently categorised as one that requires epidemiological surveys and surveillance due to its impact on livestock and public health (Bureau of Veterinary, 2009). Epidemiological surveys are conducted in two counties of every prefecture during each annual survey. Dairy farms and two villages that contain more than 20 households in each county are selected randomly for sampling. Surveillance is conducted annually in high-risk areas, and more than 1,000 animals are sampled (Tibet Bureau of Agriculture and Animal Husbandry, 2014). These samples are tested in two-stages. Firstly, screening tests, such as the RBT (Rose Bengal
Test), IELISA or FPA (fluorescence polarization assay) are used, and positive results are evaluated with a confirmatory SAT (Serum agglutination test), CFT (Complement fixation test) or CELISA test. Annual results of these tests are submitted to the MOA.
Although information is collected regarding disease prevalence, there is no investigation of risk factors for the disease. The Tibetan government implements compulsory culling 12
of infected livestock and provides monetary compensation to farmers, however compensation is generally significantly lower than the current market value (Bureau of
Veterinary, 2009).
1.4 Aims and Objectives of this study
There is little information available on the epidemiology of brucellosis in livestock in
Tibet, particularly regarding risk factors associated with its prevalence and transmission, animal movement patterns and the impact of the disease on livestock productivity. The primary aim of the study outlined in this thesis was to generate baseline epidemiological information on the prevalence of the disease in yaks in Tibet in order to strengthen and recommend changes, if required, to the existing control program.
Specific objectives of the study described in this thesis included:
To describe, using historical data, the spatial and temporal distribution of
brucellosis in yaks in Tibet
To determine the seroprevalence of brucellosis in yaks in Tibet
To identify risk factors for the disease in the agro-pastoral and pastoral regions
To determine the level of knowledge, attitudes and practices of households in
Tibet about brucellosis in yaks.
To quantify the economic impact of the disease on local herders
To provide recommendations and directions for future research for strengthening
and improving the current control program based on the findings from this study.
13
CHAPTER TWO
Literature review
2.1 Introduction
Brucellosis, caused by a Gram-negative bacteria Brucella spp., has been recognised as a zoonotic disease for centuries (Godfroid, 2002; Seleem et al., 2010). The origin of the disease can be traced back to the 5th plague of Egypt in 1600 BC (Seleem et al., 2010).
The disease has had several names including Malta fever, Mediterranean fever, undulant fever, Bang’s disease and contagious abortion (Romich, 2008).
The agent Brucella melitensis was initially isolated from the spleen of a British soldier who died from a febrile illness in Malta in 1887 by the English army surgeon David
Bruce, after whom the organism was subsequently named. In 1905 when this bacterium was isolated from the milk of a goat by Themistocles Zammit, it was recognised as a zoonotic disease (Seleem et al., 2010; Yilma et al., 2016). Brucella abortus was first isolated and identified by a Danish veterinarian, Dr. Fredrick Bang in 1897, and consequently the disease was initially known as Bang’s disease (Romich, 2008; Yilma et al., 2016). In early 1990, Brucella organisms were also discovered in marine mammals, which resulted in a change in the previous belief that the genus was restricted to terrestrial animals (Sriranganathan et al., 2008).
2.2 Aetiology
The genus Brucella is within the family Brucellaceae in the order Rhizobiales, class
Alphaproteobacteria (Godfroid et al., 2011). Brucella spp. are facultative intracellular
14
bacteria, being small Gram negative cocci, coccobacilli, or short rods (0.6-1.5µm long by 0.5-0.8 µm wide). Flagella, endospores, and capsules are absent, and thus they are non-motile. They can infect a wide range of animals, including terrestrial and marine mammals (Young, 1995; Corbel, 1997; Seleem et al., 2010; Dubal et al., 2013).
The classification of Brucellae is mainly based on their host preference and differences in pathogenicity (Alton et al., 1988). Ten Brucella species have been recognised including the six classical species of B. melitensis with Biovars 1-3 (sheep and goats),
B. abortus with Biovars 1-6, 7 and 9 (cattle and bison), B. suis with Biovars 1-5 (pigs),
B. canis (dogs), B. ovis (sheep), and B. neotomae (desert wood rat). The remaining species include B. ceti and B. pinnipedialis (infecting preferentially cetaceans and pinnipeds, respectively) (Foster et al., 2007), B. microti infecting the common vole
(Microtus arvalis) (Scholz et al., 2008), and B. inopinata which was first isolated from an infected breast implant in a woman (Scholz et al., 2010). Of these species, B. abortus, B. melitensis, B. suis, B. canis (Mirnejad et al., 2017) and B. inopinata can infect humans, although the first three species are the main pathogenic species for humans (Baldwin and Goenka, 2006).
The cell envelope of Brucella has an approximately 9nm thick outer layer of lipopolysaccharide (LPS) protein (Young and Corbel, 1989). This LPS is an important virulence factor for the genus (Cardoso et al., 2006). There are two types of phenotypes termed ʻsmoothʼ and ʻroughʼ which are dependent upon the LPS structure (Whatmore,
2009). The ʻsmoothʼ phenotype contains complete LPS with lipid A and an O-side chain polysaccharide. In contrast the ʻroughʼ strain does not contain the O-side chains
(Whatmore, 2009). Brucella abortus, B. suis and B. melitensis carry ʻsmoothʼ LPS 15
which is involved in the virulence of these bacteria (Cardoso et al., 2006).
2.3 Clinical signs, Pathology and Pathogenesis
2.3.1 Clinical signs
The most common clinical manifestation of brucellosis in livestock is reproductive loss due to abortion, birth of weak offspring or infertility (Olsen et al., 2004; Whatmore,
2009). The main species infecting cattle is B. abortus, although they can also be infected with B. suis or B. melitensis when they share pasture or facilities with other livestock such as pigs, goats or sheep (Seleem et al., 2010). Abortions in cattle usually occur at about 5 to 7 months of gestation (Geering et al., 1995; Ducrotoy et al., 2017).
Occasionally infected calves may die soon after birth, while at other times the disease can be more insidious. Secondary bacterial metritis and a retained placenta may also occur resulting in permanent infertility (Campero et al., 1990). Brucella spp. can also invade and localise in the mammary tissue resulting in mastitis and a subsequent reduction in milk production (Emminger and Schalm, 1943; Herrera et al., 2008). In bulls, the clinical signs may include fever, anorexia and depression although the infection is often inapparent (Campero et al., 1990). Acute or chronic unilateral or bilateral orchitis, epididymitis and seminal vesiculitis also can occur occasionally
(Rankin, 1965; McCaughey and Purcell, 1973). Infected bulls may become permanently infertile as a result of the chronic orchitis and associated fibrosis of the testicular parenchyma (Campero et al., 1990). In chronically infected herds, unilateral or bilateral hygromas, particularly in the carpal joints, may occur in some animals (Geering et al.,
1995).
In humans, brucellosis is generally not fatal, although it may lead to chronic infection if 16
not treated (Whatmore, 2009). The incubation period can vary from weeks to months
(Seleem et al., 2010). Brucellosis in humans is a systemic infectious disease due to invasion of various organ systems, leading to a range of clinical manifestations
(Godfroid et al., 2011). The disease, which also is known as undulant fever, has clinical signs of fever, anorexia, polyarthritis, meningitis, pneumonia and endocarditis (Saurent and Vilissova, 2002). Gorvel (2008) reported that the disease may also cause epididymo-orchitis, spondylitis, neurobrucellosis, liver abscess formation and endocarditis and can be potentially fatal.
2.3.2 Pathology
Brucella abortus has tropism for the uterus, particularly during the late gestational period because of the high concentrations of erythritol and steroid hormones in the organ at this time (Carvalho Neta et al., 2010). Erythritol can be metabolised by the organism and hence its role in the infectious process (Samartino and Enright, 1996). The bacterium primarily invades erythrophagocytic trophoblastic cells located at the base of the chorionic villi of ruminants (Anderson et al., 1986; Santos et al., 1996), resulting in an infiltration of inflammatory cells, trophoblastic necrosis, vasculitis, and ulceration of the allantochorion, consequently leading to abortion (Anderson et al., 1986). The chorion is affected to a differing extent and large parts may appear normal. Geering et al. (1995) stated that a sticky, odourless brown exudate covers the affected cotyledons.
In addition, portions of the intercotelydonary placenta are thickened, oedematous, yellowish-grey in colour and sometimes may contain surface exudates.
In some aborted foetuses, different degrees of subcutaneous oedema and blood-stained fluid in the thoracic and abdominal cavities may occur and the abomasal contents may 17
be turbid, light yellow and flaky. The liver also becomes enlarged and discoloured changing to an orange-brown colour. Fibrous pleuritis and grey-white foci pneumonia may occur; however many aborted foetuses will not show any gross pathological changes (Geering et al., 1995). After abortion there may be a mild to severe endometritis which can become chronic (Geering et al., 1995).
2.3.3 Pathogenesis
After entering the body, Brucella spp. are transported via the lymphatic and blood circulation systems to the liver, spleen, kidneys and bone marrow where they may invade both professional and non-professional phagocytic cells such as epithelial cells, placental trophoblasts, dendritic cells and macrophages (Anderson et al., 1986; Gorvel,
2008; Godfroid et al., 2011; Theron and Thantsha, 2014).
Brucella are able to survive within the host cells following phagocytosis by adapting to the intracellular conditions which results in compromising of the host immune response
(Kohler et al., 2002; Roop et al., 2009). The organism can inhibit phagosome-lysosome fusion and resist the acidified intraphagosomal environment (Pizarro-Cerdá et al., 1998;
Porte et al., 1999; Wang et al., 2001). When initially in contact with a host cell,
Brucella are internalised by activation of small GTPases of the Rho subfamily and by a moderate recruitment of actin filaments. When invading a cell, they initially localize in phagosomes avoiding fusion with late endosomes and lysosomes (Gorvel and Moreno,
2002). Then, the organism redirects its trafficking to autophagosomes and lastly reaches the endoplasmic reticulum, where it replicates extensively (Gorvel and Moreno, 2002).
Only a few ingested Brucella survive, with most being destroyed within the phagolysosomes of the host macrophages. Furthermore, intracellular trafficking and 18
survival of B. abortus are not identical in all cell types (Carvalho Neta et al., 2010). For example, neutrophils limit the intracellular growth of B. abortus (Jiang and Baldwin,
1993), while the organism survives well in other phagocytes such as macrophages
(Gorvel and Moreno, 2002).
Both human activities and ecological factors affect and induce changes in the virulence of the organism (Moreno, 2014). Compared with other pathogenic bacteria, Brucella spp. do not display classical virulence factors, such as exotoxins, cytolysins, capsules, fimbria, plasmids, lysogenic phages, resistant forms or antigenic variation (Moreno and
Moriyon, 2002). Although, until recently, little had been known about the virulence mechanisms of Brucella (Guzman-Verri et al., 2001), some virulence factors do help
Brucella invade and survive in macrophages. These include lipopolysaccharides
(LPS)/O-polysaccharides, Type IV secretion system virB, Cyclic β-1,2-glucans (CβGs),
Superoxide dismutase, Catalase, Base excision repair (BER), Urease, BvrS/BvrR, Alkyl hydroperoxide reductase (ahp C&D), Cytochrome oxidase, Nitric oxide reductase (norD) and Brucella virulence factor A (BvfA) (Seleem et al., 2008).
2.4 Epidemiology
2.4.1 Transmission
Brucellosis is transmitted either by direct or indirect contact with an infected animal or a contaminated environment, with the movement of infected animals being a key feature of disease transmission (Geering et al., 1995; Romich, 2008). The most common route of infection in cattle is via the gastrointestinal tract (Payne, 1959; Crawford et al.,
1990), although other routes, such as trans-conjunctival or inhalation, may lead to infection (Crawford et al., 1990; Ko and Splitter, 2003). The most important sources of 19
infection are aborted foetuses, foetal membranes and uterine secretions expelled after abortion or parturition (Samartino and Enright, 1993). Other routes of infection include vertical transmission to calves in utero and via milk and colostrum, and venereal transmission through both natural mating and artificial insemination (Rankin, 1965; Ray et al., 1988). Geering et al. (1995) observed that after cattle calved or aborted, the number of organisms decreased. In addition, wildlife can become reservoirs of brucellosis, making control of the disease more complicated and costly (Olsen, 2010).
Transmission of the disease to humans can also be direct or indirect. Direct transmission generally is acquired occupationally through handing infected animals or tissues or discharges from these animals. However, indirect transmission remains the highest risk, mainly through the consumption of unpasteurised milk or dairy products (Godfroid et al., 2005). Consequently human infection is predominantly food-borne or occupationally acquired.
2.4.2 Survival and resistance
Brucella do have the capacity to survive in the environment with the ability to potentially survive in a wide temperature range of -40℃ to 37℃ (Rowe, 2014; Theron and Thantsha, 2014). The organism may survive for up to eight months in an aborted foetus which is in the shade and for 3 to 4 months in faeces (Geering et al., 1995). At low temperatures Brucella can survive in soil for up to 10 weeks and in liquid manure for up to 2.5 years. Under normal storage conditions, the organism may survive in water for 60 days and in yogurt for less than a week (Falenski et al., 2011). The organism can survive for many years in frozen carcasses and may retain infectivity for years if dried, in the presence of excess protein and protected from sunlight (Young and Corbel, 1989). 20
Brucella are fairly sensitive to ionizing radiation, and also are sensitive to most commonly used disinfectants at normally recommended concentrations (Young and
Corbel, 1989).
2.4.3 Epidemiology of brucellosis
2.4.3.1 Epidemiology of animal brucellosis
Brucellosis is endemic in the Mediterranean basin, Middle East, Central Asia, China,
India, and many areas of Africa, Mexico and Latin America (Boschiroli et al., 2001;
Sanjuan-Jimenez et al., 2017). Although infection with Brucella in cattle is widespread globally, it has been successfully eradicated from some countries in northern and central
Europe, Australia, Canada, Japan and New Zealand (OIE, 2016).
In many developing counties, the seroprevalence in cattle varies from 2 to 15%
(Ghanem et al., 2009; Mekonnen et al., 2010; Lang et al., 2011; Sanogo et al., 2012;
Mamisashvili et al., 2013; Maurice et al., 2013). Major factors influencing the prevalence include stocking density and herd size, age, sex, agro-ecological zones, contact with wildlife and management and husbandry practices adopted (Omer et al.,
2000b; Muma et al., 2007b; Ghanem et al., 2009; Matope et al., 2010; Mekonnen et al.,
2010; Olsen, 2010; Sanogo et al., 2012; Borba et al., 2013; Jackson et al., 2014).
The yak, which belongs to the same genus as other domestic cattle, is also susceptible to brucellosis (Gerald et al., 2003). Epidemics of the disease have occurred in yak rearing areas of western China, India and Nepal with seroprevalences of 21.1% in India and 22% in Nepal being reported (Bandyopadhyay et al., 2009; Jackson et al., 2014). In China, the seroprevalence in yaks has ranged from 4.16 to 13.4% in Qinghai, Tibet, Xinjiang 21
and Sichuan provinces (Dilixiati. et al., 2001; Li et al., 2008; Lang et al., 2011; Gao et al., 2013). To date there have only been a limited number of studies investigating risk factors for the disease in yaks. A study conducted in Nepal highlighted the role of sex and age in the disease (Jackson et al., 2014). However, in China there have been no studies published on risk factors for brucellosis in yaks.
Brucellosis in cattle is predominantly caused by B. abortus. Cross-infection with B. melitensis can occur when herds of cattle are grazed or mixed with sheep and/or goat flocks (Verger et al., 1989). Brucella abortus biovars 1, 4 and 6 and B. melitensis biovars 1 and 3 were isolated from yaks in Tibet in the 1980s (Pan, 1992a), and Dilixiati. et al. (2001) isolated B. abortus biovar 3 from yaks in Xinjiang province of China.
Brucellosis in wildlife is associated with the disease in livestock and humans, and it is likely that interaction between wildlife and livestock is an important driver for disease transmission (Godfroid et al., 2013b). It is widely believed that the disease is introduced from domestic animals into the wildlife population where control can become an intractable situation (Ducrotoy et al., 2017). Some studies identified that bison and elk were reservoirs of B. abortus, while feral pigs can be infected with B. suis (Olsen, 2010;
Franco-Paredes et al., 2017), and hence the persistence of brucellosis in wildlife and feral animals can pose a significant risk for domestic livestock, as well as humans.
2.4.3.2 Epidemiology of human brucellosis
Human brucellosis is a common zoonotic infection worldwide and still provides challenges through its pathogenic mechanisms, progression and treatment response for scientists and clinicians (Pappas et al., 2006; Franco et al., 2007). Brucella melitensis is 22
the most virulent species for humans, although B. suis, B. abortus and B. canis can also infect people (Boschiroli et al., 2001; Rostami et al., 2016). Infection in humans is most frequently associated with consumption of contaminated foods, contact with infected animals or through laboratory exposure when working with diagnostic samples/specimens (Corbel, 1997; Wilson and Chosewood, 2009). Due to the fact that brucellosis in humans is nearly always associated with brucellosis in animals (Jennings et al., 2007; Swai and Schoonman, 2009), some occupations, such as veterinarians, herders, slaughterers and milkers, have a higher incidence of infection (Deqiu et al.,
2002).
Often the disease is misdiagnosed in humans, particularly in developing countries (Paul et al., 1995), and this has been linked to a poor general knowledge of zoonotic diseases in these locations (John et al., 2008). Although human brucellosis is rarely lethal and the disease is not reportedly transmitted from person to person, it is a debilitating disease
(Godfroid et al., 2011).
Countries in the Middle East, Mediterranean basin, Arabian Peninsula, the Indian subcontinent, Mexico, parts of central and south America have a higher incidence of human brucellosis, predominantly due to the endemicity of the disease in livestock in these regions (Pappas et al., 2006; Mirnejad et al., 2012; Rostami et al., 2015).
Consumption of raw milk is a major route of exposure in developing countries (Makita et al., 2008; Swai and Schoonman, 2009). Disease is also sporadically reported in individuals in several industrialised countries, even though animal brucellosis has been controlled (Al Dahouk et al., 2005; Al Dahouk et al., 2007).
23
2.4.3.3 Epidemiology of brucellosis in China
Brucellosis was first reported in humans in China in 1905 in Shanghai (Deqiu et al.,
2002) and the epidemiological characteristics of the disease have been investigated since the 1950’s. Both human and animal brucellosis were severe prior to 1980.
Subsequently, during the 1980s, the disease in both humans and animals reduced.
However, the level of disease in humans again rose during the 1990’s, although surprisingly brucellosis in animals did not reportedly change (Deqiu et al., 2002). The number of infected people rose from 6,448 in 2003 to 38,151 in 2011. The reasons for this were believed to be associated with frequent commercial movements to markets and a lack of disease quarantine (Liu et al., 2014).
The disease in the 1990’s in China was mainly clustered in the semi-agricultural, agricultural and pastoral provinces of Shanxi, Hebei, Henan, Shandong, Inner
Mongolia, Liaoning, Xinjiang, Tibet and Jilin. Brucella melitensis, B. abortus and B. suis have been responsible for most infections in these regions (Deqiu et al., 2002).
After the start of the 21st century, the endemic areas of human brucellosis in China were relatively extensive, mainly in the areas of Hulunbuir and Xilingule of the Inner
Mongolia autonomous region, Harbin region of Heilongjiang province and Songyuan area of Jilin province, although the disease gradually spread to the inland provinces of
Hebei, Shanxi, Henan, Guangdong and Fujian (Cao et al., 2013; Chen et al., 2013;
Zhong et al., 2013; Zhang et al., 2014).
24
2.5 Diagnosis
2.5.1 Identification of the agent
2.5.1.1 Staining
Brucella organisms stain red using Stamp’s modification of the Ziehl-Neelsen method
(OIE, 2009). Another staining technique, based on a fluorochrome or peroxidase- labelled antibody conjugate, can also be used (OIE, 2009). However, these latter techniques have lower sensitivity in milk and dairy products, where small numbers of
Brucella are likely to exist. There is also difficulty in distinguishing Brucella from other organisms that cause abortion such as Chlamydophila abortus or Coxiella burnetii on a positive Stamp’s method. Consequently samples need confirming by culture.
2.5.1.2 Culture
The culture of Brucella is generally regarded as the definitive diagnostic method for diagnosing brucellosis (Theron and Thantsha, 2014). Samples for culturing are primarily from aborted foetuses (stomach contents, spleen and lung), foetal membranes, vaginal secretions, milk, semen and arthritis or hygromal fluids. In addition, tissues from the reticulo-endothelial system (i.e. head, mammary and genital lymph nodes and spleen), the late pregnant or early post-parturient uterus and the udder are also frequently cultured (OIE, 2009).
Isolation and culture of Brucella generally is undertaken on basal media with solid media usually being more successful than liquid media (OIE, 2009). For potentially contaminated samples, selective media, such as Farrell’s medium, should be employed for primary isolation (Theron and Thantsha, 2014). Due to the lower concentration of
25
Brucella in milk, colostrum and some tissue samples, enrichment is advisable. For milk this involves centrifugation and culture from the pellet or cream (OIE, 2009).
2.5.1.3 Identification
Identification to the genus level can be confirmed on: morphology after staining; growth characteristics; results of testing for urease, oxidase and catalase; and the slide agglutination test with an anti-Brucella polyclonal serum. Speciation of the genus and identification of biovars is usually done in specialised or reference laboratories and the tests used include: CO2 requirement for growth; production of H2S; phage lysis; dye sensitivity (fuchsin and thionin); agglutination with A, M or R specific antisera; and in some cases the oxidative metabolic method (Poester et al., 2010).
2.5.1.4 Nucleic acid recognition methods
The genome of Brucella comprises two circular chromosomes of chromosome
Ⅰ(2.11Mb) and chromosome Ⅱ(1.18Mb) (Theron and Thantsha, 2014). Despite the high homology of DNA within the genus Brucella, molecular methods such as PCR- based tests, PCR restriction fragment length polymorphism (RFLP) and Southern blot have been used to differentiate between Brucella species and some of their biovars
(Corbel, 1997; Dahouk et al., 2005; OIE, 2009). The first multiplex PCR (AMOS) was developed for differentiating strains of B. abortus biovars 1, 2 and 4, B. melitensis, B. ovis, and B. suis biovar 1 (Bricker and Halling, 1994). The AMOS PCR was further improved to identify B. abortus biovars 5, 6 and 9 and genotype 3b of biovar 3 through adding a specific primer (Ocampo-Sosa et al., 2005). Subsequently a 4-primer-set multiplex PCR method was developed to discriminate B. suis biovars 1, 2 and 3
(Ferrao-Beck et al., 2006). In 2006, a new multiplex PCR assay was proposed for 26
differentiating Brucella species. However, the approach was not capable of differentiating between B. pinnipedialis and B. ceti and the various Brucella biovars
(Garcia-Yoldi et al., 2006). A novel multiplex PCR assay was subsequently developed to differentiate between all Brucella species currently recognised (Huber et al., 2009).
In addition, single nucleotide polymorphism (SNP) discrimination by either primer extension or real time PCR has recently been applied for identification as an alternative approach (Scott et al., 2007; Gopaul et al., 2008). Several specific sequences, such as the 16 S-23S gene, the IS711 insertion sequence or the bcsp31 gene encoding a 31-kDa protein, have been applied successfully to identify individual species and biovars
(Ouahrani‐Bettache et al., 1996; Baddour and Alkhalifa, 2008). However, PCR techniques have shown a lower sensitivity than culture for identification purposes
(Bricker, 2002; Leyla et al., 2003; Marianelli et al., 2008).
2.5.2 Serological tests
2.5.2.1 Common in-use serological tests
A range of serological tests have been developed to diagnose infection with Brucella spp. and these offer the advantages of being inexpensive, simple and rapid, although their accuracy is not 100% (Nielsen, 2002). Furthermore, a single serological test is inappropriate for screening animals in an epidemiological study (OIE, 2009). The O- polysaccharide (OPS) of B. abortus lipopolysaccharide is an important component that is incorporated into many serological tests (Alton et al., 1988). The OPS can induce a humoral response on infection resulting in an initial production of IgM followed by
IgG1 and IgG2/IgA (Beh, 1974; Allan et al., 1976; Nielsen et al., 1984). IgG1 is the major isotype used in serological assays as IgM has the potential for cross-reaction with other agents and the amounts of IgG2 and IgA produced are low (Allan et al., 1976; 27
Lamb et al., 1979; Nielsen et al., 1984; Butler et al., 1986). The three main species (B. melitensis, B. abortus and B. suis) with OPS are tested through using a whole cell antigen or smooth lipopolysaccharide (SLPS) prepared by chemical extraction, while B. ovis and B. canis are diagnosed by using rough lipopolysaccharide (RLPS) or protein antigens (Blasco, 1990).
The serological tests used to diagnose infection (Nielsen, 2002) include: (1) agglutination tests such as the standard tube agglutination test (SAT), acidified antigen including the rose-bengal/card test (RBT) and the buffered antigen plate agglutination test (BPAT); high salt, reducing agents including 2 mercaptoethanol (2ME) and dithiothreitol (DTT), rivanol (RIV), heat, antiglobulin tests, milk ring test (MRT); (2) complement fixation tests: warm (CFT); cold, indirect haemolysis test (IHLT), haemolysis in gel (HIG); (3) precipitation tests: agar gel immunodiffusion test (AGID) and single radial immunodiffusion test (SRD); (4) primary binding assays: radioimmunoassay (RIA), particle concentration fluorescence immunoassay (PCFIA), indirect enzyme immunoassay (IELISA), competitive enzyme immunoassay (CELISA), and the fluorescence polarization assay (FPA) (Nielsen, 2002). Of these, both the
CELISA and FPA can differentiate between animals vaccinated with Strain 19 vaccine
(S19) and animals infected naturally with B. abortus (Nielsen et al., 1989; MacMillan et al., 1990; Nielsen et al., 1995; Nielsen et al., 1996; Gall and Nielsen, 2004).
For detection of the disease in yaks, the RBT and ELISAs developed for cattle have been widely applied (Dilixiati. et al., 2001; Bandyopadhyay et al., 2009; Gao et al.,
2013), although the tests have not been validated in yaks.
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2.5.2.2 Buffered Brucella antigen tests
Both the RBT (Nicoletti, 1967; Morgan et al., 1969; Davis, 1971) and the BPAT
(Angus and Barton, 1984) are two commonly used methods using acidified antigen. The
RBT is a simple agglutination test using B. abortus S99 or S1119.3 cells stained with
Rose Bengal and is a “presumptive test” (Ducrotoy et al., 2014) while the BPAT uses B. abortus S1119.3 whole cells stained with crystal violet and brilliant green dyes (Nielsen,
2002). Antigens of both tests are used at a low pH of 3.65 which prevents some agglutination by IgM and encourages agglutination by IgG1 (Corbel, 1972, 1973; Allan et al., 1976). These tests are used widely for screening animals; however false-positive results can occur, particularly because of S19 vaccination or because of cross-reactions with antibodies induced by other agents. Consequently positive reactions should be confirmed with other tests and by undertaking a thorough epidemiological investigation
(OIE, 2009).
2.5.2.3 Reducing agents
Both DTT and 2ME are reducing agents that have been used for the serological diagnosis of brucellosis (Klein and Behan, 1981). The function of these reagents reduces disulfide bridges of IgM leading to the presence of monomeric molecules, and thereby reducing the ability to agglutinate. The reducing agents are added to serum as a diluent. For the detection of brucellosis, reactions at a 1:25 serum dilution are considered positive (Poester et al., 2010).
2.5.2.4 Milk ring test
The MRT is an adaptation of the agglutination test for testing for antibodies in milk. A difference between this test and other tests is that haematoxylin stained Brucella cells 29
are added to whole milk (Hunter and Allan, 1972; Huber and Nicoletti, 1986). If immunoglobulins are present, a portion will attach to fat globules in the milk via the Fc portion of the molecule. Agglutination will then take place, and thus a purple band will appear at the top of the milk. Otherwise, the fat band will remain buff coloured (Nielsen,
2002; Poester et al., 2010). Although the test is a relatively insensitive test and is subject to false positive results from milk derived from animals with mastitis, colostrum or milk collected late in the lactation cycle, it is a useful and inexpensive screening test
(OIE, 2009; Poester et al., 2010).
2.5.2.5 Complement fixation test (CFT)
In the CFT, diluted sera, antigen (usually whole cells) and a pretitrated amount of complement (usually guinea pig serum) are added together. Then a pretitrated amount of sheep erythrocytes coated with rabbit antibody is added. If the serum contains antibody, complement is activated and doesn’t react with the secondary immune complex of sheep erythrocytes and rabbit antibody, thus lysis of the erythrocytes will not take place. If antibody is absent, complement will lyse the erythrocytes, releasing haemoglobin which is assessed visually or by using a spectrophotometer (Nielsen, 2002;
Poester et al., 2010). Despite the fact that the CFT is a complex procedure, it is a valuable confirmatory test, although positive results do need further confirmation.
2.5.2.6 Indirect ELISA
Different types of I-ELISAs have different antigen preparations, antiglobulin-enzyme conjugates and substrate/chromogens. Three main antigens of whole cell, smooth lipopolysaccharide (sLPS) or the O-polysaccharide (OPS) have been validated in extensive field trials and used widely (OIE, 2009). However other antigens, including 30
RLPS and protein antigens, have also been used (Diaz et al., 1979; Rahaley et al., 1983;
Marin et al., 1989; Limet et al., 1993; Baldi et al., 1994; Robles et al., 2009). The I-
ELISAs generally have a very high sensitivity which should be equal to or greater than that of the RBT/BPAT (OIE, 2009; Poester et al., 2010).
2.5.2.7 Competitive enzyme immunoassays (or blocking ELISA)
In order to overcome some issues arising from residual B. abortus S19 vaccinal antibody and from cross-reacting antibody, the competitive enzyme immunoassay was developed (Poester et al., 2010). These assays are mainly based on the rationale that vaccine induced antibody has lower affinity because of shorter exposure to the antigen, while antigen from field-infection results in increased antibody affinity due to a longer period of exposure (Nielsen et al., 1989; MacMillan et al., 1990). In the assays, antigen is immobilised, a competing antibody which is specific for OPS is added at a predetermined dilution, followed by diluted test serum and by a separate detection system in some cases (Poester et al., 2010). Monoclonal antibodies (MAbs) that should be specific for a common epitope of the OPS molecule have been selected as competing antibodies due to their inherent supply and uniformity advantage (Nielsen et al., 1995;
Biancifiori et al., 2000; Silva et al., 2000; Poester et al., 2010). Two types of the immunoassays are used in brucellosis serology. The particle concentration fluorescent immunoassay is commonly used in the USA (Nicoletti and Tanya, 1993). Another type, which has been extensively adopted, involves using SLPS passively immobilised on the wall of 96-well polystyrene plates (Poester et al., 2010). The competitive enzyme immunoassay has high specificity through selecting a MAb that has higher affinity than cross-reacting antibody, but is slightly less sensitive than the indirect enzyme
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immunoassay (OIE, 2009). It is used for confirming the diagnosis of brucellosis in most mammalian species (Poester et al., 2010).
2.5.2.8 Fluorescence polarization assay (FPA)
The FPA is a homogeneous assay and can be performed in glass tubes or a 96-well plate format (Poester et al., 2010). It is very rapid and relatively inexpensive (Nielsen, 2002), and has been validated for various species, including cattle and pigs, and has been shown to have a high sensitivity. The mechanism of the assay is simple. The molecular size is related closely with the rate of rotation of a molecule in solution. For the diagnosis of brucellosis, small molecular weight subunits of OPS are labelled with a fluorochrome and used as the antigen (Poester et al., 2010). When samples, such as serum, blood or milk are tested, the rate of rotation of the labelled antigen will be reduced if antibody to the OPS is present, and the amount of antibody is proportional to this rate of reduction (Poester et al., 2010).
2.6 Treatment, prevention and control of brucellosis
Brucellosis is a reportable zoonosis in most countries and can result in significant economic losses to producers and the community as a whole. Due to its pathogenicity and intracellular niche (Kohler et al., 2002), treatment failures and relapse rates are high
(Seleem et al., 2010). Although the application of vaccines, such as S19 and RB51 in cattle and Rev1 in sheep and goats, has played an important role in controlling brucellosis in livestock, vaccination programs alone will not result in the eradication of the disease as immunity induced by vaccines are not absolute and can be overcome by high levels of exposure to infection (Seleem et al., 2010). Therefore it is important to
32
ensure good measures of husbandry and management practices and improved biosecurity are adopted in combination with a vaccination program.
2.6.1 Treatment
Brucella are intracellular bacteria, so antibiotics often cannot reach them adequately.
However, suitable treatments are possible, particularly in humans. The aims of treatment are to reduce clinical signs and symptoms, the duration of disease, complications, and to prevent relapses. In order to treat disease, antibiotic combinations have been adopted; however the reported efficacy and optimum doses of combinations has varied between researchers (Shasha et al., 1994).
In humans Glynn and Lynn (2008) recommended an oral treatment regimen of doxycycline and rifampin; however others have suggested that a combination of doxycycline with streptomycin is a better option with less side effects and this combination has been regarded by some to be the gold-standard treatment (Ersoy et al.,
2005; Alp et al., 2006; Falagas and Bliziotis, 2006). The disadvantage with this regimen is that the streptomycin must be administered parenterally. An alternative suggestion has been doxycycline with parenterally administered gentamicin (Glynn and Lynn,
2008).
In nonhuman animals, treatment is rarely suggested, with most infected animals being destroyed or slaughtered. If treatment is considered, a combination of oxytetracycline and streptomycin has been shown to be relatively successful (Nicoletti et al., 1985).
Alternative regimens evaluated have included oral administration of enrofloxacin and
33
combinations of orally or parenterally administered gentamicin, ciprofloxacin and doxycycline (Wanke, 2004; Wanke et al., 2006).
2.6.2 Prevention
Due to the serious economic consequences of brucellosis in livestock, vaccination has been extensively adopted as a preventive measure and has been demonstrated as a cost- effective approach (Nicoletti, 1990; Ducrotoy and Bardosh, 2017). Vaccines used have mainly been S19 and RB51 in cattle and Rev1 in sheep and goats (Godfroid et al.,
2011). Although these vaccines have played a key role in the eradication of the disease from some counties, there are disadvantages with their application (Godfroid et al.,
2011). Killed vaccines have been shown to be inferior to live attenuated vaccines
(Godfroid et al., 2011). Attenuated vaccines are more efficient than subunit or DNA vaccines due to the fact that they contain all the immunogenic components that can be involved in protection (Ko and Splitter, 2003), although subunit vaccines are safe and adverse reactions are rare (Yin et al., 2016). So far, no effective vaccines are available or licensed for pigs, wildlife and humans (Godfroid et al., 2011; Yang et al., 2013b).
2.6.2.1 Main vaccines used in the past
2.6.2.1.1 B. abortus strain 19 and B. melitensis Rev 1
Of the live attenuated vaccines, B. abortus S19 and B. melitensis Rev 1 are considered to be the two best vaccines developed. Both vaccines carry a S-LPS with an OPS and are able to induce effective levels of protection in cattle and goats and sheep, respectively. However, they can’t induce full protection against infection with virulent strains (Nielsen and Duncan, 1990). Again, they may cause abortion in pregnant adults, and are pathogenic to humans and induce antibodies that interfere with the commonly 34
used diagnostic tests because they possess the LPS bearing the intact O-chain (Nielsen and Duncan, 1990; Oliveira et al., 2011).
The attenuated S19 was accidentally found after extended laboratory storage of an isolate and has been the most widely used vaccine to prevent bovine brucellosis
(Nielsen and Duncan, 1990). It carries multiple genetic defects (Crasta et al., 2008).
Conjunctival inoculation of S19 presents more advantages compared with the standard subcutaneous route because it induces less side effects with less risk of abortion
(Godfroid et al., 2011). Persistently induced antibodies after vaccination with S19 mainly depend upon the age of the animals at vaccination (Seleem et al., 2010) and it is recommended that animals are vaccinated before they are six months of age to reduce this problem (Morgan, 1969). The vaccine has been shown to produce significant protection when administered to yaks (Bandyopadhyay et al., 2009).
The live attenuated strain, B. melitensis Rev 1, is a revertant of a streptomycin- dependent mutant (Cloeckaert et al., 2002). Rev 1 may induce more effective protection than S19 (Barrio et al., 2009). The vaccine is cheap and can also be administered conjunctivally; however it also has limitations including the potential for abortions if administered to pregnant animals, interference with serological diagnosis, virulence for humans, resistance to streptomycin and a tendency to dissociate into ineffective rough mutants. Vaccinating animals conjunctivally, when they are younger than 4 months of age, can minimise these limitations (Godfroid et al., 2011).
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2.6.2.1.2 Other live vaccines used in the past
Other live vaccines have also been used to control brucellosis. These include B. suis strain 2 (S2) and B. melitensis strain 5 (M5). These vaccines have been used widely in
China, especially during the early 1980s, in cattle, yaks, sheep and goats (Xin, 1986;
Deqiu et al., 2002). In Tibet S2 and M5 were used widely resulting in the effective control of the disease in yaks (Pan, 1992b; Pan et al., 1992). The S2 vaccine can be administered orally, as well as via subcutaneous injection, and is considered safe for pregnant animals (Jing et al., 2016). As it may protect animals against infection with heterologous, virulent Brucella species (Zhu et al., 2016), S2 has been the most popular and widely used vaccine for controlling the disease in livestock in China since 1982
(Gerald et al., 2003; Jing et al., 2016).
2.6.2.2 Main current vaccines
2.6.2.2.1 B. abortus RB51
Brucella abortus RB51 is a rifampin-resistant mutant attenuated strain. It is stable and less dangerous than S19 (Ashford et al., 2004; Godfroid et al., 2011), and has solved one limitation of S19 in that it is a rough strain that does not induce antibodies which interfere with serological tests (Schurig et al., 1991; Poester et al., 2006). Field studies have shown that immunity induced by strain RB51 (at least 1 year after vaccination) is more effective than that induced by S19 (Lord et al., 1998). Furthermore, subcutaneous inoculation of 109 RB51 organisms in pregnant cattle has been shown to be safe without the induction of abortions or placentitis (Palmer et al., 1997). However, its effectiveness is controversial as some studies have shown that reduced doses are ineffective and that it is less effective than S19 against severe challenges (Moriyón et al., 2004). As it has been reported that RB51 can be ineffective when the disease prevalence is high, it has 36
been suggested that repeat vaccination should be adopted (Herrera et al., 2008).
Revaccination using a reduced dose of RB51 was shown not to cause abortion but still resulted in effective protection against natural challenge in the field (Leal-Hernandez et al., 2005).
2.6.2.2.2 DNA vaccines
There have been a limited number of studies using DNA vaccines, and the majority of these have studied the vaccines in murine models of infection (Oliveira et al., 2011).
The use of combined DNA vaccines may induce significantly higher levels of protection against Brucella compared with commercial live-attenuated Brucella vaccines (Luo et al., 2006; Cassataro et al., 2007; Yu et al., 2007). However, it is not clear if DNA vaccines can induce long-term protection (Ko and Splitter, 2003) and if protection demonstrated in a mouse model can be translated to protection in larger animals or humans (Schurig et al., 2002; Oliveira et al., 2011). However Sáez et al.
(2008) reported that the DNA vaccine expressing Cu-Zn SOD induced a good protective immune response in cattle. Because of the conflicting results with DNA vaccines, more studies with these vaccines are required in large animals.
2.6.3 Control
Due to the impact of brucellosis on both animal production (abortion, reduced milk yield and infertility) and public health (cost of treatment and debility) (Seleem et al.,
2010) and its wide distribution, control measures for brucellosis have been implemented in many countries. Control strategies adopted frequently differ between developed and developing countries. In the developed world, the control mainly includes vaccination, test-and-slaughter programs, pasteurisation of milk, disease surveillance and animal 37
movement control (Godfroid et al., 2011). However in resource-limited countries, test and slaughter programs are not feasible (Zinsstag et al., 2007; Marcotty et al., 2009).
Even if a test and slaughter program could be implemented, it is unlikely to result in disease eradication if the prevalence is initially high (Zamri-Saad and Kamarudin, 2016).
Many eradication programs for brucellosis have been shown to be costly, long and difficult to carry through (Godfroid et al., 2011). Therefore the needs and perceptions of the local communities should be addressed and emphasised when control strategies are implemented (Marcotty et al., 2009). Animal husbandry and management conditions, including extensive breeding, transhumance and coexistence of several livestock species together, contribute to the difficulty in eradicating or controlling the disease. In addition, a risk assessment should be performed, taking into account the current scientific knowledge and the particular local situations (Godfroid et al., 2011), before any control procedure is implemented. Decision tree analysis, such as the probability of exposure to an infectious agent, as well as the cost and consequences of the control measures, may also be used to evaluate different methods (Godfroid et al., 2011).
Due to the low prevalence in many endemic regions, the disease is not listed as a priority disease to control, and is considered by some to be a neglected zoonosis (Adone and Pasquali, 2013; Nina et al., 2017). In order to control it effectively, a surveillance system should be developed and conducted as early as possible. The aim of surveillance is to obtain periodic information on specific epidemiological indices, including a detailed census of susceptible animals, market and slaughter data, and statistical estimates of incidence and prevalence values within the targeted area (Adone and
Pasquali, 2013). Any delay in detection of the disease will have a negative impact on control measures as this delay may result in transmission of infection into other 38
populations with the potential widespread dissemination of the disease (Godfroid et al.,
2011).
For the early detection of a disease, a surveillance system must have a high sensitivity
(Hadorn and Stark, 2008). Current surveillance strategies need to be evaluated and optimised in a local or regional setting and the cost of implementing them assessed.
Scenario tree analysis emphasised that testing of animals that aborted and the testing of animals at purchase were necessary components of surveillance. Consequently implementation of a ‘risk based’ surveillance program with an acceptable statistical confidence level should be implemented in countries and/or regions (Godfroid et al.,
2011).
The epidemiological link between brucellosis in wildlife and the disease in livestock and people is now widely recognised (Godfroid et al., 2013b). Brucella spp. have been isolated from a large number of terrestrial wildlife animals including bison, elk, feral pigs and wild boar and recently new Brucella species have been identified from some marine mammals (Godfroid, 2002; Godfroid et al., 2011). Although B. abortus has been eradicated from some countries, B. melitensis and B. suis remain major agents of infection in sheep and feral pigs, respectively in some of these countries (Corbel, 1997;
Ewalt et al., 1997). In addition, although all states of the USA are classified as free from
B. abortus in cattle, infected cattle herds have been detected. The possible reason for this is believed to be infected free ranging elk (Van Campen and Rhyan, 2010).
Consequently controlling the disease in domestic animals and humans still faces many challenges.
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Furthermore, in order to gain a deeper insight into transmission modes and if an animal species can act as a spill-over host or a true reservoir host, isolation of Brucella becomes an indispensable step in understanding the disease’s epidemiology. Although the different Brucella species do have preferred hosts, cross-species transmission is possible with sheep in Nigeria being found to be infected with B. abortus (Ocholi et al.,
2004) and camels have been demonstrated to be infected with B. melitensis and with different biovars of B. abortus (Musa et al., 2008; Gwida et al., 2012). Additionally, it is worth mentioning that any vaccination program in livestock should be implemented in reservoir hosts, not just in spill-over hosts or the key livestock species (Godfroid et al., 2011).
Improvements in: awareness about brucellosis; diagnostic capabilities; and surveillance of the disease in livestock, wildlife and humans are beneficial in the effective control of the disease. Although the disease has been controlled and even eradicated in some countries, it still causes significant losses to the livestock industries and is a threat to human health in many countries and the impact of the disease in these countries is often under-estimated. Control of the disease is a global challenge rather than restricted to being a local issue (Godfroid et al., 2005). However knowledge on brucellosis, its distribution, risk factors and impact on local communities is a key part in the local control of the disease, consequently the study outlined in this thesis was developed to improve the current information on the epidemiology of the disease in yaks in Tibet.
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CHAPTER THREE
Retrospective study of the seroprevalence of brucellosis in yaks in
Tibet
3.1 Introduction
Although brucellosis has been known of for almost 120 years ago (Seleem et al., 2008), it still remains one of the most important zoonotic diseases (OIE, 2009). The disease is widely distributed throughout the world, except for countries such as Australia, Canada and the Netherlands where bovine brucellosis has been eradicated (Seleem et al., 2010).
It has been recognised as a regionally neglected disease as it can be difficult to diagnose and requires laboratory testing for confirmation (Pappas et al., 2006; Hajia et al., 2013).
Brucellosis was first reported in China in 1905 and was considered to be a serious problem by the 1980s. Through the adoption of a vaccination program the disease was controlled in the 1990s (Deqiu et al., 2002), however early in the 21st century evidence of re-emergence of human brucellosis was reported (Liu et al., 2014). The features of brucellosis in Tibet were reported to be similar to those in other provinces of China, with evidence of the disease being a severe epidemic by the 1980s with the prevalence subsequently reducing through the implementation of a vaccination program
(Anonymous, 1985; Deqiu et al., 2002). However, there is little published information about the epidemiology of brucellosis in Tibet even though the TCADC and the Tibet
Centre for Disease Control (TCDC) have been implementing annual serological surveillance programs since 2009. Understanding the epidemiology of a disease plays an important role in developing effective control and prevention programs for that
41
disease (Jergefa et al., 2009). An initial component of this often involves examining and analysing existing data. Such retrospective studies can uncover valuable information on the spatial and temporal distribution of a disease and can also help identify susceptible species or carriers of infection (Mwebe et al., 2011). In the current chapter the findings of a study examining existing historical data are reported to explore the epidemiological characteristics of brucellosis in Tibet.
3.2 Materials and methods
3.2.1 Data
Data from the 1970s and 1980s were collected from articles published in Tibetan journals and books. The data set of recent cases/diagnoses of brucellosis in yaks, cattle, sheep, goats and pigs from 2009 to 2015 in Tibet was obtained from the TCADC. These data included results from annual surveillance of livestock in two counties from the prefectural cities or prefectures which had been randomly selected. They contained results of serological tests, species tested and geographical location of the livestock. The disease (seropositivity) was based on a positive reaction to the RBT conducted in the
TCADC laboratory. However in these data yaks and cattle were combined into a single group, as were sheep and goats. Data on human cases of brucellosis from 2011 to 2013 in Tibet were obtained from the TCDC. These data provided information on the total number of humans tested, number positive to the tube agglutination test, and the geographical locations of the samplings in the 7 counties of Damxung (Lhasa prefectural city), Zhongba (Shigatse prefectural city), Gongjue (Chamdo prefectural city), Geer (Ngari prefecture), Cuoqin (Ngari prefecture), Cuomei (Shannan prefectural city) and Anduo (Nagqu prefecture), with no information being available for Nyingchi prefectural city. The sampling sites for humans and livestock were selected by the 42
authorities, specifically the TCADC and the TCDC. Samples from livestock and humans were tested in the laboratories of these two agencies after dispatch by the relevant departments of the counties where the samples were collected.
The locations of the sampled counties in Tibet were displayed by generating two choropleth maps using ArcGIS 10.3 (Environmental System Research Institute, 2014).
3.2.2 Statistical analyses
An Excel (Microsoft Office Excel 2010) spreadsheet was developed for the data and statistical tests were undertaken using SPSS Statistics for Windows (Version 21.0, IBM
Corp., Armonk, New York, USA) and R (R Development Core Team, 2013). The data, such as spatiotemporal distribution of both prevalence of animals and humans, were tested with the Kolmogorov-Smirnov and Shapiro-Wilk tests for normality. Non-normal data, such as the spatial distribution of animal prevalence, were adjusted to normality through cosine transformation (Allen et al., 2014). Correlations between factors were investigated by calculating a Pearson’s correlation coefficient. Both the Chi-square test for independence and the odds ratios (OR) and their 95% confidence intervals were used to identify statistical differences in categorical data (Kahn and Sempos, 1989).
3.3 Results
3.3.1 Early records before the mid-1980s
From 1979 to 1985, blanket vaccination was undertaken compulsorily in all 64 counties of Tibet. More than 19 million head of livestock were vaccinated, representing 91% of the total population (Table 3.1) (Pan, 1992b). The results available from the four prefectures (prefectural cities) sampled showed a considerable reduction of the 43
seroprevalence from 13.2% (95%CI: 13.0, 13.4) prior to 1979 to 0.4% (95%CI: 0.3, 0.4) in 1985 (χ2=6195.72, df=1, p<0.001) (Table 3.2) (Pan, 1992b). In the four counties of
Anduo, Nagqu, Bange and Linzhou the seroprevalence in sheep, goats and yaks had reduced to 0.13% (95%CI: 0.1, 0.3), 0.62% (95%CI: 0.3, 1.2) and 1.07% (95%CI: 0.7,
1.6), respectively in 1983. Of these counties, the first three counties are contained within the Nagqu prefecture, while Linzhou county is part of the Lhasa prefectural city.
Prior to the vaccination campaign, sheep had the highest seroprevalence of 29.1%
(95%CI: 27.8, 30.7), followed by yaks with a seroprevalence of 22.3% (95%CI: 20.3,
24.4) and goats with a seroprevalence of 20.3% (95%CI: 18.0, 22.8). Overall there was a significant difference in the seroprevalence between the three species (χ2=51.57, df=2, p<0.001). After the control program had been implemented, yaks had the highest seroprevalence (1.1%; 95%CI 0.7, 1.6) followed by goats and sheep. Similarly, after implementing the vaccination control program, the seroprevalence was significantly different between the three species (χ2=39.96, df=2, p<0.001) (Table 3.3) (Anonymous,
1984; Zhu et al., 1987).
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Table 3.1 Results of the brucellosis vaccination program in Tibet from 1979 to 1985
Prefecture Nagqu Shannan Lhasa Shigatse Chamdo Total Number of counties sampled 11 13 9 18 13 64 Number of livestock present 2470700 7477170 5223652 1900966 4151702 21224160 Number of vaccinated livestock 2330700 7377243 4819945 1740334 3061267 19329489 Proportion of livestock vaccinated (%) 94.33 98.66 92.27 91.55 73.74 91.07
Table 3.2 Comparison of the seroprevalence to brucellosis prior to and after implementing vaccination programs in yaks, cattle, sheep and goats from 1979 to 1985
Pre-vaccination Post-vaccination p-Value No. counties Prefecture Prevalence (%) Prevalence (%) sampled No.+ve/total No.+ve/total (95%CI) (95%CI) Lhasa 3 4554/27557 16.5 (16.1, 17.0) 30/6923 0.4 (0.3, 0.6) <0.001
Shannan 1 918/2995 30.7 (29.0, 32.3) 6/10612 0.1 (0.0, 0.1) <0.001
Shigatse 6 4109/37158 11.1 (10.7, 11.4) 53/13755 0.4 (0.3, 0.5) <0.001
Chamdo 5 4379/38165 11.5 (11.2, 11.8) 70/14201 0.5 (0.4, 0.6) <0.001
Total 15 13956/105876 13.2 (13.0, 13.4) 159/45497 0.4 (0.3, 0.4)
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Table 3.3 Comparison of seroprevalence to brucellosis in three species prior to and after vaccination from 1979 to 1983
County Sheep Goats Yaks
Pre-vaccination Post-vaccination Pre-vaccination Post-vaccination Pre-vaccination Post-vaccination
No. tested 968 1051 100 45 106 572
Anduo No. positive 335 0 35 1 32 5
Seropositivity (95% CI) 34.6 (31.6, 37.7) 0.0 (0, 0.4) 35.0 (25.7, 45.2) 2.2 (0.1, 11.8) 30.2 (21.7, 39.9) 0.9 (0.3, 2.0)
No. tested 1762 2553 301 168 632 605
Nagqu No. positive 583 1 64 1 136 9
Seropositivity (95% CI) 33.1 (30.9, 35.3) 0.0 (0, 3.3) 21.3 (16.8, 26.3) 0.6 (0, 3.3) 21.5 (18.4, 24.9%) 1.5 (0.7, 2.8)
No. tested 812 2112 373 274 181 336
Bange No. positive 159 6 57 1 42 8
Seropositivity (95% CI) 19.6 (16.9, 22.5) 0.3 (0.1, 0.6) 15.3 (11.8, 19.3) 0.4 (0, 2.0) 23.2 (17.3, 30.0) 2.4 (1.0, 4.6)
Number tested 364 523 338 808 759 642
Linzhou No. positive 64 1 70 5 164 1
Seropositivity (95% CI) 17.6 (13.8, 21.9) 0.2 (0, 1.1) 20.7 (16.5, 25.4) 0.6 (0.2, 1.4) 21.6 (18.7, 24.7) 0.2 (0.0, 0.9)
No. tested 3906 6239 1112 1295 1678 2155
Total No. positive 1141 8 226 8 374 23
Seropositivity (95% CI) 29.2 (27.8, 30.7) 0.1 (0.1, 0.3) 20.3 (18.0, 22.8) 0.6 (0.3, 1.2) 22.3 (20.3, 24.4) 1.1 (0.7, 1.6)
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3.3.2 Results of recent animal surveillance (2009 to 2015)
Between 2009 and 2015 32,440 yaks, cattle, sheep, goats and pigs were sampled and tested for brucellosis with 265 positive (seroprevalence 0.82%; 95%CI: 0.7, 0.9).
3.3.2.1 Spatial distribution
In the period from 2009 and 2015 evidence of infection was found in all prefectures
(Table 3.4 and Figure 3.1). The prevalence was significantly different between the seven prefectures (χ2=75.73, df=6, p<0.001), with the highest prevalence in Ngari prefecture (1.84%; 95% CI: 1.4, 2.3) and the lowest in Nyingchi prefectural city (0.21%,
95%CI: 0.1, 0.4). The odds of being seropositive in animals from Ngari, Chamdo, Lhasa,
Shannan, Nagqu and Shigatse were all significantly higher than for Nyingchi prefectural city (Table 3.4).
Table 3.4 Seroprevalence of brucellosis in yaks, cattle, sheep, goats and pigs at the prefecture-level in Tibet (2009-2015)
Prefecture No. positive No. tested/sampled Prevalence (95%CI) Odd ratios (95%CI) Ngari 66 3580 1.84 (1.4, 2.3) 8.71 (4.47, 16.97) Chamdo 44 3863 1.14 (0.8, 1.5) 5.38 (2.71, 10.71) Lhasa 41 5864 0.70 (0.5, 0.9) 3.31 (1.65, 6.60) Shannan 40 5694 0.70 (0.5, 1.0) 3.32 (1.66, 6.65) Nagqu 34 4386 0.78 (0.5, 1.1) 3.66 (1.81, 7.43) Shigatse 30 4326 0.69 (0.5, 1.0) 3.28 (1.60, 6.71) Nyingchi 10 4727 0.21 (0.1, 0.4) 1.0
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Figure 3.1 Spatial distribution of animal brucellosis in Tibet (2009-2015)
3.3.2.2 Temporal distribution
In Table 3.5 the change in the seroprevalence for brucellosis from 2009 to 2015 in animals is summarised. No seropositive animals were detected in 2009 and 2013. The highest seroprevalence was observed in 2011 (2.34%, 95%CI: 2.0, 2.7). Overall there was a significant difference in the seroprevalence between years (χ2=302.44, df=6, p<0.001) (Table 3.5). The seroprevalences in 2010, 2011, 2012 and 2014 were significantly higher than that in 2015 (Table 3.5).
Table 3.5 Seroprevalence of animal brucellosis during the period of 2009 to 2015
Year No. positive No. sampled Seroprevalence (%) (95%CI) OR (95% CI) 2009 0 2030 0.00 (0.0, 0.2) - 2010 36 2848 1.26 (0.9, 1.7) 7.28 (4.08, 12.98) 2011 163 6957 2.34 (2.0, 2.7) 13.64 (8.27, 22.50) 2012 17 2000 0.85 (0.5, 1.4) 4.87 (2.48, 9.56) 2013 0 2780 0.00 (0.0, 0.1) - 2014 32 6142 0.52 (0.4, 0.7) 2.98 (1.65, 5.37) 2015 17 9683 0.18 (0.1, 0.3) 1.0
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In the years 2011, 2013, 2014 and 2015 surveillance was undertaken during both the spring/summer and the autumn/winter seasons. Overall the seroprevalence was significantly higher in the spring/summer seasons than in autumn/winter (p<0.001,
χ2=64.95, df=1) (Table 3.6).
Table 3.6 Seroprevalence in different seasons
Seasons Number positive Total number Prevalence (%) (95%CI) P-Value Spring/summer 156 11786 1.32 (1.1, 1.5) <0.001 Autumn/winter 56 13776 0.41 (0.3, 0.5)
3.3.2.3 Species susceptibility
The seroprevalence was similar in different species (yak and cattle data combined, and sheep and goats data combined) (p=0.91, χ2=0.18, df=2). The highest seroprevalence was detected in pigs (0.80%; 95%CI: 0.6, 1.1) and the lowest in yaks and cattle (0.75%;
95%CI: 0.6, 0.9) (Table 3.7).
Table 3.7 Seroprevalence in different susceptible species groups (2009 – 2015)
Species Number positive Total number tested Prevalence (%) (95%CI) P-Value Yaks & cattle 102 13590 0.75 (0.6, 0.9) Sheep & goats 78 9885 0.79 (0.6, 1.0) 0.91 Pigs 49 6117 0.80 (0.6, 1.1)
3.3.3 Results of surveillance for brucellosis in humans (2011 – 2013)
A total of 7421 humans were tested for brucellosis from 2011 to 2013 with 62 positives
(seroprevalence 0.84%; 95%CI 0.6, 1.1).
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3.3.3.1 Spatial distribution in humans
The seroprevalence of brucellosis in humans was different between prefectures
(p=0.038, χ2=11.80, df=5) (Table 3.8 and Figure 3.2). The highest seroprevalence was in Chamdo prefectural city (1.41%; 95%CI: 0.0, 7.6). No seropositive humans were detected in Shigatse (0%; 95% CI 0.0, 0.6).
Table 3.8 Seroprevalence of brucellosis in humans at the prefecture-level in Tibet (2011-2013)
Prefecture Number positive Total number Prevalence (%) (95% CI) OR (95%CI) Chamdo 1 71 1.41 (0.0, 7.6) 3.05 (0.34, 27.66) Shannan 27 2347 1.15 (0.8, 1.7) 2.48 (0.87, 7.12) Lhasa 25 2537 0.99 (0.6,1.5) 2.12 (0.74, 6.12) Nagqu 5 960 0.52 (0.2, 1.2) 1.12 (0.30, 4.18) Ngari 4 858 0.47 (0.1,1.2) 1.0 Shigatse 0 648 0.00 (0.0,0.6) -
Figure 3.2 Spatial distribution of human brucellosis in Tibet (2011-2013)
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3.3.3.2 Temporal distribution of seroprevalence in humans
The seroprevalence in humans varied significantly over the three years of sampling
(p<0.001, χ2=66.47, df=2), decreasing from 2.23% (95%CI: 1.4, 3.4) in 2011 to 0.05%
(95%CI: 0.0, 0.2) in 2013 (Table 3.9).
Table 3.9 Seroprevalence of human brucellosis (2011 to 2013)
Year Number positive Total number tested Seroprevalence (%) (95%CI) 2011 21 941 2.23 (1.4, 3.4) 2012 39 2539 1.54 (1.1, 2.1) 2013 2 3941 0.05 (0.0, 0.2)
3.3.4 Correlation between animal and human seroprevalence
The seroprevalence in livestock from the different prefectures was not significantly correlated with the seroprevalence in humans from those prefectures (r = 0.31, p =
0.556). However there was a positive, although not significant, correlation in the overall seroprevalence of animals and humans for the years from 2011 and 2013 (r = 0.93, p =
0.233). In Figure 3.3 the seroprevalence in humans and other animal species is charted.
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Figure 3.3 Annual seroprevalence of animal and human brucellosis in Tibet (2009-2015)
2.50
2.00
1.50
Animal 1.00
Human Percentage (%) Percentage
0.50
0.00 2008 2010 2012 2014 2016 Year
3.4 Discussion
This is the first study examining the epidemiological characteristics of brucellosis in animals and humans in Tibet using existing data.
Epidemics of brucellosis in animals were reported in Tibet as early as 1931 (Liu et al.,
1992), with outbreaks being reported in the 1970s. During the 1970s and early 1980s, three Brucella species comprising seven Biovars were isolated and identified (Biovars
1-3 of B. melitensis, Biovars 1, 4 and 6 of B. abortus and Biovars 1 of B. suis). The dominant Brucella species cultured was B. melitensis with 66 of 80 isolates (82.5%), followed by B. abortus (12 isolates) and B. suis (2 isolates). Of the B. melitensis isolates,
44 were Biovar 2 which was the most common strain cultured (Pan, 1992a). However after the implementation of a five year compulsory vaccination campaign in yaks, cattle, sheep and goats in the 1980’s using B. suis S2 vaccine and B. melitensis M5 vaccine, the seroprevalence in animals decreased significantly (Anonymous, 1985; Pan, 1992b).
52
The situation was basically consistent with the pattern of the disease in other provinces of China during that period (Deqiu et al., 2002). The two vaccines used were locally produced Chinese vaccines and were administered through nasal vaccination (sprayed into the nasal cavity), oral vaccination or subcutaneous injection (Pan, 1992b; Deqiu et al., 2002). Of these the S2 vaccine, through oral vaccination, was more widely adopted due to its convenient delivery means through drinking water, its reported safety in pregnant animals and the fact that farmers liked that it could be administered without pain or disturbance to their livestock (Anonymous, 1992; Jing et al., 2016).
After the 1980s the disease decreased in importance in China until it re-emerged in humans early in the 21st century (Deqiu et al., 2002; Zhong et al., 2013). In the face of a potential epidemic situation, the TBAA adopted the MOA’s policies of controlling and preventing diseases (i.e FMD, HPAI, PRRS, PPR, brucellosis) including annual surveillance in animals and has been actively implementing this policy since 2009
(Chapter 1). Furthermore the TCDC also initiated surveillance in humans and that data, along with historical livestock records from the 1970s and 1980s and the animal surveillance data from 2009 to 2015, were examined in this chapter.
The overall seroprevalence of brucellosis in animals from 2009 to 2015 was 0.82%
(95%CI 0.7, 0.9%) which was significantly higher than the control range of animal brucellosis in China (0.09 - 0.28%) (Wang et al., 2010). However the seroprevalence in
Tibet was similar to that reported in the neighbouring provinces of Qinghai (0.9%) (Qi and Chao, 2013) and Sichuan (1 - 2%) (Chen et al., 2011). In the 1990s, the disease was primarily restricted to the main pastoral areas of China, such as Tibet and Inner
Mongolia, however with the rapid development of animal husbandry in inland China it 53
extended into more farming-pastoral and farming regions including Helongjiang, Jilin and Shanxi provinces (Wang et al., 2010). Currently the disease results in regular epidemics in the northern, north-eastern and north-western regions of China (Li et al.,
2013).
This study demonstrated evidence of infection in animals in all prefectures of Tibet. The highest prevalence was reported in Ngari, which is an exclusively pastoral area with yaks, sheep and goats being the dominant livestock present. The lowest prevalence was reported in Nyingchi where an agro-forestry–pastoral mixed production system is adopted. In this prefecture raising livestock is just one form of income for the livestock owners with income from fruit and crops contributing significantly to the households’ income. The number of livestock in Ngari is more than three times that found in
Nyingchi (Duoji and Yang, 2014). A similar epidemiological study in Bhutan for foot and mouth disease demonstrated that the seroprevalence in yaks increased with increasing animal population (Dukpa, 2011) due to more frequent livestock movements and a greater chance of contact of naïve animals with infected animals.
In this study no infected animals were detected in 2009 and 2013. This could be due to the time of sample collection as the calving season of yaks and sheep in Tibet occurs mainly in April and May (spring/summer) (Gerald et al., 2003; Zhou et al., 2004).
Surveillance in these two years was conducted during summer and autumn when few animals calve/lamb resulting in less opportunity for the spread of infection which is associated with abortion and birthing (Al-Rawahi, 2014). The highest seroprevalence was observed in 2011 (2.34%, 95%CI: 2.0, 2.7). Zhu (2013) similarly reported that in the Inner Mongolia Autonomous Region the seroprevalence of brucellosis in animals 54
was highest in 2011, with an outbreak occurring from October 2010 to 2011. Also another study by Li (2013) demonstrated an increase in seropositivity from 2009 reaching a peak of 7.8% in 2011 in Tongliao district of Inner Mongolia (Li, 2013). The seroprevalence in humans followed a similar pattern to that of animals with a rapid increase in the number of new cases from 2003 (6448) to 2011 (38151) in China (Liu et al., 2014). The movement of infected livestock is likely linked with the occurrence and spread of brucellosis (Omer et al., 2000a; Zamri-Saad and Kamarudin, 2016). In Tibet the introduction of new breeds, including Jersey and Holstein cattle, Boer goats and Saft sheep, to improve the genetics and productivity of the local breeds (Se et al., 2010;
Axirao. et al., 2011), could have resulted in increased risk of transmission if the introduced animals were infected and then distributed into susceptible herds/flocks.
Others have shown that periodic epidemics of brucellosis can occur at 3 to 4 year intervals if vaccination is not implemented (Deqiu et al., 2002). This study highlighted the re-emergence of brucellosis in both humans and animals in China in the 21st century, similar to that reported in other areas such as the Middle East and Mongolia (Pappas et al., 2006; Seleem et al., 2010).
Although an annual program of serosurveillance is implemented in China, it often focuses on intensive sampling once or twice during each year. This study found a significantly higher seroprevalence during the spring/summer seasons than in the autumn/winter seasons due to coinciding with the calving/lambing/kidding season as described above (p<0.001). Other reports have similarly shown that higher seroprevalences mainly occur during the spring/summer periods from March to June
(Deqiu et al., 2002; Zhu, 2013). According to the national regulations of China positive
55
animals are required to be slaughtered to reduce potential for disease transmission
(Ministry Of Agriculture and National Health and Family Planning Commission, 2016).
In this study no difference in the seroprevalence between species was detected (p=0.91).
According to the national criteria, if the prevalence in a zone is < 0.5% for sheep, < 1% for cattle and < 2% for pigs, it indicates that brucellosis in the zone is under control
(Deqiu et al., 2002). In the current study the prevalence in sheep (0.79%, 95%CI: 0.6,
1.0) was higher than the recommended national value. Thus further control measures are required to control the disease in Tibet.
Although surprisingly there was no significant correlation in the spatial distribution of animal and human seroprevalence, there was a positive temporal association. This latter finding was expected given that infection in humans arises through contact with infected animals or their products (Corbel, 1997).
Although retrospective studies are cheap and easy sources of information they have a potentially serious limitation of potential bias. Initially basic information, such as gender and sex, was not collected (recorded) when the animals were sampled, however these are important host factors for the disease (Muma et al., 2006; Jackson et al., 2014).
In addition, combining data for yaks and cattle and for sheep and goats limits the ability to examine specific species data and in future data on individual species should be recorded separately. There is also the potential to introduce biases through the method of animal and herd selection (Grimes and Schulz, 2002; Beck, 2009). In Tibet, each prefecture has a different number of counties, which in turn have different numbers of townships with different numbers of villages. For routine animal surveillance two 56
counties from a prefecture and two villages from the chosen counties are selected for sampling. The counties and villages selected for sampling change between years. In contrast human surveillance is conducted in one township of a county in the prefecture and the selected county and township can either be the same one between sampling years or different ones. This has the potential to introduce selection bias in the sampling and makes comparison between the seroprevalence in animals and humans and between years challenging.
In conclusion, this chapter is the first investigation analysing existing data on brucellosis in Tibet. The study highlighted the impact of the vaccination program conducted from 1979 to 1985 in reducing the seroprevalence of brucellosis, however subsequently evidence of infection has re-emerged. The seroprevalence differed between prefectures (prefectural cities) and it is likely that the production system adopted in the prefectures was a key factor in these spatial differences.
Although the study described in this chapter summarised the historical picture of brucellosis in Tibet, there is a need to undertake current serological surveys in order to determine the current seroprevalence and to identify risk factors for infection. Linked with this is a need to understand the economic losses associated with the disease and the knowledge, attitudes and practices of local herders so that effective control programs can be implemented. These aspects will be described in the following chapters of this thesis.
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CHAPTER FOUR
Seroprevalence of bovine brucellosis in domesticated yaks (Bos
grunniens)
4.1 Introduction
Bovine brucellosis is principally caused by B. abortus, although B. melitensis and B. suis can occasionally cause disease if animals are grazed and/or managed with sheep and goats, or pigs, respectively (Ewalt et al., 1997; OIE, 2009; Praud et al., 2016). As outlined in previous chapters the disease predominantly results in abortions, with the birth of weak calves, reduced milk yield and infertility also being reported (Geering et al., 1995) and human infection can result from the consumption of non-pasteurised milk or cheese or from occupational exposure to infected animals (Corbel, 2006; Jackson et al., 2014). The disease is recognised as an important, yet neglected zoonosis, affecting livestock in many countries of the world (OIE, 2009; Seleem et al., 2010).
Although bovine brucellosis has been eradicated in some countries (Díaz, 2013; Sun et al., 2016), it remains endemic in many countries, including China (Deqiu et al., 2002;
Godfroid and Käsbohrer, 2002; OIE, 2009). As outlined previously Tibet is one of the largest pastoral regions in China and the grazing of yaks plays an important role in the lives of the pastoralist herders through the production of meat, milk, fibre and hides
(Miller, 1999a; Ji et al., 2002b; Gerald et al., 2003). Brucellosis was recognised in Tibet in the 1980’s as a disease of both livestock and humans. Through the implementation of mass vaccination the disease was reported to have been controlled (Chapter 3); however recently human infection in Tibet has re-emerged (Zhandui. et al., 2008; Liu et al.,
58
2014). As infection in humans almost always arises from contact with domestic or wild animal reservoirs and their products (Godfroid et al., 2005), in order to effectively control the disease China initiated a surveillance program of livestock in 2009 (Bureau of Veterinary, 2009). In these surveillance programs the overall prevalence of brucellosis in livestock from different prefectures/prefectural cities of Tibet was 0.82%
(95%CI 0.7, 0.9) (Chapter 3). The study reported in this chapter was designed to determine the current seroprevalence and distribution of brucellosis in yaks in selected regions of Tibet.
4.2 Materials and methods
4.2.1 Study design
A cross-sectional survey was conducted in three counties from April to May 2015. This involved collection of sera from yaks from selected herders/livestock owners and administering a questionnaire to the owners. Each herder’s yaks were categorised as an individual herd. Ethics approvals were obtained from the Human Research Ethics
Committee (HREC) (Project No: 2015/010) and the Animal Ethics Committee (AEC)
(permit No: R2728/15) of Murdoch University for this research.
4.2.2 Study sites
In this study three counties were selected as project sites for sampling based on the population and distribution of yaks in Tibet. The counties selected were Damxung,
Maizhokunggar and Yadong, representing the northern, eastern and southern areas from
Lhasa, the capital of Tibet, respectively (Figure 4.1). The locations of the sampled counties in Tibet were displayed by generating two choropleth maps using ArcGIS 10.3
(Environmental System Research Institute, 2014). Damxung and Maizhokunggar are 59
two counties of Lhasa prefectural city, whereas Yadong is a county in the prefectural city of Shigatse.
Figure 4.1 Map showing the location of the selected counties in Tibet, China
In Damxung county, which covers an area of 12,000 km2, yaks are raised exclusively under a pastoral production system. The area of grassland is 1.7 million acres, representing 68.81% of the total area of the county. The average altitude of the county is
4,300 metres above sea level. There are 8 townships including 29 villages within the county containing a human population of 41,918 (Liu, 2014a). The main livestock grazed are yaks, sheep and goats. There are estimated to be 230,000 yaks in Damxung
(Liu, 2014a). Two main transportation lines, the national highway 109 and the railway, run through the county linking Lhasa with Qinghai Province and the mainland of China.
Maizhokunggar county is located in the valley of the middle reaches of the Yarlung
Tsangpo River and is an agro-pastoral production system. The area of grassland is 0.8
60
million acres, representing 59.43% of the total county. The average altitude of this county is 4,000 metres above sea level. There are 8 townships and 43 villages in the county containing over 7,000 households and 42,700 people (Anonymous, 2014).
Nearly 130,000 yaks are found in this county distributed throughout the villages
(Anonymous, 2014). The national highway, 318, traverses the county linking Lhasa with Sichuang province.
Yadong county lies in the south of Tibet, covering approximately 4,300 km2 and has a human population of 10,000 (Anonymous, 2015). It is a frontier county situated in the mouth of the Chumbi valley near India and Bhutan. Pali, an important township of the county is situated south of Lhasa and borders Bhutan and India. It is almost exclusively a pastoral area and is considered to be the dominant yak-raising region of Tibet. The area of grassland in the township of Pali is 76,115 acres. The average altitude of the township is 4,300 metres above sea level. There are four villages (named as first, second, third and fourth villages) with 575 households and nearly 2,500 people living in the county (Anonymous, 2015). There are 6,350 yaks in total with 1,885 yaks in the 1st village, 1,603 yaks in the 2nd village, 1,894 yaks in the 3rd village and 968 yaks in the
4th village (Anonymous, 2015).
According to the farming system, Tibet is classified into four livestock production systems as described in Chapter 1. In the current study, only the pastoral and agro- pastoral production systems were represented as yaks are mainly raised under these two production systems (Tashi et al, 2005).
61
4.2.3 Sample size and sampling
The sample size to estimate the seroprevalence using a design prevalence of 10% (Gao et al., 2013) in a population of 6,350; 230,000; and 130,000 in the counties of Yadong,
Damxung and Maizhokunggar (Anonymous, 2014; Liu, 2014a; Anonymous, 2015), respectively, with 95% confidence and 5% precision was calculated using EpiTools
(Sergeant, 2015). The required number was 139. If a prevalence of 50% (worst-case scenario) was used, the sample size required was 385. In this study in order to increase the precision and accuracy of the prevalence estimate a sample size of 1523 was chosen.
The sampling strategy adopted in this study is described as follows. Based on the distribution of yaks, yaks from the three counties were randomly selected for sampling.
Given that there were 8 townships in each of Maizhokunggar and Damxung Counties and yaks were raised in all townships, four townships (Tangjia, Menba, Riduo and
Zhaxigang in Maizhokunggar County and Geda, Longren, Namucuo and Yangbajing in
Damxung County) were selected randomly from each county (Table 4.1). The township of Pali was selected from Yadong County as yaks in this county were primarily raised in this township. In Pali all yak calves had been vaccinated with S2 Brucella vaccine
(Qianyuanhao Biological Co, China), which may induce immunity providing a 2-year period of protection in cattle (Deqiu et al., 2002). A total number of 30 villages were randomly selected for sampling (Table 4.1). These villages and townships were scattered in different directions throughout the relevant counties (east, south, west and north). Both herders and villages located in remote areas were excluded from the sampling frame because of the logistical challenges in including them in the sample. At least two villages in each township and at least 160 yaks from a township were selected and sampled. At least three households were randomly selected from a village for 62
inclusion in this study with at least 20 yaks randomly sampled from each selected village. All of the selected villages in Damxung and Yadong counties adopted a pastoral production system while some villages in the three townships of Zhaxigang, Menba and
Riduo of Maizhokunggar county used an agro-pastoral production system while other villages in these townships adopted a pastoral system. Herds with at least four yaks aged one year or older were eligible for inclusion in the study. If a selected herder declined to participate in the study, another herder from the same location was invited to join the study. Geographical coordinates for the location of each sampled herd were recorded using a hand-held global positioning system (GPS) device (Garmin, USA).
Table 4.1 Summary of sampling for the cross-sectional study
Agro-pastoral Pastoral production Number of herds Number of County Township production system system sampled sera collected
Damxung Namucuo 2 villages 12 160
Longren 3 villages 9 160
Yangbajing 3 villages 13 160
Geda 2 villages 20 160
Maizhokunggar Tangjia 4 villages 37 162
Zhaxigang 4 villages 1 village 20 160
Menba 3 villages 1 village 34 161
Riduo 2 villages 1 village 14 160
Yadong Pali 4 villages 22 240
Total 9 13 villages 17 villages 181 1523
4.2.4 Serological Tests
Approximately 8 ml of whole blood was collected from the jugular vein of the selected yaks. The identity of each sampled yak was recorded on its respective vacutainer tube.
Animal attributes, such as age, sex, herd size and locality, were recorded for each
63
sampled animal. The blood samples were left at room temperature for several hours after collection and then were centrifuged at 3,000 rpm for 10 minutes. The sera were then decanted and stored at -20℃ until testing. Each serum sample was tested with a commercial RBT (Institute Pourquir, IDEXX Montpellier, France) and a C-ELISA
(INGEZIM BRUCELLA Compac 2.0, SPAIN).
The protocol for the RBT was as described by the OIE (2009) with 25 µL of serum thoroughly mixed with 25µL of antigen (B. abortus strain Weybridge 99) on a sterile glass plate. After four minutes, the test result was read by examining for agglutination under natural light conditions and compared with the results from positive and negative control sera. A sample was classified as negative if no agglutination was present. A positive sample was reported when evidence of agglutination was noticed. The negative and positive controls supplied with the diagnostic kits were used in each plate examination for comparative purposes.
The C-ELISA was conducted according to the following procedure. Briefly, 100 µL of serum was added to duplicate wells on a plate coated with purified LPS of B. abortus and incubated at room temperature. After 1 hour, 100µL of conjugate with peroxidase
(specific monoclonal antibody) was added and the plate incubated for another 1 hour at room temperature. Then 100µL of substrate was added to each well for 10 minutes and then 100 µL of stop solution added to the wells. The optical densities (OD) were read in a spectrophotometer at 450nm and were converted to a PI (Percentage of Inhibition) value following the test’s instructions. The calculation of the PI was as follows:
PI = 100 x [1 – (OD sample ÷ OD negative control)]. Any test sample with a PI > 40% was classified as positive. 64
These tests had not previously been validated in yaks, however the reported sensitivity
(Se) and specificity (Sp) in Bos taurus of the C-ELISA were 98 and 99.9%, respectively and for the RBT 81.2 and 86.3%, respectively (Gall and Nielsen, 2004). The serological status of individual yaks was determined by interpreting the tests in parallel where a sample was classified as positive if at least one of the tests was positive (Kabagambe et al., 2001; Sanogo et al., 2012). The calculated sensitivity and specificity of the tests interpreted in parallel were 99.6 and 86.2%, respectively. For a herd to be classed as positive at least one seropositive yak was required to be detected in the sampled herd.
4.2.5 Questionnaire design and implementation
A questionnaire was administered in person to the herders responsible for the yaks.
Questions were included on the animal’s basic information (sex, age, production system), the routine management and husbandry practices adopted by the herder, composition of the herd, the presence of any clinical signs and interaction of livestock with other animals. The questionnaire was initially designed in English (Appendices 1 and 2) and then translated into Chinese and Tibetan for use in the field. The questionnaire was initially read by and discussed with veterinarians serving both the
TLRI and Veterinary Department of the three counties before administering to the selected participants. After the surveys had been administered the questionnaire data were entered into Epi Info 7 (http:/www.cdc.gov/epiinfo) (CDC, Atlanta, GA, USA).
4.2.6 Preliminary analyses
Data were exported from Epi Info to Microsoft Excel (Microsoft Excel 2010, Redmond,
USA). Seroprevalence and 95% confidence intervals were calculated using the exact 65
binomial method in the contributed R package epitools in R version 3.1.2 (R
Development Core Team, 2013). Statistical methods, such as the χ2 test, Fisher’s Exact test and OR (odds ratios) and their 95% confidence intervals (CI) (Dohoo et al., 2012), were applied to the data.
4.3 Results
4.3.1 Sampling profile
A total of 1,523 yaks belonging to 181 herders originating from 30 villages were sampled in the three selected counties. Of these animals 640 belonged to 54 herders in
Damxung county; 643 to 105 herders from Maizhokunggar county and 240 to 22 herders from Yadong county (Table 4.1). The median age of the sampled yaks was 4 years with an average age of 4.6 years (range: 1 - 19 years old). The numbers of the yaks aged ≤ 2 years old, 3 - 5 years old and ≥ 6 years old were 393, 658 and 472 heads, respectively. A similar number of female and male yaks were sampled (777 and 746, respectively). The majority (67%, n=1,016) of the sera collected were from yaks raised under the pastoral production system with the remaining 33% (n=507) raised under an agro-pastoral production system. The median herd size was 40 head, with an average herd size of 48.1 (range: 4 - 170).
4.3.2 Seroprevalence at the animal level
The seroprevalence on the RBT was 1.8% (95%CI: 1.2, 2.6) which was similar to the C-
ELISA result of 1.5% (95%CI: 1.0, 2.3) (p=0.67, χ2=0.183). The results of the two tests are presented in Table 4.2. When the tests were interpreted in parallel, the seroprevalence of antibodies against brucellosis was 2.8% (95% CI: 2.0, 3.7).
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Table 4.2 Serological detection of antibodies against Brucella using the RBT and C- ELISA
Serological results Number of animals Test Prevalence (%) 95% CI RBT(+) 27 1.8 1.2, 2.6 C-ELISA(+) 23 1.5 1.0, 2.3 RBT(+) C-ELISA(+) 8 0.5 0.2, 1.0 RBT(+) C-ELISA(-) 19 1.3 0.8, 1.9 RBT(-) C-ELISA(+) 15 1.0 0.6, 1.6 RBT(-) C-ELISA(-) 1,481 97.2 96.3, 98.0 RBT(+) or C-ELISA(+) 42 2.8 2.0, 3.7
Overall, Tangjia township had the highest test seroprevalence of 4.9% (95%CI: 1.6,
11.3), followed by Zhaxigang township (3.8%; 95%CI: 1.4, 8.0). The three townships of
Menba, Riduo and Yangbajing had the same test seroprevalence of 3.1% (95%CI: 1.0,
7.1). Geda and Namucuo had the lowest seroprevalences of 1.3% each (95%CI: 0.2, 4.4)
(Table 4.3). Overall there was no significant difference in the test seroprevalences both between townships (p=0.55, χ2=6.85, df=8) and between counties (p=0.14, χ2=3.95, df=2).
Table 4.3 Animal level apparent (test) prevalence of brucellosis in yaks in the three counties sampled in Tibet
County Township Animal level
No. tested No positive Prevalence (%) 95%CI
Tangjia 162 8 4.9 2.2, 9.5
Menba 161 5 3.1 0.4, 5.8 Maizhokunggar Riduo 160 5 3.1 0.4, 5.8
Zhaxigang 160 6 3.8 0.8, 6.7
Geda 160 2 1.3 0.2, 4.4
Longren 160 4 2.5 0.7, 6.3 Damxung Namucuo 160 2 1.3 0.3, 4.4
Yangbajing 160 5 3.1 1.0, 7.1
Yadong Pali 240 5 2.1 0.7, 4.8
Overall results 1,523 42 2.8 2.0, 3.7
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4.3.3 Herd level seroprevalence results
A total of 33 herds (18.2%; 95% CI: 12.9, 24.6) contained one or more seropositive animals (Table 4.4). The highest herd-level seroprevalence of 30.8% (95%CI: 9.1, 61.4) was observed in Yangbajing township, followed by Riduo and Zhaxigang townships.
The lowest herd seroprevalence (6.7%, 95% CI: 2.1, 48.8) was detected in Namucuo township. The herd-level seroprevalence among the nine townships was not significantly different (p=0.87, χ2=3.83, df=8). The geographical distribution of the herd seroprevalence is displayed in Figures 4.2 - 4.4.
Table 4.4 Herd level apparent (test) seroprevalence of brucellosis in yaks in the three sampled counties
County Township Herd level
No. tested No positive Prevalence (%) 95% CI
Tangjia 37 6 16.2 6.2, 30.0
Menba 34 5 14.7 5.0, 31.1 Maizhokunggar Riduo 14 4 28.6 8.4, 58.1
Zhaxigang 20 4 20.0 5.7, 43.7
Geda 20 2 10.0 1.2, 31.7
Longren 9 2 22.2 2.8, 60.0 Damxung Namucuo 12 2 6.7 2.1, 48.8
Yangbajing 13 4 30.8 9.1, 61.4
Yadong Pali 22 4 18.2 5.2, 40.3
Overall results 181 33 18.2 12.9, 24.6
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Figure 4.2 Map showing the distribution of brucellosis seropositive and seronegative herds sampled in Maizhokunggar County
Figure 4.3 Map showing the distribution of brucellosis seropositive and seronegative herds sampled in Damxung County
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Figure 4.4 Map showing the distribution of brucellosis seropositive and seronegative herds sampled in Yadong County
4.3.4 Seroprevalence in yaks raised under different production systems
At the individual animal level using the data from Table 4.5, the seroprevalence in yaks raised under the agro-pastoral production system was significantly higher than those raised on pasture (4.5%; 95%CI: 2.9, 6.7; 1.9%; 95%CI: 1.1, 2.9, respectively) (p=0.003,
χ2=8.97, df=1). The highest seroprevalence of 5.0% (95%CI: 1.6, 11.3) was observed in two villages of Riduo township, whereas no seropositive animals were found in a village of Riduo and Zhaxigang townships (95%CI: 0, 6.0 and 0, 9.7, respectively).
The herd seroprevalence in yaks grazed under the agro-pastoral production system was
20.9% (95%CI: 12.9, 31.0) which was similar to that of herds grazed exclusively on a pasture system (15.8%; 95% CI: 9.1, 24.7) (p=0.371, χ2=0.80, df=1). The highest herd- level seroprevalence of 40% (95%CI: 12.2, 73.8) was observed in two villages in Riduo township which adopted an agro-pastoral production system, while two villages located
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in the townships of Riduo and Zhaxigang which adopted a pastoral production system had no evidence of infection (Table 4.5).
Through comparing the herd and animal seroprevalence in the three townships of
Menba, Riduo and Zhaxigang, which had herds adopting the two different production systems, no significant difference was observed between the seroprevalence for the two production systems (all p values ≥ 0.16) (Table 4.6).
4.3.5 Seroprevalence of vaccinated and unvaccinated yaks
All yak calves in Pali township, Yadong county had been vaccinated with S2. There was no significant difference in either the individual or herd level seroprevalence in vaccinated and unvaccinated yaks (p=0.49, χ2=0.48; p=0.99, χ2=1, respectively) (Table
4.7).
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Table 4.5 Herd and animal level apparent (test) seroprevalence of brucellosis in yaks reared under two production systems in Tibet
Production system Townships Individual seroprevalence Herd seroprevalence Ni Prevalence (%) 95%CI Nh Prevalence (%) 95% CI
Tangjia 162 4.9 2.2, 9.5 37 16.2 6.2, 30.0 Menba 121 3.3 0.9 ,8.2 24 16.7 4.7, 37.4 Agro-pasture Riduo 100 5 1.6, 11.3 10 40.0 12.2, 73.8 Zhaxigang 124 4.8 1.8, 10.2 15 26.7 7.8, 55.1 Total 507 4.5 2.9, 6.7 86 20.9 12.9, 31.0 Menba 40 2.5 0.1, 13.2 10 10.0 0.3, 44.5 Riduo 60 0.0 0.0, 6.0 4 0.0 0.0, 60.2 Zhaxigang 36 0.0 0.0, 9.7 5 0.0 0.0, 52.2 Geda 160 1.3 0.2, 4.4 20 10.0 1.2, 31.7 Exclusively pasture Longren 160 2.5 0.7, 6.3 9 22.2 2.8, 60.0 Namucuo 160 1.3 0.3, 4.4 12 6.7 2.1, 48.8 Yangbajing 160 3.1 1.0, 7.1 13 30.8 9.1, 61.4 Pali 240 2.1 0.7, 4.8 22 18.2 5.2, 40.3 Total 1,016 1.9 1.1, 2.9 95 15.8 9.1, 24.7 Overall results 1,523 2.8 2.0, 3.7 181 18.2 12.9, 24.6
Ni: number of individual yaks sampled; Nh: number of herds sampled.
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Table 4.6 Comparison of seroprevalence in individual yaks and herds adopting different production systems in three townships containing herds adopting both production systems
Number positive/total (%) Number positive/total (%) Township Category P* (Agro-pastoral) (exclusively pastoral) Menba Individual level 4/121 (3.3) 1/40 (2.5) 1.0 Herd level 4/24 (16.7) 1/10 (10.0) 1.0
Riduo Individual level 5/100 (5.0) 0/60 (0.0) 0.16 Herd level 4/10 (40.0) 0/4 (0.0) 0.25
Zhaxigang Individual level 6/124 (4.8) 0/36 (0.0) 0.34 Herd level 4/15 (26.7) 0/5 (0.0) 0.53
*Fisher’s exact test
Table 4.7 Comparison of apparent (test) seroprevalence in individuals and herds from vaccinated and non-vaccinated yaks
Individual seroprevalence Herd seroprevalence
Number Number Prevalence Number Number Prevalence OR (95%CI) P OR (95%CI) P positive sampled (%) positive sampled (%) Unvaccinated 37 1283 2.88 1.36 (0.58, 4.04) 0.49 29 159 18.24 1.02 (0.27, 3.03) 0.99
5 240 2.08 1.0 4 22 18.18 1.0 Vaccinated
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4.4 Discussion
The study further indicated that at the time of sampling brucellosis was endemic in yaks in Tibet. Although the seroprevalence in individual yaks was relatively low (2.8%), the herd seroprevalence was higher (18.2%). A higher animal level seroprevalence in yaks than that found in this study has been reported in India (21.1%) and Nepal (22%)
(Bandyopadhyay et al., 2009; Jackson et al., 2014). In China the seroprevalence in yaks has varied between locations being 6.5 to 13.4% in the Qinghai-Tibet plateau (Lang et al., 2011; Gao et al., 2013; He et al., 2015), 5.2 to 12.8% in Xinjiang autonomous region (Dilixiati. et al., 2001; Yuan et al., 2014) and 4.2% in Sichuan Province (Li et al.,
2008). One or both of the tests used in the current study were also applied in these other
Chinese studies. The different seroprevalences between regions/places could be associated with different management and husbandry practices, animal densities, geographical characteristics, transport practices adopted or the sampling protocol adopted.
In order to relieve the pressure on degraded grasslands and to improve rangeland ecosystems, Tibet has implemented regulations to restrict grazing on pasture. Pasture has been fenced and allocated to different households (Miller, 1999b; Wang et al., 2014)
(Figure 4.5), and a limit has been placed on the number of yaks that can be owned by individual households (Lan, 2013). These measures may restrict livestock movement with the potential to reduce the opportunities of contact between herds, and hence may potentially reduce the likelihood of disease transmission between herds within the region.
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Figure 4.5 Yaks grazing in fenced areas
It is apparent from this study that the disease is endemic in yaks on the Tibetan plateau.
It is possible that the introduction of yaks to improve the local genetics, and through movement of yaks for trade and nomadic grazing purposes have resulted in disease spread (Bertu et al., 2012; Boukary et al., 2013). Yaks in the study areas are recognised as of superior quality and because of this are frequently exchanged and exported to other areas for breeding purposes. This movement of animals has the potential to play a key role in the distribution of the disease to other areas of China. In addition, two national highways traverse the counties of Damxung and Maizhokunggar, as well as a railway line passing through Damxung, resulting in more trade between Tibet and mainland China. Furthermore Yadong county borders Bhutan and India allowing for potential cross-border movement of yaks. Tibet also has an overall low temperature due to its high altitude allowing extended environmental survival of Brucella organisms
(Aune et al., 2012), potentially resulting in exposure of more yaks to the pathogen.
Traditional customs and methods of raising livestock may also play a role in the disease’s spread and transmission. Herders slaughter animals themselves when the 75
livestock are old, and the higher prevalence in older animals is likely to lead to a greater risk of disease transmission (Mekonnen et al., 2010). Finally, with the development of
Tibet, nomadic practices have been replaced by an agro-pastoralist system which has significantly changed people’s diet and lifestyle resulting in more unhygienic living conditions, degraded quality of fodder and consumption of dirty water, while at the same time providing improved road transportation. These changes could increase the public health risk for zoonotic diseases, such as brucellosis (Xu et al., 2008; Jackson et al., 2014).
The seroprevalence at the individual level in animals grazed under the agro-pastoral production system was significantly higher than the seroprevalence on yaks raised under an exclusively pastoral production system (p < 0.05). However, at the herd level there was no statistical significant difference (p = 0.371). These differences may result from a variety of reasons including the management and husbandry methods adopted within each system, the herd size and its structure and the number of herds sampled.
False positive results for brucellosis can arise from cross-reactions with other agents such as Yersinia enterocolitica O:9, Escherichia coli 0157:H7, Xanthomonas maltophilia and Salmonella urbana (Alton et al., 1988; Weynants et al., 1996; Mainar-
Jaime et al., 2005; Nielsen and Yu, 2010). In the present study, the RBT and C-ELISA were adopted as diagnostic tests since they are convenient and the ELISA can detect
IgM, IgG1 and IgG2 against Brucella (Lamb et al., 1979). The diagnostic tests used in this study, however, have not been validated in yaks and this should be undertaken to ensure these tests are suitable for use in this species, even though they have been used in other studies involving yaks (Bandyopadhyay et al., 2009; He et al., 2015). In addition, 76
B. suis strain 2 vaccine was administered to yak calves in Pali township. This vaccine is locally produced and was used successfully for the control of brucellosis in the 1980s
(Pan, 1992b). As agglutinating antibodies induced by the vaccine are reported to last for approximately four months post-vaccination (Han et al., 2009), in Pali all yaks sampled were older than two years of age and consequently it is highly likely that any positive results were a result of natural infection rather than a vaccination induced one. However in this study no significant difference was observed in the seroprevalence between vaccinated and non-vaccinated yaks, indicating that the vaccine was not effective in reducing the seroprevalence. Because of this finding, vaccinated yaks were included in the overall prevalence calculations.
Conducting a serological investigation is an important component of the diagnostic techniques to explore a disease within a population (Thrusfield, 2005). Through the study the seroprevalence and distribution of brucellosis in yaks at the animal and herd levels raised under different production systems and with different histories of vaccination were explored and analysed. It was evident that the disease is a significant problem potentially affecting the development of the local industry and having an impact on the income of yak herders and their families. Therefore, in order to control disease it is important to identify risk factors for disease so that preventive measures can be adopted and practices changed, and this forms the basis of the following chapter.
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CHAPTER FIVE
Risk factor analysis
5.1 Introduction
Despite the adoption of annual surveillance for brucellosis in Tibet in 2009 and the implementation of control measures for the disease, brucellosis continues to exist in
Tibet. The epidemiology of brucellosis is complex with multiple factors affecting the distribution of the disease. These include stocking density and herd size, age, sex and contact with wildlife (Sanogo et al., 2012; Jackson et al., 2014; Alhaji et al., 2016; De
Alencar Mota et al., 2016). Furthermore the introduction of an infected animal into a previously non-infected herd is the main route for transmission of the disease (Kellar et al., 1976; Nielsen and Duncan, 1990). It is critical to understand the risk factors for a disease before developing and implementing a prevention or control program. The aim of the study reported in this chapter was to identify the risk factors for seropositivity to brucellosis in yaks in order to better understand the epidemiology of the disease to allow development of more effective control programs.
5.2 Materials and methods
In order to identify the potential risk factors for seropositivity to brucellosis in yaks in
Tibet, a structured questionnaire (Chapter 4) was used when conducting the serological survey. Data from the serological testing of yaks (Chapter 4) and the results of the questionnaire administered to the herders were entered into Microsoft Excel (Microsoft
Excel 2010, Redmond, USA) and analysis was carried out using the contributed R packages epitools (Aragon, 2012), foreign (R Development Core Team, 2015), Mass
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(Venables and Ripley, 2002), rms (Harrell, 2016) and ResourceSelection (Lele et al.,
2016) in R version 3.1.2 (R Development Core Team, 2013). Only data for 1,283 non- vaccinated yaks from Damxung and Maizhokunggar were included in the risk factor analyses.
The serological results were dichotomised as seropositive and seronegative, and the information on animals and herds were categorised by age (≤2, 3-5, ≥6 years), sex
(female or male), herd size (<50, ≥50 yaks), locality (Damxung or Maizhokunggar) and type of production system (agro-pastoral or pastoral) (Table 5.1).
Table 5.1 Characteristics of the sampled yaks and herds in the two counties (n=1,283)
Variable/category Number sampled Percentage (%) Gender
Female 606 47 53 Male 677
Age (years)
>5 432 34 3-5 551 43 23 <3 300
Herd size
≥ 50 662 52 48 < 50 621
County
Damxung 640 50 50 Maizhokunggar 643
Production system Agro-pastoral 507 40 Pastoral 776 60
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A binary logistic regression model was developed to assess the association between seropositivity to brucellosis and potential risk factors (demographic and management information). The results from the yaks from Pali township were excluded from this analysis due to a history of vaccination which may have potentially interfered with results, even though no significant difference was found between vaccinated and non- vaccinated animals and herds (Chapter 4). Univariable analyses were initially performed on the data with the χ2 test, Fisher’s exact test and OR and their 95% CI (Chapter 4).
Variables with a p ≤ 0.20 were then evaluated using a multivariable logistic regression analysis. A multivariable logistic regression model was generated using a backward stepwise process to control for confounding and to test for effect modification. Odds ratios (OR) and their 95% confidence intervals were calculated to evaluate the association between seropositivity and the variables. Variables with a p < 0.05 were retained in the final model. The model was evaluated by conducting the likelihood ratio test, and the model with the smallest AIC (Akaike Information Criterion) value was regarded as the most appropriate (Boukary et al., 2013). The model was then assessed with the Hosmer-Lemeshow goodness-of-fit test. Additionally, a Receiver Operating
Characteristic (ROC) curve was generated to display the predictive accuracy of the model (Sing et al., 2005).
5.3 Results
5.3.1 Univariable and multivariable logistic regression analyses of risk factors for seropositivity in individual yaks
1,283 yaks that were distributed in 159 herds of two counties of Maizhokunggar and
Damxung were used in these analyses. The four factors (age, herd size, county and production system) all had p values in the univariable analyses ≤ 0.20 and were selected 80
for inclusion in the saturated multivariable logistic regression model for seropositivity in individual yaks (Table 5.2).
In the final multivariable logistic-regression model only the production system and age of animals were significantly associated (p < 0.05) with seropositivity in yaks (Table
5.3). Yaks reared under the agro-pastoral production system (OR=2.90, 95% CI: 1.48,
5.86) and those that were >5 years old (OR=3.89, 95%CI: 1.23, 17.21) and between 3 and 5 years old (OR=4.51, 95%CI: 1.53, 19.29) had significantly higher odds of seropositivity than yaks raised under the pastoral production system and those that were
<3 years old (Table 5.3). The likelihood ratio test (χ2=16.37, p<0.001), AIC value of the final model (326.95) and the Hosmer-Lemeshow goodness-of-fit value (p=0.46), indicated that the model was a good fit for the data. The area under the ROC curve was
0.61, indicating that the model had moderate to good predictive ability.
Table 5.2 Univariable analysis of risk factors associated with brucellosis seropositivity among individual yaks in Tibet using the results of the Rose Bengal Test (RBT) and the competitive enzyme linked immunosorbent assay (C-ELISA) interpreted in parallel.
Variable Categories Prevalence (%) OR (95%) p-Value Age (years) >5 3.01 1.27 (0.63, 2.65) 0.06* 3-5 3.81 3.75 (1.27, 16.63)
<3 1.00 1.0
Sex Female 3.14 1.18 (0.61, 2.31) 0.61 Male 2.66 1.0
Herd size ≥ 50yaks 1.81 1.0
< 50 yaks 4.03 2.26 (1.14, 4.71) 0.02*
County Damxung 2.03 1.0 0.07* Maizhokunggar 3.73 1.86 (0.95, 3.81)
Production system Agro-pastoral 4.54 2.58 (1.32, 5.20) 0.004* Pastoral 1.80 1.0
*p ≤ 0.20 and offered to the multivariable logistic regression model
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Table 5.3 Multivariable logistic regression analysis of risk factors associated with brucellosis seropositivity among individual yaks
Risk factors Category levels Estimate SE OR 95%CI P Constant -5.28 0.64
Production system Pastoral 1.0
Agro-pastoral 1.06 0.35 2.90 1.48, 5.86 0.002
Age >5 1.36 0.65 3.89 1.23, 17.21 0.037 3-5 1.51 0.62 4.51 1.53, 19.29 0.016
<3 1.0
Estimate: Regression coefficient; SE: Standard error; CI: Confidence interval
5.3.2 Analyses of the seroprevalence data at the herd level
Herds with a history of abortion in the year preceding the survey were more likely to be seropositive (OR=4.98, 95%CI: 1.48, 16.62) than herds without abortions. No other management or husbandry variables produced a significant association with seropositive herds (all p > 0.20) (Table 5.4). Therefore, a multivariable model could not be generated for these data.
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Table 5.4 Descriptive statistics and univariable associations between seropositivity (herd level) and potential management risk factors in yaks in Tibet (n=181)
Variables Category Totala Prevalence (%) OR(95%CI) p-Value Isolate newly purchased yaks No 73 19.18 1.15 (0.37, 4.18) 0.73 Yes 82 17.07 1.0
Graze yaks with other herders' No 8 12.50 1.0 1.0* yaks Yes 151 18.54 1.43 (0.23, 37.41)
Purchased new yaks last year No 148 17.57 1.0 0.42* Yes 11 27.27 1.80 (0.36, 6.89)
Separate sick yaks from healthy No 15 20.00 1.17 (0.24, 4.09) 0.74* ones Yes 144 18.06 1.0
Wear gloves to dispose of aborted No 145 19.31 2.75 (0.51, 69.06) 0.47* foetuses Yes 14 7.14 1.0
Yaks mixed with sheep and goats No 135 18.52 1.11 (0.37, 4.18) 1.0* when grazing Yes 24 16.67 1.0
Yaks migrate to other pastures No 139 18.71 1.25 (0.38, 5.93) 1.0* Yes 20 15.00 1.0
Bull mates own or other herder's Both 46 13.04 1.0 0.28 yaks Own only 113 20.35 1.67 (0.66, 4.88)
History of brucellosis infection No 124 18.54 1.08 (0.42, 3.21) 0.86 (RBT) Yes 35 17.14 1.0
Any abortions during the last 12 No 81 11.11 1.0 0.004 months Yes 18 38.89 4.98 (1.48, 16.62) a Not all farmers answered all questions *Results from conducting a Fisher’s exact test
5.4 Discussion
The presence of a disease is associated with a range of factors including host, environmental and agent factors (Thrusfield, 2005). These factors are often confirmed by conducting epidemiological studies. This study is the first epidemiological study that explores the risk factors for brucellosis in yaks managed under different production systems in Tibet, despite the fact that there is an official brucellosis surveillance program in place for the region.
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A possible limitation of the current study is that the TBAA has started restricting the number of grazing livestock to reduce degradation of grassland regions (Lan, 2013), and herders may have been reluctant to provide details about the actual number of livestock owned if it conflicted with the new government policy.
At the individual animal level, age and production system were significant in the logistic regression model. Other studies have similarly reported an increased seroprevalence associated with age (Kebede et al., 2008). This is likely to be related to an increased chance of contact with the bacterium through environmental exposure to contaminated materials such as aborted foetuses, fluids and milk. In this study animals between 3 and 5 years old had the highest risk of infection. This period coincides with the onset of sexual maturity. If cattle are infected with Brucella species, in most cases the abortion (if it occurs) happens during the first pregnancy after infection rather than subsequent pregnancies. Therefore, it is common that first calf heifers are more susceptible to Brucella-related abortion (Muma et al., 2007a; Matope et al., 2011). This may have been responsible for the higher seroprevalence reported in the current study for yaks between 3 and 5 years of age.
The study also identified the production system as a risk factor for seropositivity, which has also been highlighted in previous studies (Jiwa et al., 1996; Jergefa et al., 2009).
The lower population density associated with a pastoral system would decrease potential exposure to Brucella, while higher stocking density in agro-pastoral systems is likely to increase the opportunity for non-infected animals to have contact with infected yaks or a Brucella-contaminated environment (Omer et al., 2000a). 84
Others have reported a higher seroprevalence in larger herds (Mekonnen et al., 2010;
Jackson et al., 2014) which contrasted with this study, where smaller herds (< 50 head) were more likely to be infected, although only in the univariable analysis (p<0.05,
OR=2.26; 95% CI 1.14, 4.71). This finding may be confounded either by the area of land owned/managed or by unreliable answers due to the local policy limiting the number of livestock as discussed previously. It is likely that stocking density would be a more appropriate method of determining risk of infection rather than herd size for future analyses.
It has been reported that brucellosis is more common in female animals, although this may be influenced by the fact that females are kept for longer periods than the majority of males and clinical signs of disease are also more apparent in females than males
(Muma et al., 2006; Mekonnen et al., 2010). The current study found no significant difference in prevalence between males and females (p=0.61). This may be explained by the similar management style the different genders are subjected to in Tibet. This included grazing on communal pastures, drinking water from communal sources and being of a similar aged (male mean age: 4.37 years old and female mean age: 5.15 years old). A similar prevalence in adult males and females has also been reported in other studies (Sanogo et al., 2012; Al-Rawahi, 2014).
In this study, a history of abortions within herds in the 12 months preceding the survey was strongly linked to seropositivity (p < 0.05). This is consistent with other studies and is not unexpected given the association between infection and abortion (Muma et al.,
85
2007a; Megersa et al., 2011a; Alhaji et al., 2016). This also explains why brucellosis was previously referred to as “contagious abortion” (Romich, 2008).
Tibet is the main pastoral province in China, and grazing animals have historically been an important aspect of the community (Lan, 2013). Mixed grazing at communal pastures is also very common in Tibet (Figure 5.1). Godfroid et al. (2013a) reported that mixed farming, particularly rearing sheep and/or goats with cattle, was a major risk factor for transmission of Brucella spp.. This may be explained by greater animal to animal contact between species or the increased potential for contact with contaminated materials on the pasture (Al-Rawahi, 2014). It is likely that the husbandry practices adopted in Tibet increased the risk of infection for other animals and herds. It was reported that in the 1970s in Tibet B. melitensis was the most prevalent Brucella species among sheep and goats. This species was also isolated from yaks, and B. abortus has similarly been isolated from sheep and goats (Laba et al., 1992; Pan, 1992a). In this study, the majority of herders (71%, n=129) raised other livestock, such as sheep and goats, as well as yaks. Some surveys conducted in other areas of China have shown that the seroprevalence in yaks is higher than in sheep or cattle. For example, a study in the
Tian mountain region of Xinjiang Autonomous Region found that the seroprevalence in yaks, sheep and cattle were 12.8, 0.69 and 0.23%, respectively (Yuan et al., 2014).
Therefore, in order to better understand the transmission and distribution of the disease, a serological survey should be extended to include other species raised by the local farmers in Tibet.
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Figure 5.1 Picture showing yaks, sheep and goats co-grazing on grasslands in Tibet
In conclusion, this study produced epidemiological data to show the association between brucellosis in domestic yaks and age and production system. It also highlighted the relationship in herds that had a history of abortion in the 12-month period that preceded the serological survey. To minimise the spread of infection between species it is recommended that the survey be extended to include sheep and goats. In order to better control brucellosis, there is also a need to better understand what herders currently know about the disease and their attitudes toward different methods of disease control to ensure that programs are relevant and delivered in a culturally acceptable manner. In the following chapter the knowledge, attitudes and practices of the farmers with respect to brucellosis in yaks is investigated to address this deficiency.
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CHAPTER SIX
A study on the knowledge, attitudes and practices of herders with
respect to brucellosis in yaks
6.1 Introduction
Brucellosis is a zoonotic disease which can affect a wide variety of terrestrial and marine mammals (Seleem et al., 2010). It is classified as a Class II reportable disease by the CDC of China due to the fact that it is an endemic disease in most provinces of
China, including Tibet (Deqiu et al., 2002; Seleem et al., 2010). Brucellosis is transmitted between animals either by ingestion of contaminated materials, such as aborted foetuses and uterine discharges, or by contact (Romich, 2008), and the main clinical signs are abortion and infertility. In humans infection is generally acquired occupationally through handling infected cows or their tissues or discharges, however indirect transmission can occur through the consumption of unpasteurised milk or dairy products. Due to its variable incubation period and chronic or asymptomatic nature, in humans the disease is frequently misdiagnosed, underreported or its diagnosis is delayed (Saurent and Vilissova, 2002; Bax et al., 2007), and it is considered to be a neglected disease globally (Pappas et al., 2006; McDermott et al., 2013).
In China brucellosis in humans was considered a severe disease from the mid-1950s until the 1970s, although the incidence subsequently decreased until the late 1990s. In the 21st century, the disease re-emerged as a problem and human brucellosis is now regarded as widespread in the country (Zhang et al., 2010; Cao et al., 2013; Zhong et al.,
2013). Studies have highlighted the higher risk of infection in farmers and pastoralists
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(Racloz et al., 2013) through their contact with livestock, and occupational groups such as veterinarians and abattoir workers (Tian et al., 2012; Liu et al., 2013; Li et al., 2014;
Liu, 2014b). The Tibetan plateau in China is one of the largest pastoral regions of the world where approximately 10.4 million sheep, 5.2 million goats, 3.9 million yaks and 1 million cattle are reared (Tashi et al., 2005). In this region livestock production plays an important role in the community (Miller, 1999a) with the yak featuring as an important animal because of its production of milk, meat, fibre and hides and its potential for use as a draft animal. The results of the survey outlined in Chapter 4 identified an individual and herd-level seroprevalence of brucellosis in yaks of 2.8% (95%CI: 2.0, 3.7) and 18.2%
(95%CI: 12.9, 24.6), respectively, together with evidence of increasing prevalence in humans (Zhandui. et al., 2008). Control programs in humans and livestock of Tibet have focussed on annual serological surveillance programs since 2009 (Chapter 3). In order to design and implement an effective disease control program in yaks, it is not only important to understand the epidemiology of the disease in the livestock, but also to understand the knowledge, attitudes and practices (KAP) of local pastoralists. Some studies to determine the awareness of farmers and herders about brucellosis have been conducted in other countries, including one conducted in Tajikistan which highlighted that farmers had poor knowledge about brucellosis and practiced high-risk practices
(Lindahl et al., 2015) and another in Uganda which reported that participants had a moderate level of knowledge about brucellosis (Kansiime et al., 2014). However prior to this study, no similar study had been conducted in China. Consequently the study that is described in this chapter was undertaken to investigate the KAP of local pastoralists with respect to brucellosis and disease control measures in the three regions previously sampled in Tibet (Chapter 4).
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6.2 Materials and methods
6.2.1 Study area
This survey was conducted in the same counties as those sampled in the serological study (Chapter 4) (Maizhokunggar and Damxung County, and Pali township of Yadong county). All households whose yaks were sampled as part of the serological study from the 9 townships were surveyed. In addition, the number of households in Pali township surveyed was increased to 58 from the 22 households who had their yaks sampled, and similarly in Ningzhong township of Damxung county 100 households were interviewed
(Table 6.1 and Figure 6.1). There are a total of 6,460, 7,000 and 575 households, respectively in Damxung County, Maizhokunggar County and Pali township of Yadong
County, for a total of 14,035 households representing approximately 83,099 inhabitants
(Zhang, 2013b; Anonymous, 2015).
6.2.2 Questionnaire design
A structured questionnaire was initially developed in English and then translated into
Chinese and Tibetan and delivered in a face to face interview with the pastoralists. The questionnaire focused on three main areas: the respondent’s socio-demographic information (age, gender, literacy, occupation, number of people in the household); their knowledge about infection, clinical signs, species infected and current treatments (if any) administered for brucellosis; and the attitudes and practices adopted including husbandry and management practices, milk consumption habits and control measures applied (Appendix 2).
Prior to the start of the actual survey, the purpose of the research was explained to the veterinarians who were conducting the face-to-face interview with the selected herders 90
at both the Tibet Livestock Research Institute and the three counties and training provided in administering the questionnaires. The questionnaire was provided to the veterinarians to ensure they were familiar with its contents and to enable discussion on its purpose prior to the training.
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Table 6.1 Distribution of households in villages of the three counties surveyed for the knowledge, attitudes and practices study
Number of villages in different production systems Number of Percentage of all County Township Agro-pastoral Pastoral households surveyed households surveyed (%)
Damxung Namucuo 2 villages 12 4
Longren 3 villages 9 3
Ningzhong 4 villages 100 32
Yangbajing 3 villages 13 4
Geda 2 villages 20 6
Maizhokunggar Tangjia 4 villages 37 12
Zhaxigang 4 villages 1 village 20 6
Menba 3 villages 1 village 34 11
Riduo 2 villages 1 village 14 4
Yadong Pali 4 villages 58 18
Total 13 villages 21villages 317 100
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Figure 6.1 Map of locations of three counties of Yadong (left), Damxung (middle) and Maizhokunggar (right) surveyed in Tibet with the number and overall percentage of households interviewed in the respective counties
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6.2.3 Procedure
The sample size was calculated using EpiTools (Sergeant, 2015) based on 95% confidence, a 5% desired precision and 20% expected knowledge (equivalent to 20% positivity) (Lindahl et al., 2015). A total of 246 households were required. Due to the fact that the number of households in the first survey was 181 including 22, 54 and 105 in Pali township, Damxung and Maizhokunggar county respectively, the second survey only focused on Pali township of Yadong county and Damxung county to increase the sample size.
A total of 317 households from 34 villages were interviewed from April to August 2015.
Of these households, yaks from 181 of them had been tested (Chapter 4) to determine the seroprevalence of bovine brucellosis. The study outlined in this chapter was approved by the Human Ethics Committee of Murdoch University (Project No:
2015/010).
Four townships (Tangjia, Menba, Zhaxigang and Riduo) in Maizhokunggar county, five townships (Geda, Yangbajing, Longren, Namucuo and Ningzhong) in Damxung county and one township (Pali) in Yadong county were selected for inclusion in the study as previously outlined in Chapter 4 and their location and details are displayed in Figure
6.1 and Table 6.1. An essential criterion for inclusion of the household was owning yaks.
Villages and households were randomly selected from the selected townships. If a selected household declined to be interviewed, a replacement was selected. Surveys were administered to the person who had the most experience with the livestock (yaks) in each household and were required to be older than 20 years of age.
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6.2.4 Data management and analyses
Data were entered into a database (Epi Info version 7 - CDC, Atlanta, GA,USA) and then converted into Microsoft Excel 2010 (Microsoft Corp., Redmond, WA,USA), and analysed for descriptive statistics and model building using the statistical software R (R
Development Core Team, 2013) as described in Chapter 5.
Descriptive statistics were used to describe the demographic and socio-demographic data (Table 6.2). Differences in responses between gender (male vs female) and between different production systems (pastoral production vs agro-pastoral production) were analysed with either the χ2 or Fisher’s exact tests.
Twelve questions on the pastoralists’ knowledge about brucellosis and eight questions on their perceptions of the disease and the practices they adopted were included in the survey (Tables 6.3 and 6.6). For these questions a correct answer was scored as 1 and an incorrect answer as 0. The results of these questions were summed and the median score for all participants determined. People with a score < 6 (the median) were categorised as having poor knowledge whereas those with a score ≥ 6 (the median) were classified with good knowledge. Similarly, people with an attitude and practice score < 6 were categorised as adopting potentially risky practices and having a poor understanding about the disease while those with a score ≥ 6 were categorised as adopting good practices and having a good understanding of the disease (Addo et al., 2011). A binary logistic regression model was then built for the binary outcome (as outlined in Chapter
5). For the independent variables, age was categorised into two groups (<50 and ≥ 50 years), and educational level was also categorised (no formal education and primary/secondary level education). 95
6.3 Results
In this study, five households from Maizhokunggar county, representing 1.6% of all respondents, declined to be involved in the survey. These households were replaced with another five randomly selected households from this county.
6.3.1 Demographic characteristics
The median age of the 317 respondents was 49.5 years (mean 50.1 years; range 20-80 years). 82% of the respondents (259) were male and only 18% female. Approximately one-third (101) of the respondents had not received any formal schooling (education).
Half of the respondents were ≥50 years old. The size of the households (number of people - < 6 and ≥ 6 people) included in this study were similar (Table 6.2).
Table 6.2 Demographic characteristics of respondents in the surveyed areas of Tibet (n=317)
Variables Category n Percentage Gender Female 58 18.3 Male 259 81.7
Age (years) <50 157 50.0 6 ≥50 157 50.0
Missing data 3
Educational level No education 106 33.4 Primary/Secondary school 211 66.6
Number of persons in <6 116 38.2 the household ≥6 188 61.8 Missing data 13
Production System Pastoral 231 72.9 Agro-pastoral 86 27.1
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6.3.2 Knowledge
In Table 6.3 the bivariate analyses of the respondents’ knowledge is summarised. In total, 58.4% of the respondents had heard that livestock of other herders in their village had abortions. Among those who had heard of abortions in livestock belonging to other herders in their village, 27.6% of the respondents had known the cause of these abortions. Approximately sixty percent (60.6%) of the respondents had heard of the disease brucellosis. Among those who had heard of the disease most people knew: the disease affected yaks as well as sheep and goats; it could be prevented in yaks; there was a control program for the disease; and vaccination was an effective way to control the disease. Almost half of the respondents knew that humans could be infected by the disease and reported that the disease in yaks could be treatable using drugs. However, few herders (25.5%) knew that the disease could be treated in humans. After categorising the responses based on the gender of the interviewed person, no difference was found in the knowledge of males and females. However, when responses were categorised according to the type of production system, there were significant differences between the response of the participants with respect to awareness of the disease (p<0.001), knowledge that organs were affected (p=0.008), that humans could be infected (p<0.001), that brucellosis could be prevented in yaks (p<0.001), that it could be treated in humans (p=0.018), that there was a control program for the disease
(p<0.001), and that yaks can be affected by the disease (p=0.007).
6.3.3 Association of risk factors with respondent’s knowledge
All demographic and socio-demographic variables relating to the respondent’s knowledge score were analysed (Table 6.4) and variables with a p < 0.20 were offered to the multivariable logistic regression model. In the final model respondents aged ≥ 50 97
years old (OR=1.98, 95%CI: 1.15, 3.46), with primary/secondary school education
(OR=2.19, 95% CI: 1.22, 3.96), with ≥ 6 persons in their family (OR=2.77, 95%CI:
1.59, 4.91), and who raised their yaks in a pastoral production system (OR=10.57,
95%CI: 5.47, 21.54) had significantly higher knowledge (Table 6.5). The results of the likelihood ratio test (χ2=77.36, p<0.001) and the Hosmer-Lemeshow test (p= 0.78) indicated that the model was a good fit of the data.
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Table 6.3 Descriptive analyses of the respondent’s knowledge for brucellosis categorised by gender and production system
Variable/category n (%) Gender p-value Production system p-value n (%) (male) n (%) (female) n (%) (pastoral) n (%) (agro-pastoral)
Have you heard of others in your village having abortions in their animals over the last 0.085 <0.001
12 month? Yes# 185 (58.4) 157 (60.6) 28 (48.3) 77 (33.3) 55 (64.0)
No 132 (41.6) 102 (39.4) 30 (51.7) 154 (66.7) 31 (36.0)
Do you know why these abortions occurred? a 0.109* 0.496
Yes# 51 (27.6) 47 (29.9) 4 (14.3) 44 (28.6) 7 (22.6)
No 134 (72.4) 110 (70.1) 24 (85.7) 110 (71.4) 24 (77.4)
Have you ever heard of brucellosis? 0.220 <0.001
Yes# 192 (60.6) 161 (62.2) 31 (53.4) 165 (71.4) 27 (31.4)
No 125 (39.4) 98 (37.8) 27 (46.6) 66 (28.6) 59 (68.6)
Do you know what organs it affects? b 0.348 0.0081
Yes# 122 (63.5) 100 (62.1) 22 (71.0) 111 (67.3) 11 (40.7)
No 70 (36.5) 61 (37.9) 9 (29.0) 54 (32.7) 16 (59.3)
Do you know if yaks can be affected by b 0.753* 0.007 brucellosis? Yes# 171 (89.1) 144 (89.4) 27 (87.1) 151 (91.5) 20 (74.1)
No 21 (10.9) 17 (10.6) 4 (12.9) 14 (8.5) 7 (25.9)
Do you know if brucellosis can affect other b 0.424* 0.309 animals like sheep and goats? Yes# 162 (84.4) 134 (83.2) 28 (90.3) 141 (85.5) 21 (77.8)
No 30 (15.6) 27 (16.8) 3 (9.7) 24 (14.5) 6 (22.2)
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Variable/category n (%) Gender p-value Production system p-value n (%) (male) n (%) (female) n (%) (pastoral) n (%) (agro-pastoral)
Do you know if humans can be infected with 0.804 <0.001 brucellosis? b Yes# 103 (53.7) 87 (54.0) 16 (51.6) 101 (71.4) 2 (5.8)
No 89 (46.3) 74 (46.0) 15 (48.4) 64 (28.6) 25 (94.2)
Do you know if brucellosis can be prevented in 0.968 <0.001 yaks? b Yes# 143 (74.5) 120 (74.5) 23 (74.2) 131 (79.4) 12 (44.4)
No 49 (25.5) 41 (25.5) 8 (25.8) 34 (20.6) 15 (55.6)
Do you know if brucellosis can be treated in 0.151 0.990 yaks? b Yes# 114 (59.4) 92 (57.1) 22 (71.0) 98 (59.4) 16 (59.3)
No 78 (40.6) 69 (42.9) 9 (29.0) 67 (40.6) 11 (40.7)
Do you know if brucellosis can be treated in 0.190 0.018 humans? b Yes# 49 (25.5) 44 (27.3) 5 (16.1) 47 (28.5) 2 (7.4)
No 143 (74.5) 117 (72.7) 26 (83.9) 118 (71.5) 25 (92.6)
Do you know if there is any control program b 0.947 <0.001 for brucellosis? Yes# 147 (77.0) 123 (76.9) 24 (77.4) 138 (84.1) 9 (33.3)
No 44 (23.0) 37 (23.1) 7 (22.6) 26 (15.9) 18 (66.7)
Do you know if vaccination is an effective way 0.696* 0.3944 to control the disease? b Yes# 176 (93.1) 147 (92.5) 29 (96.7) 153 (93.9) 23 (88.5)
No 13 (6.9) 12 (7.5) 1 (0.3) 10 (6.1) 3 (11.5) aThe responses were based on those who answered ʻyesʼ to the first question. bThe responses were based on those who answered ʻyesʼ to the third question. Due to missing data, the total numbers can’t be summed *Fisher’s exact test # Responses coded as “correct”
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Table 6.4 Association of demographic and socio-demographic variables with categorised knowledge scores of the respondents
Categorised scores Variables Odds Ratio (95% CI) P-Value Good (%)* Poor (%) Sex
Female 27 (46.6) 31 (53.4) 1.0 - Male 139 (53.7) 120 (46.3) 1.33 (0.75, 2.37) 0.328
Age
≥50 94 (59.9) 63 (40.1) 1.81 (1.16, 2.84) 0.010 <50 71 (45.2) 86 (54.8) 1.0 -
Education
Primary/Secondary school 122 (57.8) 89 (42.2) 1.93 (1.21, 3.11) 0.006 No education 44 (41.5) 62 (58.5) 1.0 -
Production System Agro-pastoral 16 (18.6) 70 (81.4) 1.0 - Pastoral 150 (64.9) 81 (35.1) 8.10 (4.52, 15.30) <0.001
No. of persons in household
<6 55 (47.4) 61 (52.6) 1.0 - ≥6 111 (59.0) 77 (41.0) 1.60 (1.00, 2.55) 0.049 * ≥ median
Table 6.5 Multivariable logistic regression model of factors associated with the respondent’s knowledge
Variable b SE p-value OR (95%CI) Constant -3.074 0.48 - - Age (years)
≥50 0.682 0.28 0.015 1.98 (1.15, 3,46) <50 1.0
Production system
Agro-pastoral 1.0 Pastoral 2.358 0.35 <0.001 10.57 (5.47, 21.54)
Education
No education 1.0 Primary/Secondary school 0.782 0.3 0.009 2.19 (1.22, 3.96)
No. of persons in household <6 1.0 ≥6 1.02 0.290 <0.001 2.77 (1.59, 4.91)
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6.3.4 Attitudes and practices
The bivariate analyses of the respondents’ attitudes and practices towards brucellosis categorised by gender and production systems are summarised in Table 6.6. The majority of respondents (> 90%) stated that: they would separate sick yaks from healthy ones; disease affected their yak’s production; and they would be willing to join training organised by the relevant veterinary department. Also, 89.4% of respondents boiled milk before drinking it, and more than half of the respondents had good attitudes and practices (e.g., isolated newly purchased yaks before mixing them with their current herd, considered that brucellosis was an important disease, and supported the existing control program). However, only 9.6% of respondents wore gloves when disposing of an aborted calf or discharges from yaks. In addition there were significant differences in the respondent’s attitudes and practices for herders using the two production systems with regards to: informing veterinary if yaks get disease; offering support for the control program and boiling milk prior to drinking it (all p<0.05) (Table 6.6). However, there weren’t any significant differences in the attitudes and practices between male and female respondents (Table 6.6).
6.3.5 Association of risk factors with the respondent’s attitudes and practices
The results of the association between demographic and socio-demographic variables and the scores of the respondent’s understanding of the disease and practices adopted are presented in Table 6.7. Of these variables, three (education, county and number of persons in the household) (p <0.20), were subsequently offered to the multivariable logistic model. Respondents with some formal education (OR=1.72, 95%CI 1.03, 2.88), with ≥ 6 persons in their family (OR=2.80, 95%CI: 1.68, 4.76), and who raised their yaks under a pastoral production system (OR=2.43, 95%CI: 1.38, 4.33) adopted better 102
practices and had a better understanding of the disease (Table 6.8). In the model evaluation, the likelihood ratio test (χ2=25.78, p<0.001) and the Hosmer-Lemeshow goodness-of-fit test (p = 0.47) indicated that the model was a good fit of the data.
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Table 6.6 Bivariate analyses of respondents’ attitudes and practices of brucellosis as influenced by sex and production system
Variable/category n (%) a Sex p-value Production system p-value n (%) (male) n (%) (female) n (%) (pastoral) n (%) (agro-pastoral)
Inform vet if yaks get disease 0.917 0.040*
Yes# 283 (89.3) 231 (89.2) 52 (89.7) 201 (87.0) 82 (95.3)
No 34 (10.7) 28 (10.8) 6 (10.3) 30 (13.0) 4 (4.7)
Separate sick yaks from healthy 0.776* 0.210* ones Yes# 293 (93.3) 238 (93.0) 55 (94.8) 210 (92.1) 83 (96.5)
No 21 (6.7) 18 (7.0) 3 (5.2) 18 (7.9) 3 (3.5)
Wear gloves to dispose of aborted calf or discharge if 0.087* 0.231
present Yes# 30 (9.6) 28 (10.9) 2 (3.4) 19 (8.3) 11 (12.8)
No 284 (90.4) 228 (89.1) 56 (96.6) 209 (91.7) 75 (87.2)
Isolate newly purchased yaks 0.777 0.952 before mix Yes# 183 (58.8) 149 (58.4) 34 (60.7) 132 (58.9) 51 (59.3)
No 127 (40.0) 105 (41.3) 22 (39.3) 92 (41.1) 35 (40.7)
Support control program 0.102 <0.001
Yes# 208 (67.5) 176 (69.6) 32 (58.2) 169 (75.8) 39 (45.9)
No 100 (32.5) 77 (30.4) 23 (41.8) 54 (24.2) 46 (54.1)
Consider disease is a factor 0.549 0.470 affecting the production of yaks Yes# 234 (73.8) 193 (74.5) 41 (70.7) 168 (72.7) 66 (76.7)
No 83 (26.2) 66 (25.5) 17 (29.3) 63 (27.3) 20 (23.3)
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Variable/category n (%) a Sex p-value Production system p-value n (%) (male) n (%) (female) n (%) (pastoral) n (%) (agro-pastoral)
Would like to join training on 0.060 0.411 brucellosis Yes# 284 (90.7) 236 (92.2) 48 (84.2) 205 (89.9) 79 (92.9)
No 29 (9.3) 20 (7.8) 9 (15.8) 23 (10.1) 6 (7.1)
Boil milk before drinking it 0.475* <0.001
Yes# 278 (89.4) 225 (88.6) 53 (93.0) 218 (95.6) 60 (72.3)
No 33 (10.6) 29 (11.4) 4 (7.0) 10 (4.4) 23 (27.7) a Not all farmers answered all questions *Fisher’s exact test # Responses coded as “correct”
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Table 6.7 Association of demographic and socio-demographic variables with the categorised scores of the respondent’s attitudes and practices
Categorised scores Variables Odds Ratio (95% CI) P-Value good (%) poor (%) Sex
Female 36 (62.1) 22 (37.9) 1.0 - Male 166 (64.1) 93 (35.9) 1.09 (0.60, 1.95) 0.772
Age
≥50 93 (59.2) 64 (40.8) 1.51 (0.96, 2.42) 0.079 <50 108 (68.8) 49 (31.2) 1.0 -
Education
Yes 144 (68.2) 67 (31.8) 1.78 (1.10, 2.88) 0.019 No 58 (54.7) 48 (45.3) 1.0 -
Production system
Pastoral 155 (67.1) 76 (32.9) 1.69 (1.02, 1.81) 0.041 Agro-pastoral 47 (54.7) 39 (45.3) 1.0 -
No. of persons in household
<6 58 (50.0) 58 (50.0) 1.0 - ≥6 131 (69.7) 57 (30.0) 2.30 (1.43, 3.72) <0.001
Table 6.8 Multivariable logistic regression model of factors associated with the level of the respondent’s attitudes and practices
Variable B SE p-value OR(95%CI) Constant -1.110 0.360
No. of persons in household <6 1.0 ≥6 1.031 0.266 <0.001 2.80 (1.68, 4.76)
Production system Agro-pastoral Pastoral 0.888 0.292 0.002 2.43 (1.38, 4.33)
Education
No formal education 1.0 Primary/Secondary school 0.544 0.262 0.038 1.72 (1.03, 2.88)
6.4 Discussion
Although methods to control brucellosis in humans are well known and focus on controlling the disease in livestock (Corbel, 1997; Seleem et al., 2010), understanding
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the knowledge, attitudes and practices of farmers is critical when developing effective control programs for the disease. Furthermore, the knowledge of farmers about the disease can provide information on the likelihood of this group acquiring infection from their livestock or from livestock products. This study is the first KAP study investigating brucellosis in a yak rearing area of the world and the results have the potential to reduce the risk of individuals from acquiring the disease, as well as providing useful information for the implementation of control measures of the disease in livestock and humans in Tibet and potentially the country.
The result of the multivariable logistic regression model showed that, not surprisingly, pastoralists with a higher level of education had better knowledge about the disease.
Other studies have shown that farmers with a lower level of education are less likely to have knowledge on brucellosis (Lindahl et al., 2015), and are more likely to acquire brucellosis (Al-Shamahy et al., 2000). A higher proportion of respondents in Uganda,
Kenya and Egypt knew of the disease (Holt et al., 2011; Kansiime et al., 2014; Obonyo and Gufu, 2015) than in the current study, and all participants in a survey of Jordan had heard of the disease (Musallam et al., 2015b). However in those studies brucellosis had a high level of endemicity in the surveyed regions. It is possible that, although most farmers knew of or had heard of the disease in the current study, their awareness of the risk of infection was low, resulting in the adoption of high-risk practices. For example, most participants handled aborted foetuses and foetal membranes during parturition without wearing protective gloves, which has previously been shown to increase the risk of infection in humans (Holt et al., 2011; Musallam et al., 2015b).
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This study found that the majority of the pastoralists had a low level of knowledge concerning the cause of any abortions they may have had in their herd. Although abortions can be caused by a variety of diseases and conditions, abortion is the key clinical sign of brucellosis, particularly in herds in which the disease has been recently introduced (Ducrotoy et al., 2017). Of those who knew about brucellosis, the majority knew that livestock, such as yaks, sheep and goats, could be infected. This finding is in agreement with other studies conducted in Tajikistan (Lindahl et al., 2015) and Egypt
(Holt et al., 2011). Although in this study just over half of the pastoralists knew that brucellosis could affect humans, few were aware that the disease was treatable in people.
Several studies have highlighted that having a good knowledge about the disease in humans resulted in the adoption of practices that reduced the potential transmission to humans (Kozukeev et al., 2006; Sofian et al., 2008). In a study in Kenya a similar lack of knowledge about the availability of treatment for affected humans was reported
(Obonyo and Gufu, 2015). A lack of knowledge about the disease or its treatability potentially could lead to a delay in seeking medical assistance and hence a delay in the diagnosis and treatment of the disease similar to that which has been reported for other diseases such as pulmonary tuberculosis in Ethiopia (Demissie et al., 2002).
In the multivariable logistic regression model, age, production system, education and number of persons in the household were all significantly associated with knowledge.
Not surprisingly older respondents (≥ 50 years) had a higher level of knowledge than younger respondents. This may be due to their longer time in the industry and hence more experience and a greater opportunity for exposure to educational material on the disease. Education is important since it can positively influence a person’s ability and inclination to acquire further knowledge (Mohamed et al., 2017). In the current study 108
herders who had at least obtained primary education had a better knowledge about the disease, which is a vital component of prevention and control of the disease in animals
(Dlamini et al., 2017). Additionally, pastoralists adopting an agro-pastoral production management system had lower levels of knowledge than those implementing a purely pastoral production system. In the seroprevalence survey the seroprevalence was higher in yaks managed under an agro-pastoral production system supporting the lack of awareness of this group about the disease (Chapter 4). This result differed from that reported in other studies where farmers living in agro-pastoral production systems have had an overall higher knowledge of brucellosis (Gele et al., 2009; Legesse et al., 2010;
Kansiime et al., 2014). These differences may be due to different management and husbandry practices and opportunities for accessing veterinary services and knowledge about diseases between different regions. In Tibet herders move their yaks in different seasons to minimise grassland degradation, as required by the local policy (Sheehy et al.,
2006). The herders are engaged with their livestock every day and are therefore more likely to be familiar with their animals and observe clinical signs of disease, if present, compared with those who undertake other agricultural activities who are likely to spend less time with their livestock. Herders may also have greater access to veterinarians located at rural veterinary stations distributed within the livestock rearing areas, than farmers in the more populated agricultural regions where there are fewer livestock and veterinarians. In this study herders from a larger household (≥ 6 persons) were more likely to have a better knowledge about brucellosis than those from smaller households.
It is probable that a larger family, potentially with the presence of older persons with experience in rearing yaks, results in more discussion and knowledge transfer between family members about disease and livestock management/husbandry.
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The pastoralist’s understanding of the disease and practices adopted play an important role in the prevention and control of brucellosis. The majority of the respondents said that when their yaks became sick they advised the local veterinary field station. A study conducted in Egypt on brucellosis reported a similar result (98%) (Holt et al., 2011).
This practice should help reduce the occurrence and spread of the disease, as well as allow the early detection of new cases of disease or incursion of the disease into previously free herds or areas (Cameron, 2012; Hennenfent et al., 2017; Pham et al.,
2017). In this study most respondents (93.3%) also said they would separate sick from healthy yaks, and 59.0% of respondents isolated newly purchased yaks before introducing them to their herd. One of the major risk factors for disease transmission is introduction of diseased animals and subsequent direct contact between susceptible and infected animals or through contact with contaminated material or fomites (Laing, 1955;
Geering et al., 1995). These practices again would help reduce the risk of a herder’s yaks contracting brucellosis, as well as other infectious diseases. However the communal grazing practices adopted routinely in Tibet may mean that although many farmers may adopt good biosecurity practices, poor practices by one farmer could impact on the other herds that the herder communally grazes with.
In the current study approximately two-thirds of the respondents (67.5%) reported that they supported the control program for brucellosis, 73.8% of respondents considered that brucellosis was a disease that affected the productivity of their yaks and most
(90.7%) would like to join in training about the disease. These aspects would be extremely beneficial when conducting a control and prevention program as support and involvement of livestock producers is critical for effective disease intervention (Ritter et al., 2016; Ritter et al., 2017). The majority of respondents also reported that they boiled 110
milk prior to drinking it, which is a key measure to control the transmission of brucellosis from animals to humans (Corbel, 2006; Kozukeev et al., 2006; Sofian et al.,
2008). In this study very few respondents (9.6%) advised that they would wear or have worn gloves to handle aborted foetuses or vaginal discharges. Similar results have been described by others (Lindahl et al., 2015; Musallam et al., 2015b). As brucellosis can be transmitted directly from aborted foetuses and discharges to humans (Doganay and
Aygen, 2003; Corbel, 2006), this practice would increase the risk of human infection
(Earhart et al., 2009). Implementation of an education program and providing low cost personal protective equipment, such as gloves, to farmers would potentially help reduce the incidence of brucellosis in yak-herders.
In the current study significant differences were found between the production systems in whether a herder would: inform a veterinarian if their yaks acquired a disease
(p=0.04); support a control program (p<0.001); and boil milk before drinking it
(p<0.001). These findings would be beneficial when formulating and carrying out a control program for brucellosis in herds adopting the different production systems.
Pastoralists who had received some formal education were more likely to adopt biosecurity measures than pastoralists without education (OR=1.72, 95%CI 1.03, 2.88), highlighting the importance of education (formal schooling) in disease control. Similar findings have been reported by others (Sofian et al., 2008; FAO, 2010). In addition, herders from households containing more people also had better understanding and adoption of safe practices than those from smaller households, most likely for the same reasons as for a higher level of knowledge in this group.
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In Tibetan society males are still considered family heads and hence in this study more males were interviewed than females. This potentially may have introduced a bias if females spent more time looking after their family’s yaks. Therefore, future studies should gather information on who in the family predominantly rears yaks or is responsible for their health and well-being. Particularly as some studies have reported less disease when women are engaged in livestock rearing because of the adoption of better hygienic practices (Fortmann, 2009).
In China, the animal diseases focused on for control have included FMD and PPR, which are diseases routinely vaccinated against (Bureau of Veterinary, 2009). Due to the fact that human brucellosis is a re-emerging disease in China and infection of humans originates from animal reservoirs (Godfroid et al., 2005; Franco et al., 2007; Li et al., 2013), since 2008 China has established 21 surveillance points for both animal and human brucellosis in 19 provinces, including Tibet (Jiang et al., 2012). Brucellosis is also now listed in the annual surveillance plan in Tibet, although compulsory vaccination is not implemented on the Tibetan plateau (Lan, 2013). The two governmental agencies, the TBAA and the TAAAS, have been organising and arranging training programs for animal disease control in townships and counties since 1982 (Lan,
2013; Luobu and Se, 2013). Farmers and pastoralists often join these activities as they receive an incentive of extra subsidies if they attend such workshops. These workshops provide information and educational materials in the form of brochures which contain pictures and material in both Chinese and Tibetan languages to reach a wider audience.
The current training program for both pastoralists and farmers living in rural areas is carried out each year for approximately one week; however in most case brucellosis has not been included in the training programs (Se and Yang, 2008). Brucellosis needs to be 112
included in future training programs and the groups targeted for such training should not be limited to pastoralists and farmers, but also should include medical physicians and veterinarians who may not be fully aware of the local problem. Díez and Coelho (2013) highlighted the importance and role of training programs in the control and eradication of brucellosis.
In conclusion, this study revealed that the pastoralists had a range of levels of knowledge, and showed diverse attitudes and practices with respect to brucellosis.
Although some showed a high level of awareness about brucellosis, there were knowledge gaps in some herders surveyed and few adopted protective practices
(wearing gloves) when dealing with potentially infectious material, such as aborted foetuses/placenta. The level of knowledge about brucellosis varied for herders in the different regions where different production systems were adopted. It is concluded that development of suitable educational material is required to ensure herders are aware of the disease and ways to minimise its impact on their herd and family. Such educational material and training will play an important component of any brucellosis control program. The financial impact of a disease is also a key factor in determining whether livestock owners will adopt control programs for a disease. In the following chapter the economic impact of brucellosis in yaks in Tibet is explored.
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CHAPTER SEVEN
Evaluation of the economic impact of brucellosis in domestic yaks of
Tibet
7.1 Introduction
Brucellosis is regarded as one of the most economically important zoonotic diseases in the world (Perry et al., 2002; Perry and Grace, 2009). The economic impact of the disease varies depending on the geographical location, livestock species reared, management system adopted and on the capacity of the country’s veterinary and medical system (McDermott et al., 2013). Although bovine brucellosis has been eradicated from some countries, it is recognised as a re-emerging zoonosis affecting many animals and people throughout the world (Seleem et al., 2010). The economic losses arising from brucellosis in animals are mainly due to: the clinical manifestation of abortions during the last trimester of gestation; the decrease in milk yield and meat production; temporary infertility; perinatal mortalities; and mortality in adult animals that abort (Singh et al., 2015). In addition it also threatens public health with infection primarily resulting from occupational exposure through the handling of infected animals, their tissues or discharges or the consumption of unpasteurized milk or dairy products
(Godfroid et al., 2005). Due to the nonspecific clinical signs in humans (Godfroid et al.,
2011), brucellosis in humans is often neglected and/or misdiagnosed (Paul et al., 1995).
Therefore, controlling the disease in livestock is an important task in many countries.
The main adopted control strategies include surveillance, vaccination, quarantine, separation and elimination of infected animals, test-and-slaughter and compensation schemes. However, implementing effective control programs is challenging in countries
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with a low gross domestic product (GDP) (Deqiu et al., 2002; European commission,
2009; McDermott et al., 2013).
As outlined in Chapter 1 the livestock sector is an important industry in Tibet which has resulted in an improved quality of life for the community and has facilitated the development of the local economy. Yaks are the most important livestock raised in the province, however they are susceptible to many of the diseases that affect cattle, including brucellosis (Gerald et al., 2003). Previous studies and the results of the studies outlined in this thesis have highlighted that the disease is endemic in yaks in Tibet
(Chapters 3 and 4). Currently annual serosurveillance surveys are carried out by the
TBAA according to the regulations of the Ministry of Agriculture of China. Although studies on the economic impact of brucellosis have been conducted in many countries
(McDermott et al., 2013; Santos et al., 2013; Singh et al., 2015), little information is available on the economic impact of the disease in China, especially Tibet. The aim of this chapter is to estimate the economic losses resulting from brucellosis in yaks in the study areas and to evaluate the financial impact of different control strategies through developing a benefit-cost analysis model.
7.2 Materials and methods
7.2.1 The study area and design
This study was conducted in Damxung county, Maizhokunggar county and Pali township of Yadong county where the seroprevalence survey was conducted. The economic investigation into brucellosis in yaks was divided into two parts. The first part involved administering a questionnaire to the farmers (n = 181) whose yaks were sampled in the serological survey conducted in the 30 villages from April to May 2015 115
(Chapter 4). The questionnaire gathered data on the price of milk, the use of milk and the cost of supplements and husbandry practices adopted (Appendix 2). In July and
August 2016 a second questionnaire was administered to households who had yaks diagnosed with brucellosis in 2015. The second survey gathered information on the price of yaks and their meat, and the households’ attitudes towards the disposal of infected yaks (Appendix 3). There are approximately 366,350 yaks in the study area:
230,000 in Damxung County; 130,000 in Damxung County; and 6,350 in Pali Township of Yadong County. The total number of households in the study areas was estimated to be 14,035; 6,460 in Damxung county; 7,000 in Maizhokunggar county; and 575 in Pali
Township (Anonymous, 2014; Liu, 2014a; Anonymous, 2015).
7.2.2 Economic model
Yaks account for 17% of all livestock in Tibet and are important for providing meat, milk, faeces (fuel) and work (carrying goods, transport and ploughing) (Ji et al., 2002b;
Yang, 2002; Ji et al., 2003). In the study described in Chapter 4 there were 8,706 yaks in the 181 households surveyed, of which 8,002 (91.9%) yaks were older than 1 year of age. It was assumed that the proportion of yaks older than one year was similar between the surveyed population and all yaks residing in Damxung and Maizhokunggar counties and Pali Township.
The data collected during the seroprevalence study and presented in Chapter 4 were used to populate the economic model. Out of a population of 1,523 yaks there were 746 males and 777 females. Of the 42 seropositive yaks detected, 22 were female. A beta probability distribution was used in this analysis to account for uncertainty in the prevalence of the disease in female yaks using the add-in to Excel, PopTools version 116
3.2.5 (Hood, 2010). The proportion of female yaks ≥ 3 years old in all yaks greater than
1 year old in the seroprevalence survey was 37.8% (576/1523) and this percentage was used to estimate the total number of female yaks (≥ 3 years old) in the three areas. Data on the impact of brucellosis on reproduction and productivity were obtained from the research of Bernués et al. (1997) in cattle. These assumptions included the following:
15% incidence of abortion in infected pregnant animals; 15% reduction in the milk yield of infected lactating animals; 5% reduction in meat yield in infected female yaks; and
10% perinatal mortality in calves born to infected yaks. A currency exchange rate of
1RMB to US$0.1540 (average rate for 2016) was used in this study (World Bank, 2016).
Values for the losses, prices and costs and the ratio of male to female yaks used in the modelling study are summarised in Table 7.1. The total amount lost per year and the average loss per head were also calculated. The price of yak meat and milk was included in the study at US$11.24/kg and $3.08/kg, respectively (derived from results of administering the questionnaires to yak herders in this study). The cost of vaccination
(S2 vaccine) was included at US$0.09 per head in this study. The cost of testing (RBT,
ELISA and blood sample consumables) was estimated at US$0.81/head. The cost of transport of testing/vaccinating teams to visit each household was calculated as approximately US$1.47 (derived from the data collected during the seroprevalence survey). The average loss from each abortion was estimated at $600 based on data from cattle (Hovingh, 2009).
In order to better understand the impact of the disease on the yak industry, data were also extrapolated to the total population of yaks in Tibet (4,711,300) (Ciren, 2006).
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Table 7.1 Parameters used in the financial analysis
Category Yaks Values included in the model Range (or ±SD) Source Prevalence (all female yaks) 22/777 1.80% 0.6%-4.0% Chapter 4 - Beta distribution Prevalence (≥4 years old female yaks) 14/466 3.20% 1.3%-5.8% Chapter 4 - Beta distribution Prevalence (≥3 years old female yaks) 21/576 3.76% 1.8%-6.2% Chapter 4 - Beta distribution Pregnancy rate (≥4 year old female yaks) 50.00% 40%-60% Pers.com. (Dawa Yangla)
Expected vaccination coverage 95.00% Bureau of Veterinary (2009)
Expected effective protection after vaccination with S2 75.00% Can and Yalcin (2014)
Average age of culling 10 years old Pers. com. (Dawa Yangla)
Proportion of infected females aborting 15% 10%-50% Bernués et al. (1997)
Value lost from an abortion US$600 US$500-900 Hovingh (2009)
Milk losses Infected female 15% 10%-25% Bernués et al. (1997) Meat losses Infected female 5% Bernués et al. (1997)
Daily milk yield of a non-infected yak 1 Kg ±0.2 Gerald et al. (2003)
Duration of lactation of a non-infected yak 184 days Gerald et al. (2003)
Calves born from Perinatal mortality 10% 5%-20% Bernués et al. (1997) newly infected females Body weight of adult female yaks 187.9 Kg ± 19.0 Gerald et al. (2003)
Body weight of adult male yaks 282.4 Kg ± 23.8 Gerald et al. (2003)
Value of a female yak (≥4 years old) US$986 US$616-1232 Questionnaire survey data
Value of a male yak (≥4 years old) US$1444 US$1000-2100 Questionnaire survey data
Value of a calf (≤ 6 months) US$123.2 US$77-184.8 Questionnaire survey data
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This survey assessed the impact of brucellosis on abortions, perinatal mortality and meat and milk production according to the formulas listed below. Data on the number of cases of still births, orchitis, epididymitis, hygromas and mastitis were not included as little information was available regarding these features of the disease in cattle or yaks.
Economic losses = Ab + Mi + Me + Pm
Where:
Ab is the cost of an abortion (Ab = number of positive female yaks × 50% (pregnancy rate) × 15% (incidence) × $600)
Mi is the milk production loss (Mi = number of positive female yaks × 50% (pregnancy rate) × 1kg/day × 15% (reduction in milk yield in infected yaks) × cost of milk [$3.08])
Me is the meat production loss (Me = number of positive female yaks × 187.9 kg × 5% (reduction in meat production in infected animals) × 14.3% (culling rate) × price of yak meat [$11.24])
Pm is the perinatal mortality (Pm = number of positive female yaks × 50% (pregnancy rate) ×10% (incidence of perinatal mortality) × value of a calf [$123.20])
In order to evaluate the impact of disease control, an economic model for a 6-year period was developed based on the fact that B. suis S2 vaccine can induce immunity providing a 2-year period of protection (Deqiu et al., 2002). In the model the input parameters for Ab, Mi and Pm were based on the number of positive female yaks aged
≥3 years. However, meat production loss was only assessed for positive female yaks aged ≥3 years old that were no longer considered productive and would have been culled (it was assumed 14.3% of the infected herd i.e. 1/7th of the herd was culled each year). It was also assumed that the meat from these yaks was either eaten or sold. The number of female calves kept every year as replacements was expected to be equal to the number of female yaks culled each year to maintain a constant population size.
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7.2.3 Strategy to control the disease
To estimate the economic impact of different brucellosis control programs, three control strategies coded as v (vaccination), t (test-and-slaughter), and c (combination of vaccination and test-and-slaughter) were evaluated (Table 7.2). In strategy v it was assumed that 95% vaccination coverage was achieved using a standard dose of B. suis
S2 vaccine (The Spirit Jinyu Biological Pharmaceutical, Co., LTD, China) in years 1, 3 and 5 of the study as this vaccine has been widely applied in China. Strategy t was a test-and-slaughter measure where test-positive yaks were slaughtered and replaced with the same number of vaccinated yaks to maintain a fixed (constant) number of yaks.
Strategy c was a combination of test-and-slaughter and mass vaccination as described above. In the c program after seropositive yaks were slaughtered, the slaughtered yaks were also replaced with the same number of vaccinated yaks to maintain a constant population size, and 95% vaccination coverage was assumed using a standard dose of B. suis S2 vaccine in years 1, 3 and 5 of the study. It was assumed that replacement yaks in the latter two control programs were of the same demographics as the slaughtered yaks, i.e. same sex and age.
Table 7.2 Description of the three control strategies
Control strategies Contents Times Vaccination alone 95% vaccination coverage Years 1, 3 and 5
Test-and-slaughter Slaughtering test-positive yaks; replacing Years 1 with the same number of vaccinated yaks
Vaccination and Slaughtering test-positive yaks; replacing Slaughter and replacement test-and-slaughter with the same number of vaccinated in year 1;Vaccination in yaks;95% vaccination coverage year 1,3 and 5
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The number of female calves kept every year for replacement purposes was assumed to be equal to the number of female yaks that died or were culled. It was also assumed that, with no disease control, brucellosis would be at an endemic equilibrium. Therefore, the number of newly infected yaks produced by one infectious yak during its infectious period was equal to 1. In this context, the number of newly infected yaks each year was the same as the number of infected yaks that died or were culled. The calculation of new cases was derived from the formula as follows (Hegazy et al., 2009; Dohoo et al., 2012):
R=R0×S*
Where: R is number of new cases that arise from an infectious individual over the course of its infectious period. R0 is the basic reproduction ratio and S* is the proportion of susceptible yaks in the total population of animals.
Due to the chronic nature of infection with Brucella (Enright, 1990; Poester et al., 2013), it was assumed that seropositive yaks became infected at 3 years of age and were culled at 10 years of age (Pers. Com. Dawa Yangla), and consequently in this analysis a duration of infection of 7 years was applied. A culling rate of 1/7th (14.3%) of the herd was applied for each year of the study, and it was assumed these were replaced with disease free animals.
The benefits derived from each control strategy were calculated as the financial savings arising from the reduced number of infected female yaks, while the costs incurred varied for the different strategies. In the vaccination strategy, the costs consisted of the vaccine purchases and associated costs of transport and accommodation of veterinary teams (in years 1, 3 and 5) and expenditure associated with employment of the 121
workforce. In the test-and-slaughter strategy, the costs included testing, the slaughter of seropositive yaks, purchase of replacement yaks, transport and accommodation of veterinary teams (twice), and employment costs of the workforce. For the combination strategy (vaccination and test-and-slaughter), costs included those listed above for the two individual control strategies, however costs for transport and accommodation of veterinary teams was required five times; twice for the test-and-slaughter strategy in year 1, and three times for vaccination (years 1, 3 and 5). The value for replacement yaks was based on the lowest price of a female yak aged between 3 and 5 years of age.
The control strategies for brucellosis in yaks in Tibet were assessed through evaluating the benefit-cost ratio (BCR) and the net present value (NPV) using the following formulas (Noordhuizen, 2001):
PV = FV ÷ (1 + r ÷ 100)n where:
PV = the present value of the cost or benefit
FV = the future value of the cost or benefit r = the real annual interest rate in percent n = the number of years in the analysis and
NPV = PVB - PVC
BCR = PVB ÷ PVC where:
PVB is the present value of the benefits and PVC is the present value of the costs
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If NPV > 0 or BCR > 1 the monetary benefits of the control strategy outweigh the costs.
A discount rate of 5.36% was set according to an average inflation rate in China from
1986 to 2017 (https://tradingeconomics.com/china/inflation-cpi).
7.2.4 Sensitivity analysis
Sensitivity analysis was conducted to assess the impact of the protection rate from vaccination on both the NPV and BCR for the control strategies v and c. The protection rate was based on both the efficacy of the available vaccine and the vaccine coverage.
Scenarios of 60% (efficacy of vaccine: 75% and vaccination coverage: 80%), 71.25%
(efficacy of vaccine: 75% and vaccination coverage: 95%) and 80.75% (efficacy of vaccine: 85% and vaccination coverage: 95%) in the protection rate were applied. To account for uncertainty in their values six input variables (abortion, meat price, milk price, calf price, transportation and price of the adult female yaks slaughtered) were expressed as probability distribution functions using @Risk 7.5 student version
(Palisade Decision Tools, Palisade Corporation) (Table 7.3). An uncertainty analysis was conducted to assess the effect of input parameter uncertainty on the NPV and BCR under the different control strategies. The median NPV and BCR values and 95% confidence intervals were calculated with 2000 iterations using the variables specified as @Risk functions. A global sensitivity analysis was carried out to identify which input parameters had the most effect on the uncertainty of the NPV, through examining the normalized regression coefficients (R2).
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Table 7.3 List of input variables used as @Risk functions
Name @ Risk function* Abortion (Per head) Normal (600; 100) Meat price (Per Kg) Pert (11.24; 10.47; 12.32) Milk price (Per Kg) Pert (3.08; 2.46; 3.69) Calf price (Per head) Pert (123.20; 77.00; 184.80) Transportation fees Pert (20634.71; 17195.59; 22927.45) Price of adult female yaks slaughtered (Per head) Pert (986; 616; 1232) * Data used in the Normal distribution: mean, standard deviation; Data used in the Pert distribution: most likely, minimum value, maximum value
7.3 Results
Based on the total number of yaks present in Damxung and Maizhokunggar counties and Pali township, the number of female yaks (≥3 years old) present in these areas was estimated at 127,350, of which 4,791 (95% CI: 3,075; 6,987) were predicted to be seropositive. The economic losses due to abortions, reduced milk and meat yield and perinatal mortality are presented in Table 7.4. The annual losses from the disease were estimated at US$ 521,042.78 (95%CI: US$334,440.75; US$759,862.80) for the population in the three regions, resulting in losses over six years of US$ 3,126,256.68
(95%CI: US$2,006,644.50; US$4,559,176.80). During the six-year period, the largest economic loss was associated with abortions (mean: US$ 1,293,595.26), followed by losses from the reduction of milk yield (mean: US$ 1,221,843.84) and reduced meat yield/production (mean: US$ 433,738.74). The lowest loss arose from perinatal mortality (mean: US$ 177,078.84). The average loss per head of yak in the study areas was estimated at US$ 1.42 (95%CI: US$ 0.91, US$ 2.07) per year. When the economic losses were extrapolated to the total population of female yaks (≥3 years old) in Tibet, a total annual loss of US$ 2,324,001.56 was estimated.
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When no control measures were adopted the number of new cases of brucellosis in female yaks during the six-year study period was predicted to be 4,107 head (95%CI:
2,636; 5,989) (Table 7.5). In contrast the number of infected female yaks decreased significantly when control strategies were adopted, particularly when the combination strategy (vaccination and test-and-slaughter) was applied (Table 7.5).
In addition, the seroprevalence varied considerably for the different strategies during the period, as illustrated in Table 7.6. The seroprevalence of brucellosis during the vaccination program decreased gradually each year over the study period. In contrast it dropped substantially in the other two control strategies in the first year, then rose slightly in subsequent years in the test-and-slaughter program and decreased slowly each subsequent year in the combination program.
The results of the benefit-cost analysis of the different control strategies are presented in
Table 7.7. For the vaccination strategy the BCR was greater than 1 and the NPV was positive. However, the other two strategies of test-and-slaughter alone and test-and- slaughter with vaccination resulted in negative NPV values and BCR values < 1. For these two latter strategies the BCR values were similar as were the NPV values.
The results of the manual sensitivity analysis (Table 7.8) show that the NPV increased with increasing protection level for the vaccination strategy. Increasing the protection level from 60% to 80.75% resulted in the NPV increasing from US$ 295,590.35
(95%CI: US$ 146,321.68; US$ 490,928.79) to US$ 327,239.52 (95%CI:
US$ 166,551.75; US$ 571,252.67). However, there was little variation in the NPV and
BCR for the combination control program as the protection rate increased (Table 7.8). 125
For the vaccination control strategy, the median NPV and BCR after the uncertainty analysis were US$ 314,935.34 (95%CI: US$ 276,019.34; US$ 354,108.26) and 3.21
(95%CI: 2.92; 3.51), respectively. In the global sensitivity analysis abortion (0.83) had the largest effect on the outcome, followed by meat price (0.39) and milk price (0.36), and calf price (0.11) with transportation costs having a negative effect (-0.12) (Figure
7.1). In the test-and-slaughter control strategy the median NPV and BCR were
US$ -9,988,848.83 (95% CI: US$ -11,568,725.75; US$ -8,118,057.50) and 0.21
(95%CI: 0.17; 0.25), respectively and the price of the adult female yaks slaughtered
(-0.98) had a negative effect on the outcome. Abortion had a slight positive value (0.19) with all remaining variables having absolute values less than 0.1 (Figure 7.2). In the combination strategy the median NPV and BCR were US$ -10,120,092.04 (95%CI:
US$ -11,711,083.38; US$ -8,273,984.15) and 0.21 (95%CI: 0.17; 0.25), respectively and the outcome was most sensitive to the price of the adult female yaks slaughtered
(-0.97), followed by abortion (0.20) with the remaining variables having absolute values less than 0.1 (Figure 7.3).
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Table 7.4 Estimated economic losses (US $ and 95%CI) of brucellosis in the study region (366,350 yaks) during the six-year study period
Parameters Average annual losses (95%CI) Losses over six years (95%CI)
Abortion 215,599.21 (138,386.26; 314,419.13) 1,293,595.26 (830,317.56; 1,886,514.78)
Milk 203,640.64 (130,710.44; 296,979.35) 1,221,843.84 (784,262.64; 1,781,876.10)
Meat 72,289.79 (46,400.51; 105,423.83) 433,738.74 (278,403.06; 632,542.98)
Perinatal mortality 29,513.14 (18,943.54; 43,040.49) 177,078.84 (113,661.24; 258,242.94)
Total 521,042.78 (334,440.75; 759,862.80) 3,126,256.68 (2,006,644.50; 4,559,176.80)
Table 7.5 Estimated numbers of newly infected female yaks in the different control strategies (95% CI) over the six-year study period
Combined test-and-slaughter No control Vaccination strategy Test-and-slaughter strategy strategy and vaccination strategy
No. of new cases 4,107 (2,636, 5,989) 1099 (703, 1611) 46 (32, 75) 12 (8, 18) over six years
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Table 7.6 Prevalence of brucellosis in each year for the different control strategies (%)
Strategies Initial First year Second year Third year Fourth year Fifth year Sixth year No control 3.76215 3.76215 3.76215 3.76215 3.76215 3.76215 3.76215 Vaccination 3.76215 3.38008 3.08615 2.77350 2.53325 2.27712 2.08051 Test-and-slaughter 3.76215 0.04075 0.04084 0.04107 0.04129 0.04152 0.04177 Combination 3.76215 0.03661 0.03350 0.03014 0.02758 0.02481 0.02270
Table 7.7 Benefit-cost analysis (US$) for evaluating three control strategies for brucellosis in the total population of yaks over a six-year period (95%CI) in Damxung and Maizhokunggar counties and Pali township in Tibet
Control strategies Category Total FV (95%) PV (95%) NPV (95%) BCR (95%)
623,972.25 456,153.50 Benefits (400,705.46; 942,331.40) (292,934.61; 688,888.74) 313,354.87 3.19 Vaccination alone 195,334.34 142,798.63 (157,678.46; 541,061.80) (2.17; 4.66) Costs (185,016.99; 202,212.58) (135,256.15; 147,826.94)
3,583,600.92 2,619,781.48 Benefits (2,300,254.63; 5,226,098.78) (1,681,594.75; 3,820,524.97) -10,100,599.08 0.206 Test-and-slaughter 17,400,217.47 12,720,380.56 (-11,033,757.50; -8,903,207.80) (0.132; 0.300) Costs (17,393,339.23; 17,404,802.96) (12,715,352.25; 12,723,732.77)
3,585,758.26 2,621,358.60 Benefits (2,301,591.37; 5,229,268.67) (1,682,571.96; 3,822,842.31) -10,241,820.59 0.204 Vaccination and test-and-slaughter 17,595,551.81 12,863,179.19 (-11,168,036.44; -9,048,717.40) (0.131; 0.297) Costs (17,578,356.22; 17,607,015.54) (12,850,608.40; 12,871,559.71)
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Table 7.8 Result of sensitivity analysis using different protection rates under the two control strategies of vaccination and combination (test-and-slaughter and vaccination)
Strategy Protection rate NPV BCR
60.00% 295,590.35 (146,321.68; 490,928.79) 3.07 (2.08, 4.32)
Vaccination 71.25% 313,354.87 (157,678.46; 514,061.80) 3.19 (2.17, 4.66)
80.75% 327,239.52 (166,551.75; 571,252.67) 3.29 (2.23, 4.86)
60.00% -10,241,371.49 (-11,167,742.79; -9,048,078.74) 0.20382 (0.13096, 0.29705)
Combination 71.25% -10,241,820.59 (-11,168,036.44; -9,048,717.40) 0.20379 (0.13093, 0.29699)
80.75% -10,242,337.84 (-11,168,371.34; -9,049,462.97) 0.20375 (0.13091, 0.29694)
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Figure 7.1 Regression coefficients of the sensitivity analysis for the vaccination control program
Abortion 0.83
Meat price 0.39
Milk price 0.36
Transportation -0.12
Calf price 0.11
-0.20 0.00 0.20 0.40 0.60 0.80 1.00 Coefficient Value
Figure 7.2 Regression coefficients of the sensitivity analysis for the test-and-slaughter control program
Price of the adult female yaks -0.98 slaughtered
Abortion 0.19
Milk price 0.08
Calf price 0.03
Meat price 0.01
Transportation 0.00
-1.00 -0.80 -0.60 -0.40 -0.20 0.00 0.20 0.40 Coefficient value
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Figure 7.3 Regression coefficients of the sensitivity analysis for the combination control program
Price of the adult female yaks -0.97 slaughtered
Abortion 0.20
Milk price 0.08
Calf price 0.03
Meat price 0.01
Transportation 0.00
-1.00 -0.80 -0.60 -0.40 -0.20 0.00 0.20 0.40 Coefficient Value
7.4 Discussion
Numerous economic studies have been conducted in other countries highlighting the potential losses for livestock producers and the general economy from brucellosis, with the largest losses mainly affecting developing nations (McDermott et al., 2013). In
Latin America losses from bovine brucellosis have been estimated at approximately
US$600 million annually (Seleem et al., 2010). However, evaluating the losses from brucellosis in China has been constrained by a lack of both field and experimental data.
The current economic evaluation was the first study providing an estimation of the impact of the disease in yaks in Tibet. It was based on a seroprevalence investigation in yaks (Chapter 4), questionnaires administered to yak herders (Chapter 5), and published parameters for calculating economic loss in cattle since no values were available for yaks (Gerald et al., 2003). Brucellosis is an insidious disease with often inapparent 131
clinical signs, with the exception when the disease is initially introduced into a naïve herd when an “abortion-storm” may result. Therefore, serological tests are important tools for the detection of infection and disease. Despite the relatively low seroprevalence (2.8%) in sampled yaks in the study (Chapter 4), the estimated economic losses were substantial. The analysis allowed calculation of potential losses arising from brucellosis and predicted the potential costs and benefits of three different control programs. The results provide valuable information for decision-makers, helping them to make informed choices on which control program to implement.
Although the Chinese central government has been investing significant funds on
Tibetan infrastructure, education and agriculture to facilitate the economic development of the region (e.g., more than 70 billion RMB investment during 2006-2010) (Yang,
2007), Tibet is one of the least developed provinces in China (Tashi et al., 2005; Xu et al., 2013). The per capita annual income of farmers and herders in the rural areas of
Shigates prefectural city was reported to be less than US$ 231 in 2011 (Xu et al., 2013), which was significantly lower than the average per capita annual income
(approximately US$ 912) of farmers in China reported in 2010 (Huo, 2013). The study also provided evidence highlighting that raising yaks is an important component of herders’ livelihood in the study area. Xin et al. (2010) concluded that animal husbandry in Tibet would not exist without the traditional yaks. The majority of products produced by yaks are consumed locally due to the large local demand (Xue et al., 2009).
Economic growth, industrialisation and urbanisation of China have created greater disposable incomes of the general Chinese population. This has resulted in a higher demand for meat, with the consumption of beef outstripping the domestic production
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(Waldron et al., 2015). Currently meat is imported from other countries to make up the deficit and there is the potential for the yak industry in Tibet to benefit from this demand resulting in a positive effect on the income of local farmers and pastoralists.
However diseases, such as brucellosis, may hinder this opportunity through reduced production and zoonotic risk.
In this study, abortions, reduction of milk and meat production and perinatal losses were included when assessing the economic losses arising from brucellosis in yaks. If no control strategy was adopted, the total economic loss over the six-year period in the study area was estimated at US$ 3,126,256.68, mainly resulting from the abortions and reduction of milk and meat yield. Other studies have also reported that losses from abortions and reduced meat and milk production were the main causes of losses to cattle from infection with brucellosis (McDermott and Arimi, 2002; Singh et al., 2015).
Abortion is one of the main clinical signs of bovine brucellosis and typically occurs between 5 and 7 months of gestation (Geering et al., 1995), potentially resulting in a significant income loss for the producer (Hovingh, 2009). The current study also demonstrated that abortions can result in major economic losses of approximately
US$0.59/head per year. When extrapolating this loss from abortions to the total population of yaks in Tibet, it results in a large loss to the yak industry and the community as a whole of US$ 2,772,628.79 every year. Although the price of yak’s milk and meat are similar to those for cattle in China (Waldron et al., 2015), yak’ products are generally more popular since their milk has a higher density, fat, protein and sugar content, and yak beef (meat) contains less fat than cattle beef (Indra and
Magash, 2002; Ji et al., 2002a; Xue et al., 2009). Milk products from yak milk, such as
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butter, cheese, yoghurt and milk residues (made by heating skimmed milk), are processed then sold locally at markets by farmers (Gerald et al., 2003). In the current survey, 22 households (12%) sold their milk products at markets and the remaining households consumed the products themselves, and any reduction in milk yield would reduce either income or protein source for these families.
Generally a higher seroprevalence results in greater losses in productivity with seropositive animals having increased rates of abortions, infertility and perinatal mortality, as well as reduced growth and milk yields (McDermott et al., 2013). An
Argentinian study found that when the prevalence of bovine brucellosis at an individual animal level was 4 to 5%, economic losses were approximate US$60 million per year or
US$1.20 per cattle head (Samartino, 2002). In Nigeria, where the prevalence ranged from 7 to 12%, losses were estimated at US$3.16 per cattle head (Ajogi et al., 1998).
However, in the current study the estimated losses were still relatively high at US$1.42 per yak per year, even though the prevalence was only 2.8% (95%CI: 2.0, 3.7). The relatively higher losses may be due to the higher current values for yak products. For example, the price of beef rose from 2.31US$/kg in 2000 to more than 7.70US$/kg in
2012 and has continued to increase (Wang and Shi, 2013).
Control of brucellosis in animals varies between countries, which results in different impacts of the disease on profitability (Godfroid et al., 2011). The USA spends approximately US$40 million on the control of brucellosis in cattle and pigs each year
(Sriranganathan et al., 2008) whereas in some low-income countries, even if the disease is endemic, almost no effective control measures are implemented (McDermott and
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Arimi, 2002; Perry et al., 2002). Furthermore, other studies in the UK and Spain have highlighted the significant economic benefit gained from controlling brucellosis in cattle (Hugh-Jones et al., 1975; Bernués et al., 1997). Since treatment of infected animals is not attempted due to the intracellular localization of Brucella and its ability to adapt to the environmental conditions encountered in the macrophage host cell
(Kohler et al., 2002; Romich, 2008), the strategy of treating infected animals was not included in the current analysis. In this study the prevalence under both strategies of vaccination and combination test-and-slaughter with vaccination declined each year during the six year period of analysis. Other studies have similarly demonstrated that a vaccination strategy can reduce the prevalence of brucellosis in cattle, goats and elk
(Alves et al., 2015; Montiel et al., 2015; Boroff et al., 2016). Through benefit-cost analysis, based on the BCR and NPV, the vaccination strategy with a BCR of 3.19 and an NPV of US$313,354.87 was the most economically profitable method to control the disease during the six-year study period. A case study conducted in Mongolia almost 15 years ago documented an average BCR of 3.2 (95%CI: 2.3 - 4.4) through adopting a vaccination control program in cattle, sheep and goats (Roth et al., 2003). Control through test-and-slaughter resulted in the greatest loss and was not profitable, primarily because of the costs of compensating for slaughtered seropositive yaks and the cost of purchasing replacement yaks. The test-and-slaughter program is also rarely achievable or viable in sub-Saharan African countries due to limited resources to compensate farmers whose animals are slaughtered (McDermott and Arimi, 2002; Godfroid et al.,
2011). Despite this approach potentially seeming to be effective in reducing the prevalence of brucellosis, it is challenging to eradicate the disease due to the inability to identify all affected animals, the lack of movement control and the lack of participation
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by farmers who doubt the reliability of the test results (Zamri-Saad and Kamarudin,
2016). If a vaccination program can be expanded to the total yak population in Tibet, it would result in considerable returns over a reasonably short period of time. Other regional studies have reported a higher seroprevalence of brucellosis in yaks of 9% in
Qinghai province, 4.2% in Sichuang province, 21% in Nepal and 21.11% in India (Li et al., 2008; Bandyopadhyay et al., 2009; Lang et al., 2011; Jackson et al., 2014). In these locations, where the prevalence is higher, it is likely that the economic benefit of brucellosis control and eradication through vaccination would be even greater than that estimated for Tibet.
The manual sensitivity analysis, not surprisingly, demonstrated that improving the protection rate of the vaccine would result in a better economic performance for the vaccination control program. Similar results have been reported in other studies (Roth et al., 2003; Alves et al., 2015). For the vaccination control program the uncertainty analysis conducted using @Risk, the NPV was positive and BCR was greater than 1 in all 2000 iterations indicating that, despite the uncertainty in some input variables, the program was economically profitable. The NPV for the vaccination control program was most sensitive to the loss from a female yak aborting, while the price of yaks that were slaughtered had the greatest effect on the NPV for the test-and-slaughter and the combination control programs. This negative regression coefficient was not unexpected as if the price of replacement animals was low; fewer funds are required to compensate farmers for culled animals. The model results could be improved by more precise empirical values for these parameters, however the results demonstrate that the
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vaccination control program is the most economically efficient approach despite uncertainty in the parameters.
Tibet is one of the largest rangeland areas in China where various livestock species are communally grazed on pasture (Chapter 4). Brucellosis may infect a wide range of terrestrial animals, including sheep, goats and cattle (Seleem et al., 2010), and consequently control should not only focus on the yaks in the province. A widespread control strategy incorporating control in other species of livestock should result in even greater economic benefits (McDermott et al., 2013; Ducrotoy et al., 2017). Finally, it is well known that the main sources of human infection are through contact with infected livestock and their products (Deqiu et al., 2002). Controlling the disease in livestock would have a flow-on effect to reducing the disease in humans resulting in an even greater economic benefit to the community (Roth et al., 2003; Zinsstag et al., 2007; Xin et al., 2010).
To effectively control the disease, the People’s Republic of China Law on Animal
Disease Prevention states that all infected animals are required to be slaughtered, and the TBAA has been implementing these regulations. For each animal that is slaughtered,
50% of the market value is compensated to the owners by the authorities (Lan, 2013).
This still causes substantial financial losses to households with infected animals. In other countries a higher level of compensation is provided for animals culled due to bovine brucellosis. For example, in Scotland, 75% of the market value is paid to owners of infected cattle (Animal Health, 2009). In contrast in some countries no compensation is provided (Sriranganathan et al., 2008), resulting in a greater financial impact for
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livestock producers who have infected animals. Such practices reduce participation by pastoralists who are unlikely to appreciate the losses to their herds and family income arising from the disease. Failure to remove infected animals further increases the likelihood of disease transmission within and between herds. Any disease control program requires interventions in a range of areas, and although this study highlighted the economic benefit of vaccination other disease-control practices such as animal identification and movement control (United States Department of Agriculture (USDA),
2003; Godfroid et al., 2011) should also be implemented in conjunction with a vaccination program.
The model developed in this study was relatively simple and did not cover all potential impacts of brucellosis in yaks or in humans. Hugh-Jones et al. (1975) found that in cattle some cows that aborted were temporarily infertile for approximately two months, resulting in an extended inter-calving interval. Due to the low reproductive capacity of yaks (Gerald et al., 2003), the impact of infertility in this study was difficult to estimate and was not included in the current model. It is likely that if all of the impacts of the infection were measured, even greater economic gains from controlling the disease and a higher BCR and NPV would be realised. This study highlights the impact of the disease on the productivity of yaks in the surveyed region of Tibet. The benefit of the vaccination program exceeded the costs of implementing this method of disease control, and since mixed grazing is still a dominant form of animal husbandry in Tibet, it is advisable to initiate a vaccination program in yaks, as well as other livestock.
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CHAPTER EIGHT
General discussion
Prior to the research reported in this thesis most epidemiological surveys of brucellosis in livestock in Tibet had been undertaken through official annual sero-surveillance studies. Historically brucellosis attracted little attention by the authorities and this, together with a lack of epidemiological information about the disease, resulted in little progress being made on its prevention and control. The study reported in this thesis was designed to gain a more thorough insight into the epidemiology of brucellosis in yaks in
Tibet and involved performing a retrospective study examining historical data, undertaking a cross-sectional seroprevalence study of yaks including assessment of risk factors for the disease, administering questionnaires to herders to determine their knowledge, attitudes and practices with respect to the disease and evaluating the economic burden of the disease in yaks.
8.1 Seroprevalence
Brucellosis is challenging to diagnose on presenting clinical signs alone even though it can lead to abortion storms, and accordingly serology, to detect antibodies against specific antigens of Brucella, has been widely used to confirm the presence of the disease in livestock (Godfroid et al., 2011). However there is no individual serological test that is appropriate for all epidemiological studies or situations (OIE, 2009).
According to the national policy of the MOA, two tests should be applied during the surveillance process for brucellosis (Ministry Of Agriculture and National Health and
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Family Planning Commission, 2016). However, only one test (RBT) was available and used in previous Tibetan sero-surveillance studies. Brucellosis is also not included in the list of animal diseases requiring compulsory vaccination in Tibet. These practices highlight the lack of emphasis placed on the control and prevention of brucellosis in livestock by the local authorities. However even with this lack of emphasis on brucellosis, in examining historical data a seroprevalence of only 0.75% (95%CI: 0.6,
0.9) was detected in yaks and cattle between 2009 and 2015 (Chapter 3). The cross- sectional serological study conducted as part of this thesis (Chapter 4) involved sampling randomly selected yaks from two counties of Lhasa prefectural city and one township of a county in Shigatse prefectural city. Two tests (RBT and C-ELISA) were used and the results interpreted in parallel to improve the sensitivity of detection
(Arruda et al., 2016). The seroprevalence when the two tests were interpreted in parallel was 2.8% (95%CI: 2.0, 3.7). The proportion of animals positive on the RBT test in this study (1.8%, 95%CI: 1.2, 2.6) (Chapter 4) was significantly higher than the official surveillance in 2015 (0.18%, 95% CI: 0.1, 0.3) (P<0.001, df=1, χ2=85.84). This difference could have arisen from the inclusion of a range of species (cattle, yaks, sheep, goats and pigs) in the official surveillance value, compared to the focus on yaks in the current study. It is possible that yaks are more susceptible to the disease than other species (Pan, 1992b). However, the result of the current study was significantly lower than the 6.5% (95%CI: 5.0, 8.5) reported in Tibetan yaks in 2011 (P<0.001, df=1,
χ2=36.47) (Gao et al., 2013). This difference could be associated with the sampling of yaks from different counties and at different times. The previous study (Gao et al., 2013) was conducted in six counties which were different to the three surveyed in the current study. In the historical data examined for human brucellosis, a high seroprevalence from
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2011 to 2013 was found in Tibet (0.84%, 95%CI: 0.6, 1.1) which was similar to the level accepted for a control zone (1% infection rate in humans) in China (Deqiu et al.,
2002). This high seroprevalence may be associated with Tibet being one of the main pastoral provinces of China where households in rural areas have frequent contact with livestock during their daily farming activities. However, the seroprevalence in humans in Tibet was lower than that of other Chinese provinces where the people sampled were mainly engaged in animal husbandry and veterinary activities (Xinjiang - 11.92%,
95%CI: 11.3, 12.6; Inner Mongolia - 9.27%, 95%CI: 9.2, 9.3; Qinghai-3.37%, 95%CI:
2.9, 3.9) (Tian et al., 2012; Muhtar et al., 2015; Fan et al., 2016) and also lower than that of rural areas in other countries including Turkey (4.85%, 95%CI: 3.6, 6.3) and
Kyrgyzstan (8.8%, 95%CI: 4.5, 16.5) (Cetinkaya et al., 2005; Bonfoh et al., 2012).
These differences could be due to a range of cultural, sanitary, socioeconomic, transport and political factors (Al Dahouk et al., 2007; Akhvlediani et al., 2010).
Undertaking routine serological surveys is essential to, not only enumerate the prevalence of brucellosis in Tibet, but also to identify infected animals. These infected animals have the potential to spread disease to susceptible animals and to previously disease-free populations (Godfroid et al., 2011). The serological study conducted as part of this thesis has provided valuable information on the current seroprevalence in the sampled counties and has helped highlight the epidemiological pattern of infection. This information can be used to assess the disease’s impact on the animals and the community and is essential to provide baseline data prior to implementing methods to control the disease (Adone and Pasquali, 2013).
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8.2 Risk factors for infection
Identifying and understanding risk factors for a disease are critical for the implementation of effective disease control programs (Porphyre et al., 2010). Despite the geographical location appearing to be associated with the seroprevalence in the retrospective study (Chapter 3), in the serological study conducted (Chapter 4) there was no difference in the individual or herd level seroprevalences between the three sampled counties (p>0.05). This may be linked to the fact that the three counties sampled were the main yak rearing areas in Tibet, with similar husbandry and management practices adopted for yaks grazing on the counties grasslands. In contrast the annual serological surveillance undertaken by the government includes sampling animals from counties distributed across Tibet and involved sampling all available livestock species, including yaks, cattle, sheep, goats and pigs (Chapter 1).
Although data resulting from surveillance of brucellosis in humans was only available for three years, the trend in the seroprevalence was aligned with the temporal distribution of animal brucellosis. The temporal distribution of animal brucellosis fluctuated peaking in 2011 at 2.34% before it dropped to 0.18% in 2015 (95%CI: 0.1,
0.3). Similarly the seroprevalance for yaks and cattle was highest in 2011 (2.7%, 95%CI:
2.0, 3.5) which then declined to 0.31% (95% CI: 0.2, 0.5) in 2015. The seroprevalence in humans in Tibet also reduced from 2.23% in 2011 to 0.05% in 2013 (Chapter 3). Zhu
(2013) highlighted that brucellosis in humans is always linked to infected animals or products from those animals, and effective control of the disease in animals will not only reduce the incidence in livestock with benefits to the farming community, but will also result in a lower incidence in humans benefiting the total community.
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In the retrospective study the seroprevalence in spring/summer was found to be significantly higher than that in autumn/winter. The spring/summer period, particularly the months of April and May, coincides with the peak calving time for yaks (Gerald et al., 2003). Brucella is primarily shed during abortions which typically occur during the third trimester of gestation (Geering et al., 1995). This stage of the pregnancy coincides with the spring/summer period resulting in a greater opportunity for disease spread resulting in a higher incidence of disease.
The age of the yaks was strongly associated with the seroprevalence in yaks, with animals between 3 and 5 years of age having the highest seroprevalence. It is during this age period that yaks reach their sexual maturity and most will become pregnant during this period. Brucella has a predilection for the pregnant uterus where the sugar erythritol is present (Carvalho Neta et al., 2010), resulting in accumulation of large numbers of bacteria at this site potentially leading to an abortion (Anderson et al., 1986).
Pastoralists that were older than or equal to 50 years of age had a higher level of knowledge about brucellosis than younger pastoralists (Chapter 6). This is likely to be linked with their experience and also increased opportunity to have seen or heard about the disease than younger pastoralists. It is important in educational campaigns to draw on the experiences and knowledge of these older pastoralists, who often are respected members of the community (Healy, 2004; Murakami et al., 2009). In addition, herders from a larger household (≥6) had higher knowledge, adoped better practices and had a
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better understanding of the disease. It could be associated with the presence of older persons potentially in their family who possess richer experience on rearing yaks.
In Chapter 5 a higher seroprevalence was found in yaks raised under an agro-pastoral production system compared to those raised under a pastoral production system
(p=0.002). In contrast in the KAP survey, pastoralists who worked within a pastoral production system had a significantly better knowledge about the disease than did pastoralists who managed their yaks under an agro-pastoral production system
(p<0.001). This negative correlation between knowledge of pastoralists and seroprevalence is not unexpected and similar findings were reported from a study conducted in Zimbabwe (Matope et al., 2010). Adesokan et al. (2013) also pointed out that a lack of awareness by livestock workers about the risks associated with the consumption of unpasteurised dairy products and contaminated beef were factors that facilitated Brucella infections in livestock workers in Nigeria. These findings highlight the need for improved awareness and knowledge of the disease by humans for the control of both livestock and human brucellosis.
The husbandry and management practices adopted by farmers also play a role in the prevalence of brucellosis (Corbel, 2006). A study conducted in Brazil of herds with more than 54 cows pointed to the proximity to wetlands as a factor that greatly increased the risk of brucellosis (Borba et al., 2013). Studies have identified practices such as feeding aborted material to dogs, introducing new animals into a herd and running multiple species of animals together as having significant effects on the seroprevalence of brucellosis in a range of species, including humans (Ghanem et al.,
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2009; Jackson et al., 2014; Musallam et al., 2015a). However, in the current study these factors did not influence the seroprevalence. This could have been associated with differences in livestock production systems, animal husbandry practices and the lifestyle of farmers between the current and previous studies. The situation in yaks may also be different to cattle resulting in differences in risk factors for the different livestock species.
Improvement in the knowledge and understanding of local farmers and pastoralists about the disease and the practices adopted can significantly reduce the prevalence of many zoonotic infections (Lindahl et al., 2015). In the KAP study, 66.6% of the pastoralists surveyed had some formal school education (primary/secondary level)
(Chapter 6). Pastoralists with higher levels of education were found to have significantly better knowledge on brucellosis and were more likely to have better understanding and adoption of safe practices to aid in disease prevention. Therefore, public health education is a critical component of reducing the spread of brucellosis and this requires further funding and development in the future in Tibet (Smits, 2013;
Kansiime et al., 2014). In particular educational material on brucellosis control in yaks which is relevant and appropriate for Tibetan pastoralists should be specifically developed and offered in a culturally fitting way.
8.3 Economic assessment
Bovine brucellosis is considered to be one of the most economically important zoonotic diseases due to its direct and indirect impact on both animals and humans (Singh et al.,
2015). Many countries have conducted economic evaluations of the disease (Bernués et
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al., 1997; Santos et al., 2013); however it is difficult to assess the economic losses arising from this disease in China due to a lack of available data, despite it being a re- emerging disease (Liu et al., 2014).
The current study was the first economic study of brucellosis in yaks performed in
China and focused on the major productivity parameters (abortion, reduction of milk yield, meat loss and perinatal mortality). The results indicated that the annual financial loss was estimated at approximately US$ 1.42 per yak even though the seroprevalence of yaks was only 2.8% (95%CI: 2.0, 3.7) (Chapters 4 and 7). Extrapolating this loss to the total population of female yaks (≥3 years old) in Tibet could result in a potential annual loss of US$ 2,324,001.56 from brucellosis. Other studies have demonstrated that brucellosis in cattle can similarly result in large economic losses which are borne by people involved in the livestock industries, as well as the general community (Santos et al., 2013; Singh et al., 2015). In the research outlined in this thesis vaccination was shown to be the most cost-effective method (BCR >1 and positive NPV) of controlling brucellosis in yaks. A similar finding had been found in other studies in cattle (Can and
Yalcin, 2014; Alves et al., 2015; Baghiyan and Shirvanyan, 2016) and control in livestock also results in benefits for humans arising from a reduced livestock seroprevalence (Roth et al., 2003). Vaccination for the prevention of brucellosis has multiple advantages including being inexpensive, convenient and effective. Historically mass vaccination of yaks in Tibet in the early 1980s was shown to have resulted in a reduction in the seroprevalence from 22.3% to 1.1% in the four counties of Anduo,
Nagqu, Bange and Linzhou (Chapter 3). This highlights the impact and potential
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economic benefit from implementing a vaccination program to control brucellosis in
Tibet.
8.4 Limitations of the present study
Although the epidemiological study reported in this thesis produced several important findings, there were limitations associated with the study. Initially, although annual official sero-surveillance data were available for analysis (retrospective data), no data on demographic information of the sampled animals had been collected. This has been a common problem with epidemiological surveys conducted on animal diseases in many provinces of China including Tibet, Shanxi and Qinghai (Jia, 2012; Xu et al., 2012;
Zhuomacuo, 2017). In contrast during the serological survey conducted as part of this study full demographic data were collected on sampled animals. The questionnaires used in the current study played an important role in the methodology of the epidemiological study and were used for: identification of the risk factors of the disease; determining the KAP of the producers; and the economic evaluation of the disease.
Several factors, such as the current government policy and reluctant interviewees, also could have impacted upon the results. For example, the authorities have a policy that limits the number of livestock which can be owned by a pastoralist in Tibet to protect the natural grasslands from overgrazing and degradation (Lan, 2013). This may have resulted in farmers and pastoralists concealing the true number of livestock and pregnant yaks owned. In addition, several farmers and pastoralists were unwilling to answer questions due to time constraints or to prior commitments. This may have resulted in the premature completion of the questionnaire, which could potentially bias the results. In addition, the two tests used in the current study have not been validated in
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yaks. These tests had, however, been previously used in other studies to determine the seroprevalence in yaks (Bandyopadhyay et al., 2009; Yang et al., 2013a). For the RBT, the agglutination intensity is affected by many factors including the amount of antigen, the temperature at which the test is run, the duration between adding the antigen and reading (interpreting the result), the experience and visual acuity of the test interpreter and potential cross-reactions with LPS of other bacteria (Cho et al., 2010). Finally, isolating (culturing) and identifying pathogens was not successfully undertaken in the current study because of time, logistical and funding constraints. However during the serological survey, half the blood sample was centrifuged for sera and tested with the serological assays whilst the other half was preserved for culture. The whole blood from the seropositive animals was cultured (results not shown), however no positive samples were detected, potentially because of the low concentration of Brucella spp. routinely found in the blood of infected animals (Young and Corbel, 1989). In the future culturing of relevant clinical specimens, such as aborted foetuses, should be undertaken to identify the species and strains of Brucella affecting yaks in Tibet.
8.5 Recommendations
Risk-based control measures
Control measures for diseases, including brucellosis in yaks, should be based on sound epidemiological reasoning. As there is a higher prevalence of brucellosis in yaks during the spring/summer season, which is when yaks calve, suitable preventive measures should particularly be adopted at or during this time. For example, if an aborted foetus is found it should be either burnt or buried to prevent it from contaminating the environment, being dispersed by dogs or being eaten by other livestock. The
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placenta/after-birth of livestock that aborts should also be burnt or buried and their contaminated holding/calving pens should be disinfected with disinfectants such as hypochlorite solutions, phenolic disinfectants, or formaldehyde (Zhao et al., 2010).
However it is important that farmers minimise their direct contact with aborted calves or placentae during disposal by wearing gloves and suitable protective clothing to reduce their risk of contracting brucellosis (Lindahl et al., 2015).
In the epidemiological study described in this thesis, age and production system were associated with brucellosis seropositivity. The age of an animal is widely acknowledged as one of the factors associated with the prevalence of infection (Megersa et al., 2011b;
Sanogo et al., 2012). Local farmers and pastoralists often have traditional ideas and are unwilling to kill and/or sell their livestock because they are too emotionally attached to them (Xue et al., 2009). If seropositive animals are detected, regardless of how long the livestock have been raised by the owner, it is necessary to implement appropriate culling strategies to prevent further transmission of the pathogen. According to regulations, authorities must compensate 50% of the animal’s market value to farmers/pastoralists if their infected livestock are slaughtered. Huang et al. (2017) pointed out that providing a reasonable compensation is an effective strategy to encourage the safe disposal of infected animals and to reduce the risk associated with black market transactions. Increasing the compensation level to close to market value is likely to increase support by pastoralists for the culling of test positive animals.
The higher concentration of livestock and humans on agro-pastoral farms results in more localised waste which can lead to an unhygienic environment, potentially allowing
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for disease transmission if Brucella is present. Ensuring a clean environment and minimising overstocking is central to control of most diseases, including brucellosis
(Jutzi et al., 1988; Provincial Infectious Diseases Advisory Committee, 2012).
As mixed grazing occurs in Tibet, in order to better explore risk factors and transmission of Brucella, the serological survey conducted in this study (Chapter 4) should be expanded to include other species such as cattle, sheep and goats.
Public educational programs
Although brucellosis is one of the most common zoonotic diseases worldwide and there has been a significant increase in the number of human cases in the 21st century (Seleem et al., 2010), many people still lack an awareness of the disease. For example, a study conducted in Tajikistan showed that the majority of small scale dairy herders surveyed had never heard of brucellosis (Lindahl et al., 2015). This leads to widespread underreporting and potential misdiagnoses of the disease in humans.
In the current study respondents who had undertaken some formal education had a significantly better understanding of the disease and implemented safer practices than farmers who had not had the opportunity to attend formal schooling (Chapter 6). If farmers had a better awareness of the risks they would be more likely to take measures both to reduce transmission of the disease in their livestock and to minimise cross- species transmission to themselves and their families. In order to increase awareness and encourage the adoption of better healthy behaviours, both the TBAA and TAAAS should conduct annual public education programs on the disease in schools and
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communities and wherever pastoral production and agro-pastoral production systems exist. Information should be provided on the clinical signs, transmission route, prevention and control measures for brucellosis. The educational message may be provided through various routes including radio broadcasts, warning signs, posters, pamphlets, newspapers, television and slides (Chen et al., 2016).
Vaccination programs
In this study, yak calves in the Pali township of Yadong had been vaccinated with S2 vaccine. However, surprisingly there was no significant difference in the seroprevalence between vaccinated and unvaccinated yaks. This could be caused by issues with the vaccine including poor storage and transport of the vaccine in and to the field (poor cold chain) (Hanson et al., 2017), or the vaccination process including leakage of the vaccine during the inoculation process resulting in less than a full dose being administered
(Zeng et al., 2009). Further studies are required to investigate the efficacy of the S2 vaccine in yaks as vaccination has been shown in many studies to be an effective means of controlling the disease in a range of species (Al-Khalaf et al., 1992; Romich, 2008;
Ding and Feng, 2013; Baghiyan and Shirvanyan, 2016). It is important that the vaccination procedure is implemented according to the vaccine instructions and further training of vaccinators may be warranted by the TBAA. The benefit-cost analysis outlined in Chapter 7 revealed that vaccination was the most cost-effective method to control and prevent the spread of brucellosis. Therefore, a vaccination program should be implemented, not only in calves but also in adult yaks, as has previously been adopted for cattle (Olsen and Tatum, 2010; Godfroid et al., 2011). Brucella suis S2 vaccine has been being popular in China as it induces a high level of immunity and is
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safe for pregnant animals when administered orally (Deqiu et al., 2002; Ding and Feng,
2013). Alton et al. (1980) also reported that pregnant cows could be successfully vaccinated with low doses of B. abortus S19 vaccine by the subcutaneous injection without affecting the pregnancy. Based on the Tibetan practice of grazing multiple species together, any vaccination program adopted should be extended to include other susceptible species such as sheep, goats and cattle.
8.6 Future research
Molecular epidemiological research
In order to obtain a better understanding of the epidemiological patterns of the disease in yaks and to determine if the yak is a spill-over host or a true reservoir, isolation and identification of Brucella spp. from yaks should be further explored. Because Brucella survives intracellularly in lymph nodes and mammary glands of ruminants after abortion, samples for identification should be taken from the lymph nodes of infected animals or from the aborted foetus (OIE, 2009). This research could provide information regarding the species and type of Brucella that is circulating within Tibet and in yaks. More importantly, research could help to identify the most effective vaccines, as not all vaccines have the same efficacy against the various Brucella spp.
(Adone and Pasquali, 2013).
Validation of serological tests in yaks
There are many serological tests to detect antibodies that are specific to the LPS of
Brucella species, such as RBT, ELISAs and FPA. These tests have been validated in some species of cattle (Bos taurus), sheep (Ovis aries) and goats (Capra hircus).
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Although Bos grunniens (domestic yak) is a species of the genus Bos, they have different digestive actions and gene expressions to cattle (Schaefer et al., 1978; Wang et al., 2016). In order to better understand the epidemiology of brucellosis in yaks, to increase the sensitivity of detection and better protect yaks from brucellosis, it is necessary to validate the commonly used serological tests in this species.
Animal experiments for the economic study
Many studies on the economic costs of brucellosis and its control have been carried out in developed countries such as the USA and the UK, however there is little information from developing countries or from China (Bernués et al., 1997; McDermott et al., 2013).
In this study (Chapter 7) the parameters applied to the economic evaluations primarily originated from studies of cattle in Spain, England and Wales. Due to different feed, animal husbandry and management practices, species and veterinary and medical capacities, the economic impact of brucellosis could vary between species, regions and countries (McDermott et al., 2013). Tibet has a unique climate with low temperatures, strong radiation and different livestock production systems and it is anticipated that the economic impact of the disease would differ in Tibet from most other countries.
Therefore, it is important to conduct further economic research into the effect of the disease on livestock in Tibet, and even other provinces of China. The results of such studies would offer more accurate evaluations of the economic impact of the disease, and provide better ideas for more effective control of the disease in the long term.
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Epidemiological study in wildlife
Brucellosis affects a variety of terrestrial animals and wildlife species (Godfroid, 2002;
Seleem et al., 2010). Brucella abortus and B. suis have been isolated from a number of wildlife species, including Bison (American Buffalo), elk/wapiti (Cervus elaphus), feral pigs (Sus scrofa), African buffalo (Syncerus caffer), eland antelope (Taurotragus oryx), waterbuck (Kobus elipsiprymnus) and reindeer (Ranfifer tarandus) (Nielsen and Duncan,
1990). Brucella melitensis has also been reported in chamois (Rupicapra rupicapra) and ibex (Capra ibex) (Garin-Bastuji et al., 1990; Ferroglio et al., 1998). Tibet has a range of ungulate wildlife species, including the Tibetan antelope (Pantholops hodgsonii),
Tibetan gazelle (Procapra picticaudata), Tibetan argali (Ovis ammon hodgsoni), blue sheep (Pseudois nayaur), Tibetan wild ass (Equus kiang) and wild yak (Bos mutus)
(Miller and Schaller, 1996). Godfroid et al. (2011) reported that wildlife can be reservoirs of brucellosis and occasionally disease can spill-over from wildlife to livestock. For example, the USA is free of B. abortus in cattle except for the Greater
Yellowstone Area (GYA). This localised focus is likely due to transmission from elk
(Cervus canadensis) (Van Campen and Rhyan, 2010). In Tibet, it is possible that frequent contact between wildlife species and livestock, in particular yaks, will increase the risks of infection in the livestock. In addition, infected placental material on pastures can be dispersed by dogs and other carnivorous animals, such as foxes, potentially resulting in exposure of Brucella to other susceptible livestock (Al-Rawahi, 2014).
In order to understand if wildlife species have been infected, and if there is a spill-over of infection from domestic animals to wild species or vice-versa, multidisciplinary research is required to study both domestic and wildlife species. If local wildlife is
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infected, it would increase the risk of the transmission of Brucella to domestic livestock and contribute to the persistence of brucellosis in the region. However, controlling brucellosis in wildlife reservoirs is complicated and costly (Olsen, 2010). Conversely wildlife species could be exposed to the bacteria shed by infected livestock, so implementing a control program in livestock could minimise transmission to wildlife species and prevent the establishment of a potential wildlife reservoir of infection.
8.7 Conclusions
In conclusion, this study focused on expanding information on the epidemiology of brucellosis in yaks in Tibet. Based on the findings, it is recommended that in order to effectively control the disease resulting in improved income for local farmers/pastoralists, an integrated approach should be implemented including adopting risk-based control measures, mass vaccination and education.
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Appendices
Appendix 1. Basic information collected while sampling
Code Owner name Village Animal Id Age Sex Breed Pregnant Abortion Remarks
Note: Sex: Male=M, Female=F; Breed: Local=L, Cross=C; Pregnant: Yes=1, No=0; Abortion: Yes=1, No=0
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Appendix 2. Farmer questionnaire for risk factors, KAP and economic survey
Information of household:
Name Village Gender Age Education Occupation Production system No of people Date No education Farmer Semi-graze Primary Dependent/housewife Full-graze Second Student Tertiary Employee University
Number of livestock:
livestock Yaks sheep goats horses Others Adult male Young Adult Young (≥1year) male(<1 year) female(≥1year) female(<1 year) Number
1 Knowledge 1.1 Have you heard of others in your village having abortions in their animals over the last 12 months? Yes [ ] No [ ]
1.2 Do you know what caused the abortion? Yes [ ] No [ ] Not sure [ ]
If yes, what caused it? (a) Disease [ ] (b) Nutrition [ ] (c) Breed [ ] (d) Other (please specify …………)
1.3 Have you ever heard of brucellosis? Yes [ ] No [ ] Not sure [ ]
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If yes, Questions Answers (a) Neighbours [ ] (b) Veterinarian at vet station of township or county [ ] (c) Newspaper [ ] Where or who did you (d) Radio hear about it from? [ ] (e)TV [ ] (f) Training program [ ] (g) Other please specify [ ] (a) Abortion [ ] (b) Retained placenta [ ] What signs does it (c) Infertility produce? [ ] (d) Orchitis, epididymitis [ ] (e) Other please specify [ ]
1.4 Do you know what organs it affects? Yes [ ] No [ ]
If yes, what organs does it affect? (a) Uterus [ ] (b) Mammary gland [ ] (c) Testis [ ] (d) Other (please specify ………………)
1.5 Do you know if yaks can be infected by brucellosis? Yes [ ] No [ ] Don’t know [ ]
If yes, how can they be infected? (a) Licking aborted foetus [ ] (b) Drinking milk [ ] (c) Urine [ ] (d) Faeces [ ] (e) Wound [ ] (f) Mate/ artificial insemination [ ] (g) Other (please specify) …………………
1.6 Do you know if it can affect other animals, including sheep or goats? Yes [ ] No [ ] Don’t know [ ]
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1.7 Do you know if humans can be infected with brucellosis? Yes [ ] No [ ] Don’t know [ ]
If yes, how can humans be infected? (a) Handling aborted foetus from animals [ ] (b) Drinking un-boiled milk [ ] (c) Other (please specify ………………)
1.8 Do you know if brucellosis can be prevented in yaks? Yes [ ] No [ ] Don’t know [ ]
If yes, how can the disease be prevented? (a) Giving medicines [ ] (b) By vaccination [ ] (c) Graze separately from other herds [ ] (d) Slaughter animals with clinical signs [ ] (e) Other (please specify) ………………
1.9 Do you know if brucellosis can be treated in yaks? Yes [ ] No [ ]
1.10 Do you know if brucellosis can be treated in humans? Yes [ ] No [ ] Not sure [ ]
1.11 Do you know if there is any control program for brucellosis? Yes [ ] No [ ]
If yes, what do you know about it? (a) Vaccine [ ] (b) Surveillance [ ] (c) Quarantine [ ] (d) Slaughter [ ]
1.12 Do you know if vaccination is an effective way to control the disease? Yes [ ] No [ ] Don’t know [ ] 159
2 Attitudes and practices 2.1 If an animal is sick what do you do? (a) Nothing [ ] (b) Inform vet in village [ ] (c) Treat it by myself [ ] (d) Ask other people in the village for help [ ]
2.2 Do you separate sick yaks from healthy ones? (a) Yes, for a couple of days [ ] (b) Yes, until the sick yaks recover [ ] (c) No, rarely [ ] (d) No, never [ ]
2.3 If you had a sick yak which had an aborted calf or discharge from the vagina, did you wear protective gloves to dispose them? (a) No, never [ ] (b) No, rarely wear [ ] (c) Yes, sometimes [ ] (d) Yes, always [ ]
2.4 Do you isolate newly purchased yaks before introducing them to your herd? Yes [ ] No [ ]
2.5 Do you consider brucellosis is an important disease affecting yaks? Yes [ ] No [ ]
2.6 Do you support the control campaign against brucellosis? Yes [ ] No [ ] Not sure [ ]
2.7 What are the key factors affecting the level of production of your yaks? Disease [ ] Grassland [ ] Breed [ ] Weather [ ] Nutrition [ ] Others [ ] Don’t know [ ] 160
2.8 Would you like to join training organized by Veterinary Departments about preventing animal diseases? Yes [ ] No [ ]
2.9 Are your yaks usually mixed with other animals when they are grazed? Yes [ ] No [ ]
If yes, which animals do they graze with? Sheep [ ] Goats [ ] Horses [ ]
2.10 Are your yaks grazed with yaks belonging to other farmers? Yes [ ] No [ ]
If yes, how many herds are they usually grazed with? ………………..
2.11 Do your yaks drink water with yaks owned by other farmers when grazing? Yes [ ] No [ ]
What is the source of the water? River [ ] Tap [ ] Other (please specify) …………
2.12 Do you move/migrate your yaks to other pastures every year? (a) Yes, every year [ ] (b) Yes, most years [ ] (c) Yes, but rarely [ ] (d) No, never [ ]
What is the main route that your herd passes through? ………………………..
2.13 Which methods do you choose to breed your yaks? (a) Natural mate (with own bull) [ ] (b) Natural mate (bull from other households) [ ] (c) Artificial insemination [ ] 161
2.14 Do you ever boil milk before you drink it? (a) Yes, all the time [ ] (b) Yes, usually [ ] (c) No, rarely [ ] (d) No, never [ ]
2.15 Have your animals ever been infected with brucellosis? Yes [ ] No [ ] Not sure [ ]
If yes and an abortion occurred, what did you do with the aborted foetus? (a) Left where it was [ ] (b) Burnt or buried it [ ] (c) Fed it to other animals (dog) [ ] (d) Consumed it [ ]
2.16 Have you ever used vaccines against brucellosis? Yes [ ] No [ ]
If yes, When was the last time you vaccinated your animals? …………………
How many animals did you vaccinate? ……………………
How much did you pay for the vaccination? ………………..
2.17 Do you know how often a vaccine should be given to yaks to prevent brucellosis? Yes [ ] No [ ]
If yes how often? (a) Once each year [ ] (b) Twice a year [ ] (c) Once every few years [ ] (d) Not sure [ ]
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3 Economic impacts 3.1 How many yaks did you sell last year? [ ] Males [ ] Females [ ] To other farmers (a) <2 years old (a) <2 years old 1 [ ] (b) Between 2 and 6 years old (b) Between 2 and 6 years old (c) >6 years old (c) >6 years old Males [ ] Females [ ] To dealers (a) <2 years old (a) <2 years old 2 [ ] (b) Between 2 and 6 years old (b) Between 2 and 6 years old (c) >6 years old (c) >6 years old Males [ ] Females [ ] To abattoir (a) <2 years old (a) <2 years old 3 [ ] (b) Between 2 and 6 years old (b) Between 2 and 6 years old (c) >6 years old (c) >6 years old
3.2 In the past 12 months have you bought or acquired new yaks? Yes [ ] No [ ] Not sure [ ]
From whom did you get them, how many did you get and how old were they? Males [ ] Females [ ] From other farmers (a) <2 years old (a) <2 years old 1 [ ] (b) Between 2 and 6 years old (b) Between 2 and 6 years old (c) >6 years old (c) >6 years old Males [ ] Females [ ] From dealers (a) <2 years old (a) <2 years old 2 [ ] (b) Between 2 and 6 years old (b) Between 2 and 6 years old (c) >6 years old (c) >6 years old Males [ ] Females [ ] From local relevant (a) <2 years old (a) <2 years old 3 animal station (b) Between 2 and 6 years old (b) Between 2 and 6 years old [ ] (c) >6 years old (c) >6 years old
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3.3 How many of your yaks were pregnant last year? ……………….
How many had calves? ………………… 3.4 How many female yaks produced milk? …………………….
Approximately how many liters of milk did each yak produce per day? (a) <1 liters [ ] (b) between 1 and 2 liters [ ] (c) between 2 and 4 liters [ ] (d) > 4 liters [ ]
3.5 What do you do with your yak milk? Sell: ………….. liters/per day Processing product: ……………….. liters/per day Consume by the household: …………………liters/per day
3.6 Which kind of products do you make from your milk? (a) Yoghurt [ ] (b) Milk sediments [ ] (c) Butter [ ] (d) Other, Please specify [ ]
3.7 If you sell milk approximately how much do you get per liter of milk? …………
3.8 In the last year did you purchase any fodder to supplement your yaks in addition to their grazing? Yes [ ] No [ ]
If yes, how often did you provide the fodder, and how much did it cost? Frequency ( Spring/summer/autumn/winter) Fodder (kg) Price/unit Period (days) Total price Every day Every two days Every week Other
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3.9 Were your yaks used for draft (animal) power last year? Yes [ ] No [ ]
If yes, on average how long did your yaks work for each day and how often did they work? (a) Number of yaks ……………………… (b) Work/day (hour) ……………………… (c) Period ………………………
3.10 Have there been any abortions in your yaks during the last 12 months? Yes [ ] No [ ] Not sure [ ]
If yes, how many? ………………….
If yes, approximately at what stage (month) of pregnancy did the abortions occur? (……………month)
3.11 Did all the abortions result in the birth of dead foetuses or did some survive for a period of time? All died [ ] Some survived [ ]
3.12 Has brucellosis ever been diagnosed in your yaks? Yes [ ] No [ ] Not sure [ ]
If yes, how many yaks were identified as infected? ……………………..
3.13 How much did you spend on your yaks over the past year to treat them for brucellosis? …………….
Thank-you for taking the time to complete this survey
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Appendix 3. Farmer questionnaire for economic survey
Information of household:
Name: ………….. Gender: ……………. Village: ………….. Date: ………………
1 Approximately what percentage of your income comes from yaks? (Approximate ... %)
2 Average weight: …………….. (Kg, male) ………………. (Kg, female)
3 Price: …………… (adult male), ………… (adult female), ……………. (calf)
4 Price/kg: ………………. (yak meat)
5 If you know there are yaks infected with brucellosis in your herd, how do you deal with them? Leave in herd [ ] Slaughter [ ] Sell [ ] Treat [ ] Other (please specify) ………………
For males If sell, what is the price? Same as original (normal) price [ ] Reduced price [ ] If price is reduced, approximately how much is the price reduced? (……… %) If treated, what medicines are used? Approximately how much is the cost? ………………………………………………….
For females If sell, what is the price? Same as original (normal) price [ ] Reduced price [ ] If price is reduced, approximately how much is the price reduced? (………%) If treated, what medicines are used? Approximately how much is the cost? ………..
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6 Do you have unfertile female yaks? Yes [ ] No [ ] Not sure [ ]
If yes, how do you deal with them? Leave in herd [ ] Slaughter [ ] Sell [ ] Treat [ ] Other (please specify) ………………
If sell, what is the sale price? Same as original (normal) price [ ] Reduced price [ ]
If price is reduced, approximately how much is the price reduced? (………… %)
If treated, what medicines are used? Approximately how much is the cost? ………………………………………………….
7 Do you have sterile bull yaks? Yes [ ] No [ ] Not sure [ ]
If yes, how do you deal with them? Leave in herd [ ] Slaughter [ ] Sell [ ] Treat [ ] Other (please specify) ………………
If sell, what is the sale price? Same as original (normal) price [ ] Reduced price [ ]
If price is reduced, approximately how much is the price reduced? (………… %)
If treated, what medicines are used? Approximately how much is the cost? .……..
Thank-you for taking the time to complete this survey
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