Presented by Yang Shibiao at the Ecohealth 2012 conference held at Kunming, China on 15-18 October 2012

1. An Integrated Approach to Control Brucellosis
in Yunnan Province China
Progressive research results from the China project team of
Ecosystem Approaches to the Better management of
Zoonotic Emerging Infectious Diseases (EID) in South East Asia
Presentation by
Dr. Yang Shibiao
Yunnan Academy of Animal Science and Veterinary, Golden Temple, Kunming, PR. China, 650224
EcoHealth Conference, Kunming China 2012
15-17 Oct, 2012, Kunming, Yunnan, China
Yang Shibiao, Yunnan Academy of Veterinary and Animal Science, Kunming, Yunnan Province, China;
Yang Guorong, Yunnan Academy of Grassland and Animal Since, Kunming, Yunnan Province, China;
Yang Xiangdong, Yunnan Institute of Endemic Disease Control and Prevention, Dali, Yunnan Province,
China;
Li Wengui, Yunnan Agricultural University, Kunming, Yunnan Province, China

2. Review on published research paper
Chinese scientific papers published from 2007 to 2012 are systemically
reviewed to focus on:
 Prevalence in animal:
1. Prevalence Increased significantly during the period from 2003 to 2005, with
a prevalence of 4.94% within 12 provinces in China.
2. In endemic area, prevalence varies greatly with species, 15.28% in captive
deer, 7.81% in goats, 1.27% in yellow cattle and 0 in other species;
3. In sporadic area, prevalence also varies greatly with species, 1.38% in goats,
but 0 in both cattle and pig.
 Host animal range: Several species of animal serve as the host of Brucella,
including:
1. Shepherd dog in pasture ecosystem,
2. Pet dogs in urbane area,
3. captured wild animals, e.g. cervidea and camelidea,
4. Yak in plateau and buffalo in south China.

3. Review on published research paper (Cont.)
 Epidemiological analysis in human: Distribution of infection cases in human is related to gender
and profession.
1. Morbidity is higher in male than female. In a retrospective analysis of 20 in-hospital patients, male
cases dominated by 70%; in another analysis, male cases took up 84.21% (80/95).
2. higher in professional groups at high risk than others, 16 farmers ordinarily feeding animal, 3 cooks
contacting fresh beef, lamb and pork, 1 worker in meat process factory previously injured by hook
for several times; and farmer cases took up 75.00% in 95 cases.
3. There are also cases of Brucellosis in non-professional human groups, 4-years-old child possibly
infected from pet dog, and 4 cases of 28 living in urbane area, of which 2 with contact to pet dog
or cat, however 2 without contact to animal.
4. Several serological surveys were also conducted in professional groups at high risk.
A summary of retrospective analysis for 3 cases survey

4. Review on published research paper (Cont.)
 Development of diagnostic technologies :
1. There are several diagnostic technologies including agglutination test covering card agglutination,
Rose Bengal test, tube agglutination test and milk ring test, complement fixation test, and
indirect/competent ELISA method for detection of antibody.
2. For serological survey, Rose Bengal test for screening and further confirmed for those that are
positive or suspect by tube agglutination or complement fixation test are set up as the national
standard for Brucellosis in animal.
3. Several new technologies for detection of the pathogen have been developed during the recent years,
including a real-time PCR assay, a biotype-specific PCR assay and a multiple PCR assay for agent
identification, and indirect ELISA for detection of antibody.
 Control measures:
1. Introducing new animal into a farm is strictly restricted, by avoiding introducing animal from
endemic area, or introducing from only a Brucellosis free farm with a 30-days quarantine.
2. Vaccination is conducted in endemic and threatening areas, an animal is vaccinated individually, and
in case of emergence, two doses are inoculated or boost vaccination performed.
3. Survey is periodically implemented to detect any positive animal individual and the confirmed one(s)
destroyed immediately.
4. Measures for sanitary and disinfection are strictly carried out ordinarily including one needle for each
individual when inoculation, proper disposal of feces and contaminated materials, and regular
disinfection.
5. Staff is properly protected by dressing work clothes and wearing gloves and masks, avoiding
treatment of aborted materials.
6. Measures for prevention of infection through fertilization and sharing pasture and water source are
also put into practice.

5. Review on national data
 Status and trend of brucellosis in China
Data was collected from officially published documents by veterinary bureau, MOA,
China Anima Disease Prevention and Control Center, and China Disease Control Center.
1. Wider distribution: Brucellosis was officially reported in cattle (including buffalo) and
goat/ sheep in total 26 provinces and autonomous regions in China since 2008, covering
Hunan Province and Guizhou Province during this period where the disease was formerly
free).
2. Higher infection rate: Infection rate increased from 3% in 2007 to 5.71% (up to 73% at
the highest) in 2009 in cattle/buffalo, and increased from 2.1% in 2008 to 4.44% (over
80% at the highest) in 2009 on individual basis.
3. Wider range of host animal: More species of host animal were involved in the infection
of the disease, e.g. dogs such as shepherd dog in pasture ecosystem and pet dogs in
urbane areas, and captured wild animals such as cervidea and camelidea, have been
confirmed to be infected with Brucellosis.
4. Co-infection: Pathogens originated from different species of host animals co-exist in a
single host animal.
5. The most important zoonosis: Total 38,151 cases in human increasing by 12.45%
compared with that in 2010 were reported by CDC China in 2011.

6. Review on national data (Cont.)
A summary of Brucellosis in the past 5 years in China

7. Review on national data (Cont.)
 Measures for prevention and control
1. Vaccination: The National Scheme of Compulsory Vaccination against Animal Diseases is
applied annually, in which Brucellosis was listed in the vaccination program. Under
approval of Veterinary Bureau, MOA, vaccination is applied to susceptible animals at
epidemic and risk areas with exemption of dairy cattle and breeding bulls in total 12
provinces
2. Surveillance: A general national animal disease surveillance scheme is undertaken annually
for Brucellosis in which all dairy and breeding cattle and goat, and a number of cattle and
goat at random are tested through Rose Bengal test for screening and further confirmed for
those that are positive or suspect in screening by tube agglutination or complement fixation
test.
3. Targeted epidemiological investigation: A targeted epidemiological investigation plans for
Brucellosis was implemented in 48 designated counties in 16 provinces.
4. Others: Other measures including reporting of notifiable diseases, emergency response,
health check at border, animal movement control and zoning inside country, stamping out,
control of wildlife reservoirs and inspection at slaughterhouse.
5. R & D: Research and development of new vaccines and diagnostic technologies are also
conducted and there is an intersectional and interdisciplinary mechanism for control of the
disease, mainly among animal health, public health and other departments.

8. Review on national data (Cont.)
 KABP questionnaire:
A KABP questionnaire was implemented nationwide in 2009, in which at least 200 herdsmen in 8
villages in 4 counties in each province were required to respond to the questionnaire questions,
and total 714 efficacious entries were collected and analyzed.
A statistical result of response to 17 questions in KABP questionnaire

9. Review on provincial data
 Status Brucellosis in Yunnan Province
Data was collected through personal communication (non-official basis) from Yunnan Province
Animal Disease Prevention and Control Center.
1. Disease status: Total 3 outbreaks of Brucella abortus and 10 of Brucella melitensis were officially
and accumulatively reported in 2011.
2. Disease monitoring: Under the general national animal disease surveillance scheme, Brucellosis
monitoring was undertaken annually in the province where sera samples were collected individually
from all dairy and breeding cattle and goat, and a number of cattle and goat were sampled at random,
and tested by following the protocol of the National Standard. Serological positive rates were
2.55%in goat/sheep, 0.76% in cattle and 0.40% in pig in 2010; and 1.09% in goat/sheep, 1.49% in
all species of bovine ( 1.90% in dairy cattle), and 0.24% in pig in 2011.
A Summary of Brucellosis status in 2011 in Yunnan Province

10. Review on provincial data (Cont.)
Result of Brucellosis monitoring in 2011 in Yunnan

11. Factors linked to the trend of change
 Increase in livestock population: Government encouragement, nongovernment investigation and
huge market requirements resulted in a stable, marked increase in livestock population, which, at
least partly, contributed to higher prevalence and wider distribution of the disease.
An increase in meat and milk
production from 2001-2010
(Unit: 10000 Ton)
 Movement of animal and its products: There is extensive movement of animal and its products, mainly
on account of market forces. In general, breeding animals move from highly developed regions or
provinces to less-developed ones, and commercial animal commodities move targeting high market
prices. Domestically, movement of this kind takes place on a large scale and at a high frequency.
 Improper set up of cooperative farms: A number of cooperative farms were set up without a
standardized protocol, a simple combination of domestic animals from backyard farms.
 Insufficient compensation mechanism: Farmers are not willing to have animals to destroyed once being
confirmed to be positive, due to that the price is much lower than market.
 Weak intersectional cooperation: Course of animal products production from animal feeding to eating is
separated into 3 sections of farming, processing and trading, and a sector is responsible for each of the
sections, and intersectional link is still weak.

12. An integrated approach to control Brucellosis
As a part of ecohealth project, the following activities were conducted by the team in ASVI, co-
funded by ILRI and Yunnan Province Agriculture Department.
 To increasing public awareness:
1. Training workshop: Total 5 training workshops on Ecohealth farming for diary cattle/buffalo,
and recognition and control for Brucellosis was carried out in Yingjiang County, Tengchong
County, Mangshi District, Eryuan County and Dali prefecture respectively in Nov-Dec 2011.
2. Trainee: Total 340 staff including farmers, veterinary officers, veterinarians and technicians
attended these workshops.
3. Training materials: Including ecohealthy farming for diary cattle/buffalo production, control of
newly-emerged animal diseases, control of zoonosis including Brucellosis and Tuberculosis in
cattle, and prevention and treatment of mastitis of kinds and hove diseases. Concepts of
ecohealth, and intersectional and transdisciplinary control measures.
4. Quality control of raw milk: Another workshop on quality control of raw milk organized by
Yunnan Dairy Cattle Association was also carried out in Kunming in Mar 2011, a training
lecture on Control of Brucellosis and Tuberculosis in Dairy Cattle was presented, and 120
technicians participated.
5. To increase public awareness: To increase public awareness for better understanding
importance of emergence and control of Brucellosis in China, A paper entitled “Brucellosis is
preventable and controllable” was well prepared and put on Yunnan Agriculture Information
website for public access in early 2010. The paper covers disease situation, transmission from
animal to human, recognition, measures for prevention and control, and this paper was
continuously cited by famous newspaper. Participating publicity to safety of meat products.

13. An integrated approach to control Brucellosis
Yingjiang County ， Oct 2011 Tengchong County ， Nov 2011
Mangshi District ， Nov 2011 Eryuan County ， Nov 2011

14. An integrated approach to control Brucellosis
Dali Prefecture ， Mar 2012 Kunming ， Mar 2012
Publicity to safety of meat products, Kunming ， Sep 2012

15. An integrated approach to control Brucellosis
 Surveillance for Brucellosis:
1. Survey in dairy cattle and buffalo (2009-2011): Serological surveillance in dairy cattle/buffalo:
Samples collected were further tested with C-ELISA by using INDEXX ELISA kit.
2. Survey in dairy and meat goats (2012)

16. An integrated approach to control Brucellosis
3. Survey in Mangshi District (2012): A serological survey in Mangshi District: This survey covered
more than 300 heads of dairy buffalo in total 7 cooperative and 3 backyard dairy buffalo farms,
for cleansing and eradication of these two diseases in Jun-Jul 2012.
Results: No positive case in total 290 sera samples and suspect agglutination was observed in 14
blood plasma samples by using Rose Bengal test. A confirmation test was subsequently
employed for the above 14 blood plasma samples and 6 additional sera samples by using C-
ELISA kit, and all the 20 samples were confirmed to be negative.
4. Survey for TB in Mangshi District (2012): A survey was also conducted for Tuberculosis in total
304 heads of by using PPD skin reaction test, total 57 cases were either suspect or positive. A
further anti-γ-IFN ELISA method was used for confirmation. Total 54 anticoagulant blood
samples in the above 57 cases, as well as 1 additional sample, were tested, and all the 55 samples
were negative.

17. An integrated approach to control Brucellosis
Sampling in buffalo and cattle, testing in laboratory, Dec 2011
Pathological Examination for TB and destroy by deep bury, Dec 2011

18. An integrated approach to control Brucellosis
 Activities jointly implemented by all partners:
1. Preparation and finalization of dossier: including draft and finalization of project report for the first
phase before Dec 2011; development, test and finalization of questionnaire and outlines for in-depth
interview (IDI) and focus group discussion (FGD), and translation into Chinese version; and
finalization of work plan for field survey in the two study sites.
2. Field survey in Liliang County: Joint field survey includes implementation of questionnaire of 104,
IDI of 18 and FGD of 9 in four villages in two townships in Yiliang County, 85 bulk milk samples
from commercial, cooperative, and backyard farms in both dairy cattle and dairy goat, as well as 126
individual human sera samples were collected during the field survey.
3. Field survey in Mangshi District: A joint field survey was undertaken by all partners, and
questionnaire, IDI and FGD were implemented in 3 townships, Mangshi District, as well as a
commercial dairy buffalo farm, a slaughterhouse and a milk processing company within the district.

19. An integrated approach to control Brucellosis
FGD in the commercial dairy cattle farm and the village, Yiliang, April 2012
Questionnaire at a backyard farm, Yiliang, April 2012

20. An integrated approach to control Brucellosis
Assignment for working groups, Mangshi ， Jul 2012 Human sera sampling, Mangshi ， Jul 2012
Questionnaire for backyard farmers, Mangshi ， Jul 2012

21. An integrated approach to control Brucellosis
 Laboratory test: The Bulk milk samples were tested by using an indirect ELISA kit for detection of
antibodies against Brucella abortus in bulk bovine milk samples, BRUCEELISA 160M produced by
Veterinary Laboratory Agency, UK.
1. Test for samples in Yiliang: Total 6 bulk milk samples were tested to be serologically positive for
antibodies against Brucella abortus with positive rate of 7.06%; 4 positive samples in Beigucheng
Township with positive rate of 9.09%, 1 in Kuangyuan Township with positive rate of 2.44%; 5 in
dairy cattle with positive rate of 11.11% and 1 in dairy goat with positive rate of 2.50%, none in
commercial farm, 2 in cooperative dairy cattle farm with positive rate of 40.00% and 4 in backyard
dairy cattle and goat farm with positive rate of 5.06%.

22. An integrated approach to control Brucellosis
Analysis of test results in Yiliang:
 Link to questionnaire: By reviewing record of the questionnaire for those whose animals being tested
as serologically positive for Brucellosis, of the above 6 positive samples, abortion occurred in all the
2 cooperative dairy cattle farms, 1 backyard dairy cattle farm and 1 backyard dairy goat farm;
however, there is no record of abortion history for the rest 2 backyard dairy cattle farms.
 Link to test results in human: By comparing the test results between animal milk samples and human
sera samples, total 5 human sera samples were tested to be serologically positive for Brucellosis, and
of which 4 distributed within the workers and owners in one of the 2 cooperative dairy cattle farms
with positive bulk milk sample; and, however, the rest one positive human sera sample belonged to a
farmer in a backyard dairy goat farm, bulk goat milk sample from whom, irrespectively and
contrarily, was serologically negative for Brucellosis.

23. An integrated approach to control Brucellosis
2. Test for samples in Mangshi: Total 41 bulk milk samples were tested to be serologically negative for
antibodies against Brucella abortus . The result accords with that in the independent survey on
individual basis performed previously in the same dairy buffalo populations in the same area.

24. Recommendations
 Confirmation test for positive samples: Sera samples should be collect individually in dairy
cattle and goat which bulk milks samples have been tested as serologically positive, and
further tested by C-ELISA to confirm positive individual animals for Brucellosis. Test
results should be further confirmed by using the national standard of diagnostic
technologies for Brucellosis in animal, or detection of pathogen in individual animal,
reported to local veterinary authority for a proper destroy of those individually confirmed
positive animals.
 Traceback the possible source: More detailed information should be collected from those
test result is serologically positive in either animal or human, to investigate the possible
source and duration of the infection. An assessment for risk analysis for both animal and
human is also needed to technologically support local veterinary and public health authority
as well as the farmers for proper dealing with the infection.
 Increase public awareness through training: A training workshop on recognition,
prevention and control should be organized for dairy cattle and goat raising farmers, village
veterinarians and village doctors to increase their awareness and change the improper ways
for dealing with sick animals.