The Current Position:Home>>Animal Health
 

White Paper on Eradication of Equine Glanders in China

DATE:2011-12-30       SOURCE:
 

Ministry of Agriculture

 People’s Republic of China

October 2009


1. Introduction

1.1 Geography of China

1.1.1 Location

Located in the Northern Hemisphere and the middle and eastern part of Asia, China is on the western shore of the Pacific Ocean. It faces the sea in the south and east and extends to the inland area of Asia on the western and northern borders. With a long coastline, it is a country endowed with both land and sea areas.

China has a vast territory. From east to west, it extends from the confluence of the Heilongjiang River and the Wusuli River at 135.03°E to the east bank of the Kara-Kul Lake on the Pamirs at 73.22°E, spanning around 62 degrees in longitude over a distance of 5,200 km and creating a maximum time difference of over 4 hours. From south to north, it stretches from the Zengmu Reef at the southernmost end of the Nansha Islands at 3.51°N to the main route of the Heilongjiang River at the north of Mohe county at 53.34°N, spanning around 50 degrees in latitude over a distance of 5,500 km.

1.1.2 Land area

China has a land area of about 9.6 million square kilometers, accounting for about one quarter of the total in Asia, and about 1/15 of the world’s total. It is the third largest country in the world, only next to Russia and Canada.

1.1.3 Land boundaries and neighboring countries

With a land boundary of some 22,800 km, China borders 15 countries, i.e. DPRK, Russia, Mongolia, Kazakhstan, Kyrgyzstan, Tajikistan, Afghanistan, Pakistan, India, Nepal, Sikkim, Bhutan, Myanmar, Laos and Vietnam. To the east and southeast, ROK, Japan, the Philippines, Brunei, Malaysia and Indonesia are also adjacent to China across the seas. .

1.2 The equine industry in China

1.2.1 Changes in the number of horses

The number of horses gradually increased after the founding of the People’s Republic of China in 1949, and reached the historic high in 1977, ranking first in the world at that time. The figure dropped sharply, however, by the end of the last century (see Table 1). In the early days after the liberation, due to of the long-term war, natural disasters and some other reasons, there were only 4.875 million horses in China in 1949, 1.615 million fewer than the number of 6.49 million in 1935, the highest on record before 1949, causing a major problem of severe shortage of animal power. So the central people’s government enacted a series of policies on the protection of farm animals, which helped the number of horses recover to 7.302 million in 1957, exceeding the highest level before 1949. This momentum of increase was maintained since then, and the horse number reached the climax of 11.445 million in 1977. However, it reduced to 7.028 million in 2007, a decrease of 4.419 million over 1977, and continues to shrink. Nevertheless, China is still a country with a big equine industry.

Table 1 Numbers of horses, donkeys and mules in stock nationwide

Unit: 10,000 heads

Year

Horse

Donkey

Mule

Note

1935

649.0

1215.0

460.0

 

1949

487.5

949.4

147.1

 

1952

613.0

1189.6

163.7

 

1957

730.2

1086.4

167.9

 

1962

632.0

645.5

132.4

 

1965

792.1

743.8

144.7

 

1970

964.8

840.0

224.5

 

1975

1129.9

812.7

335.4

 

1976

1143.8

776.6

353.6

 

1977

1144.7

763.0

371.5

The historic high

1978

1124.5

748.1

386.8

 

1979

1114.5

747.3

402.3

 

1980

1104.2

774.8

416.6

 

1981

1079.4

841.5

432.5

 

1982

1098.1

899.9

446.4

 

1983

1080.6

944.9

459.0

 

1984

1097.8

996.2

479.0

 

1985

1108.1

1041.5

497.2

 

1986

1098.8

1068.9

511.3

 

1987

1069.1

1084.5

524.8

 

1988

1054.0

1105.2

536.6

 

1989

1029.4

1113.6

593.1

 

1995

1007.2

1074.5

538.0

 

1996

871.5

944.4

478.0

 

1997

891.2

952.8

480.6

 

1998

889.1

955.8

473.9

 

1999

898.1

934.8

467.3

 

2000

876.6

922.7

453.0

A decrease of 2,681,000 over 1977

2001

826.0

881.5

436.2

 

2002

808.8

849.9

419.4

 

2003

790.0

820.7

395.7

 

2004

763.9

791.9

374.0

 

2005

740.0

777.2

360.4

 

2006

719.5

730.6

345.1

 

2007

702.8

689.1

298.5

A decrease of 4,419,000 over 1977

1.2.2 Disease control

After the Founding of the People’s Republic of China, the government stepped up the efforts in the prevention and control of animal diseases. As a result, a variety of diseases affecting the health of horses were brought under control. Equine glanders (glanders) was brought under primary control in 1985 thanks to the EIAV diagnostic preparation and vaccines successfully developed by Harbin Veterinary Research Institute in 1974, and due to massive efforts in prevention and control, isolation and culling, it was brought under control in pasture areas and even eradicated in some provinces and regions in 1985. In 2005, the criteria for eradication of glanders were met all over the country.

1.2.3 Changes in the uses of horses

During the 1970’s and 1980’s, with accelerated agricultural mechanization as well as the rapid development of transportation networks attributable to the reform and opening-up policies, horses were no longer a major power but an auxiliary one in agriculture and transportation . In the 1990’s, horses began to be increasingly used in sports competition and recreational activities.

1.2.4 New opportunities for development

The rapid economic development in China and the improvement of people’s living standards have provided market opportunities for the transformation of the equine industry, which is injected with new vitality by the development of horses for milk or meat, and by the demand for horses in sports events such as the Olympic Games, the Asian Games, the National Games, the Inter-City Games and sports activities in the ethnic minority regions.

1.2.6.1 Horse raising for milk or meat

Ethnic minorities in Xinjiang, Inner Mongolia and the southwest regions of China have the tradition of drinking horse milk and making kumiss. Horse milk has low fat and high lactose and is rich in protein, vitamins and minerals. It is able to prevent the Lactose Intolerance caused by the lack of lactase in cow milk, and is suitable for feeding infants, because its nutrients are closer to those of human milk. Horse meat is very nutritious and is the raw material for first class sausages. It has become a new direction of research in the equine industry to raise the productivity of horses for milk or meat through breed improvement.

1.3 Other equine animals

There are also considerable changes in the number of donkeys and mules as the economy grows in China. The number of donkeys and mules, as dominant draft animals, gradually increased with the economic recovery and the steady development of the rural economy after the founding of the People’s Republic of China, and reached the historic peak in 1989, i.e., 11.136 million donkeys and 5.931 million mules. Since the 1990s, with the rapid development of the transportation industry in China, donkeys and mules as tools of transportation have gradually faded into history. By 2007, the number of donkeys and mules had dropped almost by half compared to their historic peak to 6.891 million and 2.985 million respectively. Now most of them are bred for meat except those still used as draft animals in a small part of the countryside.

2. Veterinary systems

2.1 Laws and regulations on animal health

Laws and regulations related to animal health include the Law of the People’s Republic of China on Anima Disease Prevention and Control, the Law of the People’s Republic of China on the Entry and Exit Animal and Plant Quarantine, Regulations for the Implementation of the Law of The People’s Republic of China on the Entry and Exit Animal and Plant Quarantine, Regulations on Emergency Response to Major Animal Epidemics, Regulations on Bio-safety Management of Pathogenic Microorganism Laboratories, and Regulations on Administration of Veterinary Drugs. Veterinary practices in China are administrated in line with laws and regulations in terms of animal disease prevention and quarantine, regulation of laboratory bio-safety management, and administration of veterinary drugs and feeds, laying a solid legal foundation for the eradication of glanders.

2.2 Veterinary administration system

The State Council issued in 2005 the Opinions on Promoting the Reform of the Veterinary Administration System, which has strengthened the development of administrative, law enforcement and supervision, as well as technical support institutions for veterinary services at all levels. The Ministry of Agriculture (MOA) has set up a Veterinary Bureau and appointed a National Chief Veterinary Officer, and the reform of the veterinary administration system at the national and provincial levels (with autonomous regions and municipalities included) has been carried out in an all-round way. Administrative, law enforcement and supervision, as well as technical support institutions for veterinary services have been founded at different levels around the country. Stations of Animal Husbandry and Veterinary Services have been established in towns and a total of 645,000 paraveterinarians in charge of animal disease prevention and control at the village level designated. The National Animal Disease Prevention System Development Programme has been initiated. As a result, equipment and capacities of the animal health surveillance network and the disease prevention and control system have been improved, providing a good institutional guarantee for the eradication of glanders around the country.

2.2.1 Veterinary administration institutions

MOA is the national agency responsible for the administration of veterinary affairs. Its Veterinary Bureau, the national level arm of veterinary administration, together with the National Chief Veterinary Officer it appoints, assumes the specific responsibilities for veterinary administrative affairs such as animal disease prevention, quarantine and supervision. Veterinary administrative agencies also exist at the provincial, prefecture and county levels as functional departments of the local governments, which are responsible for animal disease prevention, quarantine, veterinary drug administration, residue control and supervision.

2.2.2 Veterinary administrative law enforcement system

Local veterinary administrative authorities run specialized animal health administration and supervision institutions, which are in charge of animal disease prevention and monitoring; animal and animal product quarantine; examination, registration, monitoring and administration of veterinary drugs; examination, registration, monitoring and administration of animal feeds; diagnosis and testing of animal diseases; and extension of veterinary diagnostic technologies.

2.2.3 Technical support institutions for veterinary practices

Technical support institutions for veterinary practices have been set up at the national and local levels and in colleges and universities to meet various demands. There are national reference laboratories and national diagnostic laboratories; animal disease diagnostic and quarantine laboratories at the provincial, prefecture and county levels; and laboratories for animal disease research, diagnosis and testing in agricultural colleges, universities and research institutes.

2.2.4 Competent authority for entry and exit inspection and quarantine

The General Administration of Quality Supervision, Inspection and Quarantine (AQSIQ) under the State Council of the People's Republic of China is responsible for the inspection and quarantine of animals and animal products entering and exiting China. AQSIQ has 35 direct branches or offices in provinces, autonomous regions and major ports around the country, which are engaged in inspection and quarantine of animals and animal products entering and exiting China to prevent the transboundary transmission of animal diseases.

2.2.5 Relevant social sectors and registered veterinarians

Provisions in the Law of the People’s Republic of China on Animal Disease Prevention and Control state that “any organizations or individuals engaged in surveillance, testing, quarantine, research and diagnosis of animal diseases as well as raising, slaughtering, marketing, isolation and transportation of animals that find or suspect an animal infected with a disease shall immediately report the matter to the local veterinary authority, the animal health supervision institutions or animal disease prevention institutions. The said institutions shall take prompt control measures, such as isolation, to prevent spread of the disease.” “Animal-raising farms, slaughter houses, integrated meat processing plants and other designated slaughter houses (places) shall raise and deal in animals or manufacture and deal in animal products in conformity with the requirements for animal disease prevention laid down by the administrative department for animal husbandry and veterinary services under the State Council, and place themselves under the supervision and inspection by the institutions for supervision over animal disease prevention.” “Registered veterinarians, rural veterinary support staff shall participate in prevention, control and eradication of animal diseases according to requirements of the local government or the veterinary authority.” The above mentioned previsions require active participation of relevant sectors in prevention, control and eradication of animal diseases according to relevant requirements of the government, which has laid a sound public foundation for the eradication of glanders.  

3. Eradication of glanders

3.1 History of glanders in China  

3.1.1 Distribution

Glanders existed for a long time in China. Before 1949, it mainly affected the pastoral areas with a huge equine population and some other areas. From 1950 to 1997, cases were found in

1 034 counties of 21 provinces, autonomous regions and municipalities, i.e. Beijing, Tianjin, Hebei, Shanxi, Inner Mongolia, Liaoning, Jilin, Helongjiang, Shaanxi, Gansu, Ningxia, Qinghai, Xinjiang, Shandong, Anhui, Jiangsu, Henan, Guizhou, Sichuan, Yunnan and Tibet. See Figure 1 for its specific distribution.

Figure 1 Distribution of Glanders in China

3.1.2 Features

The country went through the following five stages of the disease: high prevalence (1949-1969); strict control (1970-1979); enhanced control (1980-1992); eradication (1993-2005); and consolidation (2006-2009).

From 1949, the disease tended to spread due to increase in the population of equine animals; in 1956, the development of agricultural cooperatives put un-quarantined horses raised together, and such a practice accelerated its transmission; in 1960s, insufficient number of draft animals brought about frequent movement of horses, leading to the highest number of diseased animals; in 1970s, it was basically brought under control thanks to various prevention and control measures, but it remained an endemic in some provinces; in 1980s, only sporadic cases were detected in most of the affected provinces and autonomous regions, of which Inner Mongolia, Jilin, Helongjiang, Qinghai and Xinjiang were relatively heavily hit and Liaoning, Sichuan and Henan suffered less; from 1990s, the number of cases decreased year after year, and by 2005, it was eliminated across the country. See Table 2 and Figure 2 for details. 

Table 2: Summary of national data related to glanders 

Year

Population of equine animals (head)

Number of animals with symptoms /test-positive animals (head)

Incidence rate / rate of

positive cases (%)

Number of

dead animals (head)

Mortality rate

(%)

19491959

172230000

266040

0.15

26735

0.012

19601969

123410000

571001

0.463

51451

0.042

19701979

186590000

309946

0.166

110875

0.059

19801989

211250000

125120

0.059

53508

0.025

19901999

266378000

1911

0.0007

40

0.00015

20002005

15

0.0004

0

0

20062009

0

0

0

0

Note: 1. Since 1999, no animals with symptoms have been found clinically.

2. No test-positive animals were detected from 2005 to 2009.

Figure 2 Development of Glanders in China

3.1.3 Loss from glanders

From 1950 to 1999, 6.701 million of infected animals (clinically detected and test-positive animals) were found and 1.079 million were killed or died in 21 provinces, autonomous regions and municipalities Of China. Specifically, the number of equine animals with symptoms was nearly 1.84 million, of which nearly 520,000 died, a mortality of 28.1%. Some 4.86 million equine animals were tested positive or 35.7% of the total (see Annex II). According to the records, infected horses totaled 3,666,500 in 21 provinces, autonomous regions and municipalities from 1949 to 1989. Natural death of diseased equines and culling of positive equines resulted in direct economic losses of dozens of billion yuan (calculated at the prices in 1989).

3.1.4 Glanders prevention and control in other countries

Before the 20th century, glanders spread widely among human beings and animals, and reached all countries in the world. During the World War I, there was a high prevalence of glanders in Europe and several countries in the Balkan Peninsula. The epidemic was brought under control at last because a huge number of infected equines were killed. In recent years, many countries have eliminated this disease thanks to the quarantine and culling measures for the equine animals. However, the infection rate has remained high and the chances for human infection still exist in some countries or regions, such as Asia and South America, where draft animals are used in farming activities. Therefore it is still possible for the disease to be introduced into China once again.  

3.2 Latest developments in the prevention and control of glanders

From 1993 to 2005, MOA appraisal teams checked the results of the activities for eradication of glanders in 34 counties of 21 affected provinces, autonomous regions and municipalities in accordance with the Examination Criteria and Approval Methods for the Effects of Glanders Prevention and Control. These teams randomly sampled 3691 head of equine animals and found no clinical cases. The results of ophthalmic mallein tests were all negative (see Annex I). Qinghai province first passed the appraisal in 1993, and the Tibetan Autonomous Region did so in 2005 as the last one. By that time, all the 21 affected provinces, autonomous regions and municipalities met the requirements for eradiation enacted by MOA. MOA released the appraisal results nationwide, and issued the certificate for eradication of glanders to those provinces, regions and municipalities concerned. According to the findings of epidemiological investigations and surveillance, no animals with symptoms or tested positive were found in the ten provinces, autonomous regions and municipalities which were free from glanders in history, i.e. Shanghai, Zhejiang, Fujian, Jiangxi, Hubei, Hunan, Guangdong, Guangxi, Hainan and Chongqing.

From 2006 to 2009, the country continued surveillance on glanders, and no animals with symptoms or tested positive were detected (see Annex II).

In summary, it has been nearly five years since the country satisfied the requirements for eradication of glanders; the achievement has been consolidated; and sources of glanders have no longer existed.

3.3 Five stages toward eradication of glanders

The guideline for eradication of glanders is to adhere to the policy of prevention going first and apply the principle of giving guidance according to subjects and taking different prevention and control measures at different stages. Specifically, plans and technical standards for prevention and control were worked out; integrated measures of quarantine, isolation, disposal and disinfection were taken with special emphasis on the major steps of controlling and wiping out infection sources. To sum up, the prevention and control process could be divided into the following five stages:  

Stage one: high prevalence (1949-1969). Measures focusing on comprehensive investigation and treatment under isolated conditions were adopted. Epidemiological investigations on glanders were carried out to identify the distribution and status of the disease and other related information; and test-positive horses were treated under isolated conditions.  

Stage two: strict control (1970-1979). Integrated prevention and control measures were taken, covering quarantine, isolation, treatment, culling, and breeding of healthy animals. All detected horses with clear clinical signs were killed. All the test-positive horses to the ophthalmic mallein tests were isolated in a designated place in the infected areas. In addition, healthy foals were bred as replacements. 

Stage three: enhanced control (1980-1992). Integrated measures focusing on comprehensive quarantine, culling and disinfection were applied and feeding management and quarantine were enhanced to prevent the spread of glanders into the country.

Stage four: eradication (1993-2005). At this stage, the process of examination and approval for confirming eradication of glanders was conducted in the 21 affected provinces, autonomous regions and municipalities according to the Examination Criteria and Approval Methods for the Effects of Glanders Prevention and Control.

Stage five: consolidation (2006-2009). It was a stage of surveillance in all aspects. Various surveillance measures were carried out to consolidate the results of eradication.

3.4 Major measures

3.4.1 Strengthening government leadership through putting glanders prevention and control high on the agenda of the governments at all levels

The Sate Council established the National Committee of Glanders Prevention and Control in 1958 as the national leading institution in this regard. Accordingly, each province set up its command headquarters and offices for the same purpose. Governments at all levels put control and eradication of glanders high on their agenda, and adopted an accountability system for targeted management. The veterinary authorities enhanced management and signed responsibility pledges; agencies engaged in animal disease control gave priority to its work on glanders; the public in the infected areas also join the endeavor; and surveillance was conducted at both selected spots and the areas concerned. Veterinary workers made vigorous efforts in the implementation of prevention and control measures.

3.4.2 Enacting and improving laws, regulations and rules on veterinary service for prevention and control on a legal basis. 

In 1952, the then Northwest Military and Political Committee promulgated the Trail Measures for Key Testing and Massive Elimination of Glanders in the Northwest Region. In 1985, the State Council issued the Regulations on Prevention and Control of Animal and Poultry Diseases, classifying glanders as a disease under Category II. The Law of the People’s Republic of China on Prevention and Control of Infections Diseases intensified the prevention and control of human glanders. The Law of the People’s Republic of China on Animal Disease Prevention and Control was amended in 2007, which facilitated the process of eradicating glanders. 

MOA enacted the Technical Procedures for Glanders Diagnosis and Confirmation Criteria (Draft), and Glanders Diagnosis Techniques and Confirmation Criteria in 1956, 1972 and 1979; the National Programme for Eradication of Glanders (1996-2000) in 1996 and the National Programme for Eradication of Glanders (2001-2005) in 2001; the Examination Criteria and Approval Methods for the Effects of Glanders Prevention and Control in 1992; and the Technical Standards for Prevention and Control of Glanders in 2002 and its revision in 2007. Enaction of these laws and regulations and implementation of these programs effectively ensured successful prevention and control of glanders.

3.4.3 Carefully implementing technical measures and gradually bringing the disease under control.

3.4.3.1 General investigation conducted for understanding of actual situation. From 1951, animal disease prevention and control institutions at various levels actively carried out investigations through the ophthalmic mallein test and the complement fixation test. As a result, the infected areas and the disease situation were identified. Glanders cases were reported to the authorities at higher levels, and notified to vicinal areas immediately. A joint prevention and control mechanism integrating the regional, provincial and county-level endeavors was applied.

3.4.3.2 Treatment of infected horses in isolation. The National Meeting on Glanders Research was held in 1962, when it was decided to use Terramycin and Sulfonamides for the treatment of glanders-infected horses with apparent symptoms. From 1961 to 1963, over 30,000 infected horses were cured in Heilongjiang province. The Veterinary Research Institute of the Inner Mongolia Autonomous Region added the injection of dead glanders bacteria solution to the Terramycin application process, which successfully reduced the clinical recurrence rate of glanders among recovered horses.

3.4.3.3 Detection, culling and isolation of glanders-positive horses. From 1970, the integrated prevention and control measures of detection, isolation, breeding, treatment and culling were taken nationwide. This approach was extended nationwide gradually on the basis of pilot programs. Positive horses were marked and relevant herds were put under movement control, separately kept and restrained from draught activities. Horses with symptoms and those tested positive were isolated and killed so that the sources were eradicated. Multiple applications of the ophthalmic mallein test were adopted in Shanxi province, resulting in effective detection of infected horses. In Beijing, after the positive horses were identified through the ophthalmic mallein test, blood samples were collected for additional complement fixation test. The positive horses confirmed by these tests were put into isolation. All infected horses with apparent symptoms were culled and buried or burned without exception. The surroundings of infected horses were thoroughly disinfected.

3.4.3.4 Improved breeding of healthy herds. From 1960, measures were implemented to breed healthy herds. Raising management for foals from healthy mares was enhanced; for those from glanders-positive mares, if they proved negative after two tests, they would be raised separately to renew the herds and breed healthy foals. In Nongan county of Jilin province alone, 8 breeding farms were established, producing 226 healthy foals.

3.4.3.5 Resolute handling of outbreaks and culling of animals with symptoms or tested positive. From 1990, glanders prevention and control measures were changed successively in different regions. According to the principle of early detection, prompt response, strict standards and minimized infection, integrated prevention and control measures including detection and culling were applied. Focusing on eradication, positive horses were resolutely destroyed to cut off the spreading route.

3.4.3.6 Enhanced inspection for preventing spread of the disease. Regarding the inspection, efforts were fortified in supervising farms, marketplace, transportation and other related sectors to ensure the implementation of various measures; agricultural, financial and public security authorities coordinated with each other to contain spread of the disease; MOA dispatched many inspection teams to guide, inspect, examine and approve the work on glanders prevention and control.

3.4.4 Increasing investment to ensure the success of prevention and control efforts

In the past 6 decades, the specialized financial investment from the central and local governments was markedly increased. During the period from the Eighth Five-year Plan to the Tenth Five-year Plan, over 200 million yuan were allocated from the central budget for prevention, control and eradication of glanders, while considerable funds were invested for the same end from the provinces. According to incomplete statistics, Qinghai province invested 1.312 million yuan in the campaign. In Beijing, Hebei, and some other places, farmers were compensated for the economic loss due to culling of infected horses. These measures accelerated the eradication of glanders.

3.4.5 Strengthening R&D efforts and actively promoting research results

To strengthen the technical support, detection and cleansing were conducted as the major technical measures for the control of glanders. Many countries succeeded in control and eradication of glanders through application of the mallein test and the complement fixation test, and China also adopted these internationally accepted methods. Improved quality of diagnostic reagents, especially in terms of accuracy and sensitivity, and standardized diagnostic procedures ensured science-based diagnosis. On this basis, different treatment methods for glanders were developed in different provinces. For example, the Glanders Research Team of Shaanxi province developed a treatment method and clinically cured some infected horses in Sep. 1956. Intensified R&D efforts led to development of treatment methods, which played a positive role in containment of glanders outbreaks at that time.

3.4.6 Enhancing team-building and improving professional competence

Animal disease prevention and control authorities at various levels enhanced the capacity building for the disease prevention and control personnel. Training courses were held to help improve the accuracy of clinical diagnosis, which laid the foundation for the success of the detection work. In Heilongjiang province alone, the training covered over one million person-times. For a long time, the veterinary people fought in the forefront of the campaign to eradicate glanders, exposed to the danger of being infected and demonstrating great perseverance and dedication to the mission, while an excellent team of high integrity and professionalism was formed, making significant contribution to the eradication of glanders.

3.4.7 Intensifying publicity to encourage public participation

In the past 6 decades, the efforts were sustained in disseminating the knowledge of glanders prevention and control for increased public participation. Various vehicles including broadcasting and pamphlets were used for promotion of scientific know-how regarding glanders prevention and treatment, and equine hygiene, which enabled the efforts in glanders prevention and treatment to be well known in the infected areas and win the support of the public, creating a participatory approach for glanders control and prevention.

4. Diagnosis of glanders

4.1 Diagnosis laboratories

4.1.1 Official certification procedures

All animal disease prevention and control agencies of the county level and above in the country have affiliated laboratories that are capable of conducting glanders diagnosis. Before these laboratories went into operation, applications were submitted to the veterinary administrative authorities according to the relevant requirements of China National Accreditation Service for Conformity Assessment (CNAS); CNAS designated relevant agencies to conduct conformity inspection of these laboratories in terms of bio-safety and metrology. Those accredited laboratories can conduct glanders diagnosis.

4.1.2 Comparative experiment

The comparison of experiment results from different laboratories is based on the national standard methodology, which requires two or more separate experiment groups. Comparative analysis of the results is conducted to explore the connection between various factors and the experiment subject, and thus ensure the reliability of the diagnosis results. The comparison was carried out according to the relevant requirements of CNAS. When comparison is applied to measure the reliability, it is required:

a) to select at least three laboratories, which are accredited by CNAS or members of the multilateral agreements of the Asia Pacific Laboratory Accreditation Cooperation (APLAC) or the International Laboratory Accreditation Cooperation (ILAC);

b) to make a comparison plan and confirm its applicability, feasibility and effectiveness;

c) to analyze and evaluate the comparison results.

4.1.3 Laboratory bio-safety measures

4.1.3.1 A sound veterinary laboratory bio-safety management system. MOA promulgated the Administrative Measures for the Storage of Bacterial (Viral) Strains of Animal Pathogenic Microorganisms, and Detailed Rules for the Implementation of Bio-safety Measures for Animal Pathogenic Microorganisms Experiment Activities and identified qualified laboratories for HPAI experiments. Animal disease prevention and control agencies at various levels and all kinds of veterinary laboratories have developed and improved the regulations on the veterinary laboratory bio-safety management, and the preventive norms for laboratory bio-safety and experiment operation rules, and improved the system of recording experiment results and use of experiment apparatus.

4.1.3.2 An accountability system for veterinary laboratory bio-safety management. Veterinary administrative authorities at various levels have set up leading groups for veterinary laboratory bio-safety management, designating specialized organizations and personnel responsible for laboratory bio-safety management. The accountability system for veterinary laboratory bio-safety management has been established at various levels, with the administrative authorities signing laboratory bio-safety responsibility documents with zthe veterinary laboratories within their jurisdiction. The system with the institute which the veterinary laboratory is affiliated to and the laboratory director as the primary responsible persons has been implemented.

4.1.3.3 Full implementation of veterinary laboratory bio-safety supervision measures. Veterinary authorities at various levels carefully implement the Regulations on Bio-safety Management of Pathogenic Microorganism Laboratories and relevant requirements of the Work Safety Commission of the State Council and MOA, and fully adopt the guideline of putting safety first, focusing on prevention, and taking a comprehensive approach with the guidance of the Scientific Outlook on Development to effectively strengthen laboratory bio-safety management. Management of the storage and use of bacterial (viral) strains of pathogenic microorganisms is increasingly improved. Efforts are made to effectively strengthen the management on experimental activities concerning highly animal pathogenic microorganisms and strictly implement the system of approval in advance and laboratory qualification examination for such experimental activities. In some provinces and prefectures, a regular reporting system has been developed for such experimental activities to follow the developments of such experimental activities and improve targeted veterinary laboratory bio-safety supervision. Meanwhile, supervision on the key links for laboratory bio-safety is intensified. Veterinary authorities at various levels conduct inspections on the development of veterinary laboratory bio-safety management system, storage of bacterial (viral) strains of pathogenic microorganisms, management of specimen collection, laboratory apparatus and personnel protection, waste disposal and other important links, urge those with problems to make earnest rectification, and establish an effective supervision mechanism for veterinary laboratory bio-safety step by step.

4.1.3.4 Enhanced duty arrangements in response to veterinary laboratory bio-safety emergencies. Veterinary authorities and laboratories at various levels have prepared laboratory bio-safety emergency response plans, and developed a veterinary laboratory bio-safety accident reporting system and the duty arrangements for emergency response during major holidays and important events, ensuring early detection of potential risks, timely reporting and immediate response.

4.2 Diagnosis

4.2.1 To unify the diagnosis technology for glanders and the criteria for reading test reaction and improve their accuracy, Glanders Diagnosis Techniques and Confirmation Criteria was formulated. This document should be followed during inspection and quarantine of horses, donkeys and mules. The diagnostic test prescribed in this document is primarily the ophthalmic test. If necessary, the complement fixation test, the subcutaneous test or the intradermo-palpebral test are used. Any horse, mule or donkey with transient clinical signs of glanders should be confirmed as open glanders and not be inspected and quarantined. All records of inspection and quarantine should be kept for at least 2 years.

4.2.2 Procedures of the ophthalmic test

4.2.2.1 Materials (instruments and chemicals)

Mallein, boric acid, Lysol, absorbent cotton, gauze, ethanol, iodine, and record sheet.

Eye dropper, clip for lips (ears), boiling sterilizer, forceps, sterile trays, work clothes, gauze mask and thread gloves.

Note: Eye droppers should be disinfected before reuse if contaminated after mallein is used up or during eye instilling (contact with conjunctiva).

4.2.2.2 Before instilling eye drops, compare two eyes, carefully examine conjunctiva and see if single or both eyes are blind, and keep records. Instill the drops into the eyes with normal conjunctiva only, and then examine submaxillary lymph nodes, general body status and if there is nose leakage. For a single time of inspection, instill eye drops twice with an interval of 5-6 days (3-4 drops of undiluted mallein (0.2-0.3mL) each time). Instill the drops in the same eye twice (normally in the left eye, but if the left eye is sick and not suitable, in the right eye), and describe the operations in the record sheet. Instill eye drops in the morning, and read the results in daytime after 9 hours from instilling. The assistant sets the horse in a fixed position before instilling, and the operator inserts the forefinger of the left hand into the upper eyelid to make the haw exposed, pokes the lower eyelid with the thumb of the left hand to make the eye concave, holds the eye dropper in the right hand horizontally, supports the frontal supraorbital site with the bottom part of the right hand, places the tip of the eye dropper 1cm away from the eye concave, presses the rubber nipple with the thumb of the right hand to drip 3-4 drops of mallein. Tie the animal after instilling mallein. Prevent sand from entering the eyes and direct sunlight into the eyes, and prevent the animal from rubbing its eyes.

4.2.2.3 Reaction reading: examine 3 times at 3, 6 and 9 hours after the instilling and reexamine once after 24 hours from the injection. When reading reaction, observe the two eyes from the front side of the horse head, examine by turning over the eyelids after 6 hours, and at other observation time if necessary. Carefully examine the status of conjunctiva to see if there is secretion in eyes, and make records with confirmation symbols. Confirmation results should be recorded at each examination. The final confirmation should be concluded by the highest reaction at two consecutive instillings.

4.2.2.4 Reaction criteria for the ophthalmic test

Negative reactionno reaction or slight hyperaemia in conjunctiva and lachrymation. Recorded as “-”.

Suspected reactionred and slightly swelling conjunctiva, and greyish-white serous and grume (not mucopurulent) discharge (eye secretion). Recorded as “±”.

Positive reactioninflammation and transient swelling in conjunctivaand various purulent discharge (eye secretion). Recorded as“+”.

4.2.3 Subcutaneous test (procedures of heat reaction)

4.2.3.1 Materials (instruments and chemicals)

Undiluted mallein, Lysol, ethanol, iodine, absorbent cotton, gauze, and record sheet.

Work clothes, gauze mask, thread gloves, brush, clippers, ear clip, injector, needle, thermometer, boiling sterilizer, sterile tray, and forceps.

4.2.3.2 Conduct clinical examination a day before the subcutaneous test, and measure and record the body temperature three times in the morning, at noon and in the evening, and only animals with normal temperatures can be given the subcutaneous test. The subcutaneous test is not allowed to be conducted in following three situations: any one of the three temperatures exceeds 39; or the average of the three temperatures exceeds 38.5; or a previous subcutaneous test is done less than one and a half month before. Normally the injection site is the left neck side or next to the shoulder blade in the chest. Inject 1mL undiluted mallein after cutting hair at the injection site and disinfecting it. Within 24 hours after injection, the animals can not be used in draft activities and be fed with cold water. Inject at 0 o’clock, start to measure the temperatures 6 hours after injection with a 2 hours interval (that is 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours after injection), i.e. measure 10 times successively, and measure once again 36 hours after injection, make records carefully and draw the temperature curve. Meanwhile, record local swelling degrees for confirmation. Local swelling bigger than palmlocal swelling of 10cm in diameteris considered transient reaction.

4.2.3.3 Horses, donkeys and mules may have heat reaction, local reaction or general reaction after the subcutaneous test.

Heat reactionNormally the temperature of glandered animals will start to rise 6-8 hours after injection, reach the highest 12-16 hours after injection, and then gradually fall. The temperature of some glandered animals will rise slightly 30-36 hours after injection.

Local reactionlocal fever, swelling and pain at the injection sitetransient signs 24-36 hours after injection, local swelling can reach 10-20cm in diameter and then disappear gradually, and sometimes it can last for 2-3 days.

General reactiondepressionloss of appetitelabored breathingfastened pulsepace totter, trembleincreased times of stooling and urinatingand enlargement of the submaxillary lymph nodes. 

4.2.3.4 Reaction criteria for the subcutaneous test (heat reaction)

Negative reactiontemperature below 39℃; no local or general reaction.

Suspected reactiontemperature rising to 39℃ (not exceeding 39.6℃)and slight general reaction and local reaction; or temperature exceeding 40℃ and staying at that level, and no local reaction.

Positive reactiontemperature exceeding 40℃ and staying at that level, and slight local reaction; or temperature exceeding 39℃ and staying at that level, and transient local reaction (local swelling of more than 10cm in diameter) or general reaction.

4.2.4 Procedures of the intradermo-palpebral test

4.2.4.1 Materials (instruments and chemicals)

Mallein (diluted before use, and mix 1 portion of mallein with 3 portions of normal saline containing 0.5% phenol evenly)

1-2mL injector, needle (disinfected by boiling before use), sterile tray, boiling sterilizer, forceps, ear clip, work clothes, gauze mask, thread gloves.

Ethanol, iodine, boric acid, Lysol, gauze, absorbent cotton, and record sheet.

4.2.4.2 Before injection, examine conjunctiva and see if single or both eyes are blind. After injection, examine submaxillary lymph nodes, and see if there is nose leakage, and make detailed records. Intradermal injection at 1-2cm from the edge of the left lower eyelid and one third from the inner canthus. Disinfect the injection site with boric acid swab before injection. The assistant sets the horse in a fixed position before injection, and the operator pinches the lower eyelid with the forefinger and thumb of the left hand, holds the injector in the right hand with the bottom part of the palm (the edge of the little finger) supporting at the horse head and diagonally injects 0.1mL mallein into the injection site held in the left hand. The forefinger feels pushing a little hard and the injection site appears a little swelling. That indicates that mallein is injected intradermally. Normally inject in the morning, read test at 24, 36 and 48 hours after injection, and make detailed record.

4.2.4.3 Reaction criteria for the intradermo-palpebral test

Negative reactionno reaction or slight swelling in the lower eyelid and lachrymation. Recorded as “”.

Suspected reactionslight swelling in the lower eyelid, slight pain and fever, red conjunctiva, no discharge or only viscous discharge. Recorded as “±”.

Positive reactiontransient local swelling in the lower eyelidtransient pain and fever, inflammation and photophobia in conjunctiva, and purulent ocular discharge. Recorded as “”.

4.2.5 Clinical and differential diagnosis for open glanders

4.2.5.1 Set infected animals in a fixed position, the operator and the assistant wear work clothesnot white color),rubber gloves, gauze mask, goggles and protective mask. Clean both inside and outside of the infected animal’s nostril with 3% Lysol. In a suitable position at the front side of the infected animal, the operator opens the nostril with the hands and the assistant lightens the deep inside of the nasal cavity with a reflector or an electric torch to carefully check if there are nodule ulcers, stellate cicatrices and other abnormal signs in the septum. Disinfect clothes and instruments respectively after examination (leached for 1 hour with 3% Lysol, or boiled for 10 minutes) to avoid cross infection.

4.2.5.2 Clinical signs of nasal glanders

Nasal mucusunilateral or bilateral discharge of serous or grume nasal mucus in the beginning, which gradually turns to yellowish purulent discharge with solid protein-like materials, sometimes with blood-tinged discharge and frowziness, resulting in wheezing.

Small nodules and ulcers in nasal septum when nasal mucus is discharged or later after that, old and new grayish-white or yellowish glanders nodules of different sizes appear in nasal septum, especially nasal midriff septum, and develop into crater-shaped ulcers of different sizes and depths (nodules and ulcers mainly in the septum deep in the nasal cavity), and flat stellate or ice flower-shaped cicatrices appear when healed.

Swollen submaxillary lymph nodesin the acute or chronic form of glanders, submaxillary lymph nodes become swollen and feel pain at the beginning, and over a long time they will turn stiff and feel no pain when touched, attached to the inner side of submaxillary without moving but sometimes with activity.

4.2.5.3 Clinical signs of cutaneous glanders

Cutaneous glanders: infected horses usually develop multiple bean- or walnut-, egg-sized nodules in the skin or the subcutaneous tissue along its legs, chest, and abdominal wall. Upon rupturing, these nodules excrete infectious purulent, yellowish or red color exudates (sometimes with blood) and later form shallow round ulcers or eruptive ulcers. Around the nodules and ulcers, lymph nodes are swollen and bead-like lymph vessels are thick and rough. Resulting from edema and infiltration, skins nearby these swollen areas show hypertrophy which sometimes causes cellulitis and rubber leg. Orchitis is a complication for infected male animals.

4.2.5.4 Criteria for Open Glanders

A case showing clinical signs of nasal discharge, nasal septum nodules or ulcers, or submaxillary lymph nodule swelling described in Section 4.2.5.2 should be confirmed as open glanders.

A case showing clinical signs of nasal discharge but no nasal septum nodules and ulcers and submaxillary lymph nodule swelling, or nasal discharge and submaxillary lymph nodule swelling but no nasal septum nodules and ulcers described in Section 4.2.5.2 should be given the ophthalmic test and should be confirmed as open glanders when positive reaction is shown.

A case showing the symptoms described in Section 4.2.5.3 should be confirmed as open glanders.

4.2.5.5 A case which does not meet the criteria described in Section 4.2.5.4, but has other suspected clinical symptoms for glanders should be confirmed as suspected open glanders.

4.2.6 Test Procedures for the complement fixation test

4.2.6.1 Serum collection

Chemicals and instruments:

Lysol, carbolic acid, ethanol, iodine, gauze and absorbent cotton.

Sterile test tube, test-tube rack, test-tube label, foam box for transporting blood, boiling sterilizer, sterile trays, forceps, brush, clipper, blood lancet with rubber pipe (the lancet should be washed and thoroughly disinfected in boiling water before reuse).

Hair around the vein sulcus areas at the upper 1/3 of the neck of tested animal should be cut and that site should be disinfected. Insert the sterile blood lancet into the jugular vein and let blood flow into the tube along its wall to avoid producing foam and causing hemolysis.

In winter, blood samples should be kept indoors to avoid being frozen. In summer, they should be kept at cool and dark place, and sent to the laboratory as quickly as possible. If blood samples cannot be sent to the laboratory within 3 days after collection, serum should be transferred to another sterile tube with the addition of 1 to 2 drops of 5% carbolic acid saline for each 1ml of serum to prevent its decomposition. During transportation, the tubes should be held in a vertical position and shaking should be avoided.

4.2.6.2 Test preparation (determination of titer for hemolysin, complement and antigen)

Materials required: standard serum (negative and positive horse serum for glanders), glanders antigen and hemolysin.

Complement: Serum should be taken from a healthy guinea pig. Before collecting its blood samples, the guinea pig should not be fed with anything for at least 7 to 8 hours. Samples should be collect from the heart of the guinea pig a night before the tested. In case that large amount of complement is needed, blood can be taken from the carotid artery, and then put into Petri dish or tube to clot. The blood clot should be gently cut or separated and then refrigerated. It should be used to separate serum in the next morning. When the blood being collected on the day of testing, it can be directly poured into the centrifuge tube and placed in the thermostat for 20 minutes, then homogenized and centrifuged. The complement used each time should be prepared from pooled serum of at least 3 to 4 guinea pigs.

Sheep's red blood cell (SRBC): Collect blood from the jugular vein of sheep, defibrinate blood and centrifuge 3 times to wash red cells. First time, centrifuge for 15 minutes at 15002000rp/m. Remove and discard supernate. Add 3 to 4-fold saline to the red cell and gently mix them together before starting the same second centrifuge process. Washed red cells is diluted 1/40 before being used. Such dilution can be kept only for 1 day, but undiluted centrifuged red cell can be kept for 3 to 4 days in the refrigerator.

Saline: add 8.5g sodium chloride to 1000ml distilled water and sterilize the saline before use.

Measure the titer of hemolysin: Measure the titer of hemolysin once each month according to the following method (see Table 3). Add 0.5mL hemolysin of different dilutions (from 1:100 to 1:5000) to different tubes respectively. Add 0.5mL 1:20 complement and 0.5mL 1:40 SRBC respectively to these tube. Then set up equal volume saline control tubes which contain no complement and hemolysin. Add another 1ml saline to all the tubes and place them into 37°C to 38 °C water bath for 15 minutes. 

Interpretation of results: hemolysin titers should be recorded as the highest dilution at which complete hemolysis has occurred. This represents 1 unit. No hemolysis in all control tubes. For complement titration and formal test, 2 units (or working volume) should be applied.

Table 3

Hemolysin dilution

1100

1500

11000

11500

12000

12500

13000

13500

14000

15000

Control

Hemolysin
1:20 complement

2.5% red cell

 

Saline

0.5

0.5

 

0.5

 

1.0

 

 

 

0.5

0.5

 

0.5

 

1.0

 

 

 

0.5

0.5

 

0.5

 

1.0

 

 

 

0.5

0.5

 

0.5

 

1.0

 

 

 

0.5

0.5

 

0.5

 

1.0

 

 

 

0.5

0.5

 

0.5

 

1.0

 

 

 

0.5

0.5

 

0.5

 

1.0

 

 

 

0.5

0.5

 

0.5

 

1.0

 

 

 

0.5

0.5

 

0.5

 

1.0

 

 

 

0.5

0.5

 

0.5

 

1.0

 

 

 

0.5

 

0.5

 

1.5

 

 

 

0.5

 

0.5

 

1.5

 

 

 

 

0.5

 

2.0

 

 

 

 

Determination of complement titer:

The complement titer should be measured on that very day for conducting the complement fixation test. First, dilute the complement with saline by 1:20, and then follow the procedures in Table 4.

Table 4. Determination of complement titer              UnitmL

   Tube No.

Components  

1

2

3

4

5

6

7

8

9

10

Control tubes

11

12

13

20-fold complement

Saline

Antigen (working volume) (Add saline to tubes without antigen)

10-fold diluted positive serum or 10-fold diluted negative serum

 

0.10

0.40

0.5

 

 

0.5

 

 

 

 

0.13

0.37

0.5

 

 

0.5

 

 

 

 

0.16

0.34

0.5

 

 

0.5

 

 

 

 

0.19

0.31

0.5

 

 

0.5

 

 

 

 

0.22

0.28

0.5

 

 

0.5

 

 

 

 

0.25

0.25

0.5

 

 

0.5

 

 

 

 

0.28

0.22

0.5

 

 

0.5

 

 

 

 

0.31

0.19

0.5

 

 

0.5

 

 

 

 

0.34

0.16

0.5

 

 

0.5

 

 

 

 

0.37

0.13

0.5

 

 

0.5

 

 

 

 

0.5

1.5

1.5

 

 

2.0

 

 

 

 

 

Mix them evenly, and then place them into 37°C to 38 °C water bath for 20 minutes.

2 unit hemolysin

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

/

0.5

 

2.5% red cell supernate

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

Mix them thoroughly, and then place them into 37°C to 38 °C water bath for 20 minutes.

 

Positive serum with antigen

Positive without antigen
Negative serum with antigen
Negative serum without antigen

 

 

 

 

 

 

 

 

 

 

+++

 

+++

 

+++

 

+

 

++

 

++

 

+

 

+

 

+

 

 

 

 

 

 

 

 

 

+++

 

 

 

 

 

 

 

 

 

Complement titer: Use the dilution fluid containing 2 unit hemolysin in the experiment. Tubes with 3 different kinds of solutions are prepared. Tubes of the first group contain positive serum and antigen. Tubes of the second group contain only positive serum. Tubes of the third group contain negative serum with or without antigen. No hemolysis appears in tubes of the first group. The complement titer is given by the highest dilution of the tested serum in which complete hemolysis occurred in tubes of group 2 and group 3. For example, 0.28mL is the working volume (complement titer) when making 20-fold dilution towards tube 7 in Table 4. The formula for calculating the dilution folds of the original complement solution is as follows:

Dilution folds of complement

Complement titer

× Solution volume added per tube= Dilution folds of the original complement solution

 

 


According to this formula, the result is: 20/0.28×0.5=35.7

That means this batch of complement should be diluted by 1:35.7 and 0.5ml solution should be added to each tube as one unit complement. Given that the complement is extremely unstable and the complement titer will be reduced during the process of experiment, the actual dilution concentration should be 10% higher than the original titer. So, in the actual experiment, this batch of complement should be diluted by 1:35 and the solution volume added per tube should be 0.5mL.

The antigen titer is required to be measured at least once half a year. Detailed procedures are as follows (see Table 5):  

l  Prepare 12 rows of tubes, 9 tubes in each row. Dispense 0.5mL of antigen dilution with different concentration (from 1:10 to 1:5000 as provided in Table 5) respectively into the tubes of the first row in turn. Repeat this process for the other 11 rows;

l  Add 0.5ml of 1:10 negative horse serum dilution to tubes in the first row;

l  Add 0.5ml saline to tubes in the second row;

l  Add 0.5ml of 1:10 strong positive horse serum to tubes in the third row;

l  Add 0.5ml of 1:25 strong positive horse serum to tubes in the fourth row;

l  Add 0.5ml of 1:50 strong positive horse serum to tubes in the fifth row;

l  Add 0.5ml of 1:75 strong positive horse serum to tubes in the sixth row;

l  Add 0.5ml of 1:100 strong positive horse serum to tubes in the seventh row;

l  Add o.5ml of complement (working volume) to all these tubes and place them into 37°C to 38 °C water bath for 20 minutes;

l  Add 0.5 ml 2.5% red cell and 2 unit hemolysin to all the tubes after heating and place them again into 37°C to 38 °C water bath for 20 minutes.

The antigen titer is the highest dilution of positive serum causing the most obvious hemolysis inhibition.

Table 5

Antigen with different dilutions

110

150

175

1:100

1150

1200

1300

1400

1500

Antigen (mL)

Negative(positive)serum

Complement (working volume)

0.5

 

0.5

 

0.5

0.5

 

0.5

 

0.5

0.5

 

0.5

 

0.5

0.5

 

0.5

 

0.5

0.5

 

0.5

 

0.5

0.5

 

0.5

 

0.5

0.5

 

0.5

 

0.5

0.5

 

0.5

 

0.5

0.5

 

0.5

 

0.5

Put them into 37°C to 38 °C water bath for 20 minutes.

2.5% Red cell

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

Hemolysin (working volume

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

An Example for the observation of antigen titer

Antigen with different dilutions

110

150

1:70

1100

1150

1200

1300

1400

1500

Serum with different dilutions

1:10

1:25

1:50

1:75

1:100

+++

+++

++

++

++

+++

+++

+++

+++

+++

+++

+++

++

+++

+++

++

-

+++

++

-

-

++

+

-

-

-

According to the results of this example, the titer of the antigen is 1:150.

4.2.6.3 Test procedures

After test preparation, formal test is conducted (see Table 6). Add 0.5mL 1:10 diluted serum into one test tube, and prepare this tube for adding antigen. Add 1mL1:10 diluted serum into another tube without adding antigen as control tube. Heat horse serum at 58 ~ 59℃ for 30 minutes, and mule and donkey serums at 36~64 ℃ for 30 minutes. Add 0.5mL glanders antigen (working volume). Add 0.5mL complement (working volume). After heating, add 0.5mL 2.5% erythrocyte diluent and 0.5mL two units hemolysin for both tubes, and then put the mixture incubation into 37~38℃ water bath for about 20 minutes.

To confirm whether the above test procedures is correct, control tests are set, using healthy horse serum, positive horse serum, antigen (working volume) and hemolysin (working volume) as control.

Table 6

Formal test

Contrast

Negative serum

Positive serum

Antigen

Hemolysin

normal saline 

0.45

0.9

0.45

0.9

0.45

0.9

1.0

Serum tested

0.05

0.1

0.05

0.1

0.05

0.1

5859 (or 6364) °C water bath for 30 minutes

Antigen (working volume)

0.5

0.5

0.5

1.0

Complement (working volume

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

3738°C water bath for 20 minutes

2.5% erythrocyte

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

Hemolysin working volume

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

3738°C water bath for 20 minutes

confirmation (Example)

-

-

-

-

-

-

After incubation, observe the test for the first time immediately. If positive serums in control tubes completely inhibit hemolysis and other control tubes show complete hemolysis, the test proves correct. Keep the tubes at room temperature for 12 hours and then observe for the second time, and make detailed record for the two observations.

To determine the results of reaction correctly, the standard colorimetric tubes should be made in the following approach to determine the degree of hemolysis (see Table 7). Add 2.5% erythrocyte diluent of 0.5mL and 0.45 ~ 0.05 mL (the parameter is 0.05) into different test tubes, and add nothing into another tube. Select several test tubes with a result of complete hemolysis and mix them (the parameter is 0.25), add different volumes of hemolysin to the varied erythrocyte diluent according to the following table, then add the normal saline (2.0, 1.8 ~ 0.2mL, etc.), and make each tube have a total volume of 2.5mL.

Table 7

Hematolysis degree (%)

0

10

20

30

40

50

60

70

80

90

100

2.5% erythrocyte

Hemolysin

Normal saline 

Total

0.5

0

2.0

2.5

0.45

0.25

1.8

2.5

0.4

0.5

1.6

2.5

0.35

0.75

1.4

2.5

0.3

1.0

1.2

2.5

0.25

1.25

1.0

2.5

0.2

1.5

0.8

2.5

0.15

1.75

0.6

2.5

0.1

2.0

0.4

2.5

0.05

2.25

0.2

2.5

0

2.5

0

2.5

 

Reaction criteria for the test:

Positive reaction: erythrocyte hemolysis between 0~10% is #; erythrocyte hemolysis between 10~40% is + + +; erythrocyte hemolysis between 40~50% is + +;

Suspected reaction: erythrocyte hemolysis between 50~70% is +; erythrocyte hemolysis between 70~90% is ±;

Negative reaction: erythrocyte hemolysis between 90~100% is -.

4.2.7 Attached tables

Record sheet of the ophthalmic mallein tests         Day:   Month:  Year:

No.

Species

Gender

Age

Characters

Reaction to the first

ophthalmic test

Reaction to the second ophthalmic test

Comprehensive confirmation

Clinical examination

3

6

9

24

Confirmation

Clinical examination

3

6

9

24

confirmation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  Veterinarian:       (signature)

Record sheet of the serum test of the animals with mallein reaction

Day:   Month:  Year:

No.

Species

Clinical signs

Mallein reaction

result

Blood sampling date

Blood tube number

Complement fixation test

Note

Date of blood reception

Date of test

Result

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Veterinarian:    (signature)

5. Glanders Surveillance

In 2005, the criteria for the eradication of glanders were met in all provinces of China. In order to further consolidate the glanders free status, and ensure timely detection of potential cases, China has adopted surveillance measures combining massive surveillance in spring and autumn every year and routine clinical surveillance, which are targeted at the equine animals throughout the country.

5.1 Clinical Surveillance (passive surveillance)

According to epidemic features and clinical signs of glanders, passive surveillance is conducted for susceptible animals. Detection of suspected animals is reported to animal disease prevention supervision institutions and animal disease prevention institutions respond to the situation in the light of the procedures provided in laws and regulations.

5.1.1 Epidemic Features

Equines are the most susceptible animals to glanders, and humans and other animals, such as camels, dogs, cats, can also be infected. Equines and other animals infected are the source of transmission. Natural infection is mainly transmitted by contact with infected animals through the digestive tract or damaged skin, mucous membranes and respiratory tract. The disease is not seasonal, normally sporadic or endemic. The disease is, normally acute and aggressive in the newly infected areas; and chronic in endemic areas.

5.1.2 Clinical signs

The incubation period of this disease is 6 months.

Glanders is described clinically as acute and chronic forms.

Acute form: Glanders begins with irregular fever (39 ~ 41℃) and general signs including the enlargement of submaxillary lymph nodes. Pulmonary glanders manifests clinical signs such as dry cough, semi-voiced, voiced, and labored breathing; nasal glanders manifests clinical signs such as serous or grume unilateral or bilateral nasal discharge, nasal cavity septum with millet to sorghum kernel sized grayish round nodules bulging in the septum surface with reddish surroundings, nodules developing into crater-shaped ulcers with irregular borders and grayish or yellow bottom; cutaneous glanders manifests clinical signs such as local feverish and painful inflamed swellings in legs, chest side and lower abdomen which develop into stiff nodules, the nodules breaking with purulent discharge and developing into crater-shaped ulcers with irregular borders and grease-like bottom which are difficult to heal, the nodules extending to near tissues along the lymphatic route to form bead-like swellings, and transient cutis swelling in glandered hind legs.

Chronic form: no transient clinical signs, yellowish and purulent unilateral or bilateral nasal discharge in some cases, often erosive ulcers in nasal mucosa, and radial scars in the nasal septum in some cases.

5.2 Laboratory surveillance (active surveillance)

5.2.1 Number of animals for surveillance 

5.2.1.1 Control zone

For each county, test 200 equine animals per year (test all when there are less than 100 equine animals in a county) through the ophthalmic test. Take corresponding measures according to the requirements for control zones when positive reaction is detected.

5.2.1.2 Glanders free zone

For each county, test 100 equine animals per year (test all when there are less than 100 equine animals in a county) through the ophthalmic test.

5.2.2 Surveillance methods

According to Glanders Diagnosis Techniques and Confirmation Criteria, the Annex to Technical Standards for Glanders Prevention and Control, the ophthalmic test is the primary suveillance method. If necessary, complement fixation test, the subcutaneous test or the intradermo-palpebral test are used.

5.2.3 Reagent for surveillance

Mallein used in the surveillance activities is provided by Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

5.3 Control and Eradication requirements

5.3.1 Control Requirements

5.3.1.1 Control requirements at the county level

Counties (districts or banners) under control should meet the following three requirements:

A.   Within the county (district or banner), no clinical cases of glanders are detected in two consecutive years.

B.   Within the county (district or banner), random inspection should be carried out on 200 equine animals every year (if the population is less than 200, the whole population should be covered.), and the rate of positive reaction to the mallein test should not be higher than 0.5%.

C.   All equine animals with positive reaction to the mallein test should be culled and bio-safety measures should be taken accordingly.

5.3.1.2 Control requirements at the prefecture level

All counties (districts or banners) within the jurisdiction of a prefecture (league) should meet the control requirements.

5.3.1.3 Control requirements at the provincial level

All prefectures (leagues) within the jurisdiction of a province (municipality or autonomous region) should meet the control requirements.

5.3.1.4 Control requirements at the national level

All provinces (municipalities or autonomous regions) of the country should meet the control requirements.

5.3.2 Eradication Requirements

5.3.2.1 A glanders-free county should meet the following two requirements:

A. After meeting the control requirements, no new glanders cases are found in two consecutive years within the county (district or banner).

B.  After meeting the control requirements, no positive reaction to the mallein test is detected in the annual random inspection on 100 equine animals (if the population is less than 100, the whole population should be covered.) within the country (district or banner) in two consecutive years.

5.3.2.2 Eradication requirements at the prefecture level

All counties (districts or banners) within the jurisdiction of a prefecture (league) should meet the eradication requirements.

5.3.2.3 Eradication requirements at the provincial level

All prefectures (leagues) within the jurisdiction of a province (municipality or autonomous region) should meet the eradication requirements.

5.3.2.4 Eradication requirements at the national level

All provinces (municipalities or autonomous regions) of the country should meet the eradication requirements.

6. Prevention Measures

6.1 Border Control

6.1.1 Border surveillance stations of animal diseases. There are 146 border stations of surveillance on animal diseases, undertaking animal disease diagnosis and surveillance, information collection, compilation and reporting of disease outbreaks and response activities at the border. The stations are endowed with functions of clinic diagnosis of animal diseases, storage and transportation of biologics, collection and processing of information on animal diseases, as well as traceability for quality and safety of animal and animal products.

6.1.2 Quarantine and supervision measures

6.1.2.1 Entry-exit animal quarantine farms. These farms mostly carry out quarantine of introduced animals. During the quarantine process, the introduced animals are subject to related tests. Only with acceptable quarantine results can these animals be given entry into China. Animals that fail the quarantine procedure should be destroyed and disposed appropriately to avoid any possible introduction of animal diseases.

6.1.2.2 Entry-exit inspection, and disinfection and disposal facilities. Entry-exit disinfection and disposal facilities for disease prevention are established at major ports across the country for conducting disinfection on means of transport, persons, animals, packages and other subjects and tools entering and exiting the country that are likely to transmit animal diseases as well as bio-safety measures for articles that may transmit or carry animal pathogenic microorganisms, articles that fail quarantine inspection and goods of illegal entry. Animal product inspection and disposal facilities are set up at ports of animal product importation to address the illegal entry of animal products from infected areas.

6.2 Import Supervision

6.2.1 For equine animals imported from a glanders free country, an international veterinary certificate is required to attest that the animals: show no clinical sings of glanders on the day of shipment; and are raised in the exporting country since birth or in the six months prior to shipment.

6.2.2 For equine animals imported from a glanders-infected country, an international veterinary certificate is required to attest that the animals: show no clinical signs of glanders on the day of shipment; are raised in an officially recognized glanders-free farm in the six months prior to shipment; and are tested as prescribed in the Terrestrial Manual during the 30 days prior to shipment and the results are negative.

6.3 Inspection measures

6.3.1 Equine animals to be transported in different areas must come from glanders-free areas; equine sellers should apply for inspection before sale. Before departure of the animals, inspection of the local animal disease control supervision institution is required to attest that the animals show no clinical signs of glanders on the day of shipment; that no case of glanders occurs in the place of origin in the six months prior to shipment; and that the mallein test or the complement fixation test is conducted for the animals during the 15 days prior to shipment, with negative results obtained; and the local inspection certificate issued by this local authority is also required.

6.3.2 On the arrival of transported equine animals, they must be placed in quarantine for over 30 days and subject to two consecutive mallein tests (with an interval of 5-6 days) by the local animal disease control supervision institution before they can be mixed with other animals.

6.3.3 For the transportation of equine animals out of a county, the carrier should ask for a transportation inspection certificate issued by the local animal disease control supervision institution. The animals must be transported with the certificate. When a suspected case of glanders occurs during carriage, the owner and carrier should immediately notify the closest animal disease control supervision institution. If the diagnosis is confirmed, the owner shall take disposal measures, including killing of the animals on the spot, under the supervision of this local authority.

7. Contingency plan

7.1 Notification

7.1.1 Any organization or individual who detects suspected cases should immediately report to the local animal disease control supervision institution.

7.1.2 After the animal disease control supervision institution receives the report of suspected cases and then confirm the diagnosis, it should immediately submit the information to higher authorities in accordance with the Administrative Measures on Reporting Cases of Animal Diseases.

7.2. Response measures

7.2.1 When suspected equines are detected, the owner should immediately isolate them, restrict their movement and report to the local animal disease control supervision institution. Upon receiving the report, the authority should immediately send people to the premise to carry out diagnosis activities, including epidemiological investigation, clinical examination, pathological examination, specimen collection, and laboratory diagnosis, and take corresponding control measures based on the diagnosis result.

7.2.2 When the diagnosis of glanders is confirmed, the animal husbandry and veterinary departments of the local government above the county level should immediately send people to the field to identify the infected premise, the infected area and the threatened area, collect specimens, analyze the source of infection, apply to the government at the same level for approval of the blockade of the infected area and submit the disease information to animal husbandry and veterinary authorities of higher levels all the way to the State Council level. The government above the county level should organize the departments and institutions concerned as needed to take compulsory control and eradication measures, such as isolation, culling, destroying and disinfection, and notify neighboring areas.

7.2.2.1 Identification of the infected premise, the infected area and the threatened area

Infected premise: the location where the infected equines are held, normally the farm/farmer household or relevant abattoir or marketing place that handle the herd that contains the infected equines; or the natural village with the infected equines in the case of backyard farming.

Infected area: the area within a radius of 3 km from the infected premise. The farming environment and natural barriers, such as rivers and mountains, are given due consideration in identification of the infected area.

Threatened area: the area within a radius of 5 km from the border of the infected area.

7.2.2.2 Blockade

The infected area is blocked according to relevant regulations. During blockade, the infected and suspected equines and their products are not allowed to be sold, transferred, shifted to another herd or moved out of the infected area; equines are reproduced through artificial insemination; breeding equines in the infected area can not be mated with equines outside; suspected equines must be strictly isolated and inspected; equine markets are closed; equines from other areas are not allowed to enter the infected area; humans, vehicles and other subjects concerned entering or leaving the infected area are disinfected or other restrictive measures are taken for disease control purposes.

7.2.2.3 Isolation

When glanders cases are detected, the mallein test should be applied in a timely manner to equine animals at the infected premise. Based on the test result, the equine animals will be divided into three categories as infected animals, suspected animals and presumedly healthy animals. The infected animals should be immediately culled, and the suspected animals and the presumedly healthy animals should be isolated for observation. The isolation can be lifted only if no more cases occur based on 6 months’ observation.

7.2.2.4 Testing

In the affected areas, the suspected equine animals and their surrounding animals should be raised in isolation, and they should be tested every 6 months. For the threatened areas, serological testing (mallein test) should be conducted two times every year until the test results are all negative. For the disease free zones, serological testing should be conducted once every year.

7.2.2.5 Culling

Both clinically sick animals and the animals with positive reaction to the mallein test should be killed under the conditions of no exsanguination.

7.2.2.6 Destroying

Animals with symptoms and those tested positive as well as their fetus, afterbirth and excrement should be disposed with bio-safety measures in accordance with GB16548 Procedures of Bio-safety Measures for Bodies of Sick Animals and Poultry and Their Products. The selected venue for incineration and burying should be 1 km away from any village, township, school, water source, rangeland and animal farm. The bodies should be burned and then buried in deep pits with an earth cover of not less than 1.5 meters.

7.2.2.7 Disinfection

Any venues, tools and materials that are contaminated by the infected or suspected equine animals should be disinfected. Contaminated bedding, manure and other materials should be treated with the methods of stacking mudding fermentation, high temperature, etc. before they are used.  

7.2.2.8 Lifting of blockade

The blockade for the infected areas can be lifted based on the following conditions: no more case is found according to 90 days’ surveillance in the infected areas after the last infected equine animal is killed and thorough disinfection is carried out; no more equine animal tests positive to the mallein test after testing the equine animals one by one in half a year; contaminated sites, equipments and facilities, and other materials are thoroughly disinfected;  the practices are examined and recognized as up to the standards by the local animal disease control supervision institution; and the local veterinary authority above the county level applies to the local government that gives the order of blockade for its lifting.

8. Consistency with the International Animal Health Code

8.1 Glanders is a legally notifiable animal disease in China

Based on the varied negative impacts of animal diseases on animal farming and human health, the Law of the People’s Republic of China on Animal Disease Prevention and Control classifies the notifiable animal diseases into three categories, and glanders falls into the second category.

8.2 Current status of glanders in China

Since 1949, China has attached great importance to the prevention, control and eradication of glanders. Through unremitting efforts in nearly 6 decades, all the provinces, municipalities and autonomous regions had met the requirements for the eradication of glanders by 2005 in line with the Technical Standards for Prevention and Control of Glanders. The comprehensive surveillance from 2005 to 2009 found no clinical case and test-positive animal.

8.3 Conclusion

Glanders is a legally notifiable animal disease in China. Since all the provinces, municipalities and autonomous regions met the requirements for the eradication of glanders in 2005, no more case has been detected according to the surveillance in 5 consecutive years. China has strictly followed Article 2.5.8.3 and Article 2.5.8.4 of the International Animal Health Code in its importation of equine animals from other countries, which ensures no introduction of the disease into China.

In conclusion, the technical measures for the eradication of glanders adopted in China are consistent with the International Animal Health Code, and China have satisfied its requirements for a glanders free country .


Annex I:     

Statistics on examination and approval for eradication of glanders by the Ministry of Agriculture

Province

County/City randomly inspected

Inspection time

Number of randomly sampled horses

Observation timeh

Results

Remarks

3

6

9

24

+

Qinghai

Huangzhong, Gonghe

May 1993

322

 

139 in Huangzhong and 183 in Gonghe

Liaoning

Heshan

Oct. 1995

202

 

 

Xinjiang

Zhaosu, Chabuchaer

Sep.1996

226

 

95 in Zhaosu, 30 from farms affiliated to

Xinjiang Production and Construction

Corps, and 101 in Chabucha’er 

Henan

Yuzhou,Yanjin

Oct.1996

246

 

134 in Yuzhou and 112 in Yanjin

Ningxia

Pingluo

Oct.1997

164

 

 

Tianjin

Baodi

Jul. 1998

100

 

 

Jilin

Jiutai, Huadian

Jul. 1998

256

 

119 in Jiutai and 137 in Huadian  

Heilongjiang

Qinggang, Hailin

Aug.1998

234

 

130 in Qinggang and 104 in Hailin

Sichuan

Aba, Baoxing

Dec. 1998

159

 

87 in Aba and 72 in Baoxing

Shaanxi

Jingyang

May 1999

133

 

 

Beijing

Yanqing

May 1999

100

 

 

Inner Mongolia

Xinbaerhuyou banner, Eerguna City

Sep. 1999

231

 

128 in Xinbaerhuyou banner and 103 in Eerguna City

Gansu 

Hezuo

Sep. 1999

121

 

 

Shanxi

Pinglu 

Sep. 1999

104

 

 

Anhui

Jieshou

Nov. 1999

133

 

 

Jiangsu

Donghai

Nov. 1999

108

 

 

Guizhou

Anshun

Dec. 1999

100

 

 

Hebei

Zhangbei, Xushui

Apr. 2000

215

 

 

102 in Zhangbei and 113 in Xushui

Shandong

Huantai, Jiyang

Jul. 2000

105

 

47 in Huantai and 58 in Jiyang

Yunnan

Jinning

Dec. 2000

112

 

 

Tibet

Zhongba, Pulan, Langkazi &

Duilongdeqing

 

Aug. 2004

320

 

118 in Langkazi, 10 in Duilongdeqing, 119 in Zhongba and 73 in Pulan

Total

34

 

3691

 

 


Annex II:

National statistics on glanders surveillances 2006-2009

 

No.

Province

Number of animals tested in 2006

head

Number of animals tested in 2007

head

Number of animals tested in 2008

head

Number of animals tested in 2009

head

1

Beijing

7181

9974

7749

330

2

Tianjin

1830

500

800

3

Hebei

4915

5904

1800

 50

4

Shanxi

0

740

1800

5

Inner Mongolia

10955

11988

7603

6

Liaoning

15539

11357

4152

7

Jilin

2700

1333

975

8

Heilongjiang

4218

3586

2200

9

Jiangsu

1200

1200

3626

10

Anhui

0

0

1502

11

Shandong

0

0

800

12

Henan

20287

2586

4639

1290

13

Sichuan

0

0

1574

14

Yunnan

1384

14881

2012

15

Guizhou

2700

1000

550

16

Tibet

0

0

1526

17

Shaanxi

10146

10137

10107

18

Gansu

22000

0

2000

19

Qinghai

1312

1417

1480

20

Ningxia

0

400

818

21

Xinjiang

2099

1608

4452

22

Chongqing

0

550

500

23

Shanghai

0

523

1418

24

Guangxi

0

0

0

25

Sub-total

108466

79684

64083

1670

 

Total

253903

Notes

1.  the results were all negative.

2. These data were from the surveillance after the year 2005, when the process of   examination and approval for eradication of glanders was accomplished in China.

 

 

 

 

 

Related Accessories:
Related News
Recent Browse

Fold