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REPUBLIC OF NAMIBIA
Ministry of Agriculture Water and Forestry Directorate of Veterinary Services
April 2017
Mycobacterium bovis Surveillance Protocol
Compiled by:
Division of Epidemiology, Import & Export, Traceability, Medicine Control and Advisory Service
Private Bag 12022
Windhoek / Namibia
Tel: +264-61-2087542
Fax: 264-61-2087779
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Table of Contents 1. INTRODUCTION ......................................................................................................................... 3
2. SURVEILLANCE STRATEGY ......................................................................................................... 3
2.1 Surveillance at slaughter facilities....................................................................................... 4
2.1.1 Clinical Case definition ................................................................................................ 4
2.1.2 Post mortem Case Definition ...................................................................................... 4
2.1.3 Differential Diagnosis.................................................................................................. 4
2.2 Testing of animals during import/export ............................................................................ 5
2.3 Investigation of reported suspect cases .............................................................................. 5
3. SAMPLING FOR MYCOBACTERIUM BOVIS AT POST-MORTEM .................................................. 5
3.1 Sampling for laboratory investigation ................................................................................. 6
4. LABORATORY DIAGNOSIS ......................................................................................................... 6
4.1 Polymerase Chain Reaction (PCR) ....................................................................................... 6
4.2 Microscopic examination ................................................................................................... 7
4.3 Culture ............................................................................................................................... 7
5. TRACE BACK INVESTIGATION .................................................................................................... 7
6. TRAINING .................................................................................................................................. 7
7. REPORTING AND DISSEMINATION ............................................................................................ 8
8. ANNEXES ................................................................................................................................... 9
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1. INTRODUCTION
Bovine tuberculosis is a chronic bacterial disease of animals and humans caused by
Mycobacterium bovis. In a large number of countries bovine tuberculosis is a major infectious
disease among cattle, other domesticated animals, and certain wildlife populations. The
disease can cause production losses when poorly controlled and is classified by the World
Organisation for Animal Health (OIE) as a disease of socio-economic, public health and trade
importance. The public health risk has been alleviated in many countries through
pasteurisation of milk.
Bovine Tuberculosis, caused by the bacterium Mycobacterium bovis, is listed as a notifiable
disease in Notice No180 of Government Gazette No. 5239 of 12 July 2013. Currently, Namibia
regards itself as free from Bovine Tuberculosis. The last case diagnosed in a live bovine was
in 1994 and in a slaughtered bovine in 1995. No cases have been detected since then.
Aerosol exposure to M. bovis is considered to be the most frequent route of infection of
cattle, but infection by ingestion of contaminated material also occurs. After infection,
nonvascular nodular granulomas known as tubercles may develop. Characteristic tuberculous
lesions occur most frequently in the lungs and the retropharyngeal, bronchial and mediastinal
lymph nodes. Lesions can also be found in the mesenteric lymph nodes, liver, spleen, on
serous membranes, and in other organs.
Bovine tuberculosis infection in cattle is usually diagnosed in the live animal on the basis of
delayed hypersensitivity reactions using the single or comparative intradermal tests. Infection
is often subclinical; when present, clinical signs are not specifically distinctive and can include
weakness, anorexia, emaciation, dyspnoea, enlargement of lymph nodes, and cough,
particularly with advanced tuberculosis. After death, infection is diagnosed by necropsy and
histopathological and bacteriological techniques. Rapid nucleic acid methodologies, such as
the polymerase chain reaction (PCR), may also be used although these are demanding
techniques and should only be used when appropriately validated. Traditional mycobacterial
culture remains the gold standard method for routine confirmation of infection.
2. SURVEILLANCE STRATEGY
Mycobacterium bovis surveillance strategy is implemented through active and passive
surveillance. Active surveillance involve examination of carcasses at slaughter facilities and
testing of animals before export and after import using the intradermal tuberculin. The
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passive surveillance involves investigation of reported suspect clinical cases through
intradermal testing or post mortem examination of organs.
2.1 Surveillance at slaughter facilities
There are seven export approved abattoirs and local authority slaughter facilities distributed
throughout the country. The Directorate of Veterinary Services (DVS) provides oversight at
the export approved abattoirs while the local authority slaughter facilities are under the
supervision of Local Authorities under the Ministry of Regional and Local Government,
Housing and Rural Development and the Ministry of Health and Social Services.
Abattoir surveillance through ante and post mortem inspection is ongoing. However, in order
to collect more credible data, a sampling protocol has been developed to enable diagnosis of
all lesions that resemble bovine tuberculosis.
Meat inspection is conducted by qualified and registered meat inspectors and is done
according to the Meat Safety Act, 2000 (Act No. 40 of 2000), Red Meat Regulations (Act No.
40 of 2000) and the guidelines of the South African Meat Inspectors Manual for Red Meat. At
the export approved abattoirs meat inspection is done by Veterinary Hygiene Inspector
Assistants and Veterinary Hygiene Inspectors under the supervision of State Veterinarians. At
non-export slaughter facilities meat inspection is done by meat inspectors with a minimum of
a Diploma in Environmental Sciences.
2.1.1 Clinical Case definition
Bovine tuberculosis can be suspected when some of the following signs are observed during
ante-mortem inspection:
Weakness
Emaciation
Dyspnoea
Coughing
Enlargement of lymph nodes especially those in the neck region
2.1.2 Post mortem Case Definition
Bovine tuberculosis can be suspected when some of the following signs are observed during
post-mortem inspection
Nodular granulomas in organs including lungs, intestines, liver spleen, pleura and
peritoneum
Nodular granulomas in lymph nodes especially of head and neck
Small nodular granulomas on thoracic wall (miliary)
2.1.3 Differential Diagnosis
If the following diseases and conditions and conditions are suspected samples must be
collected to rule out bovine tuberculosis:
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Contagious bovine pleuropneumonia (with sequestra present)
Actinobacillosis
Lung abscessation
Bovine farcy (Mycobacterium farcinogenes)
Bacterial or viral bronchopneumonia
Echinococcal hydatid cysts (calcified)
2.2 Testing of animals during import/export
All cattle destined for export to neighbouring countries are subjected to the single
intradermal tuberculin test as per the Protocol on Bovine Tuberculosis Testing. Similarly all
cattle imported into the country are subjected to the single intradermal tuberculin test. Data
from such testing of cattle is compiled to give a summary of number of cattle and number of
herds tested and the results of such testing.
2.3 Investigation of reported suspect cases
State Veterinarians in the field are responsible for investigating suspected outbreaks of
disease. Any mycobacterium tuberculosis suspect whether clinical or post mortem is
investigated through appropriate intradermal tuberculin test or sample collection for
bacterial culture or PCR. The handling of suspect cases at post-mortem will be as for
investigation at slaughter facilities.
3. SAMPLING FOR MYCOBACTERIUM BOVIS AT POST-MORTEM
At post-mortem, tubercles are most frequently seen in bronchial, mediastinal,
retropharyngeal and portal lymph nodes and may be the only tissue affected. In addition, the
lung, liver, spleen and the surfaces of body cavities are commonly affected. Early nodular
pulmonary lesions can often be detected by palpation. The lesions are usually non-
odoriferous. Other anatomical sites can be infected and should be examined.
Figure 1. Typical Mycobacterium bovis granuloma.
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A tuberculous granuloma has a yellowish appearance
It has a caseous, caseo-calcereous consistency
Its appearance may be purulent
The caseous centre is usually dry, firm and covered with a fibrous connective capsule
Lesion size range from small to involvement of greater part of organ, hence serial
sectioning of tissues required to detect small lesions
3.1 Sampling for laboratory investigation
1. Lesions suggestive of bovine tuberculosis must be sampled and submitted for
diagnosis to the Central Veterinary Laboratory.
2. Collection instruments and containers should be clean and preferably sterile (use of
instruments and containers that are contaminated by environmental mycobacteria
may result in the failure to identify M. bovis infection due to the rapid growth of the
environmental mycobacteria); where feasible, single-use plastic, disposable
containers, 50 ml in capacity, may be used for a variety of specimen types.
3. The container must be CLEARLY labelled containing the following minimum
information: species, organ/tissue sampled, animal identification number.
4. A QUA from 99 must be completed to accompany the samples.
5. Specimens must be cushioned and sealed to prevent leakage, and properly packaged
to withstand breakage or crushing in transit.
6. Delivery of samples to CVL must be prompt. Prompt delivery of specimens to the
laboratory greatly enhances the chances of cultural recovery of M. bovis. If delays in
delivery are anticipated, specimens should be refrigerated or frozen to retard the
growth of contaminants and to preserve the mycobacteria.
4. LABORATORY DIAGNOSIS
The Central Veterinary Laboratory (CVL) is responsible for laboratory confirmation for
Mycobacterium bovis and forwarding samples to other laboratories for confirmation.
Precautions should be taken to prevent infection of laboratory personnel. All procedures
involving preparation of the tissue samples should be performed in a biological safety cabinet.
4.1 Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) has been successfully applied to detect members of the M.
tuberculosis complex and is especially useful for the direct detection of M. bovis in bovine
tissue samples. Any positive result should be confirmed by culture.
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4.2 Microscopic examination
Mycobacterium bovis can be demonstrated microscopically on direct smears from clinical
samples and on prepared tissue materials.
The acid fastness of M. bovis is normally demonstrated with the classic Ziehl - Nielsen stain,
but a fluorescent acid-fast stain may also be used. Immunoperoxidase techniques may also
give satisfactory results.
The presumptive diagnosis of mycobacteriosis can be made if the tissue has characteristic
histological lesions (caseous necrosis, mineralisation, epithelioid cells, multinucleated giant
cells and macrophages).
4.3 Culture
Cultures are incubated for a minimum of 8 weeks (and preferably for 10–12 weeks) at 37°C
with or without CO2. The media should be in tightly closed tubes to avoid desiccation. Slopes
are examined for macroscopic growth at intervals during the incubation period.
When growth is visible, smears are prepared and stained by the Ziehl–Nielsen technique.
Growth of M. bovis generally occurs within 3–6 weeks of incubation depending on the media
used. Characteristic growth patterns and colonial morphology can provide a presumptive
diagnosis of M. bovis; however every isolate needs to be confirmed. It is necessary to
distinguish M. bovis from the other members of the ‘tuberculosis complex’.
Currently the CVL is using PCR for M bovis diagnosis while culture and biochemical tests are
referred to South Africa.
5. TRACE BACK INVESTIGATION
Any positive result from slaughter facility must be reported to Animal Disease Control Division
to enable trace back to the farm/ village of origin by the local State Veterinarian.
6. TRAINING
All staff responsible for implementing the M. Bovis surveillance in DVS will be subjected to
training in intradermal tuberculin testing, gross pathological diagnosis and sample collection
and preservation. The gross pathological diagnosis training will also cover differential
diagnosis for M bovis aforementioned. The training for CBPP gross pathological diagnosis will
be very important for the Northern Communal Areas where CBPP freedom is one of the
objectives. Staff from Ministry of Health and Social Services and Local Authorities responsible
for meat inspection will also be trained in gross pathological diagnosis and sample collection
and preservation.
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7. REPORTING AND DISSEMINATION
All surveillance activities for Mycobacteria bovis are to be reported in Monthly Reports for
inclusion in the National Summary Reports for communication to stakeholders.
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8. ANNEXES
Annex 1. Differential Diagnosis for M bovis
1. Contagious bovine pleuropneumonia
CBPP sequestra in lung tissue
Grey hepatisation of lung due to CBPP
Marbling appearance of lung due to CBPP
2. Actinobacillosis
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Lung lesions due to actinobacillosis
3. Lung abscessation
Multiple abscesses in a lung
4. Bovine farcy (Mycobacterium farcinogenes)
Characterised by pneumonia, abscessation, mastitis, cutaneous and subcutaneous lesions
5. Bacterial or viral bronchopneumonia
Bronchopneumonia in cattle
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6. Echinococcal hydatid cysts (calcified)
Lung hydatid cyst with well defined membrane