Diagnosis of Bacterial Diseases

 

General Bacteriology

Contact details
Tel: 27 (0) 12 529 9384/9137
Fax: 27(0) 12 529 9127
Email: Dr Awoke Gelaw

Introduction

General bacteriology laboratory serves as a reference laboratory for the veterinary diagnostics service in South Africa and also provides support to the other laboratories in Southern Africa and Africa as a whole. The laboratory is organized to facilitate the recognition of the more important features of bacterial colonies and the application of biochemical, serological and other tests for the identification of pathogens from clinical specimens.

Aims and objectives

• Surveillance of diseases of national and international importance
• Identification of bacterial and fungal diseases associated with clinical and postmortem cases
• Selection of appropriate chemotherapeutic drugs
• Undertake research activities related to livestock health problems

Services

The laboratory provides a wide range of routine veterinary diagnostic tests that includes:

• Aerobic culture and identification
• Anaerobic culture and identification
• Sheathwash examination (Campylobacter and Trichomonas)
• Bacterial typing
  (1) E. coli
  (2) Klebsiella
  (3) Pasteurella
  (4) Salmonella
  (5) Actinobacillus pleuropneumoniae
  (6) Salmonella Enteritidis phage
• Clostridium botulinum toxin test
• Clostridium tetani test
• Clostridium perfringens
• Direct FA test for Clostridia
• Complete faecal examination
• Brucella culture and typing

Controlled diseases

The diagnostic tests of the following diseases are offered:

• Anthrax (Bacillus anthracis)
• Bacterial kidney disease (Renibacterium salmoninarun)
• Brucellosis (Brucella spp)
• Contagious equine metritis (Taylorella equigenitalis)
• Salmonella Enteritidis
• Glanders (Pseudomonas mallei)

Food safety

The following tests are offered in collaboration with the Feed and Food analysis and the Bacteriology PCR Laboratories

• E. coli 0157:H7
• Salmonella spp
• Salmonella typhi
• Salmonella enteritidis

FEED AND FOOD ANALYSIS LABORATORY

For more information

Feed & Food Analysis Laboratory: Dr Evelyn Madorroba

Tel: +27 (0)12 529 9384/452
Fax: +27 (0)12 529 9127

 Feed & Food Analysis laboratory is an accredited laboratory in the Bacteriology Section managed by committed and expert staff who value the need for quality results, customer service, speed and absolute confidentiality. The laboratory tests samples for individuals, private businesses and the Government Food Safety programs at highly competitive test fees.

Services:

We recognize that everyone; farmers, growers, manufacturers and processors; food handlers and consumers have a responsibility of ensuring that food is safe & suitable for consumption and that food safety and control is critical in avoiding adverse human health and economic consequences of food-borne illnesses, food-borne injury and food spoilage.

The Feed & Food Analysis Laboratory is driven on the concept of "From Farm to Fork" that involves monitoring of bacteriological quality and safety of food throughout the food chain

Our core business is to provide testing for bacteriological contaminants in animal feedstuff, food & food additives, milk and water for:

• Monitoring and verifying the bacteriological quality and safety of the feedstuff, food, milk and water
• Bacteriological surveys
• Regulatory purposes

We also provide laboratory support to investigation of food-borne outbreaks

The laboratory also provides testing of samples on contract basis depending on customer needs. We engage in collaborative research work on contract basis for food safety programs

Our Bacterial Testing Capabilities include testing for:

• Total bacterial count
• Total coliform count
• Total E. coli count
• Testing for presence and identification of the following feed and food pathogens:
• Salmonella species
• E. coli
• E. coli 0157
• Bacillus cereus
• Bacillus anthracis
• Listeria species
• Clostridium species
• Staphyloccocus aureus
• Vibrio cholerae

Feed and Food analysis list of services and tests are available in the annual Pricelist

TUBERCULOSIS

The Tuberculosis laboratory deals with Mycobacteria from tuberculosis to Johne's disease.   

Contents

• More about Tuberculosis
• Vision of the TB Laboratory
• Activities in the TB laboratory
• Training
• Important hints for collection and submission
• Protocol for the preparation of blood for Gamma Interferon Assay
• Contact Details
• Staff compliment

TB poster.pdf (BTP) (click link to access poster)

More about Animal Tuberculosis

Cause

Tuberculosis is a chronic, primarily respiratory disease which can affect all mammals including humans. It is caused by either of two bacteria of the genus Mycobacterium namely, Mycobacterium tuberculosis generally associated with human tuberculosis, and Mycobacterium bovis, the agent that cause tuberculosis in animals, generally known as bovine tuberculosis (BTB).

Lung lesions in an African buffalo with bovine tuberculosis

Lymph node abscess due to infection with Mycobacterium bovis in a kudu

Overview

Bovine tuberculosis caused by Mycobacterium bovis was most probably introduced to South Africa by European settlers and their cattle. In South Africa, bovine tuberculosis was first reported in 1880 in domestic cattle and in 1928 in wildlife in the Eastern Cape. Soon after the introduction of the "National Tuberculosis and Brucellosis Eradication Scheme" in 1969, bovine tuberculosis could be successfully controlled in commercial cattle herds. Bovine tuberculosis is a serious threat to livestock and wildlife species and is therefore controlled in many countries including South Africa, and African buffalo (Syncerus caffer) is the wildlife maintenance host. A better understanding of transmission patterns and distribution of the disease will permit more precise targeting of control measures that will potentially benefit both wildlife and livestock Molecular typing techniques are useful tools that can be applied to bovine tuberculosis epidemiology in order to underpin control of the disease. These techniques are now applied in the Tuberculosis laboratory and offered as a service.

Future prospects

Under the present budgetary constraints, effective control of bovine tuberculosis becomes increasingly difficult. The situation can in future be further complicated by the potential spillover from wild animals to livestock. If bovine tuberculosis can establish itself in a free ranging (small) mammal species which can bridge the wildlife-domestic animal interface, recurrent infections in cattle will be inevitable. Similar situations are known from Ireland, United Kingdom and New Zealand. In many countries, eradication of bovine tuberculosis from cattle and wildlife reservoirs is far out of reach. The development of vaccine in the future remains the only feasible long term solution in all ecosystems where a wildlife reservoir maintains the infection.

Vision of the TB Laboratory

To hold the status of an advisory body to the Directorate of Veterinary Services of the National Department of Agriculture with regard to bovine tuberculosis and other mycobacterioses and to become an international reference laboratory for animal tuberculosis. 

Activities in the TB Laboratory

• Diagnosis
• Research
• Production and sale of fortuitum used in the diagnosis of Tuberculosis in buffalo and cattle
• Buffalo Test Package

Diagnosis

For the diagnosis of tuberculosis the following approaches can be followed:

(1) Microscopy and Mycobacterium species isolation

• Ziehl Neelsen staining

Principle: Heat fixed impression smears are stained with carbolfuchsin, rinsed and counterstained with malachite green.

Interpretation: Acid fast organisms (mycobacteria) retain their red color and are distinguished from other material and microorganisms which absorb the counter stain.

• Culture isolation of the causative agent from fresh tissue or other specimens

Following homogenization the specimen is inoculated onto solid, egg based medium and incubated at various temperatures for a maximum of 10 weeks, and the culture slopes are evaluated weekly. Although culture is a gold standard method, the main disadvantage of this test using solid media is the long incubation time. To overcome this problem, the laboratory has purchased a VersaTREK liquid culture method for the rapid detection of mycobacteria, including M. bovis. This is an automated system that can detect M. bovis within 2-3 weeks.

M. bovis isolates on Lowenstein Jensen slopes

(2) Identification of the isolates by PCR

Purpose: to determine the mycobacterial species

PCR: is a sequence specific enzymatic amplification of small amounts of DNA material. Before PCR is performed, the acid fastness of the cultures is confirmed by Ziehl Neelsen staining. PCR methods are used for the identification of Mycobacterial species. The first PCR method is for the identification of Mycobacterium tuberculosis complex species, which is performed using primers designed to target a sequence encoding the MPB 70 antigen of M. tuberculosis complex species. PCR negative Mycobacterium species other than tuberculosis are further identified by biochemical tests if necessary. PCR positive M. tuberculosis complex species (MTBC) are further subjected to other PCR tests which make use of primers that target specific regions of difference (RD) on genomes of MTBC to differentiate them, especially M. tuberculosis and M. bovis.

Figure showing gel picture of PCR products

(3) Immunodiagnosis of tuberculosis

• The Gamma Interferon Assay

Principle: The IFNg assay is a cytokine assay designed to detect interferon gamma in the plasma of infected cattle, sheep and goat.

Application: A modified version of the IFNg assay is used to diagnose tuberculosis infection in African buffalo, bison, kudu, eland, bongo, oryx, sable antelope, and waterbuck among other members of the bovidae family

• Primagam

Principle: This test is an in vitro blood based assay of cell mediated immunity through measurements of IFNg.

Application: Primagam is used for the detection of tuberculosis in non-human primates. This test has to date shown to detect IFNg in the following species: Autos (night/owl monkey), spider monkey, chimpanzee, guerezas, gibbon/siamang, gorilla, geonon, langur, lemur, etc.

(4) Immunodiagnosis of paratuberculosis

• ELISA

Paratuberculosis or Johne's disease is a chronic infectious disease that affects all categories of domestic and wild ruminants including cattle, sheep, goats, camels, buffaloes and farmed deer. The main clinical signs are abundant diarrhea and weight loss. The infection is due to an acid fast bacillus Mycobacterium paratuberculosis.  

• CULTURE

Until recently, no culture test was done for the isolation of the causative agent of Johne's disease. The VersaTREK liquid culture system purchased by the laboratory will ensure a rapid detection of M. avium paratuberculosis

Production and sale of Fotuitum PPD (Purified Protein Derivative)

During the evaluation of the "gamma Interferon assay" in cattle and African buffalo we found that the tests produced false positive reactors in approximately 4% of samples tested. Research into the cause of the non-specific reactions led to the conclusion that animals harboring certain environmental mycobacteria, mostly of the M. fortuitum complex, become sensitized to bovine and avian tuberculin PPD resulting in some of these animals showing false positive reactions in the commercial Bovigam® assay. Following its modification into a triple comparative test by including a crude antigen mixture prepared from M. fortuitum a high specificity of > 99% was achieved. The Fortuitum antigen is offered for sale at the Tuberculosis Laboratory in ampoules containing 375µl dose each, enough to test 15 animals.

The Buffalo Test Package

Wildlife, one of South Africa's biggest assets, forms the basis of a rapidly growing industry. Conservation of African wildlife, however, goes beyond and requires more than good business management. A vast responsibility to maintain and foster a genetic diversity within healthy species rests on the shoulders of all those who want to become part of this exciting challenge. However, diseases such as bovine tuberculosis which directly affect a wide spectrum of wildlife species can cause entire conservation programs to collapse. On the other hand, there are diseases such as foot-and-mouth disease and corridor disease for which in particular the African buffalo can serve as a latent carrier and source of infection to cattle, causing devastating economic losses to the animal production sector. Therefore we have come forward with a comprehensive diagnostic "Buffalo Test Package" to better serve our clients and support Government's efforts in the control of these diseases.

Training in Laboratory Procedures relevant to the "gamma interferon assay"

Over the last years, the gamma interferon assay has established itself as the most widely used test in the diagnosis of bovine tuberculosis in buffalo. One of the major constraints, however, is the necessity for processing the blood samples within eight hours. To overcome this problem, private laboratories were given the protocol or are trained to perform blood processing (First stage of Gamma Interferon Test). The TB laboratory sells the Tuberculin PPD's. Plasma samples are then sent to the laboratory ready for stage two of Gamma interferon test.

Important notes for the submission of specimens

Specimen collection for culture isolation

• specimen is part of the affected area
• at least 5 grams where possible
• any contamination should be avoided

Packaging for transport

• avoid cross contamination of specimens
• pack in safe containers
• PREVENT ANY LEAKAGE!!
• label correctly (sender, owner, species, animal ID, specimen type, date of collection, examination required, history)
• maintain cold chain during transport

Contact details

Ms Tiny Hlokwe - Researcher and Head of Laboratory

Tel: 27 (0) 12 529 9452
Fax: 27 (0) 12 529 9127
E-mail: HlokweT@arc.agric.za   

Staff compliment

Ms Nomakorinte Gcebe - Researcher

Mr Dominic Wagner- Research technician

Ms Marcia Mogano-Research technician

Mr Boitumelo Letsoko-Research technician

Mr Petrus Manamela-Research Assistant 

Protocol for the preparation of blood for the "Gamma-Interferon" assay

"Gamma-Interferon" Assay for the diagnosis of M. bovis infections

Requirements:

1. 1 Heparin blood tube (10 ml) per animal
2. 8 sterile disposable plastic tubes (± 2 ml) per animal
3. Equipment for:
• sterile dispensing of 1.5 ml blood
  (micropipette, tuberculin syringe or pasteur pipette)
• sterile dispensing of tuberculin (25, 30 and 60 microliter)
• sterile harvesting of plasma (± 0.3 ml)

Field procedure:

1. Collect 10 ml blood in heparin per animal
2. Keep blood at ambient temperature (optimum: 15 – 25ºC) during transport
3. Submit blood to the laboratory within 8 hours after collection

Laboratory procedure:
Day 1 = day of blood collection:

1. Tilt blood tube several times to ensure proper mixing
2. Dispense 1.5 ml blood into each of 4 sterile plastic tubes per animal
3. Label tubes and add antigens as follows:

4. After adding the antigens close and tilt tubes several times to ensure sufficient mixing of the content
5. Incubate tubes at 37 ºC for 20 – 24 hours

Day 2:

1. Transfer plasma (supernatant) into sterile plastic tube (correct labelling!)

(If separation of plasma and red blood cells is not sufficient the tubes should be centrifuged in a microcentrifuge at low speed (1200 rpm) for 10 minutes

2. Freeze plasma at - 20ºC and send to Tuberculosis Laboratory Onderstepoort Veterinary Institute, Old Soutpansroad, Tel: +27 (0)12 529 9149 or 529 9452.

Note: Fortuitum PPD is available from above address

SEROLOGY

Contact: Dr Andrew Potts
Tel: +27 12 529 9396
Fax: 27 12 529 9429
E mail: PottsA@arc.agric.za

Objectives

Bacteriology Serology offers serological tests for a range of diseases with special emphasis on diseases affecting reproductive performance. The objectives of the Laboratory are to assist in the diagnosis and control of disease and to certify the disease-free status of animals for export or breeding.

Range of micro-organisms

Brucellosis (Contagious abortion or CA)

This disease causes late-term abortion in cows and can spread rapidly in a herd when other breeding cows are exposed to bacteria from the aborted foetus, membranes and fluid of an infected cow. There is no treatment and the best method of control is to identify infected animals and remove them from the herd. One of the practical ways of identifying infected animals is to test for the presence of antibodies in the blood (serum) through various serological tests. Blood that is allowed to clot will produce a clear straw-coloured liquid known as serum. The serum of infected animals will contain antibodies to the bacteria causing brucellosis (Brucella abortus).

Serological tests

• Rose Bengal Test (RBT)

This is a simple but sensitive screening test which is carried out on all sera. Equal volumes (25 µl) of serum and antigen (killed Brucella abortus organisms) are mixed in a plate for 4 minutes. In positive cases an agglutination reaction will take place between the antibodies in the serum and the antigen which can be seen as a ring in the well of the plate.

• Serum Agglutination Test (SAT)

This is also a screening test involving an agglutination reaction. The serum is serially diluted in 4 wells of a microplate (1/8, 1/32, 1/128, and 1/512) before the antigen is added. This gives an indication of the number of antibodeis in the serum and hence how positive the sample is. A reaction that only occurs in the first well indicates few antibodies. A reaction that extends to the fourth well indicates many antibodies because even after diluting the serum 1/512 there are still sufficient antibodies to cause an agglutination reaction.

• Complement Fixation Test (CFT)

The RBT and SAT are sensitive tests (no false negative results) but they can give false positive results (non-specific reactions). The CFT is a more specific test and is carried out on all RBT positive samples. The serum is also diluted to give an indication of the titre.

• Milk Ring Test (MRT)

Milk from individual cows as well as bulk tank samples can be tested. This is also an agglutination reaction and a positive reaction is indicated by a blue ring in the cream layer.

Bovine brucellosis is a difficult disease to diagnose and eradicate. In South Africa it still has a widespread occurrence and dedication and vigilance is needed if effective control is to be achieved. Serology, together with other tests, such as PCR and culture, is a means of identifying infected animals in a herd. Due to the contagious nature of the disease it is important that infected animals are detected early and removed from the herd. The long incubation period of the organism means sequential tests are essential before a herd can be declared free from brucellosis. Onderstepoort Veterinary Institute (OVI) has a well-equipped brucellosis laboratory and specialized personnel who are able to process up to 10 000 samples per month and give practical advice.

Submission of specimens

Specimens should be accompanied by the General submission form [Word Document]
Specimens submitted for Brucella serology must be accompanied by the "Brucella CA5 form" [pdf] and for dourine by the "Dourine test and agreement form" [Word Document]. On both these forms the name, farm address and telephone number of the owner must be indicated and the agreement must be signed and witnessed as any legal document.

Bovine brucellosis samples should be accompanied by a completed CA5 form. Numbering and packing of samples should be as follows:

Arrange the samples in the polystyrene holders in groups of 25 as follows:
1 2 3 4 5 26 27 28 29 30
6 7 8 9 10 31 32 33 34 35
11 12 13 14 15 36 37 38 39 40
16 17 18 19 20 41 42 43 44 45
21 22 23 24 25 46 47 48 49 50

Minimum volume – at least 0.5 ml serum especially for canine leptospirosi

PCR LABORATORY

For more information

Dr Noma Gcebe

Tel: +27 (0)12 529 9384/452
Fax: +27 (0)12 529 9127

The Laboratory focuses on Nucleic Acid based diagnosis of bacterial related animal diseases and the detection of bacterial food pathogens.

What is PCR?

The Polymerase Chain Reaction (PCR) is a technique that is used to enzymatically amplify a number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested. The technique can be used to identify disease-causing bacteria. The technique has the advantage of being rapid, sensitive and very specific. It is also relatively inexpensive. .

Services

The services of the laboratory are divided into the areas of bacterial disease diagnosis and bacterial food pathogen detection.

Contribution to the livestock producers
The following disease diagnostic tests are offered routinely:

• Anthrax (Bacillus anthracis detection and virulence detection) 
• Porcine proliferative enteritis (Lawsonia intracellularis and Salmonella spp.)
• Typing of Clostridium perfringens
• Trichomoniasis (Trichomonas foetus) and vibriosis (Campylobacter fetus fetus and C. fetus venerealis)
• Brucella (at group level and also differentiating to species level)

Tests that are under validation include tests for the detection of:

• Contagious equine metritis (Taylorella equigenitalis)

• E coli O157:H7
• Salmonella spp.
• Salmonella typhi
• Salmonella enteritidis

With the increase in the incidence of reported cases of diarrheal diseases caused by E. coli, the laboratory is now validating a test for the detection pathogenic E. coli (Shiga toxin producing E. coli, STEC). The test will be available for routine use as soon as the validation is complete.