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This article was published in 1980
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Laboratory Aspects of Bovine Brucellosis
J.E. Searson, B.V.Sc., Regional Veterinary Laboratory, Wagga
For the Veterinary Inspector to fulfil his role as an interpreter of laboratory serological results in the Brucellosis Campaign, he must have a good understanding of basic immunology. This will enable him to make logical decisions and also to explain the more unusual results to the farmer.
I will give an outline of the two different types of immune response that may occur. These concepts will then be related to the serological tests performed in the Brucellosis Campaign and finally I will make some comments on general laboratory aspects of bovine brucellosis.
Systems of Immunity
There are two systems or types of immunity:-
i. Thymus dependant - involving the T - lymphocytes - known as Cell Mediated Immunity.
ii. Bursal dependant - involving B-lymphocytes - known as Humoral Immunity.
The bursal equivalent in mammals is thought to be the bone marrow.
These two systems interact and provide the means by which an animal responds to infection (or a 'foreign' antigen). Set out below are the basic pathways in the two types of immunity.
Laboratory tests for brucellosis detect the products of the humoral response. These tests do not directly measure cell mediated immunity, which is very important in an animal attempting to free itself of a facultative intracellular organism such as Brucella abortus.
I will now concentrate on Humoral Immunity.
Antibody Production
The most accepted theory for explaining antibody production is that of clonal selection, based on the ideas of Burnet. Basically there are B-lymphocytes that each have the genetic information to enable the production of one antibody. Since different B-Lymphocytes have different specificities, a wide variety of immunoglobulins can be produced.
Some lymphocytes may produce immunoglobulin without apparent or detectable specificity. Most virgin stem cells produce immunoglobulin with well defined specificity - every individual has immunoglobulins with specificities for every conceivable antigenic determinant and contact between specific antigen and pre-existing immunoglobulin of corresponding specificity (present on the surface of virgin cells) induces full scale production of that Ig. This is the theory of the 'Generation of Antibody Diversity'.
There are two different types of response obtained to an antigen. There is a primary response, which happens after initial contact and a secondary response which happens after subsequent contact with the particular antigen.
The magnitude of the humoral response depends on:-
i. Host or Animal Factors - e.g. Age, breed, nutritional state, endocrine status.
ii. Antigen Factors - e.g. solubility, molecular weight, presentation - (living or dead organisms).
What happens in a Primary Response?
When an antigen is presented to a virgin lymphocyte population in a lymph node; the normal migration of lymphocytes through the node is virtually shut down for about one day. The degree of shutdown is dependant on mainly antigen factors. Antigen sensitive B-lymphocytes are recruited and immunoglobulin production is increased dramatically. The B-lymphocytes differentiate to form plasma cells and some memory cells. Early on, the plasmablasts predominate and produce IgM - some of these cells leave the lymph node and enter the lymph and blood circulation. IgG appears after IgM and is produced by plasma cells, the majority of which are located in the lymph nodes.
Much of the antibody produced early in the primary response has no apparent specificity i.e. it does not combine with the antigen stimulating its production. Hence the lag in the appearance of detectable antibody following infection - serological tests are not positive for a number of days following infection.
In the primary response a relatively small number of B-lymphocytes are recruited and hence the corresponding antibody response is similar to the primary but is more vigorous and events happen much more quickly as there is already an expanded pool of memory cells ready to respond. More cells are involved and antibody production is greater. IgG is produced quicker.
Brucella abortus being a facultative intracellular parasite makes cell mediated immunity (C.M.I.) vitally important to the ultimate outcome of the infection and the humoral response.
The immune status of an animal is dynamic and can vary between the extremes of complete immunity and complete tolerance. The intermediate states are far more common and are what we mainly deal with in brucellosis serology.
Progression from one state to another, in either direction is NOT surprising and in fact should be expected.
Tolerance can develop in an adult animal as an unwanted consequence of vaccination or in chronic infections. In the latter, chronic exposure to low levels of antigen can lead to 'destruction' of clones of specific antibody producing B-lymphocytes or generation of T-lymphocytes which suppress B-Lymphocyte function.
The relative unresponsiveness in freshly calved animals and during lactation is related to hormonal changes (especially steroids) rather than immunological tolerance.
Antibody production is controlled by a feed back mechanism.
Serological Tests for Brucella abortus
All these tests are designed to detect antibody to specific antigen. There is very little cross reactivity of any significance known as yet. THE TESTS DETECT ANTIBODY AND NOT INFECTION. A positive serological reactor may not have any brucella organisms present in its body.
The tests rely on detecting antibodies in the IgM and IgG classes and IgA in MRT.
In brucellosis infection IgM is produced early in the primary response followed by production of IgG.
Tests routinely used are:
i. Rose Bengal Test (R.B.T.)
ii. Complement Fixation Test (C.F.T.)
iii. Indirect Haemolysis Test (I.H.L.T.)
iv. Milk Ring Test (M.R.T.)
The RBT is performed on all samples. It is a very sensitive test and consequently we have very few false negatives when the test is diligently performed. A recent survey at the R.V.L. Wagga showed that only 2 in 10,000 samples were RBT negative CFT reactive - one at a titre of 4 and the other 8. It is appreciated that this figure is much lower than those quoted in other surveys. I question the validity of other surveys as they involve usually a single CFT on the RBT negative samples. In our experience these CFT reactions are usually not repeatable.
The higher the RBT reading or score, the more likely it is that the animal will react and have a high titre in the CFT. Low RBT scores (RBT 1) with CFT titre of 16 are unusual. These samples have both the RBT and the CFT repeated.
Whole herd CFT's appear to be of very little benefit other than as a quality control for the RBT.
The RBT mainly detects IgM and hence is positive earlier in infection that other tests.
The CFT is a complex and delicate system which detects IgM and IgG. Following S.19 vaccination in uninfected/unexposed animals, high levels of IgG do not develop and IgM levels drop quickly. Thus the test can be used to sort out some vaccination titres.
Samples are constantly checked for repeatability of CFT titres. Samples with high RBT readings and negative CFT are always repeated.
IHLT - This is a relatively new test which came into prominence with a blaze of publicity. It has failed to live up to the earlier expectations. Initially the test was developed to overcome the problem of prozoning, which was not a problem in the microtitre CFT system.
It is thought to work on the same immunoglobulins as the CFT and has no real advantage over that test. I must remind you that it is a test detecting antibodies and NOT the presence of Brucella abortus organisms in an animal. Hence positive IHLT does NOT EQUAL INFECTION. As I see it, the IHLT is used as a security blanket by some and as a modified PR tool by others. The results are used when they suit a situation and are forgotten when they do not.
The MRT has a valuable place in monitoring dairy herd status if its limitations are realised. 'I see very limited application for this test, or an individual animal basis, as there are many documented causes of false positive reactions. A negative MRT does not 'clear' an animal of brucellosis as this test detects local antibody in the udder. You can have a serological/bacteriological positive animal which returns a negative MRT.
Bacteriology
The campaign has been designed on a serological basis and thus with a knowledge of immunology and serology, sound judgements can be made as to herd or animal status. If bacteriological examinations are performed and B. abortus is not isolated, this does not ensure freedom from brucellosis. Hence, there is no place for bacteriological procedures in this campaign, excepting abortion investigations.
General Laboratory Aspects
At the Regional Veterinary Laboratory, Wagga we have a staff of 10 in Brucella Serology and are currently receiving about 100,000 blood samples a month. The laboratory procedures are repetitive and our system of performing tests is designed to limit boredom and to enhance pride in work.
The staff have been out to see bleeding teams in action, so that they know that cattle, farmers etc are involved and that their work is very important to the campaign.
In N.S.W. we have inter-laboratory check testing of samples each month and about twice a year on a national basis. There is very close agreement or repeatability of results, both within and between the laboratories. In addition we repeat the tests on approximately 5% of samples.
All tests are read twice or double checked, as are the clerical procedures, leading up to the production of the computer generated telexed results. We eliminate.99.99% of errors but no system is absolutely perfect. You are there to interpret results - if they look obviously unusual after consideration of vaccination status, herd and property history, inquire! Remember that every property and herd is different and that if you do not think about the results, we may as well dispense with interpreting vets and slaughter all serological reactors.
Do not place too much emphasis on small shifts in titre. A rise in titre from 4 to 8 can be from a 'tube' reading of 42---- to 43----. Mechanical variation can easily account for a variation of one 'tube' in titre. Before a titre is taken to have risen or fallen, a four fold change (two 'tubes') in titre, should occur - e.g. from 8 to 32 or 4 to 16 etc. Most CFT titres of 4 are going to be negative in the IHLT, as the latter test begins at a dilution of 1 in 8.
References
1. Herbert, W.J. (1974) Veterinary Immunology. Published by Blackwell Scientific Publications Ltd, Oxford
2. Morris, B. (1978) Post Graduate Committee in Veterinary Science Refresher Course in Immunology Proceedings No. 38. p. 121-141
3. N.Z. Government, Ministry of Agriculture and Fisheries (1977) 'Brucellosis - A Veterinarians Guide to the Literature.'
4. Roitt, I. (1974) Essential Immunology. Published by Blackwell Scientific Publications, Oxford
5. Tizzard, I.R. (1977). Immunology - 'Some Modern Concepts' State Vet. Journal 32:225