16 Diagnostic Tests

Allergic Tests

An allergic test is one that elicits a sensitivity response in an animal following the injection into its tissues of a protein derivative of the specific organism with which the animal is, or has been, infected. Animals in the very early or the very late stages of the disease may fail to react to such a test, in the first instance because a state of hypersensitivity has not yet been de­veloped at the time the test is applied, and in the second instance because the hypersensitivity has been lost following flooding of the tissues with antigen, a situation which is likely to occur in the advanced stages of some diseases. Certain other circumstances have been recognized to reduce the hypersensitiveness (or allergy) to a low level, the most important of which is par­turition, when the reacting tissue antibody leaves the body in the colostrum. A positive response to an allergic test, on the other hand, does not mean that the animal is actively infected at the time of the test, but only that it may have been infected, perhaps subclinically, in the past.

In general terms, allergy is a state of tissue hypersensitivity made manifest as the result of an antigen-antibody reaction which varies in degree and extent. The signs, which are usually delayed in development, are localized, although in some instances more than one tissue may be involved. Severe allergic reactions may induce a delayed febrile response. In anaphylaxis, the clinical signs are severe, with signs of shock and are general in extent.

The two most important allergic tests used in veterinary practice are those employed in the control and eradication of tuberculosis and of glanders, using tuberculin and mallein respec­tively. Because there is lack of agreement regarding standardization of the antigens, an acceptable uniform international test for either of these diseases has not been evolved. The adoption of official control and eradication pro­grammes in respect of glanders and bovine tu­berculosis in so many countries has produced a situation in which a considerable variety of diag­nostic tests are statutorily recognized and em­ployed. Allergic tests have also been applied in the diagnosis of Johne's disease but, without exception, they have been shown to have a low level of specificity.

Tuberculin Tests

There are three major methods of applying the tuberculin test to animals; these are the intra-dermal, subcutaneous and ophthalmic. The ophthalmic test is rarely used nowadays because of its comparatively low degree of specificity, while the subcutaneous test has become un­popular because it is time-consuming and, being based on temperature response, is susceptible to interference by a number of extraneous factors. As a consequence, some form of the intradermal method is employed throughout the world.

Cattle. Since cattle may become sensitized not only to the bovine type of tubercle bacillus, but also to the human and avian types, and with immunogenically related organisms such as My-cobacterium paratuberculosis, and the acid-fast bacillus associated with so-called 'skin tubercu­losis' (acid-fast lymphangitis), it has been con­sidered advisable in national bovine tuberculosis eradication programmes in Britain, Ireland and the Netherlands to use a comparative tuberculin test. Two different types of tuberculin, mamma­lian and avian, are therefore injected into the skin simultaneously, fairly close together, so that any resulting reactions may be more readily compared for interpretation purposes.

The value of the comparative tuberculin test is associated with its considerable specificity in respect of the sensitizing antigen. For this test the intradermal route of injection is used, care being necessary to place the injections at the appropriate sites on the same side of the neck as skin sensitivity varies, being greater in sites nearer the head and in the vicinity of the jugular furrow, and reduced in sites near the shoulder and in those towards the nuchal crest. The middle portion of the neck should be selected as being preferable, and the sites prepared by clipping away the hair over each area; the upper site is placed at least 10 cm below the crest and the lower 12 cm away on a line roughly parallel with the slope of the shoulder. The avian tuberculin should be injected at the upper site and the mammalian tuberculin at the lower (Fig. 240).Prior to injecting the tuberculins, the needles and syringes should be checked for efficiency; a useful way of doing this is to assemble the needle and syringe as if for use and then, after filling the syringe with water, insert the needle into a piece of good quality cork and proceed to inject 01 ml into it. If the equipment is working satisfactorily no water should escape when the injection is made into the cork. Needless to say, the needles and syringes should be sterilized before use in animals, and the needle wiped with a clean swab of cottonwool moistened with 70 % methylated spirit between each injection. The injection site should be cleaned with cottonwool soaked with methylated spirit. Prior to injecting the tuber­culin, the thickness of the skin at each site is recorded by measuring with a caliper graduated in millimetres. The equipment required for the performance of the intradermal tuberculin test is illustrated in Fig. 241.

For making the injection a short dental type needle is used, which should be attached to the syringe so that the bevel edge at the tip is facing towards the user. When held at the correct angle, i.e. about 30° to the skin surface, and properly inserted, the point of the needle will penetrate to the deeper layers of the dermis. Some degree of pressure is necessary to deposit the required amount of tuberculin (01 ml) in the skin, and evidence that this has been achieved is usually apparent in the form of a small pea-sized swell­ing, which bulges outwards. The mechanism of the syringe should incorporate an automatic, or manual control for measuring the dose. The test is read by repeating the skin measurements 72 hours after the injection. A variable degree of desensitization will occur in the skin over the area of the injection site, so that when retesting is necessary, it is advisable to allow an interval of about 2 months to elapse, and the skin on the opposite side of the neck should be used.

In Britain and Ireland the mammalian tuber­culin used is prepared from a human strain of the tubercle bacillus. The prepared product is standardized to contain 2 mg/ml purified tuber-culoprotein and of which the recommended dose is 01 ml. The avian tuberculin used in the single intradermal comparative test contains 0-5 mg/ml purified protein derivative (PPD) tuberculin and the dose is again 0-1 ml. The value of avian tuberculin, when used simultaneously with mam­malian, is that it aids in the classification of tuberculin sensitivity, an example of which is so-called non-specific infection, a category which should only be recognized in established herds of cattle under certain circumstances to be described later in this chapter.

Other European countries employ mammalian tuberculins prepared in a variety of ways from either the human or bovine types of the bacillus. In general, although there is little difference in specificity between these two types of tuberculin that of bovine origin is less potent than that of human origin. Also, because of greater specifi­city and greater ease of standardization, PPD tuberculins are more generally used.

Various modifications of the intradermal tu­berculin test are in use in different countries, although in Europe, generally, the neck site is employed. In the USA, Canada and Australia the caudal fold is the recommended injection site (in the USA the lip of the vulva at the muco-cutaneous junction is also included in routine tu­berculin tests). Tuberculin prepared from cultures of M. tuberculosis or M. bovis is injected intra-dermally, the recommended amount being 0-05 ml. Intradermal deposition of the tuberculin is more certain at these sites than in the neck, although sensitivity is at a lower level. For these tests heat-concentrated tuberculin, prepared from a human strain of tubercle bacillus, is injected in the required amount of 0-1 ml for routine tests, and 0-2 ml when retesting a herd in which positive reactors have appeared. Doubtful reac­tors, or herds with a history of repeated break­downs, are usually subjected to an intradermal tuberculin test using the neck site. In Australia a modified subcutaneous test ('short thermal test') is employed in similar circumstances.

Interpretation of results. It is necessary when reading the results of intradermal tuberculin tests to take note of the character of any swelling which appears at an injection site. In the first instance, even when a comparative test is being applied, the initial interpretation is made on an individual animal basis, hence underlying the need to apply the correct technique at all aspects. In Britain and Ireland it is officially recommen­ded that the results of the single intradermal comparative tuberculin test should be interpre­ted on the following basis: Any swelling showing oedema should be regarded as a positive reaction. In the absence of oedema no skin swelling which shows an increase in skin thickness of more than 2 mm should be regarded as a negative reaction. Skin swellings giving an increase of 3 mm should be classified as doubtful and those of 4 mm or more as positive.

The importance of making a careful clinical examination at the time of the test is highly per­tinent in the context of clinical tuberculosis, so-called 'skin tuberculosis' and Johne's disease, with particular emphasis in the case of reactor and inconclusively reacting animals.

It is necessary to clarify some of the terms used in connection with the interpretation of the test. Reaction means the changes which occur at the injection site in respect of skin thickness, or the development, or otherwise, of oedema, and is classified as negative, doubtful or positive as outlined above. A reactor, which is either posi­tive or inconclusive, is the categorization of the animal as the result of interpreting not only its local reactions to the injections of mammalian and avian tuberculins, but also those of all the other animals in the herd in the light of whether, or not non-specific sensitization exists in the group. The term doubtful is applied to a reaction, when the increase in skin thickness is more than 2 mm and less than 4 mm, in the absence of local oedema. The use of the term inconclusive is restricted to those individual animals which, on the basis of a herd or group, single intradermal comparative tuberculin test, cannot be classified as being either positive or negative reactors.

The interpretation of the comparative test is influenced by the presence of non-specific infection in the herd. It is generally accepted that the existence of non-specific infection is established during the herd test by one or more animals giving a positive reaction to avian tuberculin, and a negative reaction to mammalian tuberculin; clinical evidence of Johne's disease or of so-called 'skin tuberculosis' are also considered to establish a similar situation.

With the information derived from the herd or group test, in conjunction with the results of a clinical examination and the previous tuberculin and health record, it is possible to classify the majority of animals in respect of the comparative tuberculin test as follows:

1. Animals showing a positive or doubtful reaction to avian tuberculin and a negative re­action to mammalian tuberculin are negative reactors and can be retained in the herd.

2. Animals giving a doubtful reaction to mam­malian tuberculin and a negative reaction to avian tuberculin are inconclusive reactors and should be retested.

3. Animals showing a positive or doubtful reaction to avian tuberculin and a positive or doubtful reaction to mammalian tuberculin, pro­vided the increase in skin measurement to mam­malian tuberculin is not more than 4 mm greater than the increase to avian should be:

a. Classified as negative reactors if non-speci­fic infection is established.

b. Classified as inconclusive reactors and sub­jected to a retest if non-specific infection is not established.

4. Animals giving a positive reaction to mam­malian tuberculin and a negative reaction to avian tuberculin when the increase in skin measurement to mammalian tuberculin does not exceed the avian increase by more than 6 mm should be:

a. Classified as inconclusive reactors and sub­jected to a retest if non-specific infection is established.

b. Classified as reactors and removed from the herd if non-specific infection is not established.

5. Animals which give a positive reaction to mammalian tuberculin and a positive or doubtful reaction to avian tuberculin when the increase in skin measurement to mammalian tuberculin is 5 or 6 mm greater than the increase to avian tuberculin should be:

a. Classified as inconclusive reactors and sub­jected to a retest if non-specific infection is established.

b. Classified as reactors and removed from the herd if non-specific infection is not established.

6. In any test, animals showing a positive re­action to mammalian tuberculin and a positive, doubtful or negative reaction to avian tubercu­lin should be classified as reactors and removed from the herd when the increase in skin measure­ment to mammalian tuberculin is more than 6 mm greater than the increase to avian tuberculin.

The majority of herd or group tests can be satisfactorily interpreted on the foregoing bases but instances will occur in which some modifi­cation will be necessary. The tuberculin test record for the herd will be of value in deciding when a modified interpretation is desirable. The appearance of a number of reactors indicating bovine type infection, or a clinical case of tuber­culosis, suggests the advisability of altogether ignoring evidence indicative of non-specific in­fection. It is also recommended that when there is a high incidence of non-specific sensitivity in the absence of so-called 'skin tuberculosis', and a few animals show increases in skin measure­ment to mammalian tuberculin of 7-8 mm greater than for avian tuberculin, they may be classified as inconclusive reactors and subjected to a retest rather than being removed from the herd as reactors. Also animals with lesions of so-called 'skin tuberculosis' (acid-fast lymphan­gitis), which give reactions which would nor­mally classify them as reactors and necessitate their removal from the herd, may be regarded as inconclusive reactors and retested. If a number of such instances occur in a herd test then other animals in the reactor category, not showing clinical evidence of so-called 'skin tuberculosis', may be dealt with on similar lines. Any animal showing large increases in skin measurements to both tuberculins, even though the mammalian increase is only equal to or slightly in excess of the avian increase, should be classified as an in­conclusive reactor and subjected to a retest.

The reactions produced by the caudal fold test as employed in the USA, Canada and Australia are read between 72 and 96 hours after injection by comparison with the opposite fold. A positive reaction consists of a diffuse, oedematous, pain­ful swelling at the injection site (Fig. 242). The same standards apply when the lip of the vulva is used as the injection site.

Although the traditional subcutaneous test has been generally discarded, modified tests of this type are in use in some countries, it being claimed that it provides useful clarification in cases of doubt, particularly those due to low sensitivity. In Australia the 'short thermal test' is employed involving the subcutaneous injection in the neck area of 4 ml of intradermal mam­malian tuberculin. The rectal temperature is taken before the tuberculin is injected and the test is not proceeded with if it is higher than 39°C (102°F). If the temperature remains below this point for 2 hours after the injection and then rises and remains above 40°C (104°F) at 4 hours, 6 hours and 8 hours, the animal is classified as a positive reactor. It is claimed that the test pos­sesses a high degree of efficiency in detecting infected cattle with low-level skin sensitivity to tuberculin. Occasionally an anaphylactic reaction occurs during the acme of the thermal response.

Horses. Tuberculin tests in the horse appear to possess a much lower degree of specificity com­pared with cattle. This is particularly the case in respect of the subcutaneous test which is vir­tually useless in this species. The horse is ap­parently highly sensitive to tuberculin, so that much smaller amounts of tuberculoprotein than those customarily employed might prove more satisfactory. There is lack of authoritative infor­mation on the subject and the consensus of opinion is that a negative reaction can be accep­ted as indicating the health status of the animal, but the significance of a positive reaction is not always clear, although it is contended that the occurrence of a systemic reaction in conjunction with a positive intradermal test can be accepted as indicating the presence of infection.

The technique of the single intradermal test is similar to that in cattle, due care being necessary because of the thin skin in the neck area to ensure that the tuberculin is deposited in the dermal tissues. It is advisable to measure the skin thick­ness at the injection site at 48 and 72 hours.

Sheep and goats. The single intradermal test has been recommended for use in these species. The test is not highly specific as reports indicate that loss of skin sensitivity occurs in a proportion of tuberculous animals. The caudal (anal) fold site is probably the most satisfactory for both species. The test is read 72 hours after injecting 0-1 ml of standard mammalian tuberculin; an increase of 5 mm in the thickness of the caudal fold constitutes a positive reaction. Many infec­ted animals fail to react.

Pigs. In the majority of instances progressive tuberculosis in pigs is caused by either the avian or the bovine type of tubercle bacillus. The suc­cess of bovine tuberculosis eradication pro­grammes in many countries has considerably reduced the proportion of bovine type infections in pigs. The simultaneous injection of avian and mammalian tuberculin, one in the right and the other in the left ear, may enable the type of infection present in the herd to be differentiated. The value of the test is highest in herds with a known history and in which repeated herd tests have been performed. It appears that many tuberculous pigs lose sensitivity to tuberculin in a rather short time. Two sites on the ear have been recommended. The most acceptable is the skin towards the base of the ear on its posterior or outer surface; otherwise the skin of the an­terior border of the ear, towards its base, is selected. The dose of each tuberculin is 01 ml and the type used in cattle is satisfactory.

A positive reaction to either tuberculin con­sists of the development of a local inflammatory reaction at 24 hours, followed by haemorrhage, necrosis and ulceration at 48-72 hours. As a rule any reaction to avian tuberculin is delayed so that in this case the test should be read at 48-72 hours. In the case of either tuberculin an increase in skin thickness of 4 mm is classed as positive. It appears that in pigs maximum sensi­tivity to tuberculin occurs 3-9 weeks after infec­tion; a retest in about 8 weeks will indicate whether the lesions of the disease are actively progressing.

Dogs and cats. Tuberculin tests in dogs and cats usually fail to give unequivocal reactions in naturally infected animals. For the intradermal test tuberculin may be injected into the skin of the flank in amounts of 0,1 ml and the site re-measured at 24 and 48 hours. A subcutaneous test may be performed by injecting 0,1 ml of a 1 in 10 dilution in normal saline of standard intradermal tuberculin. The test must not be undertaken if the animal's temperature is above 39,5°C (101,5°F). A positive reaction consists of a rise of temperature of l,2-2,8°C (2-5°F) ap­pearing between the 5th and 7th hour and per­sisting for 4-5 hours. In old-established, chronic infections a fall in temperature may result, often followed by collapse and death. A proportion of infected animals which fail to evince a thermal response may show a reaction in the form of swelling in some of the joints and lymph nodes.

Mallein Tests for Glanders

Mallein as employed in tests for the diagnosis of glanders is a bacteria-free filtrate from a fluid culture of Actinobacillus {Malleomyces) mallei which has been prepared by one of a variety of methods. Although mallein has a high degree of specificity, so that by its proper use a high proportion of infected animals can be iden­tified, it must be appreciated that, as with other types of allergic sensitivity, false positive and false negative reactions can occur because of low-level sensitization, loss of sensitivity in the advanced stages of the disease and sensitization by other bacteria. Diagnostic antigens prepared from some strains of Act. mallei will cause reactions in horses sensitized to Pseudomonas pseudomallei. A number of mallein tests are in use including the intradermopalpebral, cutaneous, subcutane­ous and ophthalmic. Of these the intradermo­palpebral is most widely used.

Intradermopalpebral test. In this test, 01 ml of mallein is injected into the skin of the lower eye­lid which is then re-examined in 48 hours. The intradermal tuberculin syringe and needle for cattle will be found suitable for this test but the animal will need to be effectively restrained to ensure that the injection is correctly made. Tens­ing the skin of the lower eyelid with the ringers of the free hand will assist needle penetration. When inserting the needle into the skin, the syringe should be held at an angle of 20° to the surface of the eyelid with the needle point about 1 cm away from the mid point of the palpebral mar­gin. If correctly placed in the dermal tissues the deposited mallein produces a small, visible no­dule. Alcohol swabbing of the site should not be employed in connection with this test. A positive reaction constitutes inflammatory oedema of the eyelid or even of the whole orbital area, and frequently severe purulent conjunctivitis (Fig. 243). The swelling, which is painful, develops slowly, is most intense between 24 and 48 hours and persists for at least another 48 hours.

Cutaneous tests. A variety of cutaneous tests, including superficial scarification of the skin following clipping and cleaning of the site and then the direct application of a few drops of mallein, and an intradermal test have been em­ployed but because of comparatively low speci­ficity they have been discarded.

The subcutaneous test and the ophthalmic test have fallen into disuse for a variety of reasons. The former is prohibited in countries such as Germany and Austria, where the control of glanders is based upon serological tests, e.g. complement-fixation, because there is a significant antibody response which may persist for several months following the subcuta­neous injection of mallein. Otherwise this form of test is time-consuming and costly to perform. The ophthalmic test is of relatively low specificity.

Allergic Tests for Johne's Disease

Tests based on skin sensitivity are likely to be of value only during the later stages of the de­velopment of Johne's disease and, in the majority of cases, by the time clinical signs are fully established desensitization has occurred. In addi­tion, the value of an allergic test is minimized by the likelihood that cattle infected with tubercle bacilli of any type, or those with acid-fast lym­phangitis and those which have been vaccinated against Johne's disease, will give confusing re­actions. For these reasons, tests to determine the sensitivity status of cattle are rarely used in the diagnosis of Johne's disease.

The single intradermal test has been the most extensively employed. It involves the injection of 0,2 ml PPD johnin into the skin of the neck. The test should be read at 48 hours, a positive reaction constituting an obviously oedematous swelling. In the USA an increase of skin thickness of 3 mm or more is regarded as a positive reac­tion. The comparatively low level of specificity of the test is indicated by it being possible to substitute avian PPD tuberculin for johnin with­out serious reduction in efficiency, although in this case the test is carried out in the same way as for a double intradermal tuberculin test. In any event there is local desensitization of skin for over three months following an injection of johnin. The test is of greatest value when em­ployed on a herd basis although repeated testing will help in the identification of early clinical cases.

The short thermal test for the detection of sub-clinical Johne's disease consists of injecting 10 ml of avian tuberculin intravenously. A positive reaction consists of a rise in temperature of 1°C (2°F) between 5 and 8 hours, in conjunction with a systemic reaction including anorexia, de­pression, dyspnoea and diarrhoea in some cases. A similar test has been devised using 2-4 ml of johnin, in which case a rise in temperature of over 0,75°C (1,5°F) within 3-7,5 hours is regar­ded as a positive reaction. Sensitization to group antigens associated with heterologous bacteria will give rise to a positive reaction in both these tests so that their specificity is rather low. The development of antibodies following the intra­venous injection of either of these diagnostic antigens precludes the use of this type of test in countries where diagnosis is based on the com­plement fixation test.

Serological Tests

Although serological examination, which is based on the detection of specific antibodies in serum by employing selected specific antigenic systems, cannot, except in very few instances, be conveniently undertaken by the clinical veteri­narian, it is essential to be aware of the various tests that are available and which are of value in assisting the diagnosis of certain infectious diseases. The majority of such tests are per­formed on serum. For this purpose 10 ml of blood are usually sufficient. Care should be exercised at all stages from obtaining the sample until clotting is complete in order to prevent haemolysis. Allowing it to stand undisturbed in an environmental temperature of 20°C (68°F) or higher will aid clot retraction and separation of the serum. If the serum can then be removed into a separate sterile container prior to dispatch to the laboratory, the risk of haemolysis is entirely eliminated. In all instances when dis­patching blood samples for serological or other examination, or indeed any specimen to a labora­tory, include a history and description of the case along with a precise request for the diag­nostic service required. Each individual sample should be clearly labelled; and when a number are being submitted a separate list should be included.

Serological tests employed in this way may reveal evidence of recent or past infection, as the case may be, but in general the results obtained are often disappointing unless paired serum samples obtained at an interval of two to three weeks reveal a rising antibody titre. Such a situation provides retrospective confirmation of the diagnosis. Recognition of recent infection may also be established on a basis of clinical observation coupled with a high level of sero­logical reaction in a single test. Certain types of serological tests are of considerable assistance in the establishment of a rapid diagnosis. These include immunofluorescent techniques and the complement fixation test in canine distemper. Conversely detection of the specific cause is possible by serological means in such conditions as contagious pustular dermatitis (orf), foot-and-mouth disease and rinderpest.

The principal serological tests employed in diagnosis include the following:

Agglutination Tests

Immunological tests of this type generally have a retrospective value in diagnosis because speci­fic agglutinins only appear in the blood some time subsequent to the establishment of infection. In veterinary medicine the phenomenon of aggluti­nation has had its greatest application in relation to detecting the presence of agglutinins to Bru-cella abortus in blood serum, milk, milk-whey, vaginal mucus and seminal plasma of cattle. For this purpose either a tube or plate agglutination test may be employed. In the absence of a gener­ally accepted international standard Brucella an­tigen (some countries have adopted the WHO/ FAO Standard International Unit) it is difficult to interpret the significance of different concen­trations (titres) of agglutinins. In the USA com­plete agglutination at a serum dilution of 1 in 50 in the tube test is regarded as a suspicious titre, and at 1:100 or above as indicating positive evidence of bovine brucellosis. In the case of animals known to have been vaccinated these cri­teria are applied at 1:10 and 1:200 respectively. In Britain and Ireland any titre above complete agglutination at 1:10 (30 i.u. Brucella antibody) is considered positive in unvaccinated animals, and at 1:200 (50 i.u.) in vaccinated animals. There is a tendency for vaccinated calves to retain agglutinin titres of 1 in 40 or above when they are reared in a Brucella contaminated environment. In the case of infected cows which have calved or aborted within the preceding three weeks, withdrawal of the agglutinins from the blood and their removal from the body in colostrum and uterine excretions is more than likely to give rise to false negative results if a serum agglutination test were undertaken.

The plate or rapid agglutination test has not been widely used except in the USA and although it is not as accurate as the tube agglutination test it has a place as an initial screening test when employed on a herd basis. The results are available within a few minutes so that it can be applied to cattle of unknown brucellosis status in sale-yards. A rapid whole blood plate test has also been devised but because the erythrocytes interfere somewhat with the agglutination reac­tion the test is not entirely dependable.

A modified rapid plate agglutination test for brucellosis, the Rose Bengal Plate Test (Card test), has been extensively used in the USA and more recently in Britain and Ireland. Serum samples are examined by placing one drop of each on a white enamel tile and then placing one drop of Rose Bengal stained Brucella antigen alongside the serum. Mixing is performed with an applicator stick and the tile rotated for 4 minutes, when the result is read as positive or negative. Comparative investigations have shown that this test is about as efficient as the comple­ment-fixation test in identifying animals in the early stages of infection, surpassing the tube agglutination test in this respect. It also gives few false negative reactions and usefully excludes a significant proportion of post-vaccinal positive reactors to the tube agglutination test. The Rose Bengal test appears to meet many of the desirable requirements of a screening test in brucellosis eradication programmes.

The value of the agglutination tests on milk and milk-whey depends upon Br. abortus becoming localized in one or more quarters of the bovine udder, although transfer of blood agglutinins to milk occurs at certain times, more par­ticularly in association with parturition and when clinical mastitis exists. The milk-whey tube and plate tests will give positive results in almost all cattle in which Br. abortus has invaded the supramammary lymph node or udder, but con­fusion will occur in the case of animals either recently calved or vaccinated because of false positive reactions which will persist for three months and two weeks respectively. The whey tube agglutination test has been found to be more reliable than the milk ring test in identi­fying adult Brucella-free vaccinated animals.

The milk ring test is claimed to be more efficient than the whey test in detecting cattle infected with Br. abortus, even although the organism has not localized in the udder. Its efficiency is said to be somewhat less than that of the tube serum agglutination test but it is claimed that the milk ring test is satisfactory for screening bulk herd samples. Milk from a cow which gives a positive ring-test reaction can be diluted with negatively reacting milk from 4 cows and still give a positive reaction; greater dilution would be likely to invalidate the result by causing a negative reaction. The milk ring test has been extensively employed in bovine brucellosis era­dication programmes as a herd screening test, being of greatest value where only calfhood vac­cination is permitted. On the basis of the disparity between the milk ring test reactions shown by milk samples from individual quarters infected cattle can, in many instances, be differentiated from those with vaccinal agglutinins in which case the quarter reaction is more or less uniform. The proportion of false positive milk ring test reactions is highest in recently calved and late lactation cows.

The test is simple to perform, requiring only limited materials. The procedure consists of adding two drops of tetrazolium-stained Brucella abortus antigen to 2 ml of well-mixed milk in a small tube. After thorough mixing of the contents the tube is incubated at 37°C for 1 hour. If Brucella agglutinins are present the stained antigen is agglutinated and carried to the surface with the rising fat globules to form a deep-blue creamline. In the absence of specific agglu­tinins the milk remains bluish with a normal cream layer. In doubtful reactions both cream layer and milk are uniformly stained.

The vaginal mucus agglutination test will, in general, give positive reactions only when the uterus is infected. The value of the test is largely offset by the technical complexities associated with acquiring and handling the mucus samples, and also the periodicity of agglutinin secretion. A pipette or tube technique should be used in preference to a tampon for collecting the mucus. In bulls Br. abortus may localize in the genital organs without the appearance of agglutinins in the blood. Testing the seminal plasma by means of a tube dilution technique similar to that of the serum agglutination test will invariably yield positive results in such cases.

Because brucellosis is a chronic disease the type of antibody present in the blood and else­where varies at different stages. During active infection and soon after vaccination with an agglutinogenic antigen, agglutinating (IgM) anti­bodies are detectable; at a later stage only non-agglutinating (IgG) antibodies exist. Treatment of serum samples with 2-mercaptoethanol des­troys IgM type immunoglobulins thereby pro­viding a means of differentiating between serum titres resulting from recent infection or vaccina­tion and those associated with old-standing infection.

Some specific antibodies, even in high titre, are incapable of agglutinating Br. abortus; they have been classified as incomplete agglutinins. The presence of such immunoglobulins can be detec­ted by means of a modified agglutination reaction. This is achieved by employing an anti-species (antiglobulin or Coombs') serum. When bacterial cells which have adsorbed non-agglu­tinating antibody are prepared by being washed twice in fresh saline solution after prior centri-fugation and finally resuspended in saline con­taining the appropriate species antiglobulin they will be agglutinated by reason of the antiglobulin linking the Brucella antibodies together. The term antiglobulin and Coombs' test have been used for the procedure, but the latter has a special connotation in relation to blood grouping in human pathology and should not be employed in the context of bacterial agglutination pheno­mena.

The serum agglutination test also has a prac­tical application in the diagnosis of brucellosis in other species of animals including goats, sheep, pigs, horses, camels, deer, etc. Because of the considerable variation in the serum aggluti­nin titre in individual animals, even when infec­ted, it is recommended that for goats, sheep and pigs the test results should be interpreted on a herd basis, and that the presence of a single positive reactor in a herd or flock in which clinical brucellosis has been suspected justifies a positive diagnosis.

A serum agglutination test, although the sim­plest method, appears to have serious limitations in the diagnosis of brucellosis in pigs caused by Brucella suis. Following infection a positive titre is unlikely until about eight weeks have elapsed, and the majority of infected pigs evince only a low titre. The test should, therefore, be used to determine the herd status. As a guide to the interpretation of test results, herds with no history of infection and in which no individual pig has a titre greater than 1 :100 are classified as negative; herds with a record of Br. suis infection in which any one pig has a titre greater than 1:25 are classified as positive.

Brucellosis in goats, cattle and sheep caused by Brucella melitensis may be diagnosed by means of a serum agglutination test. In assessing the status of individual animals the test has limitations because of the transient character of the infection in many cases. Interpreting the results on a herd basis gives more satisfactory results. Also, because of the prolonged persis­tence of mammary gland infection, at least in the goat, it is likely that a milk ring test would give a more accurate reflection of the disease status of individual animals.

Serum agglutination tests are of some value in detecting animals which have been infected by a particular Salmonella spp. As a general rule, agglutinins do not appear in the blood until about 2 weeks after the appearance of clinical signs of the disease. Also in young animals including foals and calves, infection tends to be eliminated and positive agglutinin titres usually disappear within 2-3 months. Conversely, infec­ted adult animals, which may or may not evince overt signs of disease, tend to become 'carriers' and retain positive serum agglutinin titres. The application of agglutination tests in the diagnosis of salmonellosis in animals is precluded by the lack of knowledge regarding positive and nega­tive criteria for the serum agglutinin titres in the different species. It appears that positive flagellar agglutinin titres are more likely to be significant than those obtained with somatic antigen. The efficiency of bacteriological methods in detecting the presence of salmonella infection in both clinically affected and carrier animals has reduced the significance of serological methods as diagnostic aids. Following isolation, specific identification and classification is achieved by means of somatic and flagellar agglutinin adsorp­tion tests.

The presence of Salmonella infection in adult fowl can be determined by means of agglutina­tion tests. The two most important serotypes S. pullorum and S. gallinarum, are antigenically identical so that a suspension of S. pullorum will detect infection with either of these bacteria. A tube dilution or a whole-blood plate test may be employed. In the tube test complete aggluti­nation at 1:40 or a higher serum dilution is indicative of infection. The rapid whole-blood plate agglutination test has the advantage that it can be performed in the field, only a small quantity of blood is required, and reactor birds can be removed from the flock immediately on detection. For the test the wing vein is pierced and blood collected with a standard loop (002 ml), this is mixed with twice its volume of methyl violet stained antigen on a white porce­lain tile. Agglutination within 2 minutes indicates a positive reaction. Some degree of agglutination will occur with S. pullorum antigen when it is admixed with serum from birds infected with other species of Salmonellae possessing some of the same somatic antigens. Under modern in­tensive systems of poultry production the inci­dence of pullorum disease and fowl typhoid is extremely low. Avian salmonellosis is nowadays more usually caused by individual members of a variety of serotypes which in many instances gain entrance to poultry units as contaminants of foodstuffs.

A serum agglutination test has been widely used in the detection of herds of cattle affected with vibriosis. Because of considerable variation in agglutinin titres and in their persistence in infected cattle, the test has been found to be of greatest value when applied on a herd basis. The local production of agglutinins by the female reproductive tract lends value to a modified form of agglutination test performed on vaginal mucus which can be collected by means of a pipette or by tampon. In the performance of the test the mucus is diluted with sodium chloride and formalin and then stored at 4°C for 24 hours before testing. Some possible anomalies, such as uncertainty in selecting infected cattle, varia­bility in the time of appearance of the aggluti­nins, false positive reactions during oestrus and tendency for the titre to fall with time, all mili­tate against the test being used on an individual animal basis. A similar situation prevails in respect of bovine trichomoniasis. The serum agglutination test may be of value in the diag­nosis of vibrionic abortion in sheep; similar reservations apply as in the case of cattle.

Serum agglutination (tube or plate) or haemagglutination-lysis tests are the methods most commonly used to detect antibodies against Leptospirae spp. Recent infection may be con­firmed when paired sera reveal a rising agglutinin titre. Otherwise positive titres at a serum dilution of 1:500 or over in cattle, 1:500 in pigs and 1 :1000 in dogs may be taken to indicate recent or past infection. The specific serotype of Leptospira may be indicated by noting the antigen which gives a reaction at the highest dilution of serum; confirmation can be obtained by means of agglutinin-absorption tests. In leptospirosis, agglutinins are at a peak about 4 weeks after generalized infection has occurred and then begin to subside. They persist at significant levels for over three months in sheep, for over a year in cattle and pigs and even longer in horses.

The agglutination test employs an antigen composed of a formalized culture, whereas in the haemagglutination-lysis test the antigen con­sists of living organisms. The serum agglutinin titre is taken to be the dilution in which about 50% of the organism are agglutinated or lysed. Both tests necessitate the use of dark-ground microscopy for the final reading. Both tube and plate agglutination tests are available for the diagnosis of tularaemia. In pigs, because of uni­lateral cross-agglutination with Brucella spp. antigens, the tularaemia tube dilution test gives more satisfactory results when it is interpreted in conjunction with the herd history. A positive serum titre of 1:50 is regarded as being indica­tive of tularaemia in pigs while a titre of 1:200 is similarly interpreted in sheep in which species cross-agglutination between Pasteurella tularen-sis and Brucella abortus is much less common. Following infection agglutinin titres in sheep range from 1 :640 to 1:1500; values around 1:320 7 months later are usual while in horses the titre reverts to normal values within 21 days.

Complement-fixation Test

The principle of the complement-fixation test is that complement, which is a normal constitu­ent of serum, is taken up (fixed) on the formation of a specific antibody-antigen complex, and lysis of the antigenic component then occurs. In com­plement-fixation tests unknown sera are heated to 56°C for 30 minutes to destroy all the com­plement and then a known amount of comple­ment in the form of guinea pig serum is added to each serum under test. Following the addition of known antigen the mixture is incubated at 37°C and if a specific complex is formed all the complement is used up, so that on the addition of the haemolytic system (sheep erythrocytes plus homologous red cell antiserum) no haemoly­sis will occur. The complement-fixation test can be adopted to detect either antibody, which shows a rising titre during the course of certain diseases, or antigen. The latter form of the test yields rapid diagnostic results and is particularly useful in dealing with suspected cases of foot-and-mouth disease and rinderpest.

The complement-fixation test is regarded as being the most accurate of the serological tests for glanders in all species. Sufficient antibody develops to give a positive reaction within about seven days, following infection, and this situation exists for a prolonged period in chronically affected animals. A horse is considered to be infected with Actinobacillus mallei when 0-1 ml or less of its serum completely inhibits haemoly­sis. Similar values in relation to the quantities of serum required to produce positive and negative complement-fixation reactions are employed in the diagnosis of dourine and certain other types of trypanosomiasis.

The complement-fixation test is of value in detecting Brucella infected cattle; it is more efficient in the early stages of infection than any of the agglutination tests. Although the test is capable of distinguishing between post-vaccinal and post-infective antibodies this differentiation is only satisfactory when vaccination has ante­dated the test by a considerable period. Accep­table criteria for the complement-fixation test in bovine brucellosis are positive results at serum dilutions of 1:4 or less in animals with serum agglutination titres of 1:20 (50 i.u.).

The complement-fixation tests has been applied rather intermittently in the diagnosis of Johne's disease in both sheep and cattle. The virtual eradication of bovine tuberculosis has in recent years given a fresh impetus towards evaluating the efficiency of this test. At the present time its greatest usefulness is in confirming clinically suspect cases of Johne's disease (accuracy about 90%) which give positive reactions, even in the early phase. Under field conditions, however, some months may elapse between the establish­ment of infection and the appearance of detec­table antibodies. The degree of specificity of the test in non-clinically affected animals and in animals with tuberculosis is rather low (25%) and it gives a considerable proportion of false positive results in normal animals. A number of countries throughout the world allow the impor­tation of cattle only on the basis of a negative complement-fixation test in respect of Johne's disease.

Although the virus responsible for enzootic abortion in ewes has common antigens with other viruses of the psittacosis-lymphogranuloma group, fixation of complement by the serum of an ewe which has aborted, in conjunction with a history of the disease in the flock, may be regarded as being diagnostically significant. Retrospective diagnosis of canine infectious hepatitis, or canine distemper, can be established by means of the complement fixation test.

Precipitin Test

The principle of this test is somewhat similar to that of the agglutination test except that the reaction occurs at the interface between soluble antibody, in the form of globulin and a prepared soluble extract of the antigen. The precipitation reaction may occur at room temperature but, in some cases, is hastened by exposure to tem­peratures from 37°C to 55°C (98-8-131°F). The test can be done as a gel diffusion test (immuno-diffusion) using small wells cut in an agar gel or in small tubes.

The tube precipitin test has had its greatest application in the classification of streptococci. More recently agar gel diffusion tests are exten­sively used in the study and diagnosis of many virus diseases, e.g. rinderpest, mucosal disease, swine fever. This type of test can be adopted to detect either antigen or antibody. A positive result takes the form of a white line of precipita­tion at the interface between antigen and anti­body.

Haemagglutination Tests

These tests are not dependent on the formation of an antigen-antibody complex. Many viruses, when mixed with washed chicken erythrocytes, cause them to agglutinate. Homologous virus antibody will prevent the appearance of this phenomenon. In performing the test a standard suspension of erythrocytes is mixed with virus in a tube which is then allowed to stand for a few hours. If the red cells remain unagglutinated they sediment and collect as a mass in the bottom of the tube; when aggregated the cells form a thin film over the whole bottom of the tube. The method of the test can be adopted for the detection of either virus or antibody (haemagglu­tination inhibition test), the latter usually appears in the blood of affected animals and birds within a few days after the development of clinical signs of disease and has reached a high titre within a few weeks. Other factors apart from virus antibody are known to inhibit haemagglutination; for myxoviroses these factors can be removed by treatment with trypsin and for arboviruses by extraction with acetone.

Virus neutralization Test

This type of serological test is only applicable in those instances in which homologous virus antibody is produced by the animal following infection. In this form the test is of retrospective value in diagnosis although it may be used for virus identification at an earlier stage. Because viruses will only grow in living cells the employ­ment of this type of test depends upon the availa­bility of suitable tissue cell cultures, chick embryos or a susceptible laboratory animal. A number of viruses are capable of causing micro­scopic cytopathogenic lesions in some of the cells in tissue cultures and/or in chick embryoes. Adding homologous antibody to the virus in an appropriate amount by serially diluting the latter will prevent virus multiplication when it is intro­duced into susceptible cells. By this means the presence of homologous virus antibody is indi­cated when cytopathogenic lesions fail to de­velop.

Viruses which can be recognized by this means include those causing louping-ill, viral encephalo-myelitis of pigs, pseudorabies (Aujeszky's disease), equine encephalomyelitis, infectious bovine rhinotracheitis, swine influenza, African horse sickness, etc.

Toxin Neutralization Test

The principle of this test is the detection of specific toxin in the tissues or intestinal contents of animals by means of injecting a filtrate of the unknown material together with one of a series of purified antitoxins into laboratory animals of a suitable species. Conversely antitoxin can be identified in the sera of recovered animals fol­lowing initial titration with a range of known toxins. Diseases which can be recognized by this method include lamb dysentery, pulpy kidney, ovine enterotoxaemia, infectious necrotic hepa­titis, tetanus, malignant oedema and ulcerative lymphangitis.