|
SUMMARY
Definition of the disease: Pullorum disease of chickens is caused by Salmonella enterica subspecies enterica serovar Pullorum (Salmonella Pullorum)* and, in its acute form, is almost exclusively a septicaemic disease of young chickens. However, the organism may also be associated with disease in turkey poults. Ovarian transmission is a major route by which the organism can spread. Game birds and 'backyard' poultry flocks may act as reservoirs of infection, and wild birds may act as vectors for the organism and as such are important in the epidemiology of the disease.
*See the note in Chapter 2.10.3. Salmonellosis for the principles followed concerning the nomenclature of Salmonella.
Fowl typhoid in chickens and turkeys is caused by S. Gallinarum and is more often observed in the later growing period and in mature stock.
Description of disease: Clinical signs in chicks and poults comprise anorexia, diarrhoea, dehydration, weakness and death. In mature birds disease is less severe but decreased egg production, poor hatchability and some increased mortality may occur.
Identification of the agent: Samples should not be taken from birds or eggs that have recently been treated with antimicrobial drugs. They can be swabs or aseptically collected samples from infected tissues, or intestinal and cloacal contents. Other materials that may be sampled include eggs, embryos, faecal droppings and hatcher debris, especially fluff, dust and broken eggshells and chick box linings. Samples of tissues such as caecal tonsils and spleen from infected birds are preferable to faecal and environmental samples. Tissue samples should be inoculated into nonselective and selective enrichment broth and into selective agar medium, such as brilliant green agar, as soon as possible after collection. In case of delay, samples should be stored at 4°C. Typical colonies can be identified by serological and biochemical tests.
Serological tests: These are satisfactory for establishing the presence and estimating the prevalence of infection within a flock. The test used in the field is the rapid whole blood plate agglutination test. This test is unreliable in turkeys and ducks, many uninfected turkeys may give positive reactions. In the laboratory a serum agglutination test is used, either as a rapid plate test or as a tube test. These can be applied as macro- or microagglutination tests though the latter may be more likely to give false-positive results with turkey sera. Any positive reactors should be confirmed as being infected by culture at post-mortem examination. Enzyme-linked immunosorbent assays have been reported but no commercial test is available.
The use of vaccines to control S. Enteritidis infections in chickens may cause problems in the interpretation of serological results.
Requirements for vaccines and diagnostic biologicals: Live and inactivated vaccines are available for fowl typhoid in some countries. The most commonly used vaccine is a live vaccine derived from the stable rough strain of S. Gallinarum known as '9R'.
A. INTRODUCTION
Fowl typhoid and pullorum disease, caused by Salmonella enterica subspecies enterica serovars Gallinarum and Pullorum, respectively, are widely distributed throughout the world but they have been eradicated from commercial poultry in many developed countries of Western Europe, the United States of America (USA), Canada, Australia and Japan. Salmonella Pullorum remains in wild and game birds however. Salmonella Gallinarum and S. Pullorum are host adapted to avian species and are considered to pose a minimal zoonotic risk (11).
Clinical signs are typical of a septicaemic condition in poultry and include increased mortality and poor quality in chicks hatched from infected eggs. Older birds show signs of anaemia, depression, laboured breathing and diarrhoea causing adherence of faeces to the vent. The highest mortality occurs in birds of 2-3 weeks of age. In older birds disease may be mild or inapparent. In breeding flocks reduced egg production and hatchability may be the only signs, and trans-ovarian infection resulting in infection of the egg and hatched chicks or poults is one of the most important transmission routes for the disease.
Post-mortem signs of pullorum disease in newly hatched chicks are those of peritonitis with generalised congestion of tissues and inflamed unabsorbed yolk sac. Longer standing infections commonly lead to typhlitis with development of necrotic caecal casts and small necrotic foci in the liver, lungs and other viscera. Adult birds may develop misshapen or shrunken ovaries. In fowl typhoid, as well as generalised signs of septicaemia, the liver is usually enlarged, dark and friable with a distinctive coppery bronze sheen. The bone marrow is also often dark brown. Although clinical signs and post-mortem findings of pullorum disease and fowl typhoid may be highly suggestive of the conditions, they are not sufficiently distinct from other causes of septicaemia to be pathognomic. It is therefore necessary to confirm disease by isolation of the organisms. Serological tests can be used to establish the presence of the disease in a flock.
B. DIAGNOSTIC TECHNIQUES
In its acute form, pullorum disease is almost exclusively a disease of young chickens, and the agent can be recovered from almost all organs, tissues and faeces. In older birds that have become carriers, S. Pullorum is most commonly recovered from the ova, and only exceptionally from other organs and tissues, including the alimentary tract. In the acute phase of fowl typhoid the organism is also widely distributed, but in carrier birds, the organism is found most often in the liver and caecae.
| . | Culture
|
| | Salmonella Pullorum and S. Gallinarum belong to the Kauffmann-White scheme serogroup D, along with S. Enteritidis, which is thought to be closely related. The organisms are Gram negative nonsporogenic rods 1.0-2.5 µm in length and 0.3-1.5 µm in width. They are considered to be nonmotile under normal conditions but inducement of flagellar proteins and motility has been shown in special media (8).
|
| | To recover the organisms, the birds should not have been treated with antimicrobial drugs for approximately 2-3 weeks previously.
|
| | Samples may be obtained from live birds, fresh or freshly frozen carcasses, egg materials, fresh faeces, or any contaminated materials from housing, incubators or transport boxes. Swabs may be taken from the cloaca of live birds. Samples from visibly abnormal tissues are preferable to faecal and environmental samples. Aseptic samples can be taken from the spleen, liver, gall-bladder, kidneys, lungs, heart, ova, testes, alimentary tract or joint lesions. The surface is seared with a hot spatula and a sample is obtained by inserting a sterile cotton swab or sterile loop through the heat-sterilised surface. The demonstration of infection in serological reactor birds that are apparently normal may require the culture of large volumes of homogenised tissues as well as direct swabbing. Tissue pools may be made from tissues collected from a number of birds.
|
| |
When floor litter or faecal material is sampled, it should be remembered that S. Pullorum and S. Gallinarum are more difficult to isolate from faecal and environmental samples than other salmonellae. Samples should include floor faeces, moist and dry litter and swabs from open drinkers. These samples should be cultured by direct inoculation of a selective enrichment broth such as selenite cysteine, followed by plating on selective media such as brilliant green agar.
|
| | Both S. Pullorum and S. Gallinarum grow well on nonselective media, but selective and enrichment media have been described that contain substances to inhibit the growth of extraneous organisms. The efficiency of recovery of Salmonella varies according to circumstances, and experience in the use of a medium is an important but unquantifiable factor. Some complex media may have an inhibitory effect on these organisms, so that it is advisable to use both nonselective and selective media for isolation from tissues. Both solid media and broths can be employed. As the toxic properties of selective media may vary, it is preferable to monitor these by comparing growth of control cultures on both types of medium. The inhibitory media should grow at least 75% of the colonies of the corresponding non-inhibitory medium (3, 4, 7, 10).
|
| | All the media mentioned below are examples of commonly used media, but there are many others found to be equally satisfactory.
|
| | Non-inhibitory media include nutrient agar and blood agar, on which colonies are seen to be smooth, translucent, slightly raised, and about 2 mm in diameter. Broths include buffered peptone water and nutrient and meat infusion broths.
|
| | . | Selective media include:
|
| | | MacConkey agar: The agar is inhibitory to non-enteric organisms, it differentiates lactose fermenters (pink colonies) from nonlactose fermenters (colourless colonies). NaCl is omitted to limit the spread of Proteus colonies. Salmonella colonies are smooth and colourless. Salmonella Pullorum produces smaller colonies than other salmonellae. MacConkey is the agar of choice for direct plating from tissues.
|
| | | Xylose lysine deoxycholate agar: The agar is inhibitory to non-enteric organisms. Salmonella Pullorum grows sparsely as small red colourless colonies. S. Gallinarum colonies are small, dome-shaped, and may have a central black spot due to H2S production, but this reaction may be delayed or variable.
|
| | | Brilliant green agar (BGA): The agar is inhibitory to coliforms and most Proteus strains; useful for distinguishing enteric organism colonies. Salmonellae form low, convex, pale red, translucent colonies of 1-3 mm in diameter, similar to Citrobacter. Proteus forms pin-point colonies, Pseudomonas aeruginosa appears as small red colonies, and lactose fermenters are green. Salmonella Pullorum produces smaller more pale colonies than other salmonellae. BGA is the agar of choice following enrichment.
|
| | | Brilliant green sulphapyridine agar: The agar is inhibitory to coliforms and Proteus strains. The sulphapyridine is added to stabilise selectivity in the presence of egg materials. Salmonella Pullorum produces small colonies.
|
| | | Salmonella Pullorum and Gallinarum grow poorly and do not produce typical colonies on newer chromogenic agars such as Rambach agar.
|
| | . | Liquid enrichment and selective media include:
|
| | | Selenite F broth: Inhibitory to coliforms but not Proteus, improved by addition of brilliant green. Loss of activity after 24 hours. Selenite cysteine broth is more stable. Although selenite broths are considered to be preferable for isolation of S. Pullorum and S. Gallinarum from faeces, if there are difficulties with issues of toxicity or shelf life in particular laboratories the other enrichment broths mentioned below may be used.
|
| | | Tetrathionate/brilliant green broth: Inhibitory to coliforms and Proteus, but may also inhibit some strains of S. Pullorum/ Gallinarum.
|
| | |
Rappaport-Vassiliadis soya peptone broth: For selective enrichment following pre-enrichment, use 1 part inoculum to 100 parts medium. Salmonella Pullorum and Gallinarum are more likely to be overgrown by other organisms during pre-enrichment of faeces or intestinal contents than salmonellae that are not host-adapted.
|
| . | Recovery of salmonellae
|
| | The methods for recovering S. Pullorum and S. Gallinarum vary according to the origin of the samples. Although their isolation from cloacal swabs and faeces may be unrewarding, examination of tissues taken at post-mortem is usually more successful. The methods are as follows:
|
| | Cloacal swabs and fresh faeces from live birds: Swabs dipped in nutrient broth are suitable, small swabs being used for young chickens. The swabs are streaked on selective media, and placed in enrichment broth. The plates and the broth are incubated at 37°C. At this temperature, some Proteus and Pseudomonas organisms tend to be inhibited. Higher temperatures may be used with some broths, e.g. 41.5°C for Rappaport-Vassiliadis (RV), but care needs to be exercised because some enrichment media may be too inhibitory at high temperatures and RV broth is more inhibitory than selenite cysteine broth for S. Pullorum and S. Gallinarum. Subcultures are made on to selective media after 24 and 48 hours.
|
| | Gall-bladder contents: Swabs of gall-bladder contents are streaked on to nonselective and selective agars and placed in inhibitory and non-inhibitory broths, followed by incubation at 37°C and subculture on to selective agar after 24 and 48 hours.
|
| | Organs and tissues: Swabs or segments of tissues are taken in an aseptic manner from individual tissues and lesions and cultured on to nonselective and selective media, and into similar broths. These are incubated at 37°C and subcultured on to selective agar after 24 and 48 hours. Intestinal material in selective broths may also be incubated at 40°C; S. Gallinarum grows well but there may be some inhibition of S. Pullorum at this temperature.
|
| | Carrier birds: Larger amounts of material are required to identify the carrier birds. The ovary is the tissue of choice for S. Pullorum, and the liver and gall-bladder should be tested for S. Gallinarum. In practice it is usually best to pool samples from a variety of tissues. Tissues are homogenised in a small volume of broth and directly plated out. Approximately 10 ml of homogenate is also added to 100 ml of nonselective enrichment broth (e.g. buffered peptone water) and selective enrichment broth (e.g. selenite cysteine broth, brilliant green broth), and incubated at 37°C. These broths are subcultured on to nonselective and selective agar after 24 hours.
|
| | Alimentary canal, including the caecal tonsils and intestinal contents: After grinding or homogenisation in a small volume of broth, 10 ml of the homogenate is incubated in 100 ml of selective enrichment broth at 37°C. In general better isolation is achieved with selenite cysteine broth.
|
| | Eggshells: The broken eggshells are placed in a tenfold volume of enrichment broth (e.g. selenite cysteine broth). The broth is incubated at 37°C and subcultured on to selective agar after 24 and 48 hours.
|
| | Egg contents: Aseptically taken contents of fresh eggs are homogenised and mixed with 200 ml of buffered peptone water or nutrient broth, incubated at 37°C, and subcultured on to nonselective and selective agar after 24 and 48 hours. Incubated eggs, whether infertile or containing small embryos, can be similarly treated.
|
| | Embryos: Homogenised viscera and swabs from the yolk sacs of well developed embryos may be streaked on to nonselective and selective agar, one swab being placed in 10 ml of both nonselective and enrichment broth (e.g. selenite cysteine broth, brilliant green broth). Incubation is carried out at 37°C, and subcultures are made on to nonselective and selective agars after 24 and 48 hours.
|
| | Environmental samples: These include hatcher fluff, debris and macerated egg/chick waste samples and chick box liners or floor faecal or litter samples; 25 g is mixed with 225 ml of enrichment broth (e.g. selenite cysteine broth, brilliant green broth), incubated at 37°C, and subcultured on to selective agar after 24 and 48 hours.
|
| 1. | Identification of the agent
|
| | Typical S. Gallinarum colonies on nonselective media are round, translucent, glistening, domed, smooth, and 1-2 mm in diameter after 24-48 hours' incubation. Salmonella Pullorum colonies are slightly smaller and transluscent. On selective media their appearance varies with the medium, but suspect colonies can be investigated serologically by testing for 'O'9 somatic antigens, observing for motility and testing biochemically.
|
| |
After incubation for 20-24 hours, the plates should be examined carefully for the presence of typical S. Pullorum and S. Gallinarum colonies. If growth is slight after 24 hours' incubation, the plates should be reincubated for a further 24 hours and examined again. For biochemical and serological confirmation from each plate, five typical or suspect colonies should be chosen for further examination. If there are fewer than five typical or suspect colonies, all of them should be taken for further examination. Selected colonies should be streaked on to the surface of nutrient agar, in a manner that allows the growth of separate colonies. For biochemical confirmation, only pure cultures taken from nonselective media should be used. The following media should be streaked using an inoculating loop: triple sugar iron (TSI) agar; lysine iron agar (or l-lysine decarboxylation medium); urea agar according to Christensen; tryptone/tryptophan medium for indole reaction; glucose with an inverted Durham tube for acid and gas production; dulcitol, maltose, ornithine decarboxylation medium and semisolid agar, for motility. The reactions shown in Table 1 occur.
|
| | Identification kits are commercially available, for example Analytical Profile Index (API) system for Enterobacteriaceae. However, care must be taken when using API because S. Pullorum may be misidentified as Hafnia spp. Molecular tests using ribotyping techniques and polymerase chain reaction have been developed in research laboratories (9), but are not generally available for confirmation of S. Gallinarum and S. Pullorum.
|
| | For serological confirmation as to serogroup, colonies from nonselective media (nutrient or blood agar) are used. The first stage is elimination of autoagglutinable strains. For this, material taken from a single colony of pure culture is transferred to a glass slide and mixed with a drop of sterile saline. The slide is rocked gently or the drop stirred with a loop for 30-60 seconds and observed for agglutination against a dark background, preferably with the aid of a magnifying glass or dissecting microscope. If the bacteria have clumped into more or less distinctive units, the strain is considered to be autoagglutinable and must not be submitted to the following tests. If the bacterial sample is recognised as non-autoagglutinable, it is tested with a polyvalent 'O' (A-G) antiserum. For this purpose, the material from a single colony is dispersed in the drop of polyvalent 'O' antiserum on the glass slide to obtain a homogenous and turbid suspension. After gently rocking for 30-60 seconds, the reaction is observed against a dark background for agglutination. Alternatively the slide agglutination test may be carried out with smaller volumes of suspension under a dissecting microscope. In this case a portion of the colony to be checked is added to a loopful of saline on the microscope slide to produce a light suspension to check for autoagglutination ('rough strains'). If no agglutination takes place, one or two loops of antisera are added, the drop stirred with a loop and observed for agglutination. Salmonella Pullorum and S. Gallinarum should agglutinate with polyvalent 'O' antisera but not with polyvalent flagella (poly 'H' phase 1 and phase 2) antisera. If the reaction is positive, the single colony is tested further in the same manner using group-specific sera for S. Pullorum and S. Gallinarum serotypes ('O'9 antiserum). After serogrouping, isolates may be sent to a reference laboratory for serotyping.
|
Table 1. Biochemical investigation of Salmonella Pullorum and S. Gallinarum
|
Salmonella Pullorum
|
Salmonella Gallinarum
|
TSI glucose (acid formation)
|
+
|
+
|
TSI glucose (gas formation)
|
v
|
-
|
TSI lactose
|
-
|
-
|
TSI saccharose
|
-
|
-
|
TSI hydrogen sulphide
|
v
|
v
|
Gas from glucose (medium with Durham tube)
|
+
|
-
|
Urea hydrolysis
|
-
|
-
|
Lysine decarboxylation
|
+
|
+
|
Ornithine decarboxylation
|
+
|
-
|
Maltose fermentation
|
- or late +
|
+
|
Dulcitol
|
-
|
+
|
Motility
|
-
|
-
|
+ = 90% or more positive reaction within 1 or 2 days; - = No reaction (90% or more); v = Variable reactions.
| | . |
Test procedure for culture of visceral, faecal, intestinal and environmental samples for S. Pullorum and S. Gallinarum
|
| | | i) | Where possible, begin laboratory procedures on the same day as samples are collected.
|
| | | ii) | Homogenise the material as much as possible by manual mixing, macerating or stomaching with a small volume of sterile saline if the material is dry.
|
| | | iii) | Stir the mixture with a small rectal swab or loop and streak thickly on to one-quarter of a brilliant green agar plate. (Swabs from uncontaminated tissues sampled in an aseptic manner can also be streaked on to blood agar.)
|
| | | iv) | From this deposit of material on the plate, streak the rest of the plate to obtain individual colonies.
|
| | | v) | Add 5-25 g of the homogenised sample to freshly made selenite cysteine broth (see note on liquid enrichment and selective media above) to make a 1:10 sample to broth ratio. Shake or stir to disperse the sample in the broth.
|
| | | vi) | Incubate the brilliant green agar plates and selenite cysteine broth at 37°C for 24 hours.
|
| | | vii) | Examine the plate after 24 hours' culture. Carry out agglutination tests on up to five suspect colonies with polyvalent 'O' (A-G) antisera and polyvalent H (phase 1 and phase 2) antisera. If agglutination is unclear subculture suspect colonies on to nutrient agar or blood agar and repeat tests after 24 hours' incubation of those media.
|
| | | viii) | If poly 'O' is positive then check with 'O'9 antiserum. If 'O'9 is positive and poly 'H' is negative, this is indicative of the possible presence of S. Pullorum or S. Gallinarum.
|
| | | ix) | If there are no positive colonies on the brilliant green agar plate, streak out a 10 µl loop of incubated selenite cysteine broth onto brilliant green agar as in step iv above.
|
| | | xi) | Incubate the brilliant green agar plates at 37°C for 24 hours and re-incubate the previous (negative) brilliant green agar plates and the selenite cysteine broths for a further 24 hours.
|
| | | xi) | Repeat examination of plates as in step vii above.
|
| | | xii) | If plates are still negative, replate from selenite cysteine broth and incubate second brilliant green agar plate for a further 24 hours and examine as in step vii above.
|
| | | xiii) | Confirm S. Pullorum and S. Gallinarum using biochemical tests as shown in Table 1. Isolates can be sent to a Salmonella reference laboratory for confirmation of serotype and for phage typing of S. Pullorum.
|
| | . | Molecular epidemiology
|
| | |
Standard molecular 'fingerprinting' techniques used for Salmonella, such as plasmid profile analysis or pulsed field gel electrophoresis can be used for investigating outbreaks of S. Pullorum or S. Gallinarum.
|
| 2. | Serological tests
|
| | Serological tests are best applied as a flock test as results for individual birds will vary according to the stage of infection. It is therefore necessary to take sufficient individual samples to determine infection in the flock. If the test is to be used for detecting individual infected birds for culling, it should be repeated at least twice and preferably until the whole flock has given at least two negative tests.
|
| | The tests that are most readily applied include rapid whole blood agglutination, rapid serum agglutination (RST), tube agglutination and micro-agglutination (13). Other invasive Salmonella such as S. Enteritidis and S. Typhimurium may give false-positive results in serological tests for S. Pullorum.
|
| | Both S. Pullorum and S. Gallinarum possess 'O' antigens 9 and 12 and may also posess O antigen 1. However, in the case of S. Pullorum, there is a variation in the ratio of 121, 122 and 123; the standard strain contains more 123 than 122, while the reverse is true of the variant form. Intermediate forms also exist. (There appears to be no such form variation in the case of S. Gallinarum.) As this variation occurs, it is necessary to use a polyvalent antigen in the agglutination tests. The same antigen is used to detect both S. Pullorum and S. Gallinarum.
|
| | a) | Rapid whole blood agglutination test
|
| | | The rapid whole blood agglutination test can be used under field conditions for detecting both S. Pullorum and S. Gallinarum, and the reactors can be identified immediately. However, it is not reliable in turkeys as the test results in a significant proportion of false-positive results. Chickens can be tested at any age, although some authorities specify a minimum age of 4 months (13, 14) and positive results from chicks less than 4 weeks of age may be due to maternal antibodies.
|
| | | . | Preparation of stained antigen for the rapid whole blood or rapid serum agglutination test
|
| | | Incubate one standard form strain of S. Pullorum (antigenic structure 9, 121, 123) and one variant form (antigenic structure 9, 121, 122) at 37°C and harvest separately until final mixing for the complete antigen.
|
| | | Sow strains on to separate agar slopes, incubate at 37°C for 24 hours, emulsify growth with sterile normal saline and spread an inoculum over an agar plate to produce easily selected discrete colonies. For this the plates are incubated for 48 hours, a number of colonies are marked out and each is tested for agglutination on a slide with 1/500 acriflavine in saline. Smooth-phase colonies do not produce agglutination. Pick off typical colonies that do not produce any agglutination, seed on to agar slopes, and incubate for 24 hours. Emulsify the growth in saline and evenly distribute 2 ml over the surface of the medium (200 ml) in a Roux or similar flask. Incubate the flasks for 60 hours.
|
| | |
For harvesting the bacterial growth, flood the surface of each flask with enough sterile buffered formol saline, pH 6.5 (8.5 g/litre sodium chloride, 10 ml/litre neutral formalin, 4 ml/litre 0.5 M sodium phosphate: made up to 1 litre with distilled water, pH adjusted to 6.5 using 1 M orthophosphoric acid or 1 M sodium hydroxide), to give dense cell suspensions (about 10 ml per flask). Add 12-15 sterile glass beads of 3-5 mm diameter and rock the flasks until all the growth is in even suspension; leave in a vertical position for at least 15 minutes. Check the morphology and purity of the suspensions by preparing and examining Gram-stained films. Bulk the suspension from each flask containing the same strains. To each 100 ml of suspension, add 200 ml of absolute alcohol. Shake the mixture and allow to stand for 36 hours or until precipitation is complete. Check the agglutinability of the standard and variant precipitate by first centrifuging a sample to separate the alcohol, which is removed, dilute with normal saline and test with a known positive and negative serum. If satisfactory, remove the clear supernatant alcohol (centrifugation at 2000 g for 10 minutes may be helpful in precipitation), and add sufficient phosphate buffered saline (PBS) containing 10% (v/v) glycerol to standardise the density to 75 x No. 1 Wellcome opacity tube (or 50 x tube No. 1.0 on the McFarland scale). Add equal volumes of standard and variant strains, and add 1% (v/v) of 3% (w/v) alcoholic crystal violet solution to the final mixture, and allow to stand for 48 hours at room temperature. Store in a tightly closed container at 0-4°C for up to 6 months. To assess safety, carry out a culture test on blood agar for nonviability of the unwashed antigen before standardisation. Each bottle of antigen must be tested after alcoholic precipitation and before standardisation against standard titre antisera for S. Pullorum and S. Gallinarum, and against a negative serum. If possible, also test with known positive and negative serum and blood from positive and negative chickens.
|
| | | Stained antigen products for the whole blood plate agglutination test are available commercially, and although there seems to be some slight differences in their sensitivity (5), it is unlikely that poultry flocks infected with the different variants of S. Pullorum would be missed.
|
| | | . | Test procedure
|
| | | i) | Use a clean white tile marked into squares of about 3 x 3 cm. If a tile with 3 x 4 squares is used, up to 12 blood samples can be tested at the same time.
|
| | | ii) | Place 1 drop (about 0.02 ml) of crystal-violet-stained antigen in the centre of each square.
|
| | | iii) | Obtain a sample of fresh whole blood. This is conveniently done by making a stab of a wing vein using a needle with a triangular point.
|
| | | iv) | Place an equal size drop of fresh whole blood next to a drop of antigen.
|
| | | v) | Mix the drops of antigen and blood using a fine glass rod, which is wiped clean between samples.
|
| | | vi) | Use a gentle rocking motion to keep the drops agitated for up to 2 minutes. Several tests may be carried out simultaneously on the same tile, but the drops should not be allowed to dry out during this time. In very warm conditions, larger drops may be required to avoid drying out.
|
| | | vii) | A positive reaction is indicated by easily visible clumping of the antigen within 2 minutes.
|
| | | viii) | A negative reaction is indicated by absence of clumping of the antigen within 2 minutes.
|
| | | ix) | Include known positive and negative control sera on each testing occasion, using them in the same way as the blood.
|
| | | x) | On completion of a set of tests, the tile is washed and dried, ready for further use.
|
| | | In the absence of positive reactions, any doubtful reactions can only be interpreted in the light of the previous Salmonella testing history of the flock. Where there are positive reactors, any doubtful reactor should be regarded as positive. Also, recently infected birds may not show a typical positive reaction until they are retested after 3-4 weeks.
|
| | b) | Rapid serum agglutination test
|
| | | The RST is performed in the same manner, except that serum is substituted for whole blood. For export test purposes an initial screening of sera by RST followed by confirmation of positives by the tube aglutination test is the optimal approach. Ideally serum samples tested by any method should be tested within 72 hours of collection as nonspecific reactions may increase in older samples. Fresh samples can be frozen for later testing if a delay is unavoidable.
|
| | c) | Tube agglutination test
|
| | | Fresh serum from chickens, turkeys or other birds is used at an initial dilution of 1/25, obtained by mixing 0.04 ml of serum with 1.0 ml of antigen (for preparation of small volumes of somatic antigens see Chapter 2.10.3. Salmonellosis). Positive and negative control sera are included in each test. The antigen is prepared from unstained S. Pullorum or S. Gallinarum cultures diluted to a concentration of No. 1 on the McFarland scale (as described above). The mixture is incubated at 50°C for 18-24 hours before reading. A positive reaction consists of a granular white deposit with a clear supernatant fluid; a negative reaction shows uniform turbidity. Samples positive at a dilution of 1/25 are retested at a higher range of dilutions and a titre of 1/50 is usually considered to be positive, although this figure seems to vary in the literature.
|
| | d) | Micro-agglutination test
|
| | | This resembles the tube agglutination test, but requires much smaller volumes of reactants. The test is performed in microtest plates. Sera are first diluted by adding 10 µl of serum to 90 µl of normal saline, and then adding 100 µl of previously standardised stained microtest antigen to give a final dilution of 1/20. By titrating the serum in doubling dilutions and adding an equal volume of standardised stained antigen, an end-point (titre) can be obtained. The plates are sealed and incubated at 37°C for 18-24 or 48 hours. A positive reaction consists of a fine diffuse precipitation, whereas a negative reaction shows a button-like precipitate. Titres of 1/40 are usually considered to be positive but this test is more liable to produce false-positive results with turkey sera.
|
| | Other serological tests include micro-antiglobulin (Coombs), immunodiffusion, haemagglutination and enzyme-linked immunosorbent assay (ELISA).
|
| | ELISA techniques have been described for detecting antibodies to S. Pullorum and S. Gallinarum. The indirect ELISA using lipopolysaccharide antigen is likely to be the most sensitive and specific serological flock test for Salmonella, including S. Gallinarum and S. Pullorum. It is relatively easy to perform with serum or yolk, and can be used for quantifying the titre of antibody (1, 2, 14). No commercial ELISA kits for S. Pullorum and S. Gallinarum are currently available.
|
C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
Although both live and inactivated vaccines have been prepared for use against S. Gallinarum, the vaccine most widely used is made from the rough 9R strain (6). It has only been employed in chickens. The number of viable organisms per dose is important; these organisms can survive in vaccinated birds for many months and may be transmitted through the egg (and perhaps from bird to bird). Vaccination may reduce flock losses, but will not prevent infection with field strains. In addition, vaccination with 9R may sometimes precipitate high mortality in infected birds (12), and may stimulate the production of transient antibodies. It is usual to vaccinate at 8 weeks and again at 16 weeks of age. Antimicrobials should be avoided before and after vaccination.
Currently available vaccines, however, have only a minor role to play in the control of fowl typhoid. This can best be achieved by biosecurity, hygiene in management, monitoring and removal of infected flocks.
| 1. | Seed management
|
| | a) | Characteristics of the seed
|
| | | Live fowl typhoid vaccine is a suspension of suitably attenuated living organisms of a rough strain of S. Gallinarum, e.g. 9R. The organisms in the vaccine give the biochemical reactions characteristic of S. Gallinarum. Colonies of a 24-hour culture prepared from the vaccine on nutrient agar plates are rough when examined by the acriflavine slide test. The culture does not contain the somatic antigens characteristic of the smooth forms of S. Gallinarum.
|
| | b) | Method of culture
|
| | | Salmonella Gallinarum is grown on or in a suitable medium, such as nutrient agar or broth, for 24 hours at 37°C.
|
| | c) | Validation as a vaccine
|
| | | There is no satisfactory method of assessing the protection afforded by the vaccine in the field. However, experience has shown that the vaccine can provide some benefit in situations where control cannot be achieved by hygiene and management alone. The potency test described below may be used to provide evidence of efficacy.
|
| 2. | Method of manufacture
|
| | The vaccine may be prepared by inoculation of a suitable medium, such as nutrient broth, with a fresh culture of S. Gallinarum (9R) and incubation at 37°C for 24 hours, with or without aeration. The organisms are harvested by sedimentation or centrifugation.
|
| | Alternatively the organisms may be grown on and harvested from a solid medium, such as nutrient agar. In either case, the suspension is diluted in PBS solution, pH 7.0, and may be freeze-dried. The dose used per bird is between 5 x 106 and 5 x 107 organisms.
|
| 3. | In-process control
|
| | The culture used for inoculation of the production cultures and the harvested cells are examined microscopically using Gram staining to check for purity.
|
| 4. | Batch control
|
| | a) | Sterility
|
| | | Tests for sterility and freedom from contamination of biological materials may be found in Chapter I.1.5.
|
| | b) | Safety
|
| | | Six healthy, susceptible (preferably specific pathogen free [SPF]) chickens, 8-16 weeks of age, are each injected subcutaneously with ten doses of vaccine, and are observed for at least 7 days; no local or systemic reaction should develop.
|
| | c) | Potency
|
| | | Fifteen healthy chickens, 8-16 weeks of age, of the Light Sussex or Rhode Island Red breeds, or crosses of these, and taken from a stock that is free from S. Pullorum infection, are each injected subcutaneously with a quantity of vaccine corresponding to one field dose, i.e. 5 x 107 viable organisms. After an interval of 21-28 days, the vaccinated chickens and an equal number of similar unvaccinated chickens are deprived of food for approximately 18 hours. The chickens are then challenged by oral administration of 1 ml of a broth suspension containing 5 x 107 organisms of a virulent strain of S. Gallinarum mixed with 300 mg of a powder consisting of chalk (40%), light kaolin (43%) and magnesium trisilicate (17%). All the chickens are observed for 14-21 days. The vaccine passes the test if at the end of this period the number of surviving vaccinated chickens that show no macroscopic lesions of fowl typhoid at post-mortem exceeds by eight or more the number of similarly defined control chickens.
|
| | d) | Duration of immunity
|
| | | The vaccine should provide protection throughout the laying period, and this can be measured by potency (efficacy) tests at stages during lay. A booster dose during lay may be required, but should not be used during lay in flocks providing eggs for human consumption.
|
| | e) | Stability
|
| | | The shelf life of the vaccine can be measured by conducting potency tests at periods after manufacture. These should be done on at least six samples. Potency should remain satisfactory for at least 1 year.
|
| | f) | Preservatives
|
| | | No preservatives are used.
|
| | g) | Precautions (hazards)
|
| | | The organism is not known to be pathogenic to humans, and there are no special risks associated with the manufacture of either the vaccine or the antigen. However, the vaccine may establish a persistent infection in carrier birds and can precipitate disease in already infected chickens.
|
| 5. | Tests on the final product
|
| | a) | Safety
|
| | | The safety test described in Section C.4.b. should be used on a representative sample from each batch of final product.
|
| | b) | Potency
|
| | | The potency test described in Section C.4.c. should be used on a representative sample from each batch of final product.
|
REFERENCES
| 1. | Barrow P.A. (1992). ELISAs and the serological analysis of salmonella in poultry: a review. Epidemiol. Infect., 109, 361-369.
|
| 2. | Barrow P.A. (1994). Serological diagnosis of Salmonella serotype enteritidis infections in poultry by ELISA and other tests. Int. J. Food Microbiol., 21, 55-68.
|
| 3. | Ellis E.M., Williams J.E., Mallinson E.T., Snoeyenbos G.H. & Martin W.J. (1976). Culture Methods for the Detection of Animal Salmonellosis and Arizonosis. Iowa State University Press, Ames, Iowa, USA.
|
| 4. | Fricker C.R. (1987). The isolation of salmonellas and campylobacters. J. Appl. Bacteriol., 63, 99-116.
|
| 5. | Gast R.K. (1997). Detecting infections of chickens with recent Salmonella Pullorum isolates using standard serological methods. Poult. Sci., 76, 17-23.
|
| 6. | Harbourne J.F., Williams B.M., Parker W.H. & Fincham I.H. (1963). The prevention of fowl typhoid in the field using a freeze-dried 9R vaccine. Vet. Rec., 75, 858-861.
|
| 7. | Harvey R.W.S. & Price T.H. (1975). Isolation of Salmonellas. Public Health Laboratory Service, London, UK.
|
| 8. | Holt P.S. & Chaubal L.H. (1997). Detection of motility and putative synthesis of flagellar proteins in Salmonella pullorum cultures. J. Clin. Microbiol., 35, 1016-1020.
|
| 9. | Itoh Y., Hirose K., Miyake M., Khan A.Q., Hashimoto Y. & Ezaki T. (1997). Amplification of rfb and flic gene by polymerase chain reaction for identification and detection of Salmonella serovar enteritidis, dublin and gallinarum-pullorum. Microbiol. Immunol., 41, 791-794.
|
| 10. | Mallinson E.T. & Snoeyenbos G.H. (1989). Salmonellosis. In: Isolation and Identification of Avian Pathogens, Third Edition, Purchase H.G. et al., eds. American Association of Avian Pathologists, Kendall Hunt Publishing, Iowa, USA, 3-11.
|
| 11. | Shivaprasad H.L. (2000). Fowl typhoid and pullorum disease. Rev. sci. tech. Off. int. Epiz. 19, 405-424.
|
| 12. | Silva E.N., Snoeyenbos G.H., Weinack O.M. & Smyser C.F. (1981). Studies on the use of 9R strain Salmonella Gallinarum as a vaccine in chickens. Avian Dis., 25, 38-52.
|
| 13. | United States Department of Agriculture (1996). Auxiliary Provisions on National Poultry Improvement Plan. Code of Federal Regulations, Title 9, Part 147, 717-727.
|
| 14. | Wray C. & Wray A. (eds) (2000). Salmonella in Domestic Animals. CAB International, Wallingford, Oxon, UK, 407-427.
|
*
* *
|