|
SUMMARY
Anthrax is primarily a disease of herbivorous animals, although all mammals, including humans, and at least some avian species can contract it. Mortality can be very high, especially in herbivores. The aetiological agent is the spore-forming, Gram-positive rod-shaped Bacillus anthracis. The disease has world-wide distribution and is a zoonosis. Anthrax is of importance to the livestock industry, certain populations of wildlife, and humans, especially those who are occupationally exposed.
The disease is mediated by exotoxins. Peracute, acute, subacute and, rarely, chronic forms of the disease are reported. Ante-mortem clinical signs may be virtually absent in peracute and acute forms of the disease. Subacute disease may be accompanied by progressive fever, depression, inappetence, weakness, prostration and death. Chronic disease may show localised swelling, fever and enlarged Iymph nodes; death may occur if the airway becomes obstructed. Post-mortem examination of recently dead animals (which must be performed with great care to avoid infection of the operator or contamination of the environment) may show any number of lesions, none of which is pathognomonic or entirely consistent. Lesions most commonly seen are those of a generalised septicaemia often accompanied by an enlarged spleen having a 'blackberry jam' consistency and poorly clotted blood. Haemorrhage from the nose, mouth and/or anus at death is not a common sign.
Identification of the agent: Visualisation of the encapsulated bacilli, usually in large numbers, in a blood smear stained with polychrome methylene blue (M'Fadyean reaction) is fully diagnostic. Bacillus anthracis is readily isolated in relatively high numbers from blood or tissues of a recently dead animal that died of anthrax, and in pure culture, on any nutrient agar incubated aerobically at 37°C. Its characteristic appearance on blood agar makes this the medium of choice. As the carcass decomposes, especially in a warm climate, the putrefactive bacteria out compete and eventually eliminate the infective organism within the carcass. Confirmation of anthrax in these cases may depend on isolation from soil contaminated by the terminal discharges.
Colony morphology of B. anthracis is quite characteristic after overnight incubation on blood agar. The colony is relatively large, measuring approximately 0.3-0.5 cm in diameter. It is grey-white to grey, nonhaemolytic with a rough, ground-glass appearance and has a very tacky, butyrous consistency. Prominent wisps of growth trailing back toward the parent colony, all in the same direction, are sometimes seen. This characteristic has been described as a 'medusa head' appearance.
The vegetative cells of B. anthracis are large, measuring 3-5 µm in length and approximately 1 µm in width. Ellipsoidal central spores, which do not swell the sporangium, are formed at the end of the exponential cell-growth phase. The cells stain strongly Gram positive and long chains are often seen in vitro, while paired or short chains are seen in vivo. The bacilli in infected tissue are encapsulated, but this characteristic is lost when the bacterium is grown aerobically in vitro. The capsule can be induced by incubating in defibrinated horse blood for at least 5 hours, or by culturing the isolate on nutrient agar containing 0.7% sodium bicarbonate with incubation at 37°C in the presence of CO2.
Additional useful laboratory tests are the absence of motility, susceptibility to the specific diagnostic 'gamma' bacteriophage and sensitivity to penicillin. Caution is advised when using these tests, as it has been shown that some genotypes are resistant to the gamma phage and penicillin. Primers are now available that can be used to show the presence of the toxin and capsule genes by polymerase chain reaction as confirmation of virulence, replacing animal inoculation. A thermoprecipitin test described by Ascoli in 1911 is still used in some countries to supply retrospective evidence of anthrax in decomposed carcasses or animal products.
Serological tests: Antibody detection in serum from infected animals is rarely used for diagnostic purposes and is essentially a research tool. The predominant procedure today is the enzyme-linked immunosorbent assay (ELISA).
Requirements for vaccines and diagnostic biologicals: The most widely used livestock anthrax vaccine, developed by Max Sterne in 1937, is a live, non-encapsulated, spore former held in suspension. In Russia and some Eastern European countries, an equivalent type of vaccine is used (strain 55). The Pasteur vaccine is no longer used in Italy. A new vaccine, Carbosap, has been developed that retains both plasmids and exhibits very low virulence. A list of producers is given in the World Health Organization anthrax guidelines (14).
There are no standardised requirements for diagnostic biologicals. Diagnostic gamma phage may be obtained from, for example, the United States Centres for Disease Control and Prevention (The Division of Bacterial and Mycotic Diseases, Centers for Disease Control, 1600 Clifton Road, Atlanta, Georgia 30333, United States of America) or various central veterinary or anthrax reference laboratories.
A. INTRODUCTION
Anthrax is primarily a disease of herbivorous animals, although all mammals, including humans, and at least some avian species can contract it. Mortality can be very high, especially in herbivores. The aetiological agent is the spore-forming, Gram-positive rod-shaped Bacillus anthracis, the only obligate pathogen within the genus Bacillus. Most of the other species of Bacillus are common ubiquitous environmental saprophytes, although a number, notably B. cereus, B. licheniformis and B. subtilis, are occasionally associated with food poisoning in humans and with other clinical manifestations in both humans and animals.
Animal anthrax occurs in at least three different forms: peracute or apoplectic form, acute form, and subacute to chronic form. Ruminants are most likely to manifest the peracute and acute forms, horses the acute form, and dogs, cats, and pigs a subacute to chronic or localised condition. In the peracute disease, signs preceding death often go unobserved. The clinical history usually describes the animal to be in good health a few hours before death. If the animal is observed shortly before death, fever up to 42°C (107°F), muscle tremors, dyspnoea, and mucosal congestion are the most common signs. Shortly afterwards, the animal will often have terminal convulsions, collapse and then die. Following death, unclotted blood may sometimes be seen to exude from the anus, vulva, nostrils, and/or mouth. Incomplete rigor mortis is also common.
The acute form may occur in cattle and clinical signs, such as depression, anorexia, fever, rapid respiration, increased heart rate, congested mucous membranes, or oedematous swellings, may be observed up to 48 hours before death. The acute form is usually seen in horses and varies with the site of exposure. Enteritis and colic are accompanied by high fever and depression. Death usually occurs within 48-96 hours. Spores introduced subcutaneously, for example by biting insects, result in a hot, oedematous swelling at the site that spreads to the throat, thorax, abdomen, prepuce or mammary gland. Dyspnoea due to throat swelling with resulting compression of the trachea may also be apparent. The course of the disease is usually 1-3 days, with some animals surviving for 1 week or more.
The subacute to chronic form of anthrax occurs in domestic and wild pigs, dogs and cats. The infectious bacteria are usually ingested when the host feeds on a contaminated source. The organism tends to localise in the regional lymph nodes of the pharyngeal area, where severe swelling may occur, resulting in death by occlusion of the airway. In cases where this does not occur, a fatal bacteraemia may develop, although recovery after a few days of illness is not uncommon. An intestinal form with severe acute gastroenteritis is also seen in carnivores and omnivores.
Suspicion of anthrax will depend on signs, such as sudden death with or without haemorrhaging from the orifices and incomplete rigor mortis, or on the history of the site, herd, etc. If anthrax is suspected, a thin smear is made on a microscope slide from a small drop of blood. This may be obtained by making a small cut in an ear vein or with a syringe from any available vein (care must be taken to avoid contaminating the environment or the operator). The blood smear is air-dried, fixed by immersion in 95-100% alcohol for 1 minute and stained with polychrome methylene blue (further details below). The presence of the encapsulated bacilli resembling railway 'box-cars' in pairs or short chains, usually in large numbers, is definitive for anthrax. Smears made from swabs of the blood emerging from any of the orifices will also reveal the capsulated bacilli, but may be contaminated with other organisms or artefacts. Samples of the blood can also be taken for culture.
To prevent environmental contamination, it is usual not to perform a necropsy on the carcass of suspected or confirmed cases. If the carcass is opened by necropsy or scavengers, the vegetative form of B. anthracis is released from the acidic environment of decay and produces spores that create foci of contamination. In some countries post-mortem is forbidden. Post-mortem findings are well documented, however. There are no consistent pathognomonic lesions and considerable similarities are seen to other infectious and toxic causes of acute death. Poorly clotted dark blood, enlarged pulpy spleen with a 'blackberry jam' consistency, and multiple petechial haemorrhages characteristic of a septicaemia are usual. In horses, post-mortem findings may be similar to those of ruminants, but sometimes may just consist of oedematous lesions confined to the throat and neck with no involvement of internal organs. In omnivores (pigs) and carnivores, findings of septicaemia as described for ruminants may occur, but more often there is extensive oedema and inflammation in the pharyngeal area. If the focus of infection is in the gastrointestinal tract, severe inflammation, sometimes with haemorrhage and necrosis, may be seen in the stomach, intestines, and mesenteric lymph nodes, accompanied by peritonitis and excessive peritoneal fluid.
Natural decomposition of a carcass destroys most of the vegetative organisms through the action of putrefactive bacteria. This can occur within a day or two in hot climates if the carcass is left undisturbed. The capsulated bacilli may not be readily seen in smears of blood samples taken after this has occurred, though the blood may remain culture positive for a day or so more. Some sporulation may have occurred in fluids exuded through natural body openings, and, particularly if a carcass has been opened by scavengers, many spores may be dispersed into the environment. Swabs of congealed fluids or samples of soil contaminated by the fluids are likely to yield B. anthracis on culture. Culture of soil samples may be the best way to confirm that death was due to anthrax in well putrefied carcasses.
| . | Disposal after sample collection
|
| | Proper disposal of an infected carcass by incineration (although labour intensive and fuel consumptive) is the most desirable method and is often required by law. The contaminated soil, bedding, etc. should be incinerated together with the carcass and, ideally, the site should be chemically disinfected for extra certainty. Where incineration is not feasible, deep burial (preferably with quicklime) is the alternative, although periodic recovery of anthrax spores from some old carcass burial sites have shown that this is the least satisfactory alternative.
|
| . | Risk factors for handlers of anthrax carcasses
|
| | The risk of inhaling infectious doses becomes significant in occupations involving the processing of animal by-products for manufacturing goods (industrial anthrax). These include the tanning, woollen, carpet, bone processing, and other such industries, where the potential for aerosolisation of substantial numbers of spores increases the risk of exposure to infectious doses. Laboratory workers should use good laboratory practice when working with specimens from suspected anthrax cases and when culturing B. anthracis. A biological safety cabinet is required for the manipulation of broth cultures or spore suspensions. Decontamination of all contaminated materials and surfaces should be accomplished by either autoclaving, incineration, or 10% sodium hypochlorite (bleach). The use of laboratory animals for diagnostic purposes is no longer required, but if they are used, the bedding and cages should be autoclaved after use and the bedding incinerated afterwards. Care should be taken not to create dusty aerosols when handling the bedding. Care should also be taken to avoid skin wounds from sharp instruments or animal bites and scratches.
|
B. DIAGNOSTIC TECHNIQUES
| 1. | Identification of the agent
|
| | Demonstration of encapsulated B. anthracis in smears of blood or tissues from fresh anthrax-infected carcasses and growth of the organism on blood agar plates is relatively uncomplicated and within the capability of most bacteriology laboratories. Difficulty may be encountered in the case of pigs and carnivores in which the terminal bacteraemia is frequently not marked, or in animals that received antibiotics before death.
|
| | Recovery of B. anthracis from old decomposed carcasses, processed specimens (bone meal, hides), or environmental samples (contaminated soil) is also often difficult, requiring demanding and labour-intensive procedures.
|
| | a) | Fresh specimens
|
| | | . | Capsule visualisation
|
| | | As described above, virulent encapsulated B. anthracis present in tissues and blood and other body fluids from animals that have died from anthrax should be looked for in smears of these specimens that have been dried, fixed in absolute alcohol for 3 minutes and stained with polychrome methylene blue (M'Fadyean's reaction) or Giemsa stain. The capsule stains pink, whereas the bacillus cells stain dark blue. The cells are found in pairs or short chains and are often square-ended (the chains are sometimes likened to a set of railway carriages - so-called 'box-car' appearance). The Gram stain does not reveal the capsule. The capsule is not present on B. anthracis grown aerobically on nutrient agar or in nutrient broths, but can be seen when the virulent bacterium is cultured for a few hours in a few millilitres of blood (defribrinated horse blood seems to work best). Alternatively, the capsule is produced when the virulent B. anthracis is cultured on nutrient agar containing 0.7% sodium bicarbonate and incubated in the presence of CO2 (20% is optimal, but a candle jar works well). The agar is prepared by reconstituting enough nutrient agar base powder for 100 ml of agar in 90 ml of water. It is then autoclaved and cooled to 50°C in a water bath; 10 ml of a filter-sterilised (0.22-0.45 µm filter) 7% solution of sodium bicarbonate is added and mixed. The solution is then poured into Petri dishes. The encapsulated B. anthracis will form mucoid colonies and the capsule can be visualised by making thin smears on microscope slides, fixing, and staining with polychrome methylene blue as before.
|
| | | . | Polychrome methylene blue (M'Fadyean's stain)
|
| | | Polychrome methylene blue is prepared as follows: 0.3 g of methylene blue is dissolved in 30 ml of 95% ethanol; 100 ml of 0.01% potassium hydroxide (KOH) is mixed with the methylene blue solution. Ideally, this should be allowed to stand exposed to the air, with occasional shaking, for at least 1 year to oxidise and mature. Addition of K2CO3 (to a final concentration of 1%) hastens the 'ripening' of the stain, but before it is regarded as diagnostically reliable, its efficacy should be established by testing it in parallel with an earlier, functional batch of stain on bona fide samples. It has now been found that stains that give positive reactions with cultures of B. anthracis cultured artificially in horse blood sometimes do not give positive results in the field. A commercial preparation of polychrome methylene blue (M'Fadyean) stain is becoming increasingly hard to obtain.
|
| | | In making smears for staining, only small drops of blood or tissue fluid are needed and a thin, small smear is best. After fixing in ethanol and drying, a small (approximately 20 µl) drop of stain is placed on the smear and spread over it with an inoculating loop. After 1 minute, the stain is washed with water, blotted, air-dried and observed initially using the x10 objective lens under which the short chains appear like short hairs; once found, these can be observed under oil immersion (x1000) for the presence of the pink capsule surrounding the blue/black-staining bacilli. To avoid laboratory contamination, the slide and blotting paper should be autoclaved or left for some hours in a 10% sodium hypochlorite solution.
|
| | | . | Culture and identification of Bacillus anthracis
|
| | | Bacillus anthracis grows readily on most types of nutrient agar, however, 5-7% horse or sheep blood agar is the diagnostic medium of choice. Inclusion of polymyxin (100,000 units per litre of medium) will suppress contaminant bacteria and aid in isolation of B. anthracis. Blood is the primary clinical material to examine. Swabs of blood, other body fluids or swabs taken from incisions in tissues or organs can be spread over blood agar plates. After overnight incubation at 37°C, B. anthracis colonies are grey-white to grey, 0.3-0.5 mm in diameter, nonhaemolytic, with a ground-glass moist surface, and very tacky when teased with an inoculating loop. Tailing and prominent wisps of growth trailing back toward the parent colony, all in the same direction, are sometimes seen. This characteristic has been described as a 'medusa head' appearance. Confirmation of B anthracis can be accomplished by the demonstration of a capsulated, spore-forming, Gram-positive rod in blood culture. Absence of motility is an additional test that can be done.
|
| | | The susceptibility of B. anthracis to the gamma bacteriophage was first described by Brown & Cherry in 1955 (3). The phage is available from CDC, various national central veterinary laboratories, and other anthrax reference laboratories. The procedure for the test is simply to streak a lawn on a blood or nutrient agar plate, or portion of a plate (several tests can be done on one plate) with the suspect organism and place a 10-15 µl drop of the phage suspension on one side of the streaked area and place a 10-unit penicillin disk to the other side. Allow the drop of phage suspension to soak in and incubate the plate at 37°C. A control culture should be included; the Sterne vaccine can be used for this. If the culture is B. anthracis, the area under the phage will be devoid of bacterial growth, due to lysis, and a clear zone will be seen around the penicillin disk after overnight incubation. (Note: phage-resistant B. anthracis isolates are encountered very occasionally; similarly, there are a few reports in the literature of penicillin-resistance.)
|
| | | Induction of the capsule is described above.
|
| | | . | Confirmation of virulence with the polymerase chain reaction
|
| | | Full confirmation of virulence can be carried out using the polymerase chain reaction (PCR). The following instructions are taken from ref. 14. Template DNA for PCR can be prepared from a fresh colony of B. anthracis on nutrient agar by resuspension of a loopful of growth in 25 µl sterile deionised (or distilled) water and heating to 95°C for 2 minutes. It is important that a fresh colony be used otherwise there may be some live bacteria in the lysate. Following cooling to approximately 4°C, and brief centrifugation, the supernatant can be used for the PCR reaction.
|
| | | Suitable primers (2, 6) for confirming the presence of the pX01 and pX02 plasmids are given in the table below.
|
Target
|
Primer ID
|
Sequence 5'-3'
|
Product size
|
Concentration
|
Protective antigen (PA)
|
PA 5
3048-3029
|
TCC-TAA-CAC-TAA-CGA-AGT-CG
|
596 bp
|
1 mM
|
|
PA 8
2452-2471
|
GAG-GTA-GAA-GGA-TAT-ACG-GT
|
|
|
Capsule
|
1234
1411-1430
|
CTG-AGC-CAT-TAA-TCG-ATA-TG
|
846 bp
|
0.2 mM
|
|
1301
2257-2238
|
TCC-CAC-TTA-CGT-AAT-CTG-AG
|
|
|
| | | PCR can be carried out in 50 µl volumes using the above primers, 200 µM each of dATP, dCTP, dTTP and dGTP, 1.5 mM MgCl2 and 2.5 units of amplitaq polymerase, all in NH4 buffer, followed by the addition of 5 µl of template DNA. A 2% agarose gel has been found to work best with these small fragments.
|
| | | Alternatively, 'Ready-To-GoT' beads are available from Pharmacia Biotech (Uppsala, Sweden, product number 27-9555-01). These are premixed, predispensed, dried beads, stable at room temperature, containing all the necessary reagents, except primer and template, for performing 25 µl PCR reactions. The template can be added in a 2.5 µl volume.
|
| | | The following PCR cycle can be used: 1 x 95°C for 5 minutes; 30 x 95°C for 0.5 minutes followed by 55°C for 0.5 minutes followed by 72°C for 0.5 minutes; 1 x 72°C for 5 minutes; cool to 4°C.
|
| | | It should be noted that, in use for some years now in an anthrax reference facility, the primers given in the table above have worked well in confirming the presence or absence of pXO1 and/or pXO2 in pure cultures of isolates from animal (including human) or environmental specimens or samples. They are unsuitable, however, for direct detection of B. anthracis in such specimens or samples. A choice of alternatives can be found in ref. 7. For the rare possibility that an isolate may lack both pXO1 and pXO2, a chromosomal marker should also be run; primers for these are also supplied in ref. 7.
|
| | b) | Agent identification from old, decomposed specimens, processed materials, and environmental samples, including soil
|
| | | These specimens more often than not have saprophytic contaminants that outgrow and obscure B. anthracis on nonselective agars. The following procedure is suggested:
|
| | | i) | The sample is blended in two volumes of sterile distilled or deionised water and placed in a water bath at 62.5 +/- 0.5°C for 15 minutes.
|
| | | ii) | Tenfold dilutions to 10-2 or 10-3 are then prepared. From each dilution, 10-100 µl are plated on to blood agar and 250-300 µl on to PLET agar (polymyxin, lysozyme, EDTA [ethylene diamine tetra-acetic acid], thallous acetate) (8, 14). All plates are incubated at 37°C.
|
| | | iii) | Blood agar plates are examined for typical colonies as previously described after overnight incubation, and the PLET plates are examined after 40-48 hours. Confirmation of the identity of suspect colonies as B. anthracis is done as described above.
|
| | | PLET medium (8, 14) is prepared by using heart-infusion agar base (DIFCO) made up to the manufacturer's instructions with the addition of 0.25-0.3 g/litre EDTA and 0.04 g/litre thallous acetate. (NOTE: thallous acetate is poisonous and should be handled with care.) The mixture is autoclaved and uniformly cooled to 50°C before adding the polymyxin at 30,000 units/litre and lysozyme at 300,000 units/litre. After mixing thoroughly, the agar is dispensed into Petri dishes.
|
| | | Reports of procedures for direct detection of B. anthracis in soils and other environmental specimens using the PCR are emerging. None of these has become routinely applicable at the present time.
|
| | | Animal inoculation may be considered for recovery of B. anthracis if all other methods fail. Examples of when this might occur are specimens from animals that received antibiotic therapy before death or environmental samples containing sporostatic chemicals. Due to the increasing concern to eliminate the use of animals for biological testing, this approach should be used as a last resort and only if justified. Adult mice or guinea-pigs are the animals of choice. If the samples involved are soils, the animals should be pretreated, the day before testing, with both tetanus and gas gangrene antiserum. The samples are prepared as described for culturing (Section B.1.a. above), including heat-shocking at 62.5°C for 15 minutes. Mice are injected subcutaneously with 0.05-0.1 ml; guinea-pigs are inoculated intramuscularly with up to 0.4 ml (0.2 ml in each thigh muscle). Any B. anthracis present will result in death in 48-72 hours and the organism can be cultured from the blood as described above.
|
| | c) | Immunological detection and diagnosis
|
| | | It needs to be borne in mind that B. anthracis is antigenically very closely related to B. cereus, which is an almost ubiquitous component of the environmental microflora. The only unshared antigens that lend themselves to differentiating these two species by immunological approaches are the anthrax toxin antigens, produced during the exponential phase of growth, and the capsule of B. anthracis. This places considerable constraints on the extent to which immunological methods can be used in routine detection methodology.
|
| | | . | Ascoli test (1)
|
| | | In 1911, Ascoli (1) published a procedure for the detection of thermostable anthrax antigen in animal tissue being used for by-products. This uses antiserum raised in rabbits to produce a precipitin reaction. The test lacks high specificity, in that the thermostable antigens of B. anthracis are shared by other Bacillus spp., and is dependent on the probability that only B. anthracis would proliferate throughout the animal and deposit sufficient antigen to give a positive reaction. Nowadays, it appears to be used in Eastern Europe only.
|
| | | To perform the Ascoli test, put approximately 2 g of sample in 5 ml of saline containing 1/100 final concentration of acetic acid and boil for 5 minutes. The resultant solution is cooled and filtered through filter paper. A few drops of rabbit antiserum (see preparation below) are placed in a small test tube. The filtrate from the previous step is gently layered over the top of the antiserum. A positive test is the formation of a visible precipitin band in less than 15 minutes. Positive and negative control specimen 7suspensions should be included.
|
| | | Antiserum is prepared in rabbits by the subcutaneous inoculation of Sterne anthrax vaccine on days 1 and 14. On days 28 and 35, the rabbits receive 0.5 ml of a mixture of several strains of virulent B. anthracis not exceeding 105 colony-forming units (CFU)/ml suspended in saline. Alternatively, the live virulent bacteria can be inactivated by prolonged suspension in 0.2% formalised saline, but the antigen mass needs to be increased to 108-109 CFU/ml. The suspension should be checked for inactivation of the B. anthracis before animal inoculation by culture of 0.1 ml into 100 ml of nutrient broth containing 0.1% histidine and, after incubation at 37°C for 7 days, subculture on to blood or nutrient agar. The dose regimen for the formalised suspension after initial vaccination on days 1 and 14 is increasing doses of 0.1, 0.5, 1, and 2 ml given intravenously at intervals of 4-5 days. Following either procedure, a test bleed at 10 days after the last injection should determine whether additional 2 ml doses should be administered to boost the precipitin titre.
|
| | | . | Immunofluorescence
|
| | | While some success has been achieved with immunofluorescence for capsule observation in the research situation (4), it does not lend itself to routine diagnosis.
|
| 2. | Serological tests
|
| | Historically, there has been little need for serological support for the diagnosis of anthrax in animals. Either the animal had anthrax, recognised from the recent history of the herd or site, and was treated accordingly, or it died. Most of the interest in developing serological testing has been for research on humoral responses in humans, and to a lesser extent in animals, for evaluating vaccines and for epidemiological studies involving naturally acquired seroconversion in humans, livestock and wild mammals.
|
| | Currently accepted as the best serological procedure is the enzyme-linked immunosorbent assay (ELISA) in microtitre plates coated with the protective antigen (PA) component of the anthrax toxin at 3-5 µg/ml in high pH (9.5) carbonate-coating buffer. The toxin antigens appear to be truly specific for B. anthracis, although there is at present no commercial source of these. This means that anthrax serology is currently confined to a few specialist laboratories. Various versions of the ELISA exist and can be found in standard laboratory manuals; any version will do for anthrax serology, although certain sera appear to be more 'sticky' than others. A useful tip is to use reconstituted dried milk as the blocking agent and to raise its concentration until control negative sera are giving reliable negative results. For bovine sera, this may be a 10% suspension or higher.
|
| | Examples of the successful field application of anthrax serology are given elsewhere (5, 11, 15).
|
| 3. | Hypersensitivity test (AnthraxinT)
|
| | In Central and Eastern Europe, a skin test using AnthraxinT (V.O. Medexport, 113461 Moscow, Russia), first licensed in the former USSR in 1962, has become widely used for retrospective diagnosis of human and animal anthrax and for vaccine evaluation (12). This is a commercially produced heat-stable protein/polysaccharide/nucleic acid complex, derived from oedematous fluid of animals injected with the vaccine STI or the Zenkowsky strains of B. anthracis. The test involves intradermal injection of 0.1 ml of Anthraxin and inspection after 24 hours for erythema and induration at the site lasting for 48 hours after the injection. This delayed-type hypersensitivity is seen as reflecting anthrax cell-mediated immunity and was the test reportedly capable of diagnosing anthrax retrospectively some 31 years after primary infection in up to 72% of cases. It was used with success in a retrospective investigation of a series of cases occurring in a spinning mill in Switzerland where synthetic fibres were combined with goat hair from Pakistan (10). The diagnostic reliability of Anthraxin, like Ascoli test antigen, depends on the nature of anthrax rather than on the specificity of the antigens involved.
|
C1. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS: VACCINES
The most widely used vaccine for prevention of anthrax in animals was developed by Sterne in 1937 (13). He derived a rough variant of virulent B. anthracis from culture on serum agar in an elevated CO2 atmosphere. This variant, named 34F2, was incapable of forming a capsule and was subsequently found to have lost the pX02 plasmid, which codes for capsule formation. It has become the most widely used strain world-wide for animal anthrax vaccine production. In Central and Eastern Europe, an equivalent pX02- derivative, Strain 55, is the active ingredient of the current livestock vaccine. A list of manufacturers of anthrax vaccine for use in animals is given in Appendix V of ref. 14.
The following information concerning preparation of the anthrax vaccine for use in animals is based on refs 9 and 16. Generalised procedures are given; national regulatory authorities should be consulted in relation to Standard Operating Procedures that may pertain locally.
| 1. | Seed management
|
| | a) | Characteristics of the seed
|
| | | Anthrax vaccine production is based on the seed-lot system. A seed lot is a quantity of spores of uniform composition processed at one time and maintained for the purpose of vaccine preparation. Each seed lot is no more than three passages from the parent culture and must produce a vaccine that is efficacious and safe for use in animals. It is recommended that a large seed lot be prepared from the parent strain and preserved by lyophilisation for future production lots. The parent culture can be purchased (National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom). The seed lot is acceptable for anthrax vaccine if a vaccine prepared from the seed lot or a suspension harvested from a culture derived from a seed lot meets the requirements for control of final bulk with respect to freedom from bacterial contamination, safety and efficacy (immunogenicity).
|
| | b) | Preparation of the master seed
|
| | | Seed lots are cultured on solid media formulated to promote sporulation of the organism (see Section C1.2. below). The solid medium formula given in ref. 9 is: 50 g tryptic digest of casein; 10 g yeast extract; 0.1 g CaCl2.6H2O; 0.01 g FeSO4.7H2O; 0.05 g MgSO4.7H2O; 0.03 g MnSO4.4H2O; 5.0 g K2HPO4; 1.0 g KH2PO4; 22 g agar; 1000 ml deionised or distilled water. The ingredients are dissolved in the water with the appropriate amount of heating; the solution is adjusted to pH 7.4, distributed into Roux bottles (120 ml per bottle) or other appropriate container, sterilised by autoclaving and cooled in the horizontal position. After the agar has solidified, excess liquid should be removed aseptically and the bottles left in an incubator (37°C) for at least 2 days to dry and to check them for sterility.
|
| | | Volumes of 2 ml of vaccine seed from a reference laboratory should be spread across the agar in Roux bottles, which should be incubated at 37°C until at least 80% sporulation is apparent by microscopic examination of aseptically extracted loopfuls (at least 72 hours). The growth is harvested with 10 ml per bottle of sterile deionised or distilled water and checked for purity. After washing three times in sterile deionised or distilled water with final suspension, also in sterile deionised or distilled water, sterilised lyophilisation stabiliser is added and the suspension is dispensed into lyophilisation vials and freeze-dried.
|
| | c) | Preparation and testing of the working seed
|
| | | Reconstitute a vial of seed stock and inoculate several slants (approximately 10 ml) of sporulation (casein digest) agar. Incubate at 37°C for 72 hours and store in a refrigerator. Test the slants for purity by culture on to nutrient agar plates and in nutrient broth (0.1 ml in 100 ml of nutrient broth). The latter should be subcultured on to nutrient agar after incubation at 37°C for 7 days and should be a pure culture of B. anthracis. A sample of the broth culture should also be checked for lack of motility.
|
| | | Volumes of seed needed for a production run should be calculated on the basis of harvesting the spores from each slant with 10 ml of sterile deionised or distilled water and using this to inoculate five Roux bottles.
|
| | d) | Safety of the seed lot
|
| | | Not less than 5 x 109 culturable spores should be injected subcutaneously into each of three healthy, 1-2-year-old, unvaccinated sheep, which must survive an observation period of at least 10 days.
|
| | e) | Immunogenicity of the seed lot
|
| | | At least 10 healthy guinea-pigs, 300-500 g in weight should be inoculated with 5 x 106 viable spores and observed for 21 days. At least 80% of the animals should survive. The immunised animals, together with three unimmunised controls, should then be challenged with 10 median lethal doses (LD50) of the strain 17 JB of B. anthracis. During a 10-day observation period, none of the immunised animals should succumb to the challenge while all the controls should die from anthrax. The test should be repeated if one of the immunised animals dies.
|
| 2. | Method of production
|
| | a) | Preparation of vaccine concentrate
|
| | | Roux bottles with casein digest agar are prepared as for the master seed in Section C1.1.b. above. One Roux bottle can be expected to yield about 2000 doses of vaccine. Each Roux bottle is inoculated with 2 ml of working seed suspension and incubated at 37°C with porous plugs for several days until small loopfuls of culture from randomly selected bottles show at least 90% of the organisms to be in sporulated forms when examined in wet mounts by phase contrast (phase bright spores) or following staining for spores. The growth from each bottle is then harvested with 20 ml of physiological saline. Tests for contaminants should be carried out by subculture to nutrient agar plates and inoculation of 100 ml nutrient broth with 0.1 ml of harvested spores followed by subculture to nutrient agar after 7 days at 37°C and by tests for motility. Acceptable harvests (i.e. those showing no evidence of contaminants) are pooled.
|
| | b) | Glycerination
|
| | | Twice the volume of sterile, pure, neutral glycerol should be added to the bulk pool. Saponin (0.1% final concentration) may also be added at this point if it is to be included as an adjuvant. Mix thoroughly (the inclusion of sterilised glass beads may be helpful). Carry out a purity test as before and hold for 3 weeks at ambient temperature to allow lysis of any vegetative bacteria, determine the viable spore count and store under refrigeration thereafter.
|
| | c) | Determining titre and dilution for use
|
| | | The number of culturable spores in the product is then calculated by spreading tenfold dilutions on nutrient agar plates. The suspension is diluted so that the final bulk contains the number of culturable spores desired. The diluent should contain the same proportions of saline, glycerol and (if being included) saponin as present in the vaccine concentrate. The vaccine should contain not less than 10 million viable spores per dose for cattle, buffaloes and horses, and not less than 5 million spores per dose for sheep, goats and pigs.
|
| | d) | Safety
|
| | | Safety testing is performed on two healthy sheep or goats and consists of inoculating subcutaneously twice the recommended vaccination dose. The animals are observed for 10 days. The final bulk passes the test if no systemic reactions develop and if not more than a transient oedema is observed at the injection site. If the test is carried out in sheep only, a progressive oedema indicates that the vaccine may be unsuitable for goats.
|
| | e) | Filling the containers
|
| | | Distribution of aliquots of vaccine into single and multidose containers is performed as outlined in World Health Organization Technical Report No. 363 series entitled General Requirements for Manufacturing Establishments and Control Laboratories (Requirements for Biological Substances No. 1), 1965, 16-17. Basically, the final bulk is distributed to containers in an aseptic manner in an area not used for production, and any contamination or alteration of the product must be avoided. The vaccine may be lyophilised after distribution into appropriate dosage containers. Containers are sealed as soon as possible with a material that is not detrimental to the product and that is capable of maintaining a hermetic seal for the life of the vaccine.
|
| 3. | In-process control
|
| | Purity tests consist of microscopic examination of stained smears with culture and motility tests as in Section C1.2.a.
|
| 4. | Batch control and tests on the final product
|
| | a) | Sterility
|
| | | The vaccine is a live culture of B. anthracis spores; sterility does not apply, but the batches must be tested for freedom from contamination (see Chapter I.1.5.).
|
| | b) | Efficacy
|
| | | Efficacy or immunogenicity is tested on the final bulk as follows: at least ten healthy 300-500 g guinea-pigs are inoculated with a sheep dose of the vaccine. The guinea-pigs are observed for 21 days, and at least 80% of the animals must survive the observation period. Surviving immunised guinea-pigs and three nonvaccinated controls are challenged with an appropriate dose of virulent B. anthracis. A recommended challenge is 200 LD50 of the Pasteur II strain (17JB), which is available from the same source as the Sterne 34F2 vaccine strain. If, by 10 days after challenge, all vaccinated guinea-pigs survive and control animals die, the final bulk is deemed to be satisfactory. If any vaccinated animals die during the post challenge observation period from a cause other than anthrax, and death is not associated with the vaccine, the test may be repeated.
|
| | c) | Dose
|
| | | The recommended dose for cattle and horses is a minimum of 10 x 106 culturable spores; for sheep, goats and pigs, it is 1-5 x 106 culturable spores. The vaccine should contain these spores in an appropriate volume, e.g. 1 x 107/ml.
|
| | d) | Duration of immunity
|
| | | Most experts agree that immunity is good for at least 1 year and it is recommended that an annual booster be given. Horses may be slow to develop immunity following initial vaccination; some manufacturers therefore recommend a two-dose initial vaccination, administered 1 month apart, followed by a single annual booster.
|
| | e) | Stability
|
| | | As there is no generally acceptable test for stability of anthrax vaccines, it is recommended that, in each filling lot, the number of culturable spores be determined before and after holding at an appropriate temperature for an appropriate period. There should be no evidence of a fall in the number of culturable spores.
|
| | f) | Preservatives and storage
|
| | | Bacillus anthracis spores are stable in unlyophilised or lyophilised vaccine and preservatives are not required. Storage under refrigeration is recommended (4°C).
|
| | g) | Precautions (hazards)
|
| | | The vaccine has been shown to cause disease in some goats and llamas; this may be related to the saponin adjuvant. The vaccine is not recommended for use in pregnant animals, nor in animals destined for slaughter within 2-3 weeks of vaccination. Local regulations may specify other time periods in some countries or regions, but there is no scientific reason for regarding meat from clinically healthy animals as unfit for human handling or consumption after a holding period of 2 weeks following vaccination. Concurrent administration of antibiotics to vaccinated animals is contraindicated as the antibiotic will interfere with the vaccine. Antibiotics should not be given for several days before and after vaccination. Left over vaccine, empty vials, and equipment used for vaccinating are contaminated with the live spores and should be autoclaved, disinfected, or incinerated. Accidental human inoculation is treated by expressing as much of the inoculum as possible from the injection site and washing the wound thoroughly with soap and water. Medical attention should be sought if infection develops.
|
| 5. | Tests on the final product
|
| | a) | Safety
|
| | | Every batch of vaccine will be tested for safety as described in Section C1.2.d.
|
| | b) | Potency
|
| | | Every batch of vaccine will be tested for potency, as described in Section C1.4.b.
|
C2. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS: DIAGNOSTIC BIOLOGICALS
| . | Propagation of the diagnostic 'gamma' bacteriophage
|
| | 1. | Prepare a 'lawn' of growth of attenuated B. anthracis on 150 x 15 mm Mueller-Hinton plates with 5% sheep blood.
|
| | 2. | Incubate for 4-6 hours at 37°C, if there is sufficient vegetative growth (heavy and visible to the naked eye) then seed the growth with phage. If the growth is thin, then incubate overnight before seeding.
|
| | 3. | The seeding is done using a sterile transfer pipette. Approximately 2 ml of the stock gamma bacteriophage is dropped over the surface of the vegetative growth. The plate is tilted to allow the phage to cover the whole area of growth. This may be repeated until the whole surface of the vegetative growth has been covered with phage.
|
| | 4. | Incubate the plates at 37°C overnight.
|
| | 5. | Place the incubated plates in a 20°C freezer overnight.
|
| | 6. | The next day remove the plates from the freezer and allow to thaw for 2 hours.
|
| | 7. | Pour off the brownish red liquid and pre-filter it through Whatman No. 3 filter paper.
|
| | 8. | Carry out a final sterile filtration into a sterile storage vessel using a 0.22 µ filter.
|
| | 9. | To confirm the potency of the phage do a serial dilution 1/1, 1/10, through 1/10,000 and test with B. anthracis for susceptibility.
|
| | 10. | Store the phage in a refrigerator at 2-8°C. Do not freeze.
|
REFERENCES
| 1. | Ascoli A. (1911). Die Präzipitindiagnose bei Milzbrand. Centralbl. Bakt. Parasit. Infeckt., 58, 63-70.
|
| 2. | Beyer W., Glockner P., Otto J. & Bohm R. (1996). A nested PCR and DNA-amplification-fingerprinting method for detection and identification of Bacillus anthracis in soil samples from former tanneries. Salisbury Med. Bull., No. 87, Special Suppl., 47-49.
|
| 3. | Brown E.R. & Cherry W.B. (1955). Specific identification of Bacillus anthracis by means of a variant bacteriophage. J. Infect. Dis., 96, 34-39.
|
| 4. | Ezzell J.W. & Abshire T.G. (1996). Encapsulation of Bacillus anthracis spores and spore identification. Salisbury Med. Bull., No 87, Special Suppl., 42.
|
| 5. | Harrison L.H., Ezzell J.W., Abshire T.G., Kidd S. & Kaufmann A.F. (1989). Evaluation of serologic tests for diagnosis of anthrax after an outbreak of cutaneous anthrax in Paraguay. J. Infect. Dis., 160, 706-710.
|
| 6. | Hutson R.A., Duggleby C.J., Lowe J.R., Manchee R.J. & Turnbull P.C.B. (1993). The development and assessment of DNA and oligonucleotide probes for the specific detection of Bacillus anthracis. J. Appl. Bacteriol., 75, 463-472.
|
| 7. | Jackson P.J., Hugh-Jones M.E., Adair D.M., Green G., Hill K.K., Kuske C.R., Grinberg L.M., Abramova, F.A. & Keim P. (1998). PCR analysis of tissue samples from the 1979 Sverdlovsk anthrax victims: The presence of multiple Bacillus anthracis strains in different victims. Proc. Natl Acad. Sci. USA, 95, 1224-1229.
|
| 8. | Knisely R.F. (1966). Selective medium for Bacillus anthracis. J. Bacteriol., 92, 784-786.
|
| 9. | Misra R.P. (1991). Manual for the Production of Anthrax and Blackleg Vaccines. Food and Agriculture Organisation of the United Nations (FAO) Animal Production and Health Paper 87, FAO, Rome, Italy.
|
| 10. | Pfisterer R.M. (1990). Retrospective verification of the diagnosis of anthrax by means of the intracutaneous skin test with the Russian allergen 'Anthraxin' in a recent epidemic in Switzerland. Salisbury Med. Bull., No. 68, Special Suppl., 80.
|
| 11. | Redmond C., Hall G.A., Turnbull P.C.B. & Gillgan J.S. (1996). Experimentally assessed public health risks associated with pigs from farms experiencing anthrax. Vet. Rec., 141, 244-247.
|
| 12. | Shlyakhove E., Rubinstein E. & Novikov I. (1997). Anthrax post-vaccinal cell-mediated immunity in humans: kinetics pattern. Vaccine, 15, 631-636.
|
| 13. | Sterne M. (1937). The effect of different carbon dioxide concentrations on the growth of virulent anthrax strains. Onderstepoort J. Vet. Sci. Anim. Ind., 9, 49-67.
|
| 14. | Turnbull P.C.B., Boehm R., Cosivi O., Doganay M., Hugh-Jones M.E., Lalitha M.K. & De Vos V. (1998). Guidelines for the Surveillance and Control of Anthrax in Humans and Animals. WHO/EMC/ZDI/98.6. World Health Organization, Geneva, Switzerland.
|
| 15. | Turnbull P.C.B., Doganay M., Lindeque P.M., Aygen B. & McLaughlin J. (1992). Serology and anthrax in humans, livestock and Etosha National Park wildlife. Epidemiol. Infect., 108, 299-313.
|
| 16. | World Health Organization Expert Committee on Biological Standardization (1967). Requirements for Anthrax Spore Vaccine (Live - for Veterinary Use) (Requirements for Bioiogical Substances No. 13). World Health Organization (WHO) Technical Report Series No. 361. WHO, Geneva, Switzerland.
|
FURTHER READING
| A. | Logan N.A. & Turnbull P.C.B. (1998). Bacillus and recently derived genera. In: Manual of Clinical Microbiology, Seventh Edition, Murray P.R., Baron E.J., Pfaller M.A., Tenover F.C. & Yolken R.H., eds. American Society for Microbiology, Washington DC, USA, 357-369.
|
| B. | Quinn C.P. & Turnbull P.C.B. (1998). Anthrax. In: Topley & Wilson's Microbiology and Microbial Infections, Vol. 3, Ninth Edition, Collier L., Balows A. & Sussman M., eds. Arnold, London, UK, 799-818.
|
| C. | Turnbull P.C.B., (ed.) (1996). Proceedings of the International Workshop on Anthrax. Salisbury Med. Bull., Special Supple. No. 87, 139 pages.
|
| D. | Turnbull P.C.B. (1998). Anthrax. In: Zoonoses. Biology, Clinical Practice, and Public Health Control. Palmer S.R., Soulsby E.J.L. & Simpson D.I.H., eds. Oxford University Press, Oxford, UK, 3-16.
|
| E. | Whitford H.W. & Hugh-Jones M.E. (1994). Anthrax. In: Handbook Series in Zoonoses, Second Edition, Beran G.W., editor-in-chief. CRC Press, Boca Raton, Florida, USA, 61-82.
|
*
* *
NB: There are OIE Reference Laboratories for Anthrax (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).
---------------------------------------------
[top]
Contact : scientific.dept@oie.int
|