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SUMMARY
Paratuberculosis (Johne's disease) is a chronic enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) (34).
Identification of the agent: The diagnosis of paratuberculosis is divided into two parts: the diagnosis of clinical disease and the detection of subclinical infection. The latter is essential for control of the disease at the farm, national or international level.
Diagnosis of paratuberculosis is made on clinical grounds confirmed by the demonstration of M. paratuberculosis in the faeces by microscopy, culture, or by the use of DNA probes and the polymerase chain reaction. Diagnosis is made at necropsy by the finding of the pathognomonic lesions of the disease in the intestines, either grossly with the demonstration of typical acid-fast organisms in impression smears of the lesions or histologically, and by isolation of M. paratuberculosis in culture.
The detection of subclinical infection depends on the detection of specific antibodies by serology, or culture of M. paratuberculosis from faeces or tissues collected at necrosy, or the demonstration of cell-mediated response through the use of the gamma interferon assay. The choice of test depends on the circumstances and the degree of sensitivity required at individual animal or herd level.
Cultures of M. paratuberculosis may be obtained from faeces or tissues, after treatment to eliminate contaminants, by inoculation into artificial media with and without the specific growth factor - mycobactin - that is essential for the growth of M. paratuberculosis.
Serological tests: The single largest problem in paratuberculosis control is the difficulty of detecting subclinically infected ruminants. The serological tests commonly used for paratuberculosis in cattle are complement fixation (CF), absorbed enzyme-linked immunosorbent assay (ELISA) and gel immunodiffusion. Sensitivity and specificity are often determined by reference to results of faecal culture, which itself has unknown sensitivity in subclinically infected cattle. When used to confirm diagnosis of paratuberculosis in cows with typical clinical signs, some tests, for example CF and absorbed ELISA, perform very well.
Requirements for vaccines and diagnostic biologicals: Vaccines for paratuberculosis may be live attenuated or killed bacteria either incorporated with an adjuvant or lyophilised and adjuvanted on reconstitution. Bacterial counting is difficult and bacterial content of vaccines may be based on weight, while vaccine potency may be judged by batch tests for sensitising ability in guinea-pigs.
Vaccine safety or abnormal toxicity may also be tested in guinea-pigs.
For diagnostic skin tests, Johnin and avian tuberculin are purified protein derivatives (PPD) of a heat-treated culture of M. paratuberculosis or M. avium, respectively. Johnin is standardised for content of PPD by chemical assay and its biological activity is identified in guinea-pigs sensitised with M. paratuberculosis. Avian tuberculin activity is determined in guinea-pigs sensitised with M. avium by comparison with a reference preparation calibrated in international units.
A. INTRODUCTION
Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is an organism first observed by Johne & Frothingham in 1895. Mycobacterium paratuberculosis causes paratuberculosis or Johne's disease, an intestinal granulomatous infection. First recognised in cattle, then in sheep and later in goats, paratuberculosis is found most often among domestic and wild ruminants and has a global distribution. The disease has also been reported in horses, pigs, deer and alpaca, and recently in rabbits, stoat, fox and weasel (3, 10). Under natural conditions, the disease in cattle spreads by ingestion of M. paratuberculosis from the contaminated environment. The disease persists after the introduction of infected animals. Infection can be spread vertically to the fetus (16) and semen can be infected with the organism (31). The primary source of infection in calves is milk from infected cows or milk that is contaminated with the faeces of diseased cattle.
The identification of M. paratuberculosis is based on its mycobactin requirement and its pathogenicity in the host. Mycobactin dependence has long been used as a taxonomic characteristic for M. paratuberculosis because most mycobacteria are able to make mycobactin for themselves. Mycobacterium paratuberculosis, M. silvaticum and some primary isolates of M. avium lack this capacity, however, and require mycobactin to grow in the laboratory. Thus, the mycobactin requirement is not confined to M. paratuberculosis; this characteristic exists to various degrees within the M. avium group (33).
Clinical signs of paratuberculosis are a slowly progressive wasting and diarrhoea, which is intermittent at first, becoming progressively more severe until it is constantly present in bovines (9). Diarrhoea is less common in small ruminants.
Early lesions occur in the walls of the small intestine and the draining mesenteric lymph nodes, and infection is confined to these sites at this stage. As the disease progresses, gross lesions occur in the ileum, jejunum, terminal small intestine, caecum and colon, and in the mesenteric lymph nodes. Mycobacterium paratuberculosis is present in the lesions and, terminally, throughout the body. The intestinal lesions are responsible for a protein leak and a protein malabsorption syndrome, which lead to muscular wasting. Clinical signs usually first appear in young adulthood, but the disease can occur in animals at any age over 1-2 years.
Within a few weeks of infection, a phase of multiplication of M. paratuberculosis begins in the walls of the small intestine. Depending on the resistance of the individual, this infection is eliminated or the animal remains infected as a healthy carrier. The proportion of animals in these categories is unknown. A later phase of multiplication of the organisms in a proportion of carriers leads to the extension of lesions, interference with gut metabolism and clinical signs of disease. Subclinical carriers excrete variable numbers of M. paratuberculosis in the faeces. In most cases larger numbers of organisms are excreted as clinical disease develops.
Delayed-type hypersensitivity (DTH) is detectable early in the infection and remains present in a proportion of the subclinically infected carriers, but as the disease progresses, DTH wanes and may be absent in clinical cases. Serum antibodies are detectable later than DTH. They may also be present in carriers that have recovered from infection. Serum antibodies are present more constantly and are of higher titre as lesions become more extensive, reflecting the amount of antigen present. In sheep, there may be a detectable serological response in clinical cases.
Other mycobacterial diseases and infections, including mammalian and avian tuberculosis, cause DTH and the presence of serum antibodies. It follows therefore that these diseases need to be differentiated from paratuberculosis and M. paratuberculosis infection, both clinically and by the use of specific diagnostic tests. Exposure to environmental saprophytic mycobacteria may also sensitise livestock, resulting in nonspecific DTH reactions.
Animals vaccinated against paratuberculosis develop both DTH and serum antibodies. Vaccination is an aid to the prevention of clinical disease, but does not necessarily prevent infection. It also interferes with programmes for the diagnosis and control of bovine tuberculosis. Thus, if it is necessary to attempt a diagnosis of infection in vaccinates, only tests to detect M. paratuberculosis in the faeces can be used (14).
In individual animals, especially from a farm in which the disease has not previously been diagnosed, a tentative clinical diagnosis must be confirmed by laboratory tests. However, a definitive diagnosis may be warranted on clinical grounds alone if the clinical signs are typical and the disease is known to be present in the herd. Confirmation of paratuberculosis depends on the finding of either gross lesions with the demonstration of typical acid-fast organisms in impression smears or microscopic pathognomonic lesions and the isolation in culture of M. paratuberculosis.
B. DIAGNOSTIC TECHNIQUES
To diagnose the presence of paratuberculosis in an individual clinically suspect animal, a number of laboratory tests can be used including: faecal smears, faecal and tissue culture, DNA probes using faeces or tissues, serology, necropsy and histology.
Herd tests to detect subclinical infection are carried out to determine the prevalence of the infection, usually so that control measures can be instituted. As no test is 100% sensitive or specific, control of the disease by the disposal of positive reactors depends on repeated tests at 6-month or yearly intervals over a number of years and the elimination of reactors to serological tests or faecal shedders; the removal of offspring from female reactors is also considered to be prudent. Even these procedures are not always successful without changes in hygiene and livestock management to reduce the transmission of infection within a herd (2).
| 1. | Identification of the agent
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| | a) | Necropsy
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| | | Paratuberculosis cannot be diagnosed on superficial examination of the intestines for signs of thickening. The intestines should be opened from the duodenum to the rectum to expose the mucosa. There is not a close correlation between the severity of clinical signs and the extent of intestinal lesions. The mucosa, especially of the terminal ileum, is inspected for pathognomonic thickening and corrugation. Early lesions are seen by holding the intestine up to the light, when discrete plaques can be visualised. The mesenteric lymph nodes may be enlarged and oedematous. Smears from the affected mucosa and cut surfaces of lymph nodes should be stained by Ziehl-Neelsen's method and examined microscopically for acid-fast organisms that have the morphological characteristics of M. paratuberculosis. However, acid-fast organisms are not present in all cases. Diagnosis is therefore best confirmed by the collection of multiple intestinal wall and mesenteric lymph node samples into fixative (10% formol saline) for subsequent histology. Both haematoxylin-and-eosin-stained sections and Ziehl-Neelsen-stained sections should be examined. The pathognomonic lesions consist of infiltration of the lamina propria, Peyer's patches and the cortex of the mesenteric lymph nodes with large, pale-staining epithelioid cells and multinucleated Langhans' giant cells, in both of which clumps or singly disposed acid-fast bacilli are usually, but not invariably, found. Langhans' giant cells are not uncommon and contain few organisms.
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| | | Lesions in sheep and goats are similar to those observed in cattle. There is often only slight thickening and inflammation of the mucosa, but nodules of caseation and calcification sometimes form in the intestine and in associated lymph nodes. Enlargement of the mesenteric lymph nodes is seen in alpaca. Sometimes the pigmented form of paratuberculosis is observed in sheep.
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| | b) | Bacteriology (microscopy)
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| | | Ziehl-Neelsen-stained smears of faeces are examined microscopically. A diagnosis of paratuberculosis can be made if clumps (three or more organisms) of small (0.5-1.5 µm), strongly acid-fast bacilli are found. The presence of single acid-fast bacilli in the absence of clumps does not indicate a definitive diagnosis. The disadvantage of this test are that only about one-third of cases can be confirmed on microscopic examination of a single faecal sample.
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| | c) | Bacteriology (culture)
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| | | Mycobacterium paratuberculosis infection mainly involves the lower small intestine and adjacent caecum. Mycobacterium paratuberculosis organisms are vastly outnumbered by other bacteria in faecal and intestinal tissue specimens.
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| | | Primary colonies of M. paratuberculosis may be expected to appear any time from 5 to 14 weeks after inoculation. Primary colonies on Herrold's medium containing mycobactin are very small (1 mm in diameter), colourless, translucent and hemispherical. (Mycobactin can be obtained commercially (mycobactin J) from Allied Monitor, P.O. Box 71, 201 Golden Drive, Fayette, MO 65248, United States of America, or Symbiotic Society, 299 av. Jean Jaurès, 69007 Lyon, France.) Their margins are round and even, and their surfaces are smooth and glistening. The colonies become more opaque and increase in size (4 or 5 mm) as incubation continues. The colonial morphology changes with age from smooth to rough, and from hemispherical to mammilate (32).
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| | | The uncommon, bright yellow pigmented sheep strain is difficult to grow on artificial media. It has been reported that unpigmented sheep strains grow less well than cattle strains, and no cultures should be discarded as negative without prolonged incubation.
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| | | For identification of M. paratuberculosis, small inocula of suspect colonies should be subcultured on the same medium with and without mycobactin, to demonstrate mycobactin dependency. (The test is not reliable if large numbers of bacilli are present.)
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| | | There are two basic methods in use for the culture of M. paratuberculosis from clinical specimens: the method using oxalic acid and NaOH for decontamination and Löwenstein-Jensen medium for growth, and the method using hexadecylpyridinium chloride (HPC) for decontamination in combination with Herrolds's egg yolk medium (HEYM) for growth. Both media contain mycobactin.
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| | | . | Media
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| | | Examples of suitable media are:
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| | | i) | Herrold's egg yolk medium with mycobactin (19)
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| | | | For 1 litre of medium: 9 g peptone; 4.5 g sodium chloride; 2.7 g beef extract; 27 ml glycerol; 4.1 g sodium pyruvate; 15.3 g agar; 2 mg mycobactin; 870 ml distilled water; six egg yolks (120 ml); and 5.1 ml of a 2% aqueous solution of malachite green. Measure the first six ingredients and dissolve by heating in distilled water. Adjust the pH of the liquid medium to 6.9-7.0 using 4% NaOH, and test to ensure the pH of the solid phase is 7.2-7.3. Add the mycobactin dissolved in 4 ml ethyl alcohol. Autoclave at 121°C for 25 minutes. Cool to 56°C and aseptically add six sterile egg yolks and sterile malachite green solution. Blend gently and dispense into sterile tubes. (Use fresh eggs not more than 2 days old from a flock that is not receiving antibiotics. With a brush, scrub the eggs with water containing a detergent. Rinse with water and place the eggs in 70° alcohol for 30 minutes. Dry by inserting between two sterile towels. With sterile rat-tooth forceps, crack one end of the eggshell, making a hole of approximately 10 mm, and remove the egg white with the forceps and gravity. Make the hole larger and break the yolk. Mix the egg yolk by twirling the forceps, and remove the yolk sac. Pour the mixed egg yolk into media.)
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| | | | It is permissible to add 50 mg chloramphenicol, 100,000 U penicillin and 50 mg amphotericin B.
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| | | ii) | Modified Dubos's medium (28)
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| | | | For 1 litre of medium: 2.5 g Difco casamino acids; 0.3 g asparagine; 2.5 g anhydrous disodium hydrogen phosphate; 1 g potassium dihydrogen phosphate; 1.5 g sodium citrate; 0.6 g crystalline magnesium sulphate; 25 ml glycerol; 50 ml of a 1% solution of Tween 80; and 15 g agar. Dissolve each salt in distilled water with minimum heat and make up to 800 ml. Add mycobactin in alcoholic solution at 0.05% (2 mg dissolved in 4 ml ethyl alcohol), heat the medium to 100°C by free-steaming, and then sterilise by autoclaving at 115°C for 15 minutes. Cool to 56°C in a water bath, add antibiotics (100,000 U penicillin; 50 mg chloramphenicol; and 50 mg amphotericin B) and serum (200 ml of bovine serum sterilised by filtering through a Seitz 'EX' pad and inactivated by heat at 56°C for 1 hour). The medium is kept thoroughly mixed and then dispensed into sterile tubes. An advantage of this medium is that it is transparent, which facilitates the early detection of colonies.
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| | | iii) | Middlebrook 7H9, 7H10 and 7H11 media (Difco), and 7H12 Bactec medium enhanced with mycobactin in the same proportion as for Herrold's medium can also be used. The advantage of this media is that it is transparent, which facilitates the early detection of colonies.
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| | | iv) | Löwenstein-Jensen medium with or without mycobactin (13).
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| | | . | Sample preparation
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| | | Although faecal culture is technically difficult and time-consuming to carry out, it is still a good method for diagnosis of paratuberculosis in live animals. It is the only test that does not produce false-positive results (100% specificity). It will detect infected animals 6 months or more before they develop clinical signs, and during the clinical stage its sensitivity approaches 100%.
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| | | . | Processing tissue specimens
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| | | | Chemical preservatives should not be used. The tissues can be frozen ay -20°C.
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| | | | To avoid contamination, the faeces should be rinsed from portions of intestinal tract before shipment to the laboratory.
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| | | i) | Digestion of tissues
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| | | | Approximately 4 g of mucosa from the ileocaecal valve or 4 g of mesenteric node are placed in a sterile blender jar containing 50 ml of trypsin (2.5%). The mixture is adjusted to neutrality using 4% NaOH and pH paper, and stirred for 30 minutes at room temperature on a magnetic mixer. The digested mixture is filtered through gauze. The filtrate is centrifuged at approximately 2000-3000 g for 30 minutes. The supernatant fluid is poured off and discarded.
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| | | ii) | Decontamination of inoculum
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| | | | The sediment is resuspended in 20 ml of 0.75% HPC and allowed to stand undisturbed for 18 hours at room temperature. The particles that settle to the bottom of the tube are to be used as the inoculum and are removed by pipette without disturbing the supernatant fluid. Other methods of decontamination can be used, such as treatment with 5% oxalic acid.
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| | | iii) | Inoculation of culture media and incubation
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| | | | Approximately 0.1 ml of inoculum is transferred to each of three slants of Herrold's medium containing mycobactin and to one slant of Herrold's medium without mycobactin. The inoculum is distributed evenly over the surface of the slants. The tubes are allowed to remain in a slanted position at 37°C for approximately 1 week with screw caps loose.
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| | | | The tubes are returned to a vertical position when the free moisture has evaporated from the slants. The lids are tightened and the tubes are placed in baskets in an incubator at 37°C.
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| | | | The egg in Herrold's medium contributes sufficient phospholipids to neutralise the bactericidal activity of residual HPC in the inoculum. The other media (Modified Dubos and Middlebrook) do not have this property. Other treatments can be used for sample decontamination, for example oxalic acid at 5%.
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| | | | HPC is relatively ineffective in controlling the growth of contaminating fungi. Amphotericin B (fungizone) was found by Merkal & Richards (22) to control effectively fungal overgrowth of inoculated media. Fungizone may be incorporated in the Herrold's medium at a final concentration of 50 µg per ml of medium. Due to loss of antifungal activity, storage of Herrold's medium containing fungizone should be limited to 1 month at 4°C.
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| | | | The slants are incubated for 15-20 weeks and observed weekly from the sixth week onwards.
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| | | . | Processing faecal specimens
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| | | | No refrigerant or chemical preservative is used. The faecal specimens can be frozen at -70°C.
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| | | i) | Suspension and decontamination of faeces
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| | | | 1 g of faeces is transferred to a 50 ml tube containing 20 ml of sterile distilled water. The mixture is shaken for 30 minutes at room temperature. The larger particles are allowed to settle for 30 minutes. The uppermost 5 ml of faeces suspension is transferred to a 50 ml tube containing 20 ml of HPC. The tube is inverted several times to assure uniform distribution and allowed to stand undisturbed for 18 hours at room temperature.
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| | | ii) | Inoculation of culture media
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| | | | 0.1 ml of the undisturbed sediment is transferred to each of four slants of Herrold's medium, three with mycobactin and one without mycobactin. A smear may be made from the sediment and stained by the Ziehl-Neelsen method.
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| | | iii) | Incubation and observation of slants
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| | | | The same as for tissue specimens.
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| | | Variations in the above methods have been described (4, 15, 21, 24, 27, 38, 39). The sensitivity of culture may be enhanced using liquid media and with centrifugation rather than sedimentation techniques. The double incubation method assists with decontamination of the inoculum (30).
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| | | A more rapid technique for the isolation of M. paratuberculosis employs the use of a radiometric-based detection system, the Bactec 460. Growth of mycobacteria is measured by the release of 14CO2 from palmitate as a consequence of bacterial metabolism. However, as this system is radiometrically based, it is not feasible for use in some laboratories and has been phased out in others. Recently three other fluorescence-based rapid methods were introduced, the Bactec 960 system, the MGIT (Mycobacterial Growth Indicator Tube) system (Becton Dickinson) and the MBBact system (Organon Technica). Severe problems were encountered during initial experiments in which these methods were tested on faecal samples due to overgrowth by other bacteria (spore forms and fungi); however, these methods have been further developed and are now used with some success in many laboratories.
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| | d) | DNA probes
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| | | DNA probes are being developed that offer a means of detecting M. paratuberculosis in diagnostic samples and of rapidly identifying bacterial isolates (8, 20). They have been used to distinguish between M. paratuberculosis and other mycobacteria, especially those of the M. avium group.
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| | | McFadden et al. have identified a sequence (17, 18), termed IS900, which is an insertion sequence specific for M. paratuberculosis. It has been reported that a small number of isolates other than M. paratuberculosis have produced amplified products the same size as expected from M. paratuberculosis. A restriction enzyme digest may be applied to positive IS900 products to confirm that their sequence is consistent with M. paratuberculosis (5). The use of IS900 as a DNA probe for specific identification of M. paratuberculosis in faecal samples from cattle by enzymatic amplification of DNA using the polymerase chain reaction (PCR) has been reported (36). A commercial diagnostic test based on the detection of IS900 sequences following isolation of mycobacteria from faecal samples and enrichment of a DNA fraction from IS900 sequences by PCR has been developed. The test is available in kit form suitable for use in laboratories (IDEXX DNA Probe test kit for the detection of Mycobaterium paratuberculosis or the PCR Adiavet Paratb kit). It has poor diagnostic sensitivity compared with faecal culture. Another kit, PCR Adiavet Paratb, is now available (IDEXX DNA Probe test kit for the detection of Mycobaterium paratuberculosis or the PCR Adiavet Paratb kit).
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| 2. | Serological tests
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| | The serological tests commonly used for paratuberculosis in cattle are complement fixation (CF), enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) (29) corresponding to humoral immunity, and the gamma interferon assay corresponding to cellular immunity.
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| | a) | Complement fixation test
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| | | The CF test has been the standard test used for cattle for many years. The CF test works well on clinically suspect animals, but does not have sufficient specificity to enable its use in the general population for control purposes. Nevertheless, it is often demanded by countries that import cattle. A variety of CF test procedures are used internationally. An example of a microtitre method for performing the CF test is as follows:
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| | | i) | The antigen is an aqueous extract of bacteria from which lipid has been removed (strain M. paratuberculosis 316F). Mycobacterium avium D9 may also be used.
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| | | ii) | All sera are inactivated in the water bath at 60°C for 30 minutes and diluted at 1/4, 1/8 and 1/16. A positive control serum and a negative control serum should be included on each plate. The following controls are also prepared: antigen control, complement control and haemolytic system control.
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| | | iii) | Reconstituted, freeze-dried complement is diluted to contain six times H50 (50% haemolysing dose) as calculated by titration against the antigen.
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| | | iv) | Sheep erythrocytes, 2.5%, are sensitised with 2 units of H100 haemolysin.
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| | | v) | All dilutions and reagents are prepared in calcium/magnesium veronal buffer; 25 µl volumes of each reagent are used in 96-well round-bottom microtitration plates.
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| | | vi) | Primary incubation is at 4°C overnight and secondary incubation is at 37°C for 30 minutes.
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| | | vii) | Reading and interpreting the results: Plates may be left to settle or centrifuged and read as follows: 4+ = 100% fixation, 3+ = 75% fixation, 2+ = 50% fixation, 1+ = 25% fixation and 0 = complete haemolysis. The titre of test sera is given as the reciprocal of the highest dilution of serum giving 50% fixation. A reaction of 2+ at 1/8 is regarded as positive. Results should be interpreted in relation to clinical signs and other laboratory findings.
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| | b) | Enzyme-linked immunosorbent assay
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| | | The ELISA is, at present, the most sensitive and specific test for serum antibodies to M. paratuberculosis. Its sensitivity is comparable with that of the CF test in clinical cases, but is greater than that of the CF test in subclinically infected carriers. The specificity of the ELISA is increased by M. phlei absorption of sera.
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| | | The absorbed ELISA, designed by Yokomizo et al. (43, 44) and modified by Milner et al. (23), was developed into a commercial kit by Cox et al. (6). This kit was evaluated by Ridge et al. (25), and was found to have a sensitivity in clinical cases of 88.3%, and in subclinical cases of 48.8%, and a specificity of 99.8% in cattle, and in sheep a sensitivity of 35-54% and a specificity of 98.2-98.5%. However, other workers have found a lower sensitivity and specificity (40).
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| | | The absorbed ELISA combines the sensitivity of ELISA with the added specificity of an absorption step. Sera to be tested are diluted with buffer containing soluble M. phlei antigen prior to testing in an indirect ELISA. This procedure eliminates nonspecific cross-reacting antibodies. In early versions, sera were absorbed with whole M. phlei, which were removed by centrifugation prior to testing.
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| | | A microtitre plate format has been developed in which M. paratuberculosis antigen is coated on to 96-well plates. Samples are diluted in sample diluent containing M. phlei to remove cross-reacting antibodies. On incubation of the diluted sample in the coated well, antibody specific to M. paratuberculosis forms a complex with the coated antigens. After washing away unbound materials from the wells, horseradish peroxidase (HPRO)-labelled anti-bovine immunoglobulin is added. This reacts with immunoglobulins bound to the solid-phase antigen. The rate of conversion of substrate is proportional to the amount of bound immunoglobulin. Subsequent colour, measured (at 450 nm) spectrophotometrically is proportional to the amount of antibody present in the test sample.
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| | | The antigen used to coat the ELISA plates is available commercially.
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| | | An anti-bovine IgG labelled with HRPO is used as conjugate. The substrate chromogen solution is hydrogen peroxide tetramethyl benzidine. A solution of 0.5 M H2SO4 is used to stop the reaction when the absorbance of the positive control serum reaches a predetermined point.
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| | | Several absorbed ELISA kits are commercially available. The method and test materials needed, the interpretation of the results and calculations are fully described in the instructions accompanying the commercial kit. The procedure is the same as for any routine ELISA. Screening tests are usually performed in one well per serum, but are unreliable, and diagnostic tests should be performed in duplicate wells.
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| | c) | Agar gel immunodiffusion test
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| | | The AGID test is useful for the confirmation of the disease in clinically suspect cattle, sheep and goats (26). Several variations of the method are in use.
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| | | The antigen employed is a crude protoplasmic extract of laboratory strain M. avium 18 (formally M. paratuberculosis 18) prepared by disruption of cells in a hydraulic press cell fractionator. Disrupted cells are centrifuged at 40,000 g for 2 hours to remove cell wall debris, and the supernatant fraction is retained and lyophilised. This antigen is resuspended in water at a concentration of 10 mg/ml.
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| | | Agarose is dissolved in barbital buffer, pH 8.6, containing sodium azide, to give a final agarose concentration of 0.75%. Agarose may be poured into Petri dishes or on to glass slides. Wells are cut in a hexagonal pattern. Wells are 4 mm in diameter, 4 mm apart, and the agar should be 3-4 mm deep. Antigen is added to centre wells. Test, positive and negative control sera are added to alternate peripheral wells.
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| | | Plates are incubated in a humid chamber at room temperature. Gels are examined for precipitation lines after 24 and 48 hours' incubation. The appearance of one or more clearly definable precipitation line(s), showing identity with that of a control positive serum, before or at 48 hours, constitutes a positive test result. Absence of any precipitation lines is recorded as a negative test result. Nonspecific lines may occur.
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| 3. | Tests for cell-mediated immunity
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| | a) | Gamma interferon assay
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| | | The assay is based on the release of gamma interferon from sensitised lymphocytes during an 18-36-hour incubation period with specific antigen (avian purified protein derivative [PPD] tuberculin, bovine PPD tuberculin or johnin) (42). The quantitative detection of bovine gamma interferon is carried out with a sandwich ELISA that uses two monoclonal antibodies to bovine gamma interferon. The sensitivity and specificity may be comparable with the tuberculin skin test. A commercial diagnostic test based on the detection of gamma interferon has been developed. The method and test materials needed, the interpretation of results, and calculations, are fully described in the instructions accompanying the commercial kit (Mycobacterium paratuberculosis gamma-interferon test kit, CSL Limited (Veterinary), 45 Poplar Road, Parkville, Victoria 3052, Australia).
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| | b) | Delayed-type hypersensitivity
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| | | The test for delayed-type hypersensitivity (DTH) is a measure of cell-mediated immunity, but has limited value. The test is carried out by the intradermal inoculation of 0.1 ml of avian PPD tuberculin (avian tuberculin can be obtained from VLA Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom, or from Symbiotic Society, 299 av. Jean Jaurès, 69007 Lyon, France) or johnin (avian tuberculin and johnin are of comparable sensitivity and specificity) into a clipped or shaven site, usually on the side of the middle third of the neck. The skin thickness is measured with calipers before and 72 hours after inoculation. Increases in skin thickness of over 2 mm should be regarded as indicating the presence of DTH. It should be noted that positive reactions in deer may take the form of diffuse plaques rather than discrete circumscribed swellings, thus making reading of the test more difficult. The presence of any swelling should be regarded as positive in this species. However, sensitisation to the M. avium complex is widespread in animals, and neither avian tuberculin nor johnin are highly specific (12). A herd test gives only an indication of the number of sensitised animals and may thus be used only as a preliminary test prior to the initiation of a control programme.
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C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
Vaccines: Vaccines used against paratuberculosis are: live, attenuated, incorporated with oil and pumice; lyophilised, live, attenuated, which may be adjuvanted with, for example, oil after reconstitution; and heat-killed bacterins. Vaccines may be prepared from one strain of M. paratuberculosis 316F or 2E (Weybridge) or M. paratuberculosis 3 and 5 or II (Canadian strains), or as many as three strains may be used. The information below applies to a live, attenuated vaccine adjuvanted with oil and pumice (7, 35, 41). Vaccination may cause a reaction at the site of injection. Vaccination may also interfere with eradication programmes based on elimination of infected animals and can interfere with the interpretation of DTH skin tests for bovine tuberculosis.
Diagnostic products: Johnin PPD is a preparation of the heat-treated products of growth and lysis of M. paratuberculosis. Avian tuberculin PPD is a preparation of heat-treated products of growth and lysis of M. avium D4ER or TB 56. Details of avian tuberculin PPD are in Chapter 2.7.8. Avian tuberculosis. These two preparations are used, by intradermal injection, to reveal DTH as a means of identifying animals infected or sensitised with M. paratuberculosis.
Guidelines for the production of veterinary vaccines are given in Chapter I.1.7. Principles of veterinary vaccine production. The guidelines given here and in Chapter I.1.7 are intended to be general in nature and may be supplemented by national and regional requirements.
| 1. | Seed management
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| | a) | Characteristics of the seed
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| | | Vaccine: Seed strains should be of a prevalent type, which may be checked by biotyping or genetic analysis. They should have been demonstrated to be innocuous when administered by the recommended route of vaccination to intended target species.
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| | | Johnin: Strains of M. paratuberculosis used to prepare seed cultures should be identified by biotyping or genetic tests. They should be shown to be free from contaminating organisms.
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| | b) | Method of culture
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| | | Vaccine: Seed cultures may be made on potato slants partly immersed in a suitable medium, such as Reid's synthetic medium (L-asparagine, 5.0 g; potassium dihydrogen phosphate [KH2PO4, anhydrous], 2.0 g; magnesium sulphate [MgSO4-7H2O], 1.0 g; ammonium citrate [(NH4)3 C6H5O7], 2.0 g; sodium chloride, 2.0 g; ferric ammonium citrate, 0.075 g; dextrose monohydrate B.P., 10 g; glycerol B.P. [48 ml], 60 g; distilled water to 1000 ml; pH [not adjusted] 5.6-5.8. When required, the above medium is solidified by the addition of 1.5% granulated agar [Difco]. Sterilised at 121°C for 15 minutes) (37). Cultures may be stored lyophilised. Active cultures are normally incubated at 37°C.
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| | | Johnin: The culture substrate should be shown to be capable of producing a product free from substances known to cause toxic or allergic reactions. A suitable medium for seed culture is that of Reid, solidified with 1.75% agar, in screw cap tubes. Cultures may also be stored lyophilised.
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| | c) | Validation as a vaccine
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| | | Vaccine: Purity tests should be carried out on seed cultures and final harvest by stained smears.
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| | | The vaccine should be used as part of a control programme and will not on its own provide complete protection against disease caused by M. paratuberculosis (41). There is usually good control of clinical disease, but subclinical infection persists in vaccinated herds, albeit at a reduced level. Vaccine should be administered to animals in early life only, e.g. calves in their first month of life. It should be inoculated subcutaneously and causes a small inflammatory swelling. This is gradually replaced by a cold, painless, fibro-caseous nodule, which varies in size and which may persist for years. Vaccination has been used to control the disease in sheep and goats, including older animals. In order to get the best results from vaccination, management practices to control the disease should also be in place.
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| | | The use of vaccines may interfere with the outcome of diagnostic skin tests for tuberculosis, and this should be remembered when planning a control programme (11).
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| | | Johnin: Cultures should be checked by staining smears for the presence of contaminating organisms.
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| | | To test for lack of sensitising effect, three guinea-pigs that have not previously been treated with any material that could interfere with the test, are each injected intradermally on each of three occasions at 5-day intervals, with 0.01 mg of the preparation under test in a volume of 0.1 ml. Each guinea-pig, together with each of three control guinea-pigs that have not been injected previously, is injected intradermally 15-21 days after the third injection with the same dose of the same johnin. The reactions of the two groups of guinea-pigs should not be significantly different when measured 24-48 hours later.
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| 2. | Method of manufacture
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| | Vaccine: For vaccine batches, the organisms may be grown on a liquid synthetic medium, such as Reid's synthetic medium. The organisms grow as a pellicle on the liquid surface. To ensure a good surface area, it is convenient to use vessels such as conical flasks containing one-third of their nominal volume of liquid medium. These flasks may be seeded directly from potato slant cultures, but with some strains, one or more passages on liquid medium may be necessary to ensure adequate pellicle growth for the final, vaccine batch passage. Such passaging should usually take place at 2-week intervals as longer periods may result in over-maturation and sinking of the pellicle. Incubation is at 37°C.
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| | To prepare the vaccine, the pellicle growth from 2-week-old cultures of each strain to be included may be separated from the liquid medium by decantation, filtration and pressing between filter paper pads. The moist M. paratuberculosis culture is blended with an adjuvant, such as liquid paraffin, olive oil and pumice (7).
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| | Johnin: Johnin for skin test diagnosis is a PPD prepared from one or more strains of M. paratuberculosis (available from VLA Weybridge or CDI, Lelystad, the Netherlands). It may be prepared by the following method.
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| | Mycobacterium paratuberculosis strains are grown as a pellicle on liquid Reid's medium. Production cultures are usually inoculated from liquid seeding cultures rather than directly from seed on solid medium (Reid's synthetic medium). Production cultures are incubated at 37°C for 10 weeks.
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| | At the end of the incubation period, the culture medium has a pH of about 5 and little or no johnin will be obtained unless the pH is raised, using sodium hydroxide, to about 7.3 before steaming. After thorough mixing, the cultures are free steamed for 3 hours. The bulk of the killed organisms is removed by coarse filtration and the filtrate is clarified by further filtration. Protein in the filtrate is precipitated chemically with 40% trichloroacetic, washed and redissolved (alkaline solvent). The product is sterilised by filtration. An antimicrobial preservative that does not give rise to false-positive reactions, such as phenol (not more than 0.5% [w/v]), may be added. Glycerol (not more than 10% [w/v]) may be added as a stabiliser. The product is dispensed aseptically into sterile glass containers, which are then sealed.
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| 3. | In-process control
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| | Vaccine: Adequate growth of culture and cultural purity need to be checked. Presence of contaminating organisms may be detected by conventional sterility tests on harvests. Tests for pathogenic mycobacteria are carried out by injection of moist culture, taken prior to blending with adjuvant and diluted tenfold in saline, into two guinea-pigs, each receiving 1 ml. These are observed for 8 weeks, killed humanely, and examined for any abnormal lesions.
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| | Johnin: After final filtration the sterility of each filtrate of the PPD solution is checked.
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| | Sterile filtrates are tested for protein content by a Kjeldahl method (1). The protein content is adjusted to give between 0.475 and 0.525 mg/ml of protein in the final product. The pH is adjusted to the range 6.5-7.5.
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| 4. | Batch control
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| | a) | Sterility
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| | | Tests for sterility and freedom from contamination may be found in Chapter I.1.5. The vaccine organism will not normally grow to a detectable level in conventional sterility tests.
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| | b) | Safety
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| | | Vaccine: These tests are normally performed in laboratory animals, although multidose tests in target animals would also be satisfactory. A typical laboratory animal test would be as follows. Each of two guinea-pigs is inoculated, subcutaneously, with an acceptable batch of vaccine at a fraction of the cattle dose previously determined to give a nodule but no overt necrosis at the injection site. Animals are observed for 8 weeks, killed humanely and examined for any abnormal lesions.
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| | | Johnin: Two guinea-pigs should each be injected subcutaneously with 0.5 ml of the johnin under test. No significant local or systemic lesions should be seen within 7 days (1).
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| | | Tests on johnin for living mycobacteria may be performed either on the material immediately before it is dispensed into final containers or on samples taken from final containers themselves. A sample of at least 10 ml should be taken, and this should be injected intraperitoneally or subcutaneously into at least two guinea-pigs, dividing the volume to be tested equally between the guinea-pigs. It is desirable to take a larger sample, say 50 ml, and to concentrate any residual mycobacteria by centrifugation or membrane filtration. The guinea-pigs are observed for at least 42 days and post-mortem examinations are carried out. Any macroscopic lesions are examined microscopically and culturally.
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| | c) | Potency
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| | | Vaccine: As protection tests appear to be impractical, a test of sensitising ability may be used. This may then be related to bacterial content based on weight. A typical test would be as follows: guinea-pigs are sensitised by intramuscular injection of 0.5 ml of a 100-fold dilution in liquid paraffin of the vaccine under test. Skin tests are performed 6 weeks after sensitisation using intradermal inoculations of 0.2 ml of at least three serial dilutions of an M. paratuberculosis antigen, such as johnin PPD, the dilutions being chosen to give expected skin reactions of from 8 mm to 25 mm diameter. Each guinea-pig receives several dilutions per flank, their distribution being chosen by a Latin square design. After 24-48 hours, skin reactions are measured. A reference preparation for tests of this type has not yet been fully established. Avian tuberculin PPD of known international unitage may be used as a skin test antigen in tests of this type to ensure that the vaccine is capable of producing adequate sensitisation (corresponding to the vaccination).
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| | | Johnin: The potency of johnin is currently determined by chemical assay for protein using a Kjeldahl method. A PPD content of 0.5 +/- 0.025 mg/ml of final product is recommended (1).
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| | | The identity of the material should be confirmed by injecting intradermally into guinea-pigs sensitised by injections of killed M. paratuberculosis (100 mg powder mycobacteria + 25 ml vaseline + 100 mg pumice stone) 6 weeks previously.
|
| | | It is possible to perform a potency test using dilutions of johnin in guinea-pigs sensitised with M. paratuberculosis, similar to such tests for the potency of bovine and avian tuberculin, but a standard preparation for this type of test has not yet been fully established.
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| | d) | Duration of immunity
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| | | Vaccine: After vaccination at the age of 14-30 days, the vaccination effect is expressed as the reduction in the rate of excretors among vaccinated animals as compared with nonvaccinated bovines (14).
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| | | There is usually good control of clinical disease, but a reduced level of subclinical infection persists. The favourable results probably reflect a diminishing exposure to infection resulting from a reduction in the number of heavy excretors in the herd.
|
| | e) | Stability
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| | | Vaccine: The vaccine may be stored at 2-8°C for 9-12 months without loss of potency. It should not be frozen.
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| | | Johnin: Johnin should be protected from light and stored at 2-8°C. Under these conditions it should retain its potency for at least 5 years.
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| | f) | Preservatives
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| | | A preservative is normally included for vaccine in multidose containers. For johnin, the phenol used is no more than 0.5% (w/v). The concentration of the preservative in the final product and its persistence through shelf life should be checked.
|
| | g) | Precautions (hazards)
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| | | Vaccine: The vaccine causes some side-effects, nodule formation and sensitisation of animals to the tuberculin test (11). In humans, accidental injection of vaccine has resulted in chronic inflammatory reactions requiring surgical treatment (14).
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| 5. | Tests on the final product
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| | a) | Safety
|
| | | See Section C.4.b.
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| | b) | Potency
|
| | | See Section C.4.c.
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REFERENCES
| 1. | Anon (1985). Johnin purified protein derivative. British Pharmacopoeia (Veterinary), 184-185.
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| 2. | Argente G. (1988). Utilisation de la culture fécale dans un plan de prevention de la paratuberculose dans 500 troupeaux; justifications techniques et économiques. In: Second International Colloquium on Paratuberculosis. Laboratoire Central de Recherches Vétérinaires, 94703 Maisons-Alfort Cedex, France, 30-35.
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| 3. | Beard P.M., Henderson D., Daniels M.J., Pirie A., Buxton D., Greig A., Hutchings M.R., McKendrick I., Rhind S., Stevenson K. & Sharp J.M. (1999). Evidence of paratuberculosis in fox (Vulpes vulpes) and stoat (Mustela erminea). Vet. Rec., 145, 612-613.
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| 4. | Collins M.T., Kenefick K.B., Sockett D.C., Lambrecht R.S., McDonald J. & Jorgensen J.B. (1990). Enhanced radiometric detection of Mycobacterium paratuberculosis by using filter-concentrated bovine fecal specimens. J. Clin. Microbiol., 28, 2514-2519.
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| 5. | Cousins D., Whittington R., Masters A., Evans R. & Kluver P. (1999). Investigation of false positives in the IS900 PCR for identification of M. paratuberculosis. Proceedings of the Sixth International Colloquium on Paratuberculosis, Melbourne, Australia. International Association for Paratuberculosis, 259-264 and Mol. Cell. Probes, 14, 431-442.
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| 6. | Cox J.C., Drane D.P., Jones S.L., Ridge R. & Milner A.R. (1991). Development and evaluation of a rapid absorbed enzyme immunoassay test for the diagnosis of Johne's disease in cattle. Aust. Vet. J., 68, 157-160.
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| 7. | Doyle T.M. (1945). Vaccination against Johne's disease. Vet. Rec., 57, 385-387.
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| 8. | Ellingson J.L.E., Bolin C.A. & Stabel J.R. (1998). Identification of a gene unique to Mycobacterium avium subspecies paratuberculosis and application to diagnosis of paratuberculosis. Mol. Cell. Probes, 12, 133-142.
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| 9. | Gilmour N.J.L. (1954). Mycobacterium paratuberculosis. In: Handbuch der Bakteriellen Infektionen bei Tieren, Blobel H. & Schliesser T., eds. Gustav Fischer Verlag, Jena, Germany, 281-313.
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| 10. | Greig A., Stevenson K., Henderson D., Perez V., Hugues V., Pavlik I., Hines M.E. 2nd, McKendrick I. & Sharp J.M. (1999). Epidemiological study of paratuberculosis in wild rabbits in Scotland. J. Clin. Microbiol., 37, 1746-1751.
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| 11. | Halgaard C. (1984). Adverse effects of vaccination against paratuberculosis: tuberculin sensitization and nodule formation. In: Paratuberculosis: Diagnostic Methods, Their Practical Application and Experience with Vaccination. Commission of the European Communities Agriculture Publication, Luxembourg, 145-153.
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| 12. | Inderlied C.B., Kemper C.A. & Bermudes L.E.M. (1993). The Mycobacterium avium complex. Clin. Microbiol. Rev., 6, 266-310.
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| 13. | Jorgensen J.B. (1982). An improved medium for the culture of Mycobacterium paratuberculosis from faeces. Acta Vet. Scand., 23, 325-335.
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| 14. | Jorgensen J.B. (1984). The effect of vaccination on the excretion of Mycobacterium paratuberculosis. In: Paratuberculosis: Diagnostic Methods, Their Practical Application and Experience with Vaccination. Commission of the European Communities Agriculture Publication, Luxembourg, 131-136.
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| 15. | Lagadic M., Le Menec M. & Argente G. (1983). Techniques de culture de Mycobacterium paratuberculosis : leur utilisation en routine dans un laboratoire de diagnostic. Recueil Med. Vet., 159, 801-807.
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| 16. | Larson A.B. & Kopecky K.E. (1970). Mycobacterium paratuberculosis in reproductive organs and semen of bulls. Am. J. Vet. Res., 31, 255-258.
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| 17. | McFadden J.J., Butcher P.D., Chiodini R. & Hermon-Taylor J. (1987). Crohn's disease - isolated mycobacteria are identical to Mycobacterium paratuberculosis, as determined by DNA probes that distinguish between mycobacterial species. J. Clin. Microbiol., 25, 796-801.
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| 18. | McFadden J.J., Butcher P.D., Thompson J., Chiodini R. & Hermon-Taylor J. (1987). The use of DNA probes identifying restriction fragment length polymorphisms to examine the Mycobacterium avium complex. Mol. Microbiol., 1, 283-291.
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| 19. | Merkal R.S. (1970). Diagnostic methods for the detection of paratuberculosis (Johne's disease). In: Proceedings of the 74th Annual Meeting of the US Animal Health Association, 620-623.
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| 20. | Merkal R.S. (1984). Paratuberculosis. In: The Mycobacteria: A Sourcebook, Kubica G.P. & Wayne L.G., eds. Marcel Dekker, New York, USA, 1237-1249.
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| 21. | Merkal R.S., Larsen A.B., Kopecky K.E. & Ness R.D. (1968). Comparison of examination and test methods for early detection of paratuberculosis in cattle. Am. J. Vet. Res., 29, 1533-1538.
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| 22. | Merkal R.S. & Richards W.D. (1972). Inhibition of fungal growth in the cultural isolation of mycobacteria. Appl. Microbiol., 24, 205-207.
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| 23. | Milner A.R., Mack W.N., Coates K., Wood P.R., Sheldrick P., Hill J. & Gill I. (1988). The absorbed ELISA for the diagnosis of Johne's disease in cattle. In: Johne's Disease, Milner A. & Wood P., eds. CSIRO Publications, Melbourne, Australia, 158-163.
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| 24. | Ridge S.E. (1993). Cultivation of Mycobacterium paratuberculosis from bovine fecal samples by using elements of the Roche MB Check system. J. Clin. Microbiol., 31, 400-405.
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| 25. | Ridge S.E., Morgan I.R., Sockett D.C., Collins M.T., Condron R.J., Skilbeck N.W. & Webber J.J. (1991). Comparison of the Johne's absorbed EIA and the complement fixation test for the diagnosis of Johne's disease in cattle. Aust. Vet. J., 68, 253-257.
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| 26. | Shermann D.M., Markham R.J.F. & Bates F. (1984). Agar gel immunodiffusion test for the diagnosis of clinical paratuberculosis in cattle. J. Am. Vet. Med. Assoc., 185, 179-182.
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| 27. | Singh S.N., Maddux R.L. & Kadel W.L. (1991). Comparative evaluation of conventional sedimentation versus new double incubation culture method for the isolation of Mycobacterium paratuberculosis from bovine fecal samples. In: Proceedings of the Third International Colloquium on Paratuberculosis. International Association for Paratuberculosis, Providence, USA, 84-93.
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| 28. | Smith H.W. (1953). Modification of Dubos's media for the cultivation of Mycobacterium johnei. J. Pathol. Bacteriol., 66, 375-381.
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| 29. | Sockett D.C., Conrad T.A., Thomas C.D. & Collins M.T. (1992). Evaluation of four serological tests for bovine paratuberculosis. J. Clin. Microbiol., 30, 1134-1139.
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| 30. | Stabel J.R. (1997). An improved method for the cultivation of Mycobacterium paratuberculosis from bovine fecal samples and comparison to three other methods. J. Vet. Diagn. Invest., 9, 357-380.
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| 31. | Sweeney R.W., Whitlock R.H., Buckley C.L & Spencer P.A. (1995). Evaluation of a commercial enzyme-linked immunosorbent assay for the diagnosis of paratuberculosis in dairy cattle. J. Vet. Diagn. Invest., 7, 488-493.
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| 32. | Thorel M.F. (1984). Identification of Mycobacterium paratuberculosis. In: Paratuberculosis: Diagnostic Methods, Their Practical Application and Experience with Vaccination. Commission of the European Communities Agriculture Publication, Luxembourg, 61-64.
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| 33. | Thorel M.F. (1991). Taxonomic and genomic research on mycobactin-dependent mycobacteria. In: Paratuberculosis: Diagnostic Methods, Their Practical Application and Experience with Vaccination. Proceedings of the Third International Colloquium on Paratuberculosis. International Association for Paratuberculosis, Orlando, Florida, USA, 222-235.
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| 34. | Thorel M.F, Krichevsky M. & Vincent Levy-Frebault V. (1990). Numerical taxonomy of mycobactin-dependent mycobacteria, emended description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov. Int. J. Syst. Bacteriol., 40, 254-260.
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| 35. | Vallee H., Rinjard P. & Vallee M. (1941). Sur la prémunition de l'entérite paratuberculeuse due au bacille de Johne. Bull. Acad. Méd., 125, 195-198.
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| 36. | Vary C.P.H., Anderson P.R., Green E., Hermon-Taylor J. & McFadden J.J. (1990). Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's disease. J. Clin. Microbiol., 28, 268-275.
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| 37. | Watson E.A. (1935). Tuberculin, johnin and mallein derived from non-protein media. Can. J. Public Health, 26, 268-275.
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| 38. | Whipple D.L., Callihan D.R. & Jarnagin J.L. (1991). Cultivation of Mycobacterium paratuberculosis from bovine fecal specimens and a suggested standardised procedure. J. Vet. Diagn. Invest., 3, 368-373.
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| 39. | Whitlock R.H., Rosenberger A.E., Sweeney R.W., Hutchinson L.J. (1991). Culture techniques and media constituents for the isolation of Mycobacterium paratuberculosis from bovine fecal samples. Proceedings of the Third International Colloquium on Paratuberculosis. International Association for Paratuberculosis, Providence, USA, 94-111.
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| 40. | Whitlock R.H., Wells S.J., Sweeney R.W. & Tiem J. Van (2000). ELISA and fecal culture for paratuberculosis (Johne's disease): sensitivity and specificity of each method. Vet. Microbiol., 77, 387-398.
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| 41. | Wilesmith J.W. (1982). Johne's disease: A retrospective study of vaccinated herds in Great Britain. Br. Vet. J., 138, 321-331.
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| 42. | Wood P.R., Billman-Jacobe H., Milner A.R. & Carrigan M. (1990). Serology and cellular assays for the diagnosis of mycobacterial infections in cattle. Proceedings of the Third international Colloquium on paratuberculosis. International Association for Paratuberculosis, Providence, USA, 12-19.
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| 43. | Yokomizo Y., Merkal R.S. & Lyle P.A.S. (1983). Enzyme-linked immunosorbent assay for detection of bovine immunoglobulin G antibody to a protoplasmic antigen of Mycobacterium paratuberculosis. Am. J. Vet. Res., 44, 2205-2207.
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| 44. | Yokomizo Y., Yugi H. & Merkal R.S. (1985). A method for avoiding false-positive reactions in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of bovine paratuberculosis. Jpn J. Vet. Sci., 47, 111-119.
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*
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NB: There are OIE Reference Laboratories for Paratuberculosis (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).
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Contact : scientific.dept@oie.int
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