A review of tests available for use in the diagnosis of tuberculosis in non-bovine species.

Bovine tuberculosis is an important disease that has impacts on regional and international trade. The disease can affect both social and economic stability and have a deleterious affect on species diversity. The intradermal tuberculin test has been in use for almost a century and, despite the technological advances of the last two decades, is still the only prescribed test for the diagnosis of tuberculosis in cattle. Many other species of animal, including humans, can be infected with Mycobacterium bovis. This paper reviews the various tests that have been used by researchers for detecting infection with M. bovis in a variety of animal species, and attempts to prioritise or comment on the importance of having appropriately validated diagnostics for the different species. The difficulties of test validation using small numbers of animals, especially when tuberculosis occurs in only a few instances or the species of animal affected is rare and/or valuable, are discussed.

Australia's campaign to eradicate bovine tuberculosis: the battle for freedom and beyond.

In 1970, voluntary State-based TB control programs in Australia were replaced by a coordinated national campaign to eliminate both brucellosis and tuberculosis from the cattle population. The campaign was funded and managed under tripartite agreement by State/Territory and Commonwealth governments and Industry. The tuberculosis component of the campaign relied on test and slaughter with surveillance for the disease in abattoirs and trace-back to property of origin an essential component. Because of the moderate sensitivity of the skin test ( approximately 70%), testing was repeated at prescribed intervals over a number of years. In the more hostile environment of northern Australia, novel strategies were developed to maximize musters and remove 'at risk' animals. Australia is fortunate it did not have a feral host for M. bovis (apart from buffalo, which were included in the campaign) to complicate eradication. A national granuloma submission program was implemented in 1992 to increase the intensity of abattoir monitoring. Selective or total depopulation was used in some herds to achieve the requirements of the national Standard Definitions and Rules of the Campaign and achieve the status of 'TB Free Area' in December 1997. Monitoring for tuberculosis has continued under the 5-year Tuberculosis Freedom Assurance Program and measures to further reduce the risk of new cases have been implemented.

Genetic differentiation of Australian isolates of Mycobacterium tuberculosis by pulsed-field gel electrophoresis.

As part of an epidemiological study of tuberculosis in Australia, 84 isolates of Mycobacterium tuberculosis from patients were analysed by pulsed-field gel electrophoresis (PFGE). The isolates were genetically heterogeneous, with 66 different DNA banding patterns obtained following digestion of genomic DNA with Dra1 and 53 patterns with Xba1. When the results were compared with those previously obtained in restriction fragment length polymorphism analysis (RFLP), in 87% of cases the results with Dra1 were consistent with those obtained with insertion sequence IS6110 as a probe in RFLP. However, PFGE was able to differentiate four of eight isolates which were identical with IS6110 typing. The high polymorphism amongst strains and the high average age of the patients (51 years) suggested that most organisms were cultured from patients who had reactivation of existing infections. Isolates with identical DNA patterns were found in different states of Australia, but no one strain predominated in any area. This suggests that tuberculosis has been introduced into Australia from various sources.

Tuberculosis due to Mycobacterium bovis in the Australian population: cases recorded during 1970-1994.

OBJECTIVE: To determine the contribution of Mycobacterium bovis to active tuberculosis in the Australian population during 1970-1994, and to collate and analyse demographic data from bacteriologically proven cases. DESIGN: Summary data for tuberculosis cases notified by Australian public health agencies during 1970-1985 and 1991-1994 were obtained from the database of notifiable diseases maintained by the Department of Health and Family Services. More detailed demographic data for cases confirmed by bacteriology during 1970-1994 were supplied by the Australian Mycobacterium Reference Laboratory Network. RESULTS: At least 236 cases of bovine tuberculosis (TB) occurred in the Australian population during 1970-1994 (mean 9.4 cases; range 3-22 cases annually). The bovine strain has accounted for around 1% of Australian cases of TB during this period. Laboratory sources provided demographic data for 150 cases with positive bacteriology. For this group, the mean age was 54 years (range 22-86), and the male:female ratio was 2.4:1. The majority of cases (74%) involved pulmonary disease. Australian-born persons accounted for 68% of the total cases and typically had histories of employment in meat and/or livestock industries. CONCLUSION: M. bovis was responsible for less than 1.5% of cases of TB in the Australian population during 1970-1994. Most cases were apparently due to reactivation of infection acquired through occupational exposure. Thus, although virtual eradication of M. bovis from Australia's cattle herds has now reduced the risk of exposure, it can be expected that human cases of bovine TB will continue to be detected for years to come. The bovine strain should be considered as the possible agent of TB in foreign-born Australians.

Tuberculosis due to Mycobacterium bovis in the Australian population: DNA typing of isolates, 1970-1994.

SETTING: Bacteriologically confirmed cases of Mycobacterium bovis in the Australian population. OBJECTIVE: To evaluate the DNA fingerprinting techniques commonly used for M. bovis on isolates from humans and determine whether they were useful for determining the origin of human infection. DESIGN: M. bovis strains isolated between 1970 and 1994 were obtained from five Australian Reference Laboratories. Four DNA fingerprinting techniques, comprising Southern hybridisation with three different probes (the insertion sequence [IS]6110, the polymorphic guanine-cytosine-rich sequence [PGRS] and the direct repeat [DR]) and a PCR-based method (spoligotyping) were used. RESULTS: The PGRS, DR and IS6110 RFLP methods identified 32, 22 and 14 different types respectively from the 45 isolates available. Spoligotyping identified 18 different types. When all methods were combined 41 different strains were identified. Clear differences were found between many isolates from Australian-born patients and those from patients born overseas. CONCLUSIONS: The PGRS RFLP method was the most effective method for typing the human strains, but a combination of methods is recommended for maximum sensitivity. Most Australian-born patients that had worked in the meat and livestock industries were infected with strains similar to those that are commonly found in Australian cattle, confirming the occupational risk in these industries. Patients born overseas were typically infected with strains genetically different from those of patients born in Australia. This suggests that patients born overseas identified with M. bovis were presenting with reactivation of infection acquired outside Australia.

Towards a standardized approach to DNA fingerprinting of Mycobacterium bovis. International Union Against Tuberculosis and Lung Disease, Tuberculosis in Animals Subsection.

The Tuberculosis in Animals Subsection of the International Union Against Tuberculosis and Lung Disease (IUATLD) recently identified a need to standardize the deoxyribonucleic acid (DNA) strain typing of Mycobacterium bovis. The standard method for strain typing of M. tuberculosis isolates cannot be directly extrapolated to M. bovis due to the low copy number of IS6110 identified in the majority of M. bovis strains, particularly from cattle. To improve the resolution of M. bovis strains, alternative methods and additional DNA probes have been investigated. In combination with studies of published literature, laboratories performing M. bovis DNA fingerprinting were surveyed. Results of these surveys allowed us to reach consensus and to make recommendations for DNA typing of M. bovis isolates, which hopefully will lead towards a standardized approach to the DNA fingerprinting of this organism. This approach, in conjunction with conventional epidemiological traceback approaches, should facilitate more accurate and effective investigations into the epidemiology, maintenance and transmission of M. bovis within and between man and domesticated, feral and wild animals, both at a local and a global level.

Rapid identification of Haemophilus somnus, Histophilus ovis and Actinobacillus seminis using the API ZYM system.

The API ZYM system, a commercially-available technique that measures bacterial enzyme activity was used to test 43 isolates identified as H. somnus, H. ovis or A. seminis and 19 from related genera. The enzyme patterns resulting from the API ZYM differentiated H. somnus and H. ovis from A. seminis and related genera but not from each other. An identification scheme based on 9 of the enzymes in the API ZYM and a few simple biochemical tests is proposed for the rapid and reliable identification of these bacteria in a diagnostic bacteriology laboratory.

Preparation, assessment and preservation of antigen from Brucella abortus strain 45/20 for use in the rough antigen complement fixation test.

Two methods of extraction were used to prepare antigens from Brucella abortus rough strain 45/20. The antigens were assessed for use in the complement fixation test. A suitable antigen was prepared using the saline extraction method of Miller et al. (1976) and used extensively in CF tests. Four methods of preservation were compared; -20 degrees C, -196 degrees C, 0.5% phenol at 4 degrees C, and lyophilisation. The antigen could be stored at -20 degrees C or -196 degrees C for up to 2 years.

The use of the enzyme-linked immunosorbent assay (ELISA) to detect the IgM and IgG antibody response to Leptospira interrogans serovars hardjo, pomona and tarassovi in cattle.

Leptospira interrogans serovars pomona, hardjo and tarassovi were each used to inoculate 6 cattle. Three-hundred and ninety-nine sera collected from the inoculated animals and from a control group over a 3-month period were tested using the microscopic agglutination test (MAT) and the enzyme-linked immunosorbent assay (ELISA). Leptospiruria was monitored by microscopic examination and culture. The ELISA detected specific IgM antibody against the serovars in all infected cattle 1 week after inoculation. This IgM antibody persisted in most of the animals for 3-5 weeks. Specific IgG antibody appeared at the same time or just after IgM, but persisted for much longer. Levels of antibody detected by the ELISA and the MAT did not correlate with each other, nor with the periods of leptospiruria found in the infected cattle.

Use of DNA amplification for the rapid identification of Mycobacterium bovis.

DNA amplification using the polymerase chain reaction technique was evaluated for rapid identification of Mycobacterium bovis. Two oligonucleotide primers of 20 bases in length were constructed to target a region of the gene encoding the M. bovis secretory protein, MPB70. The amplification reaction produced a single product 372 bp in size which was readily detected by agarose gel electrophoresis. All 84 strains of M. bovis tested produced a positive signal in the amplification reaction. In addition all isolates fro the M. tuberculosis complex tested, with the exception of M. microti, gave a single band at 372 bp. No amplified product was detected when 24 other species of mycobacteria and species from four other genera were tested. The sensitivity of the test was such that a single viable cell could be detected in the reaction. This technique provides a simple and extremely sensitive method of identifying isolates of M. bovis and other pathogenic M. tuberculosis complex organisms.

Bovine IgM and IgG response to Leptospira interrogans serovar hardjo as measured by enzyme immunoassay.

The enzyme-linked immunosorbent assay (ELISA) was used to detect specific IgG and IgG antibodies in the sera of cattle infected or immunized with Leptospira interrogans serovar hardjo. IgM appeared first but was quickly followed by IgG which persisted longer than IgM. The levels of antibody detectable by ELISA and by the microscopic agglutination test (MAT) did not correlate, suggesting that the two techniques measured different antigen--antibody systems. The transient nature of the IgM response as measured by ELISA indicates potential usefulness as a serodiagnostic test for detecting current leptospiral infections.

Advantages of a new agar medium in the primary isolation of Mycobacterium bovis.

Suspect tuberculous lesions from 116 cattle were examined histologically and cultured for Mycobacterium bovis using 5 different media. The media used were: B83, an agar medium incorporating bovine blood and sodium pyruvate; Middlebrook's agar; 2 variations of Stonebrink's medium; Löwenstein-Jensen medium. The B83 medium and a modification of Stonebrink's medium which had a lowered concentration of malachite green were most successful, detecting 95.2% of tuberculous animals when used together. The B83 medium detected isolates approximately 1 week earlier and had more colonies than the Stonebrink's modification. A combination of 2 slopes of B83 and 2 slopes of modified Stonebrink's medium is recommended for routine culture of samples.

Tuberculosis in imported hyrax (Procavia capensis) caused by an unusual variant belonging to the Mycobacterium tuberculosis complex.

Tuberculosis was diagnosed in an adult female hyrax (Procavia capensis) imported from South Africa and held in a captive colony at the Perth Zoo. An organism similar to Mycobacterium microti was isolated from the lung of this animal and the lung of an adult male hyrax in the colony. The organism was not pathogenic to rabbits or guinea pigs. Protein profiles and RFLP patterns using the probes IS6110 and pTBN12 showed both hyrax isolates were identical. These isolates were similar to a M. tuberculosis complex strain isolated from dassies (hyrax) in the late 1950s in South Africa and to M. microti, but appeared to be more closely related to the "dassie bacillus". It is likely that at least one of the hyrax was infected at the time of collection in South Africa. The finding of tuberculosis in these imported animals highlights concern over the lack of suitable tests for the detection of tuberculosis in unusual animal species such as these, and the problems that can arise as a result of the importation of infected animals.

Use of a repetitive element isolated from Mycobacterium tuberculosis in hybridization studies with Mycobacterium bovis: a new tool for epidemiological studies of bovine tuberculosis.

Typing of M. bovis isolates for epidemiological purposes is possible using restriction endonuclease analysis (REA). However, the DNA fragment patterns obtained are complex and difficult to analyse due to the large number of bands produced. In an attempt to develop a less complicated typing scheme two DNA probes were used in hybridization studies to detect restriction fragment length polymorphisms (RFLP) in M. bovis. An oligonucleotide probe which matches part of the insertion sequence IS6110 produced few bands and failed to discriminate between bovine isolates of M. bovis. A probe prepared from a highly repeated DNA sequence, cloned from M. tuberculosis when used on southern blots of AluI digested M. bovis DNA, resulted in a discriminating typing scheme which was easier to perform and analyse than the REA. The RFLP typing scheme identified 27 different strains from a total of 36 isolates of M. bovis and 7 reference strains from the M. tuberculosis complex. Using REA, 24 types were identified using BclI and PvuII digests and 23 different types using BstEII digests. When results of all 3 enzyme digests were combined, the REA identified 27 types from the same strains. Ten isolates of M. bovis from 5 properties involved in an outbreak of bovine tuberculosis were all identified as the same type with both techniques.

Tuberculosis in captive seals: bacteriological studies on an isolate belonging to the Mycobacterium tuberculosis complex.

Culture of tuberculous lesions from six of 14 captive seals yielded an organism which, on the basis of biochemical and drug sensitivity tests, was identified as Mycobacterium bovis, although the organism showed a weak cording pattern and was glycerol tolerant. It was pathogenic in rabbits and guinea pigs and after passage the organism exhibited strong cord formation and was glycerol intolerant. Restriction endonuclease analysis and sodium dodecyl-sulphate polyacrylamide gel electrophoresis indicated that the organism belonged to the Mycobacterium tuberculosis complex. Restriction patterns indicated that infection in the six seals was from a single source. Western blotting with monoclonal antibody to M bovis identified antigens at 23 and 27 kDa in M bovis which were absent from the seal isolates.

Mycobacterium bovis infection and control in domestic livestock.

Bovine tuberculosis, caused by Mycobacterium bovis, is a well-known zoonotic disease which affects cattle world-wide. The public health risk has been alleviated in many countries by the introduction of pasteurisation, but the disease continues to cause production losses when poorly controlled. The Office International des Epizooties classifies bovine tuberculosis as a List B disease, a disease which is considered to be of socio-economic or public health importance within countries and of significance to the international trade of animals and animal products. Consequently, most developed nations have embarked on campaigns to eradicate M. bovis from the cattle population or at least to control the spread of infection. The success of these eradication and control programmes has been mixed. Mycobacterium bovis infects other animal species, both domesticated and wild, and this range of hosts may complicate attempts to control or eradicate the disease in cattle.

Tuberculosis in a wild Australian fur seal (Arctocephalus pusillus doriferus) from Tasmania.

Tuberculosis was found in a wild, mature male Australian fur seal (Arctocephalus pusillus doriferus) at Hobart, Tasmania on 8 March 1992. We observed fibrogranulomatous and pyogranulomatous lesions in the lung, pleura, lymph nodes and spleen. The SDS/PAGE profile of this Tasmanian isolate was similar to other seal strains; however, differences were detected using pTBN12 and insertion sequence IS6110 probes.

Use of enzyme immunoassay in a serological survey of leptospirosis in sheep.

A total of 731 serums, all from Merino rams from 20 farms, were tested for antibodies against Leptospira interrogans serovars hardjo, pomona and tarassovi using the microscopic agglutination test (MAT). The enzyme immunoassay (EIA) technique was used to test all serums for IgM and IgG antibodies to serovar hardjo. In the MAT, reactions to serovar hardjo were most common with 236 rams (32.3%) reacting at 1/100 or greater. Only 1.9% of serums reacted against serovar tarassovi and 1.1% against serovar pomona. The percentage of sheep with positive MAT reactions to serovar hardjo ranged from 0 0 to 94.9 between farms. When using EIA, 46 (6.2%) of the serums were positive for IgM antibody and 246 (33.6%) were positive for IgG antibody. Correlation of the EIA for detection of IgG antibody with the MAT was good. The EIA detection of IgG antibody was considered to be a good alternative test to the MAT for epidemiological studies in sheep.

DNA fingerprinting of Australian isolates of Mycobacterium avium subsp paratuberculosis using IS900 RFLP.

OBJECTIVES: To evaluate additional restriction enzymes for IS900 RFLP of Mycobacterium avium subsp paratuberculosis and examine the genetic diversity among Australian isolates for epidemiological studies of Johne's disease. DESIGN AND PROCEDURE: Seventy-one isolates of M paratuberculosis from cattle, sheep, goat, alpaca and rhinoceros in six Australian States and the Northern Territory, reference strains and reference DNA from previously characterised strains were tested for genetic variation. Bst EII, Pvu II and Pst I restriction enzymes were used, and four others (Bam HI, Alu I, Xho I and Dra I) were assessed for their ability to detect polymorphisms. Multiple isolates from some animals were tested. RESULTS: Bam HI, was the most effective enzyme for identifying polymorphisms (12 types), followed by Bst EII (11 types). Both Pvu II and Pst I were relatively ineffectual. Fifteen different types were identified, 12 in clinical isolates. Most isolates were cattle (C) strains and fell into the C1 (n = 28) and C3 (n = 32) groupings. All isolates from alpaca were type C1, and bovine isolates were commonly C1 (n = 15) or C3 (n = 28). All of the sheep were infected with sheep (S) strains; no S strains were identified in cattle. Two of six isolates from one animal had single band differences. CONCLUSION: The epidemiological features of M paratuberculosis in Australia are similar to those reported in New Zealand, where cattle and sheep are commonly infected with different strains. However, because of the lack of polymorphism identified within the major groups, it is unlikely that DNA fingerprinting will have a significant role in epidemiological studies of Johne's disease, unless an unusual strain in being studied.

Specificity of absorbed ELISA and agar gel immuno-diffusion tests for paratuberculosis in goats with observations about use of these tests in infected goats.

OBJECTIVE: To determine the specificity of serological tests that are currently used in veterinary diagnostic laboratories in Australia for detection of Mycobacterium avium subsp paratuberculosis infection in goats. DESIGN: A laboratory study. PROCEDURE: Four tests were studied, comprising AGID with M. a. paratuberculosis antigen derived from cattle isolates of caprine or bovine origin, the EMAI caprine Johne's disease absorbed ELISA and the CSL PARACHEK Johne's absorbed EIA. The specificities of AGID and ELISA for paratuberculosis (Johne's disease) were estimated after examining a panel of 1000 serum samples collected from goats in Western Australia, a region free of paratuberculosis. In addition a comparison was made of test performance in a small number of paratuberculous goats from New South Wales using sera from two archival collections. RESULTS: The specificity of the AGID tests was 100% while the specificities of the two absorbed ELISA were 99.7 to 99.8% at appropriate positive-negative cut-offs. Based on testing the small sample of sera from infected goats, the absorbed ELISA tests detected about twice as many goats with Johne's disease as the AGID. Each test detected paratuberculous animals regardless of whether infection was caused by cattle or sheep strains of M. a. paratuberculosis. CONCLUSIONS: Both ELISA and AGID tests for paratuberculosis have high specificity and can be used in a market assurance program without risk of generating large proportions of false positive test results. However, the results suggested the ELISA is more sensitive for detection of infected goats and should be used in preference to the AGID. The two formats of ELISA evaluated in this study have similar characteristics and could be used in paratuberculosis control programs for the goat industries, but further data on sensitivity would increase confidence in their application.