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After a potential biological incident the sampling strategy and sample analysis are crucial for the outcome of the investigation and identification. In this study, we have developed a simple sandwich ELISA based on commercial components to quantify BSA (used as a surrogate for ricin) with a detection range of 1.32-80 ng/mL. We used the ELISA to evaluate different protein swabbing procedures (swabbing techniques and after-swabbing treatments) for two swab types: a cotton gauze swab and a flocked nylon swab. The optimal swabbing procedure for each swab type was used to obtain recovery efficiencies from different surface materials. The surface recoveries using the optimal swabbing procedure ranged from 0-60% and were significantly higher from nonporous surfaces compared to porous surfaces. In conclusion, this study presents a swabbing procedure evaluation and a simple BSA ELISA based on commercial components, which are easy to perform in a laboratory with basic facilities. The data indicate that different swabbing procedures were optimal for each of the tested swab types, and the particular swab preference depends on the surface material to be swabbed.

An adult female white-tailed trogon (Trogon viridis) was presented with abdominal enlargement and hard subcutaneous masses. Necropsy findings included bony masses extending from skeletal structures, disseminated pale foci in the liver, and a pale mass in the kidney. Histological examination revealed multifocal to coalescing granulomatous inflammation in the bone, liver, kidney, lung and spleen. Mycobacterium celatum was isolated from the liver and identified by DNA sequencing. This is the first report of M. celatum infection in an avian species.

Milk and faeces samples from cows with clinical symptoms of paratuberculosis were examined for the presence of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by culture and PCR. M. paratuberculosis was cultivated in variable numbers from faeces or intestinal mucosa in eight of 11 animals. In milk from five cows (all faeces culture positive), we cultivated a few colonies of M. paratuberculosis (<100 CFU per ml). Milk samples from two cows were PCR positive (both animals were faeces culture positive, and one cow was milk culture positive). One cow was culture negative on intestinal mucosa, but culture positive in milk, and two cows were negative in culture and PCR from both faeces and milk. In conclusion, the presence of M. paratuberculosis could be detected in raw milk by PCR, but cultivation of milk was more sensitive.

In the present study restriction fragment length polymorphism analyses with the recently described insertion sequence IS1245 as a probe was performed with clinical Mycobacterium avium complex strains cultured in Denmark during a 2-year period. The overall aim of the study was to disclose potential routes of transmission of these microorganisms. As a first step, the genetic diversity among isolates from AIDS patients and non-human immunodeficiency virus (HIV)-infected patients was described. In addition, a number of isolates from nonhuman sources cultured during the same period were analyzed and compared to the human isolates. A total of 203 isolates from AIDS patients (n = 90), non-HIV-infected patients (n = 91), and nonhuman sources (n = 22) were analyzed. The presence of IS1245 was restricted to Mycobacterium avium subsp. avium isolates. The majority of human isolates had large numbers of IS1245 copies, while nonhuman isolates could be divided into a high-copy-number group and a low-copy-number group. Groups of identical strains were found to be geographically widespread, comprising strains from AIDS patients as well as strains from non-HIV-infected patients. Samples of peat (to be used as potting soil) and veterinary samples were found to contain viable M. avium isolates belonging to genotypes also found in humans.

Diagnostic findings in 218 aborted bovine foetuses are reported. The materials were examined in a matched case-control study of 69 Danish dairy herds with a sudden increase in the number of abortions and a corresponding 69 control herds. Foetuses aborted during the subsequent 6-month period were examined to identify the cause of abortion if possible. A total of 186 specimens were submitted from case herds and 32 from control herds. A likely cause of abortion was diagnosed in 73 foetuses. The most common cause was bovine viral diarrhoea virus (BVDV: 13%) followed by Neospora caninum infection (10%), mycosis (5%) and Bacillus licheniformis infection (4%). Foetal and/or placental lesions were found in a further 27 cases. Only BVDV infection and neosporosis were diagnosed in more than one foetus per herd and only protozoal associated abortions occurred significantly more frequently in the case, rather than in the control, herds.

A monoclonal antibody raised against a 40 kDa protein present in certain M. avium strains (IS901/IS902 positive) was used for developing a blocking ELISA. Sera from experimentally infected sheep were evaluated by indirect ELISA, AGID and blocking ELISA. The blocking assay proved to be highly specific for differentiation of sheep infected with different subspecies of M. avium.

The aim of the present study was to characterize all strains of the Mycobacterium avium complex isolated in Denmark in 1993. A total of 141 M. avium complex strains (86 from man, 38 from animals, and 17 from peat) were analysed by serotyping, ELISA specific for a 40 kDa protein, and IS901-specific PCR. Serotype analysis showed that the most frequent serotypes among human strains were serotype 4 (27%) and serotype 6 (19%), which differs from an earlier survey where serotype 1 was most prevalent. The most frequent serotypes in animals were serotype 2 (53%) and serotype 6 (13%), whereas the most prevalent serotypes among strains isolated from peat were serotype 4 (29%) and serotype 9 (18%). There was a concurrent appearance of IS901 and p40 in all strains. Only M. avium complex strains isolated from animals, and belonging to serotype 1 or serotype 2, contained the IS901/p40 markers. The different distribution of serotypes of M. avium complex strains in animals and man, and the presence of IS901/p40 exclusively in animal strains, suggests that transmission of M. avium from animals to man is not of significance in Denmark.

The pathogenicity of 13 strains of Bacillus licheniformis was studied in immunodepressed mice. The strains had been isolated from cases of bovine abortions (n = 5), bovine feedstuffs (n = 3), soil (n = 1), and grain products (n = 2). The origin of two strains was unknown. Groups of 10 mice were inoculated intravenously with B. licheniformis bacteria at doses from < 10(6) to 10(10) colony-forming units. Following 7 days of infection, the animals were euthanized and examined bacteriologically, histologically, and immunohistochemically using a PAP technique based on primary polyclonal rabbit anti-B. licheniformis antibodies. B. licheniformis bacteria were reisolated from the liver, spleen or kidneys of mice in all groups. Inflammatory lesions were present in mice of all immunodepressed groups, but only brain and pulmonic lesions were definitely attributed to B. licheniformis infection, as strong immunostaining was found within these lesions. It is concluded that all strains of B. licheniformis examined were pathogenic for immunodepressed mice, and that spontaneous infections may be established by bacterial strains to which susceptible individuals are accidentally exposed.

The occurrence of two markers, a newly identified 40-kDa protein (p40) and the insertion sequence IS901-IS902, in strains of Mycobacterium avium subspp. was evaluated. Analysis of 184 type and field strains of the M. avium complex from human, animal, and environmental sources by PCR specific to IS901 and by a monoclonal antibody specific to p40 demonstrated the presence of the two molecular markers in all of the M. avium subsp. silvaticum strains examined and also in a number of M. avium subsp. avium strains (the latter isolated mainly from pigs). The appearance of the two markers was completely concurrent in all strains. Further, the marker-positive M. avium subsp. avium strains were mainly serotype 2, whereas M. avium complex strains of serotypes 4, 6, 8, 9, and 10 were marker negative. The M. avium subsp. avium type strains ATCC 25291 and approximately 50% of the M. avium subsp. avium field strains isolated from animals contained the markers, while only one strain of human origin was found to be marker positive. Therefore, IS901 and p40 appear to have substantial potential to differentiate among isolates of the M. avium complex. This observation raises new issues regarding classification of strains, since the presence of the markers was found to be inconsistent with the present taxonomic grouping of M. avium subspp.

Danish isolates of Mycobacterium avium complex were serotyped by the use of seroagglutination. The most prevalent serovars among patients with AIDS (n = 89) were 4 and 6, while among non-AIDS patients the most prevalent serovars were 1, 6, and 4, with no major differences between those in patients with pulmonary disease (n = 65) and those in patients with lymph node infection (n = 58). The results suggest a Scandinavian distribution of serovars with a predominance of serovar 6 and fail to demonstrate any selective protection against different serovars by Mycobacterium bovis BCG vaccination.