Makino Department of Veterinary Microbiology, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan (e-mail: email@example.com.).
Aims: To detect and isolate Bacillus anthracis from meat and tissue by rapid and simple procedures.
Methods and Results:Bacillus anthracis Pasteur II cells were added to 1 g lymph node and pig meat, which were then cut into small pieces and suspended in PBS. Aliquots were spread on Bacillus cereus selective agar (BCA) plates to isolate B. anthracis cells, and incubated in trypticase soy broth. The enrichment culture was used for nested PCR with B. anthracis specific primers, which were to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids.
Conclusions: One cell of B. anthracis was detected by nested PCR from 1 g of the samples, and was also isolated on BCA plates according to colony morphology within two days.
Significance and Impact of the Study: These results could be useful for detecting animals with latent anthrax, and meat contaminated with B. anthracis, rapidly and simply.
Bacillus anthracis is the causative agent of anthrax in humans and animals. The median lethal dose (LD50) of anthrax depends on the animal species. For example, the minimum infectious dose estimates are approximately 100 and 35 000 spores for impala and sheep, respectively, but are as high as 109–10 spores for dog and pig (Watson and Keir 1994). When animals are infected with B. anthracis, detecting and diagnosing anthrax in sensitive animals is easy, but it is difficult in resistant animals. Therefore, direct and rapid detection of B. anthracis in resistant animals, especially pigs, is essential for meat hygiene, because pigs with latent anthrax could be a potential and dangerous source of human anthrax. Generally, the polymerase chain reaction (PCR) is a simple, rapid method for diagnosing infectious disease; a single cell of B. anthracis was detected by PCR using bacterial culture (Johns et al. 1994; Reif et al. 1994). However, detecting B. anthracis DNA directly from meat samples is difficult unless the samples contain more than 103 cells g–1 of B. anthracis (Tebbe and Vahjen 1993). In this study, the focus was on improving the PCR detection method for B. anthracis from samples of meat and lymph node. Also, a simple method is shown for the isolation of B. anthracis cells from meat using a selective medium.
MATERIALS AND METHODS
Bacillus anthracis Pasteur II (Uchida et al. 1985) was mainly used in this study. The spore forms were prepared by the method of Uchida et al. (1985) to estimate the exact numbers of B. anthracis. To 1 g of lymph node and pig meat, 0, 1, 10 and 100 spores were added. To isolate B. anthracis cells directly from these samples, 1 g sample was cut into small pieces and mixed with 10 ml phosphate-buffered saline (PBS). The suspension was centrifuged and the pellet was spread on trypticase soy agar (TSA; Becton Dickinson), Bacillus cereus selective agar (BCA; Oxoid) or PLET plates (Knisely 1966). Another pellet was used for PCR. In addition, 1 g sample was inoculated into 9 ml trypticase soy broth (TSB; Becton Dickinson). After 12 h of incubation at 37°C with shaking, 100 μl of the culture was spread on the above plates and 1 ml was used for PCR.
Template DNA for the PCR was prepared using the FastPrep™ FP120 (Bio 101, Inc. CA, USA) according to the manufacturer’s instructions. Total DNA was precipitated with ethanol and suspended in 50 μl sterile water; 100 ng were used for PCR. The following PCR cycle was used: 1 × 95°C for 2 min; 35 × (95°C for 15 s, followed by 60°C for 15 s, followed by 72°C for 30 s); 1 × 72°C for 5 min; cool to 4°C. Nested PCR with the primers listed in Table 1 was performed in a reaction mixture (25 μl), as previously described (Makino et al. 1993), using model 9600 (Applied Biosystems Japan Ltd, Tokyo, Japan). These primers are specific to B. anthracis. BA813 and S-layer specific primers were included to confirm the presence of B. anthracis chromosomal DNA (Etienne-Toumelin et al. 1995; Ramisse et al. 1999). PA and CAP specific primers confirmed the presence of plasmids pXO1 and pXO2, respectively, which are normally contained in fully virulent B. anthracis strains (Mikesell et al. 1983; Green et al. 1985; Uchida et al. 1985). The primers only amplified the right sizes of DNA fragments using B. anthracis; no DNA fragments were detected in other Bacillus strains (Table 2) and other bacterial species (Makino et al. 1993) (data not shown).
Table 1. Primers for the nested PCR used in this study
Table 2. Bacillus strains used in this study and their characteristics
When DNA extracts for PCR were prepared from 1 g samples artificially contaminated with 1 spore of B. anthracis, no DNA fragments were amplified (Fig. 1). However, using DNA extracts from 1 g of the samples with more than 10 spores, the right sized DNA fragments were amplified (Fig. 1). Therefore, to increase the sensitivity of the detection of B. anthracis DNA, each 1 g of sample with 1 and 10 spores of B. anthracis was inoculated into 9 ml TSB, followed by incubation for 12 h at 37°C with shaking. When DNA extracts were prepared from the enrichment cultures, DNA fragments were amplified (Fig. 2).
In order to isolate bacterial cells from the samples, clarifying the infection process of B. anthracis is essential. First, a 1 g aliquot of the sample was spread directly onto the agar plates. As 103–104 bacterial cells g–1 were generally detected in meat from the market using TSA plates as a non-selective medium (data not shown), direct detection of B. anthracis cells from the samples on the TSA plates would be difficult. Therefore, PLET and BCA plates were used as the selection plates for B. anthracis. On the BCA plates, B. cereus was easily distinguished from other Bacillus strains because colonies of B. cereus were peacock or turquoise blue and turbid rings were formed around the colonies by the production of lecithinase. Therefore, BCA plates were chosen to select B. anthracis as B. anthracis is closely related to B. cereus by biochemical characteristics and DNA homology (Sneath 1986).
All Bacillus strains were spread on PLET plates, with the result that only B. anthracis and B. licheniformis grew (Table 2). However, the number of B. anthracis cells on the PLET plates was about 10- to 100-fold less than those on the TSA and BCA plates (data not shown), indicating that isolation of B. anthracis from samples containing 1–10 cells of B. anthracis would be difficult. Moreover, B. anthracis grew well experimentally on the PLET plates (Table 2), but when the samples were spread on the PLET plates, large numbers of unspecified bacterial colonies were detected. Bacillus anthracis colonies were rarely indistinguishable from such bacterial colonies because their colony morphologies were almost identical (data not shown). Finally, it was concluded that the PLET plates would be unsuitable for detecting B. anthracis directly from the samples.
Next, BCA plates were examined. All Bacillus strains, except for seven strains of six species, grew on the plates (Table 2). Among them, B. mycoides, B. thuringiensis, B. cereus and B. anthracis formed large rough colonies on the BCA plates after 20 h of incubation at 37°C (Fig. 3a, b, c, and h), but the colony morphology of B. anthracis (Fig. 3g, h) was distinguishable from that of B. cereus, B. thuringiensis and B. mycoides (Fig. 3a, b and c) because turbid rings around the colonies were detected only in those three species. Other Bacillus strains formed small yellow colonies on the plates, which were simply distinguishable from those of the above four species (Fig. 3d, e and f as representative examples).
The number of B. anthracis cells on the BCA plates were almost the same as the inoculum size (data not shown). Thus, 1 g of sample was directly spread on BCA plates. From 1 g of samples with more than 10 spores of B. anthracis, B. anthracis was clearly detected as large colonies (Fig. 3i), but from samples with 1 spore, B. anthracis was rarely detected (data not shown). Therefore, to detect B. anthracis, 1 g meat with 1 spore of B. anthracis was inoculated into TSB. After 12 h of incubation at 37°C, 0·1 ml of the culture was spread on the BCA plates. As several large rough colonies in the lawn of small colonies were found (Fig. 3j), they were examined by PCR to determine whether they were B.anthracis; all the large colonies were B. anthracis (date not shown). Therefore, BCA plates are useful for isolating B. anthracis from meat.
In this study, a rapid, simple and reliable method, combining nested PCR and selective media, for detecting B. anthracis in meat samples is described. Although a direct detection method for B. anthracis in mouse spleen using PCR had already been established (Makino et al. 1993), its detection sensitivity was about 10 times lower than the detection system described in this study, and B. anthracis cells were not isolated from the samples. However, the present system enables monitoring of meat with 1 cell of B. anthracis using PCR, and can also be used to isolate live B. anthracis cells from samples using an enrichment procedure. When pigs have latent anthrax, B. anthracis cells are often found in their mesenteric lymph nodes. At that time, as shown by unusual histological changes in the lymph nodes of the carcasses at the slaughter inspection, the system described here might be useful for rapidly checking whether pigs are infected with B. anthracis.
As a first inspection, nested PCR, direct plating on selective plates and enrichment of the samples must be carried out at the same time. If the samples contain more than 10 cells of B. anthracis, the animals with latent anthrax can be selected and diagnosed the following day. When the samples contain fewer than 10 cells of B. anthracis, diagnosis can be made within two days. In addition, with the PCR system using all the primer sets shown in Table 1, it would be possible to confirm the presence of virulent strains in the samples.
The BCA plates, which were originally developed to isolate B. cereus in food (Daffonchio et al. 1999), were used to distinguish B. anthracis from other Bacillus species. The results suggested that the BCA plates are excellent for isolating B. anthracis cells from samples, such as meat, tissue and the atmosphere, which were contaminated with low numbers of Bacillus species. However, detecting B. anthracis in soil, which is one of the most important sources of B. anthracis, would be too difficult, because soil contains a huge number of Bacillus species. Bacillus anthracis might be efficiently identified and separated from all environmental samples if the method described here could be improved.
This work was supported in part by grants from the Ministry of Health, Labour and Welfare (Research on Emerging and Re-emerging Infectious Diseases). H.I.C. is a research scientist of the Japan Health Sciences Foundation.