Deer 1, mediastinal lymph node. H-57/91. 7. Deer 1, mesenterial lymph node. H-82/91. 7. Deer 1, pooled sample. S:202/92. 7. Deer 2, retropharyngeal lymph.
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1995, p. 3183–3185 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 33, No. 12
Molecular Epidemiological Studies of Mycobacterium bovis Infections in Humans and Animals in Sweden ¨ RAN BO ¨ LSKE,5 REGINE SZEWZYK,1† STEFAN B. SVENSON,2,3 SVEN E. HOFFNER,1,4 GO 5 6 5 ¨ ¨ HELENE WAHLSTROM, LENA ENGLUND, ANDERS ENGVALL, AND GUNILLA KALLENIUS1* Department of Bacteriology1 and Department of Vaccine Research,2 Swedish Institute for Infectious Disease Control, S-105 21 Stockholm, Department of Bacteriology and Epizootology, Biomedicum, Swedish Agriculture University, S-75123 Uppsala,3 Microbiology and Tumorbiology Centre, Karolinska Institute, S-1717 Stockholm,4 National Veterinary Institute, S-75007 Uppsala,5 and Swedish Board of Agriculture, S-551 82 Jo ¨nko ¨ping,6 Sweden Received 21 February 1995/Returned for modification 20 June 1995/Accepted 19 September 1995
Forty-nine isolates of Mycobacterium bovis from humans and animals in Sweden were analyzed by restriction fragment length polymorphism (RFLP) patterns probed by the insertion element IS6110. Most isolates had patterns indicating the presence of only one or two genomic copies of the IS6110 insertion element. This simple type of pattern was found in all human isolates. In contrast, isolates from M. bovis infections in five herds of farmed deer in Sweden showed a specific RFLP pattern with seven bands, indicating seven copies of the IS6110 sequence. In 1958, Sweden was declared free from M. bovis in cattle. However, in 1987, M. bovis was reintroduced with imported farmed deer, and since 1991, 11 outbreaks in deer herds, but not in other livestock or wildlife, have been diagnosed. Continued RFLP studies of the new Swedish M. bovis isolates can reveal possible transmission of this deer strain to other animals or humans. uation of the banding pattern found. With this method, the relationship between different strains can be revealed, provided there are a sufficient number of copies of the insertion elements present in the bacterial DNA. The mycobacterial reference laboratory at the Swedish Institute for Infectious Disease Control (SIIDC) participates in an ongoing molecular epidemiological surveillance of human tuberculosis, supported by the European Union, with a standardized RFLP assay (with the insertion sequence IS6110 as the target), which was also chosen in the present study.
Mycobacterium tuberculosis complex isolates from domestic animals are almost always Mycobacterium bovis. In Europe, M. bovis is, next to the classical human M. tuberculosis, the most frequent variant within the M. tuberculosis complex that causes disease in humans. In 1958, Sweden was declared free of bovine tuberculosis after an extensive national eradication campaign. Since then, sporadic cases of M. bovis infections in cattle have occurred, the latest being an outbreak in 1978 in which a farmer with renal tuberculosis infected his cattle. More recently, however, starting in 1991, M. bovis infections have occurred in 11 herds of farmed deer (1). All animals in these herds have been slaughtered. In Sweden, few cases of M. bovis infections have occurred in humans (11), mainly in the elderly, who are likely to have been infected with M. bovis before the infection was eradicated in cattle. Since 1978, no obvious transmission between animal and humans was found until 1991, when Hillerdal et al. reported M. bovis infections in a household also involving the family’s cat (7). In 1993, two camels exported from Sweden were diagnosed with M. bovis infection while in import quarantine in the United States. Molecular genetic methods have recently been introduced in studies of the epidemiology of tuberculosis. Outbreaks of multi-drug-resistant tuberculosis have been traced by DNA fingerprinting of M. tuberculosis isolates by restriction fragment length polymorphism (RFLP) (4). In Sweden, the spread of classical human tuberculosis between different animals in a zoological garden was demonstrated by RFLP analysis, showing that bacteria of the same M. tuberculosis clone caused disease in two tapirs and one gibbon (9). The advantage of the RFLP method, with labeled probes, compared with restriction fragment analysis of the whole genome lies in the easier eval-
MATERIALS AND METHODS M. bovis isolates. Forty-nine Swedish M. bovis-M. bovis BCG strains isolated during 1990 to 1993 were analyzed by RFLP patterns (Tables 1 and 2). Eleven isolates came from humans (age, 48 to 78 years): 6 from women and 5 from men. The isolates from humans were referred to SIIDC, Stockholm, Sweden, for species identification. One isolate (strain 2698/89) came from the man in the family in which the cat was also infected (described above). To our knowledge, all had been infected in Sweden. In addition, seven M. bovis BCG isolates came from the same number of children with local BCG infections. Twenty-eight M. bovis isolates came from 13 deer, originating from five different herds involved in an outbreak of bovine tuberculosis described elsewhere (1). Most deer isolates were isolated at the National Veterinary Institute, Uppsala, Sweden; during the investigation of the outbreak, some were isolated at SIIDC from material obtained from the National Veterinary Institute. In addition, one isolate from a cat (described above) and two isolates from two different camels were included. The camel strains were isolated by the U.S. Department of Agriculture National Veterinary Services Laboratories, Ames, Iowa, from camels exported from Sweden and held in U.S. import quarantine. Both strains were kindly donated to the National Veterinary Institute by J. Payeur at the U.S. Department of Agriculture, Ames, Iowa. All isolates were tested at SIIDC with the M. tuberculosis complex-specific nucleic acid probe by the Accuprobe system (Gen-probe, San Diego, Calif.). The isolates were further characterized by standard biochemical methods (10). Resistance to thiophen-2-carboxylic acid hydrazide (5 mg/liter) was determined by radiometric respirometry (BACTEC, Becton Dickinson, Cockeysville, Md.). The BACTEC method has previously been shown to agree with results obtained with LJ medium (3, 8). Detection of niacin was performed according to the method of Wayne (14), and the nitrate reductase test and pyrazinamidase test were performed according to the method of Kent and Kubica (10). By these tests, all strains conformed with the criteria for the species M. bovis. All isolates were positive by the M. tuberculosis complex-specific nucleic acid probe. RFLP analysis. Bacterial growth, DNA extraction, digestion of DNA, and Southern blotting were performed essentially as described previously (12). After
* Corresponding author. Mailing address: Department of Bacteriology, Swedish Institute for Infectious Disease Control, S-105 21, Stockholm, Sweden. Phone: 46 8 7351188. Fax: 46 8 7351040. † Present address: Umweltbundesamt, Institute for Water, Soil and Air Hygiene, 14191 Berlin, Germany. 3183
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TABLE 1. RFLP patterns of M. bovis and M. bovis BCG isolates from humans and animals other than deer Origin
M. bovis Human
Isolate
RFLP pattern (no. of bands)
2698/89 3869/90 69/91 244/91 3040/91 2180/92 S:27/91 S:183/91 S:37/92 S:9/93 S:49/93
2 1 1a 1 1 1 1 1 1 1 1
Camel
KAM 1 KAM 2
1 1
Cat
S:174/89
2
7 isolates
1
M. bovis BCG a
Band in a different position compared with those of the other isolates.
the genomic M. tuberculosis DNA had been extracted, it was digested with the restriction endonuclease PvuII and separated by electrophoresis in 0.8% agarose gels. After electrophoresis, the DNA fragments were transferred from the gel to a nylon membrane (Hybond N1; Amersham). The blot was hybridized with a 245-bp peroxidase-labeled (6) fragment of the IS6110 sequence, which had been produced by amplification by PCR with the primers INS-1 (5-CGTGAGG GCATCGAGGTGGC) and INS-2 (5-GCGTAGGCGTCGGTGACAAA), and nonradioactively labeled with an enhanced chemiluminescence kit (Amersham International Plc., Little Chalfont, United Kingdom), as instructed by the manufacturer. The probe was detected by the enhanced chemiluminescence direct nucleic acid detection system (Amersham). An M. tuberculosis reference strain, Mt.14323, from the strain collection of the Laboratory of Bacteriology and Antimicrobial Agents (National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands) was included in each gel as a standard.
RESULTS The M. bovis isolates were analyzed with a standardized RFLP assay with the insertion sequence IS6110 as the target. The RFLP patterns of all isolates consisted of one or more band, showing that the IS6110 element was present in one or more copies in the genome of all of the M. bovis isolates (Fig. 1 and Tables 1 and 2). All M. bovis BCG isolates, the two camel isolates, and all but one of the human M. bovis isolates had an RFLP pattern with only one band at an identical position. One of the human M. bovis isolates (69/91) had one band with a different molecular weight than those of the other isolates. One human isolate (strain 2698/89) and the cat isolate (strain S:174/89) from the same household had RFLP patterns with two bands at identical positions. In contrast, all M. bovis deer isolates but one had an identical RFLP banding pattern, characterized by seven IS6110 insertions (Fig. 1 and Table 2). One of two isolates from a deer in herd 2 repeatedly showed an identical pattern, except for lacking the band with the highest molecular weight. DISCUSSION In this study of RFLP patterns, all human M. bovis isolates showed simple RFLP patterns containing only one or two copies of the IS6110 insertion elements in the genome. However, M. bovis isolates from an outbreak of M. bovis infections in five herds of farmed deer in Sweden had a highly specific
RFLP pattern, characterized by seven copies of the IS6110 sequence. The IS6110 insertion element is present in 1 to 20 copies per genome of mycobacterial clones belonging to the M. tuberculosis complex (6). In classical human M. tuberculosis variants, the IS6110 element is usually present in 8 to 20 copies, while in M. bovis BCG vaccine strains, this element is present in only one copy (5, 13). M. bovis strains in general show low copy numbers, in the range of one to five copies (13). This was also true for our isolates from humans, the cat, and the two camels. In a report about the IS6110 RFLP patterns of 24 M. bovis strains from The Netherlands, all strains had fewer than six copies and one-third of the strains had only a single copy (13). In another study, from New Zealand (2), only 5 of 160 strains of M. bovis had more than one copy of IS6110; three of these strains came from a group of 10 strains originating from countries other than New Zealand. On the basis of the single- or very-low-copy marker of IS6110 in most M. bovis strains, Collins et al. (2) suggested that the IS6110 RFLP could only be used to distinguish M. bovis strains into wide groups rather than clones. This was true for the camel strains and the human strains in our study that had RFLP patterns with one to two bands. Thus, no conclusion about clonal identity among human and camel strains can be drawn. Strain S:174/89, isolated from a cat that contracted tuberculosis from its owner, had two insertions of IS6110, which was
TABLE 2. RFLP patterns of M. bovis deer isolates Origin
Herd 1 Deer Deer Deer Deer Deer node Deer Deer Deer Deer Deer Deer Deer node Deer Deer Deer Deer Deer Deer Deer Deer Deer
Isolate
RFLP pattern (no. of bands)
1, 1, 1, 1, 2,
liver mediastinal lymph node mesenterial lymph node pooled sample retropharyngeal lymph
H-1/91 H-57/91 H-82/91 S:202/92 H-13/91
7 7 7 7 7
2, 2, 2, 3, 3, 3, 3,
mediastinal lymph node mesenterial lymph node pooled sample lung mesenterial lymph node mediastinal lymph node retropharyngeal lymph
H-28/91 H-55/91 S:141/92 H-24/91 H-38/91 H-62/91 H-63/91
7 7 7 7 7 7 7
3, 3, 3, 4, 4, 5, 5, 6, 7,
inguinal lymph node prescapular lymph node pooled sample lung periportal lymph node mesenterial lymph node pooled sample pooled sample pooled sample
H-64/91 H-65/91 S:204/92 H-53/91 H-54/91 H-85/91 S:203/92 S:63/92 S:65/92
7 7 7 7 7 7 7 7 7
Herd 2 Deer 1, lymph node Deer 1, lung Deer 2, pooled sample
H-115/91 H-116/91 S:64/92
6 7 7
Herd 3 Deer 1, pooled sample Deer 2, pooled sample
S:19/92 S:66/92
7 7
Herd 4; deer 1, pooled sample
S:40/92
7
Herd 5; deer 1, pooled sample
S:75/92
7
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ACKNOWLEDGMENTS We thank Eva Lindell and Solomon Ghebremichael for skillful technical assistance. This study was supported by grants to SIIDC from the Swedish Heart Lung Foundation and by the Commission of the European Communities, Directorate General XII, Biomedical and Health Research, Biomed 1, contract BMH1-CT93-1614.
REFERENCES
FIG. 1. Comparison of IS6110 RFLP patterns of M. bovis isolates. Lanes: 1 to 8 are isolates from deer. Lane 1, H-55/91; lane 2, H-62/91; lane 3, H-64/91; lane 4, H-115/91; lane 5, S:203/91; lane 6, S:204/91; lane 7, S:63/92; lane 8 S:75/92. Lanes 9 and 10 and 12 to 14 are isolates from humans. Lane 9, S:183/91; lane 10, 3040/91; lane 12, 2698/89a; lane 13, 2698/89b; lane 14, S:104/93. Lane 11 is an isolate from a cat (S:174/89).
also the case for the strain from the owner. Those two strains differed from the other strains presented in this survey. Because of the low copy number, it is not possible to conclude that these two strains belong to the same clone, but our results support the connection indicated by the epidemiological investigation (7). To our knowledge, the M. bovis clone causing outbreaks in the Swedish deer herds has one of the most complex IS6110 RFLP patterns so far described. The high number of copies and the identity of the banding patterns make it certain that all isolates belong to the same clone of M. bovis. This confirms the results from the epidemiological tracing and the restriction fragment analysis reported earlier by Bo ¨lske et al. (1). The RFLP pattern of one isolate from a deer in herd 2 repeatedly showed only six bands and lacked the band with the highest molecular weight. The fact that the remaining six DNA bands had exactly the same molecular weights as the bands of the other isolates strongly suggests that this isolate also belongs to the same M. bovis clone, particularly since another isolate from the same animal had the common seven-band pattern. This illustrates that in spite of the described stability of RFLP patterns, minor changes may occur within a clone. It is therefore of value to analyze several different isolates from the same individual. The unique pattern of this M. bovis clone makes it possible on a long-term basis to detect any spread of this M. bovis clone to other wild and domestic animals as well as to humans.
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