Molecular Fingerprinting of Mycobacterium bovis subsp. caprae Isolates from Central Europe

Abstract

To study the dissemination of Mycobacterium bovis subsp. caprae, 79 European isolates from cattle, humans, and other hosts were examined by spoligotyping and IS6110 restriction fragment length polymorphism (RFLP) analysis. Among a total of 11 different spoligotypes identified, type C1 proved to be predominant (n = 62). Five of the spoligotypes are described for the first time. A total of 43 different RFLP types were identified, thus allowing further differentiation for epidemiological tracking. Isolates from a series of outbreaks in one village proved to be of the same spoligotype and of identical or closely related RFLP types.

Mycobacterium bovis subsp. caprae is a member of the M. tuberculosis complex, which also includes M. tuberculosis, M. africanum, M. bovis, M. bovis BCG, M. microti, and M. canettii. General interest in this pathogen, which was isolated from tuberculous lesions in humans, cattle, goats, sheep, and deer, has been steadily increasing since its first description (3). Thus, the prevalence of M. bovis subsp. caprae was extensively studied in Spain (4, 7) and France (8). There have been several reports about its occurrence in animals and humans in Austria (18) and the Czech Republic (15, 16, 17) and also in Germany (13), where it is reported to account for about one-third of human M. bovis-associated cases of tuberculosis (10).

Identification of the various M. tuberculosis complex organisms by spoligotyping (9, 12) is performed on the basis of the presence or absence of certain combinations of 43 spacers which are interspersed among a high number of conserved repeats in the chromosomal direct-repeat region. Additional intraspecies genetic differences can be revealed by IS6110 fingerprinting or IS6110 restriction fragment length polymorphism (RFLP) analysis (19), which takes advantage of the mobility of insertion element IS6110 along the mycobacterial chromosome.

The combination of spoligotyping and IS6100 RFLP can be expected to improve the possibilities of revealing epidemiological relationships because of the different discriminatory potential of both methods (18). Recent studies of serial isolates of M. tuberculosis clearly showed that due to the relatively frequent occurrence of IS6110 transposition events, IS6100 RFLP patterns were more likely to change within a few years of a strain's appearance than spoligotyping patterns (1, 6, 20).

In the present study, 79 isolates of M. bovis subsp. caprae collected from tuberculous lesions in cattle (n = 55), humans (n = 14), wild boar (n = 7), camels (n = 2), and deer (n = 1) from five European countries were subjected to molecular typing by spoligotyping and IS6100 RFLP analysis.

The isolates described in Table 1 were grown in cultures according to standard procedures (2). Chromosomal DNA of mycobacterial strains was isolated using the cetyltrimethylammonium bromide method (12) and subjected to direct-repeat-specific PCR and hybridization with membrane-bound spacer oligonucleotides and a spoligotyping kit (ISOGEN Bioscience, Maarsen, The Netherlands). IS6100 RFLP analysis, which included PvuII digestion of mycobacterial DNA, separation by agarose gel electrophoresis, Southern blotting, and hybridization with a digoxigenin-labeled IS6110 probe, was conducted according to a previously described method (19).

TABLE 1.

Classification of M. bovis subsp. caprae isolates from different herds or groups according to genotype and host organism

Genotype	Yr of isolation	Host organisma					Total no. of:
		Bovine	Bactrian camel	Red deer	Wild boar	Human	Herds or localities	Isolates
C1-rc1	1998	GER(Bt06-10)					5	19
C1-rc2	1998	GER(Bt09)					1	1
C1-rc3	1998	GER(Bt10)					1	1
C1-rc5	1995	GER(Bt02)					1	1
C1-rc6	1995	GER(Bt01)					1	1
C1-rc7	1996, 1998	GER(Bt04, -05)					2	2
C1-rc10	1997, 2002	GER(Bt03, -17)					2	2
C1-rc11	1999	GER(Bt11)					1	2
C1-rc12	1999	GER(Bt11)					1	1
C1-rc14	1999, 2000	GER(Bt12, -13)					2	2
C1-rc15	1999					CZE(H01)	1	1
C1-rc16	2000	GER(Bt14)					1	1
C1-rc17	2000	GER(Bt15)					1	1
C1-rc18	2001	GER(Bt16)					1	1
C1-rc24	2001					GER(H01)	1	1
C1-rc25	2001					GER(H06)	1	1
C1-rc26	2000				HUN(Ss01)		1	1
C1-rc27	1999, 2000	HUN(Bt01)				GER(H12)	2	2
C1-rc28	1999			CZE(Ce01)			1	1
C1-rc29	2001	GER(Bt18)					1	1
C1-rc32	2002		CZE(Cf01)				1	2
C1-rc33	2001				HUN(Ss03)		1	1
C1-rc35	2001				HUN(Ss04)		1	1
C1-rc37	2001	HUN(Bt02)			HUN(Ss02)		2	2
C1-rc38	2001				HUN(Ss05)		1	1
C1-rc39	2001	CRO(Bt01)					1	9
C1-rc40	2001	CRO(Bt01)					1	1
C1-rc41	1995	CZE(Bt01)					1	1
C1-rc42	2001	CRO(Bt01)					1	1
C2-rc4	1998					GER(H03)	1	1
C2-rc20	1999					GER(H04)	1	1
C2-rc22	2000					GER(H05)	1	1
C2-rc23	2001					GER(H02)	1	1
C2-rc26	2000				HUN(Ss06)		1	1
C3-rc10	1996	GER(Bt19)					1	1
C4-rc21	1991	GER(Bt20)				GER(H07)	2	2
C5-rc30	2001					GER(H08)	1	1
C5-rc34	2001				HUN(Ss07)		1	1
C5-rc36	2001	HUN(Bt03)					1	1
C6-rc13	2000					GER(H09)	1	1
C7-rc19	1998					GER(H10)	1	1
C8-rc8	1998	GER(Bt21)					1	1
C9-rc9	1983	GER(Bt22)					1	1
C10-rc31	2002					GER(H11)	1	1
C11-rc43	2001					SLO(H01)	1	1
Total		25	1	1	7	14	55	79

^aCRO, Croatia; CZE, Czech Republic; GER, Germany; HUN, Hungary; SLO, Slovenia; Bt, Bos taurus (cattle); Cf, Camelus ferus (bactrian camel); Ce, Cervus elaphus (red deer); Ss, Sus scrofa (wild boar); H, human (Homo sapiens).

Spoligotyping of the 79 isolates revealed 11 different spoligotypes (designated C1 to C11), the profiles of which are schematically depicted in Fig. 1. Spoligotype C1, which is defined by the presence of spacers 2, 17 to 27, and 29 to 38, represented by far the most abundant pattern (62 isolates). Alongside previous reports showing this spoligotype to be occurring in individual European countries (8, 10, 13, 15, 16, 17, 18), the present data suggest that C1 is indeed disseminated throughout the continent and appears to be the predominant spoligotype of M. bovis subsp. caprae in cattle and wild boar and also in humans. The closely related spoligotype C2 (distinct from C1 by the absence of spacer 2) was detected in five isolates. Type C5, which (in contrast to type C1) lacks spacer 32, was found three times, and type C4 (lacking spacers 33 and 34) was identified twice. All other spoligotypes (C3 and C6 to C11) were detected only in single isolates. Types C2, C4, C5, C8, and C9 have been found before in Europe (8, 10, 13, 14, 18), but isolates of C3, C6, C7, C10, and C11 are described here for the first time. Interestingly, C10 [represented by the human isolate GER(H11)] is the only spoligotype to harbor spacer 14 (Table 1).

FIG. 1.

Spoligotype patterns obtained from 79 isolates of M. bovis subsp. caprae. Spacer numbers are given in the first line. The designation of the spoligotype is given in the left-hand column. Open or solid squares indicate the absence or presence of a specific spacer sequence, respectively.

The considerable extent of genetic diversity among the present 79 isolates was revealed by IS6100 RFLP analysis, which identified 43 different RFLP types (designated rc1 to rc43) (Fig. 2 and Table 1). The most prominent RFLP types among all isolates were rc1, whose four-band pattern was encountered 19 times, and rc39 (six bands; n = 9). Type rc10 was found three times, and rc7, rc11, rc14, rc21, rc26, rc27, rc32, and rc37 were found two times. All other RFLP types represented only single isolates. Another report on RFLP typing of 10 isolates of M. bovis subsp. caprae (18) mentioned six different RFLP patterns, one of which was identical to type rc14 identified in this study.

FIG. 2.

Dendrogram showing relative IS6110 band sizes of all isolates belonging to spoligotype C1. Chemiluminescent hybridization images were normalized using Phoretix 1D software, and cluster analysis was done with the unweighted pair group method with arithmetic mean algorithm. The RFLP type is given at the right-hand margin. The most abundant type (rc1) is highlighted.

Generally, the isolates combined in a spoligotype proved to be rather diverse in terms of RFLP patterns. Thus, there were 29 different RFLP types among the representatives of spoligotype C1 (n = 62). The two isolates of spoligotype C4 were of the same RFLP type, i.e., rc21. In all other instances, there were as many RFLP types as isolates. A classification of isolates according to genotype and host organism is given in Table 1.

Isolates having matching genotypes are of particular interest for epidemiological considerations. Thus, bovine isolates from herds GER(Bt12) and GER(Bt13) originating from different farms of the same village in southern Germany and collected in 1999 and 2000, respectively, both belonged to cluster C1-rc14 (Table 1). It seems obvious that an epidemiological relationship between them exists. Interestingly, Prodinger et al. (18) have detected isolates of this particular genotype from cattle and a red deer calf in an Austrian region bordering southern Germany in 1999. Therefore, it is probable that transmission occurred through wildlife, even more so as the respective herds and the deer had access to the same Alpine pasture.

The remarkable homogeneity in terms of genotypes among the isolates from five German herds of cattle clearly indicates transmission of the pathogen between neighboring farms. In this panel of isolates, 19 out of 21 were identified as genotype C1-rc1 (Table 1). There was proven contact among four of these herds, i.e., GER(Bt06) to GER(Bt09), which were all from the same village in southern Germany. A relationship to the fifth herd, GER(Bt10), which was located in northern Germany, appeared probable but was not verifiable.

An interesting genetic grouping was formed by isolates from herds GER(Bt04/Bt05) and GER(Bt12/Bt13), which harbored the closely related clusters C1-rc7 and C1-rc14, respectively; although the isolates were collected during different outbreaks in the years 1996, 1998, 1999, and 2000, respectively, a connection between them seems likely, as the herds were based in the same region.

In another case of a common genotype shared by two herds of cattle in the same region, i.e., C1-rc10 in GER(Bt03) and GER(Bt17), the time difference between isolations was 5 years, thus raising the issue of how the causative agent might have persisted during that period.

The identification of genotype C1-rc37 in both cattle HUN(Bt02) and wild boar HUN(Ss02) of the same district in Hungary indicates transmission between animal species. The infection of cattle from wildlife reservoirs was already reported from Spain (3), Italy (5), and Slovakia (11). Transmission of genotype C4-rc21 from cattle to a tuberculosis patient, i.e., from GER(Bt20) to GER(H07), was presumed but could not be substantiated by further evidence.

The relatively rapid evolution of IS6100 RFLP profiles as a result of insertion events may be the main reason for the great variety of the patterns encountered. As the transposition rate was shown to be dependent on the M. tuberculosis genotype (1), however, IS6110 fingerprinting-based epidemiological tracking over a period of a few years can be difficult in the case of rapidly evolving strains. In an investigation of the rate of change involving serial isolates from 544 patients, the half-life (t_1/2) of a given RFLP pattern was extrapolated to be 3.2 years (6). Warren et al. (20) observed evolutionary changes of banding patterns in 4% of their M. tuberculosis strains and calculated a mean t_1/2 of 8.74 years. They suggested that this value may be composed of a high rate of change seen during the early disease phase (t_1/2 = 0.57 years), i.e., during active growth prior to therapy, and a low rate in the late disease phase (t_1/2 = 10.69 years), i.e., during or after treatment.

Assuming levels of genetic variability of M. bovis subsp. caprae in the same range, the higher rate of change should apply for the present panel of isolates, as the animals did not get treatment. Indeed, rapid genetic variation during acute infection in the absence of therapy is indicated by the identification of closely related RFLP types in several sets of related isolates, namely, rc1 and rc2, rc1 and rc3, rc5 and rc6, rc11 and rc12, rc39 and rc40, and rc39 and rc42 (Dice similarity index of all pairs > 80%; data not shown). Although coinfection cannot be ruled out, the close genetic relatedness within these pairs of RFLP types suggests microevolution of the prevailing strains during persistence of the pathogen in a herd and/or upon transmission. Interestingly, two isolates from GER(Bt11), representing related RFLP types rc11 and rc12, respectively, were collected from the same cow, the latter originating from a lymph node, the site of active growth of mycobacteria.

We conclude that the combination of data from spoligotyping and IS6100 RFLP analysis provided additional insight into epidemiological processes and the evolution of genotypes of M. bovis subsp. caprae.