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Restriction endonuclease analysis of plasmid DNA of Yersinia pseudotuberculosis infections in Shimane Prefecture, Japan
Zbl. Bakt. 282, 498-506 (1995) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Restriction Endonuclease Analysis of Plasmid DNA of Yersinia pseudotuberculosis Infections in Shimane Prefecture, Japan HIROSHI FUKUSHIMA and MANABU GOMYODA Public Health Institute of Shimane Prefecture, 582-1 Nishihamasada, Matsue, Shimane 690-01, Japan Received October 12, 1994 . Accepted January 14, 1995
Summary The epidemiology of Yersinia pseudotuberculosis infections in a limited area of Shimane Prefecture, Japan, was examined by serotyping and restriction endonuclease analysis of virulence plasmid DNA of Y. pseudotuberculosis strains isolated from humans, wildlife animals and river water. Almost all isolates from three sources belonged to serotype Ib REAP pattern D and serotype 4b REAP patterns B, G and L. The identity of the distribution of serotype and REAP patterns among isolates from humans, wildlife animals and river water shows that Y. pseudotuberculosis is transmitted to humans through environmental substances contaminated by wildlife animals infected with this species. Introduction Yersinia pseudotuberculosis is an important causative agent of sporadic and epidemic human enteric disease and is widely distributed in wildlife and domestic animals and environmental substances (13, 14). Wildlife animals are thought to be the principal reservoir of infection by Y. pseudotuberculosis (11). We (7, 9) have pointed out that Y. pseudotuberculosis is transmitted to humans through environmental substances contaminated by wildlife animals. In this study, we used restriction endonuclease analysis of virulence plasmid DNA (REAP) isolated from this species. The relationship between Y. pseudotuberculosis in wildlife animals and human Y. pseudotuberculosis infections was not clear as isolates from environmental substances were heavily contaminated with other bacteria. We (6) then developed an effective method for the selective isolation of Y. pseudotuberculosis from river water and the circumstances of contamination of river water were examined. In thus article, we present our findings of Y. pseudotuberculosis infections in the eastern part of Shimane Prefecture, Japan, an area where the epidemiology of human yersiniosis has been studied for 15 years, based on REAP of many strains isolated from humans, wildlife animals and environmental substances, particularly river water samples obtained from mountain rivers.
Epidemiology of Yersinia pseudotuberculosis Infection
499
Materials and Methods Strains of Y. pseudotuberculosis. One hundred and fifty six Y. pseudotuberculosis strains harbouring a 40 to 50 MDa virulent plasmid and belonging to serotypes Ib, 2b, 3, 4a, 4b, 6 and 10 were from patients (8), wildlife animals (4), well water and river water over a wide area in the eastern part of Shimane Prefecture (Table 1). Isolation of Yersinia pseudotuberculosis from river water was done using direct KOH and HeLa cell treatments (3, 5, 6). These strains were isolated from the area shown in Fig. 1. Serotypes Ib and 4b showed a similar distribution among wild animals, river water and humans. Strains of serotypes 2b, 2c, 3, 4a, 6 and 10 were rarely isolated from humans, river water and wild animals; therefore, distribution of these strains among the three sources could not be assessed. Serotype Ib strains were isolated from river water and wildlife animals in a limited area of the Chugoku Mountainous District, an area in which serotype 1b strains were prevalent among humans. Serotype 4b strains were widely isolated from river water in the Shimane Peninsula and the Chugoku Mountainous District, areas in which serotype 4b strains were predominantly isolated from humans and wild animals. REAP. REAP was done using the enzyme BamHI (9).
Results REAP. REAP of Y. pseudotuberculosis strains using the enzyme BamHI is shown in Table 2. REAP patterns of Y. pseudotuberculosis revealed 16 (A to P) distinct patterns (9). The predominant serotypes Ib and 4b were classified into 6 and 7 patterns, respectively (Fig. 2). Serotype 1b strains isolated from humans belonged to four REAP patterns (D, E, F and H), and almost all isolates from river water samples and wildlife animals belonged to REAP pattern D, except for one strain each which belonged to patterns B and H. Serotype 4b strains isolated from humans belonged to four REAP patterns (B, G, Hand K) and almost all isolates from river water samples and wild animals beTable 1. Serotypes of Y. pseudotuberculosis isolated from humans, river water and wild animals in Shimane Prefecture No. of isolates of serotype: Source Patients a River water Well water Wild miceb Raccoon dogs Hares b Deerb Marten b Wild duckb Total a b
Total
Ib
43 65 1 9 29 1 6 1 2
13 16 1 1 3 1
157
36
Data from reference 7. Data from reference 4.
2b
3
4a
4b
1
2 2
1
28 45 7 24
1
6
1
10
1
6 1 1
1 1
5
1
112
1
1
500
H. Fukushima and M. Gomyoda
Chugoku Mountain District
10km Fig. 1. Regional distribution of Y. pseudotuberculosis among wildlife animals, river water and humans in the eastern Shimane Prefecture. Symbols: A, serotype 1b; 0, serotype 2b; ~, serotype 3; 0, serotype 4a; e, serotype 4b; 0, serotype 6; -k, serotype 10.
Total
Patients River water Well water Wild mice Raccoon dogs Hares Deer Marten Wild duck
Source
1
157
6 1 2
43 65 1 9 29
Total
1
1
B
29
1
8 15 1 1 3
D
1
1
E
1b
2
2
F
3
2 1
H
1
1
H
2b
4
1
1 2
B
3
1
1
D
1
1
L
4a
33
1 3
52
1
2
11
3
7 5
12
G
25
D
6 15
B
4b
4
6
4
8
2
K
H
No. of strains (with indicated REAP pattern by BamHI) of serotype:
Table 2. REAP patterns of Y. pseudotuberculosis isolates in Shimane Prefecture
12
4
3
5
L
1
1
B
6
1
1
D
10
tTl
~
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....
V> 0
0 i:I
8.
i:I ~
'"0;'
-
0-
;:
"
...
~ <::so:.
0
;: \:l...
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~.
;i.
~
0
....,
~
0"
o·
(1)
a
0.:
'"0
502
H. Fukushima and M. Gomyoda
Serotype 1b
o
Fig. 2. REAP patterns of BamHI digestion of plasmid DNA within serotypes 1b and 4b of Y. pseudotuberculosis. Lane MW, lambda DNA digested with HindlIl as a molecular marker. The strains of serotype 1b are represented as follows. Lanes: B,RD43; D,Pa1994; E,Pa3492; F,Pa9198; H, Pa12116; and 0, 510-1. The strains of serotype 4b are represented as follows. Lanes: B, Pa3422; D, Wla643; G, Pa3450, H, 87-11, K, 143; L, W134; and M, 81-286.
longed to REAP patterns Band G. Several isolates from raccoon dogs belonged to REAP patterns D and K. REAP pattern L was observed in twelve strains isolated from river water and wildlife animals but not from humans. Serotypes 2b, 3, 4a, 6 and 10 were previously classified as belonging to 5, 4, or 2, or a single different REAP patterns, respectively (9), and in this study, the isolates fell into one or two REAP patterns. Almost all isolates from humans, river water samples and wild animals belonged to serotype 1b REAP pattern D and serotype 4b REAP patterns B, G and L.
Regional distribution of REAP patterns The regional distribution of predominant REAP patterns of serotypes 1b and 4b was closely related among wildlife animals, river water and humans (Fig. 3). Sertoype Ib REAP pattern D was frequently observed among isolates from river water and wild animals in the limited area of the Chugoku Mountainous District, an area in which serotype Ib REAP pattern D was observed among human isolates. The distribution of serotype 4b REAP pattern B was mainly observed among isolates from humans, river water and wild animals on the southern border of the epidemic area of serotype Ib REAP pattern D in the Chugoku Mountainous area. The distribution of serotype 4b
Epidemiology of Yersinia pseudotuberculosis Infection
503
Chugoku Mountain District
••
10km
Fig. 3. Regional distribution of REAP patterns of Y. pseudotuberculosis serotypes Ib and 4b among wildlife animals, river water and humans in eastern Shimane Prefecture. Symbols: e, Humans; A, River and well water; 0, Wild mouse; 1cr, Raccoon dog; D, Deer; !c" Marten; ., Hare; <), Wild duck.
REAP pattern G was mainly observed among isolates from humans, river water and wildlife animals in Shimane Peninsula and some strains were found on the western border of the epidemic area of serotype Ib REAP pattern D in the Chugoku Mountainous area. Serotype 4b REAP pattern L was observed among isolates from river water and deer in the western part of the Shimane Peninsula but not from humans.
Discussion An effective and solid barrier against the spreading of certain subtypes of Y. pseudotuberculosis by a predominant type of Y. pseudotuberculosis among wildlife animals was suggested by findings that certain subtypes were prevalent among humans and wildlife animals in a limited area. The predominant type strains are widely prevalent among humans and wildlife animals surrounding the area in which these subtypes are prevalent among wildlife animals (Figs. 1 and 3). The possibility of this phenomenon was noted in experimentally induced infections with pathogenic Yersinia in mice (1, 12, 15) and wild mice (2). Wild mice convalescing from infection with Y. pseudotuberculosis are protected upon subsequent challenge with homologeneous and heterogeneous serotypes of Y. pseudotuberculosis (2). This protection is commonly 34
Zbl. B,kt. 282/4
504
H. Fukushima and M. Gomyoda
observed in a subsequent challenge with virulent Yersinia organisms, such as Y. pestis, Y. pseudotuberculosis and pathogenic Y. enterocolitica, of mice convalescing from infection with Y. pseudotuberculosis or pathogenic Y. enterocolitica (1, 12, 15). This acquired resistance to virulent Yersinia organisms is due to a cross-immunity (1), and factors linked to the protection against re-infection with virulent Yersinia organisms may be V and W antigens (15). This phenomenon may dominate the distribution of species and serotype of virulent Yersinia organisms in nature. Alonso et al. (1) pointed out that Y. enterocolitica, demonstrated to be widely spread among mammalian populations in various parts of the world, might act as a silent but efficient barrier against Y. pestis from natural foci. In the present study, this hypothesis was given support. Almost all isolates from humans and wildlife animals belonged to serotype 1b REAP pattern D and serotype 4b REAP patterns B, G and L, and the most predominant type was serotype 4b REAP pattern G. The majority of serotype 1 b REAP pattern D strains was prevalent among humans and wildlife animals in a limited area of Chugoku Mountain district (Fig. 3). In much the same area with serotype 1b REAP pattern D strains, serotype 4b REAP pattern B strains were prevalent. However, the majority of serotype 4 b REAP pattern G strains were prevalent in Shimane Peninsula, west of the area in which the strains of serotype 1b REAP pattern D and serotype 4b REAP pattern B were prevalent. Both strains of serotype 1b REAP pattern D and serotype 4b REAP pattern B were rarely isolated in Shimane Peninsula in which strains of serotype 4b REAP pattern G were prevalent. These findings suggest that Y. pseudotuberculosis serotype 4b REAP pattern G, which was widely spread among wildlife animals in the examined area, might act as an efficient barrier against other types of Y. pseudotuberculosis. Y. pseudotuberculosis is an important causative agent of zoonosis. Infection with Y. pseudotuberculosis is acquired by direct or indirect contact with animals (11). Unchlorinated drinking water from wells, springs and streams in mountainous areas of Japan is considered to have caused many sporadic and epidemic infections (9, 10, 13). However, the actual circumstances of contamination of environmental substances such as river water and soil remained unknown because an effective method for selective isolation of Y. pseudotuberculosis had not been developed. Isolation of Yersinia pseudotuberculosis in river water was done using direct KOH and HeLa cell treatments (3,5,7). Details on the distribution of Y. pseudotuberculosis in water from rivers in the eastern part of Shimane Prefecture, Japan, an area in which Y. pseudotuberculosis serotype 1b and 4b strains are prevalent among wild animals and humans, were first obtained using direct KOH and HeLa cell treatments. Thus, a detailed epidemiological link among humans, wildlife animals and environmental substances such as water can be examined by serotyping and REAP. The role of unchlorinated drinking water as a route of human infection was suggested by the following observations. Patients, wildlife animals and river water strains had the same cultural, biological, pathogenic and molecular characteristics and the same epidemiological aspects. The majority of water strains belonged to serotypes 1band 4b, types most prevalent among wild animals and found most frequently in patients with gastroenteritis due to Y. pseudotuberculosis. The distribution of each serotype in river water was the same as that in humans and wild animals. Namely, serotype 1b strains were mainly isolated from river water in the limited area of Chugoku Mountain district where serotype 1b strains were prevalent among humans and wildlife animals, although the serotype 4b strains were widely distributed in· river water and in humans and wild animals, regardless of the area (Fig. 1).
Epidemiology of Yersinia pseudotuberculosis Infection
505
In the present work, REAP of the Y. pseudotuberculosis strains isolated from humans, wildlife animals and river water in the eastern part of Shimane Prefecture demonstrated for the first time the close link between human Y. pseudotuberculosis infections, harbouring of Y. pseudotuberculosis by wildlife animals and contamination of environmental substances with Y. pseudotuberculosis. The distribution of serotype and REAP patterns of the isolates from river water was much the same as those of the isolates from humans and wildlife animals (Table 2 and Fig. 3). Serotype 4b REAP pattern L strains were frequently isolated from water samples obtained from rivers in a limited area of the western part of Shimane Peninsula in which this type is prevalent in deer; patients with this type have not been identified in this area. These findings strongly suggest that environmental substances such as water and soil are contaminated with Y. pseudotuberculosis excreted from wildlife animals infected with this species.
References 1. Alonso, J. M., E. Vilmer, D. Mazigh, and H. H. Mollaret: Mechanisms of acquired resistance to plague in mice infected by Y. enterocolitica 03. Current. Microbiol. 4 (1980) 117-122 2. Fukushima, H.: Susceptibility of wild mice to Yersinia pseudotuberculosis and Yersinia enterocolitica. Zentralbl. Bakteriol. 275 (1991) 530-540 3. Fukushima, H.: Direct isolation of Yersinia pseudotuberculosis from fresh water in Japan. Appl. Environ. Microbiol. 58 (1992) 2688-2690 4. Fukushima, H. and M. Gomyoda: Intestinal carriage of Yersinia pseudotuberculosis by wild birds and mammals in Japan. Appl. Environ. Microbiol. 57 (1991) 1152-1155 5. Fukushima, H., M. Gomyoda, and S. Aleksic: Isolation of Yersinia pseudotuberculosis in river waters in Japan and Germany using direct KOH and HeLa cell treatments. Zbl. Bakt. (in press) 6. Fukushima, H., K. Hoshina, and M. Gomyoda: Selective isolation from HeLa cell lines of Yersinia pseudotuberculosis, pathogenic Y. enterocolitica and enteroinvasive Escherichia coli. Zentralbl. Bakteriol. 280 (1994) 332-337 7. Fukushima, H., M. Gomyoda, K. Shiozawa, S. Kaneko, and M. Tsubokura: Yersinia pseudotuberculosis infection contracted through water contaminated by a wild animal. J. Clin. Microbiol. 26 (1988) 584-585 8. Fukushima, H., K. Hoshina, R. Nakamura, Y. Ito, and M. Gomyoda: Epidemiological study of Yersinia enterocolitica and Yersinia pseudotuberculosis in Shimane Prefecture, Japan. Contrib. Microbiol. Immunol. 9 (1987) 103-110 9. Fukushima, H., M. Gomyoda, S. Kaneko, M. Tsubokura, N. Takeda, T. Hongo, and F. N. Shubin: Restriction endonuclease analysis of virulence plasmids for molecular epidemiology of Yersinia pseudotuberculosis infections. J. Clin. Microbiol. 32 (1994) 1410-1413 10. Inoue, M., H. Nakashima, T. Ishida, and M. Tsubokura: Three outbreaks of Yersinia pseudotuberculosis infection. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe B186 (1988) 504-511 11. Mair, N. S.: Yersiniosis in wildlife and its public health implication. J. Wildl. Dis. 9 (1973) 64-71 12. Pearson, A. D., I. D. Ricciardi, D. H. Wright, and W. G. Suckling: An experimental study of the pathology and ecology of Yersinia enterocolitica infection in mice. Contrib. Microbiol. Immunol. 5 (1979) 335-345 13. Sato, K. and M. Komazawa: Yersinia pseudotuberculosis infection in children due to untreated drinking water. Contrib. Microbiol. Immunol. 12 (1992) 5-10
506
H. Fukushima and M. Gomyoda
14. Tsubokura, M., K. Otsuki, K. Sa to, M. Tanaka, T. Hongo, H. Fukushima, T. Maruyama, and M. Inoue: Special features of distribution of Yersinia pseudotuberculosis in Japan. J. Clin. Microbiol. 27 (1989) 790-791 15. Uchida, I., K. Kaneko, and N. Hashimoto: Cross-protection against fecal excretion of Yersinia enterocolitica and Yersinia pseudotuberculosis in mice by oral vaccination of viable cells. Infect. Immun. 36 (1982) 837-840 Hiroshi Fukushima, Ph. D., Public Health Institute of Shimane Prefecture, 582-1 Nishihamasada, Matsue, Shimane 690-01, Japan
Restriction Endonuclease Analysis of Plasmid DNA of Yersinia pseudotuberculosis Infections in Shimane Prefecture, Japan HIROSHI FUKUSHIMA and MANABU GOMYODA Public Health Institute of Shimane Prefecture, 582-1 Nishihamasada, Matsue, Shimane 690-01, Japan Received October 12, 1994 . Accepted January 14, 1995
Summary The epidemiology of Yersinia pseudotuberculosis infections in a limited area of Shimane Prefecture, Japan, was examined by serotyping and restriction endonuclease analysis of virulence plasmid DNA of Y. pseudotuberculosis strains isolated from humans, wildlife animals and river water. Almost all isolates from three sources belonged to serotype Ib REAP pattern D and serotype 4b REAP patterns B, G and L. The identity of the distribution of serotype and REAP patterns among isolates from humans, wildlife animals and river water shows that Y. pseudotuberculosis is transmitted to humans through environmental substances contaminated by wildlife animals infected with this species. Introduction Yersinia pseudotuberculosis is an important causative agent of sporadic and epidemic human enteric disease and is widely distributed in wildlife and domestic animals and environmental substances (13, 14). Wildlife animals are thought to be the principal reservoir of infection by Y. pseudotuberculosis (11). We (7, 9) have pointed out that Y. pseudotuberculosis is transmitted to humans through environmental substances contaminated by wildlife animals. In this study, we used restriction endonuclease analysis of virulence plasmid DNA (REAP) isolated from this species. The relationship between Y. pseudotuberculosis in wildlife animals and human Y. pseudotuberculosis infections was not clear as isolates from environmental substances were heavily contaminated with other bacteria. We (6) then developed an effective method for the selective isolation of Y. pseudotuberculosis from river water and the circumstances of contamination of river water were examined. In thus article, we present our findings of Y. pseudotuberculosis infections in the eastern part of Shimane Prefecture, Japan, an area where the epidemiology of human yersiniosis has been studied for 15 years, based on REAP of many strains isolated from humans, wildlife animals and environmental substances, particularly river water samples obtained from mountain rivers.
Epidemiology of Yersinia pseudotuberculosis Infection
499
Materials and Methods Strains of Y. pseudotuberculosis. One hundred and fifty six Y. pseudotuberculosis strains harbouring a 40 to 50 MDa virulent plasmid and belonging to serotypes Ib, 2b, 3, 4a, 4b, 6 and 10 were from patients (8), wildlife animals (4), well water and river water over a wide area in the eastern part of Shimane Prefecture (Table 1). Isolation of Yersinia pseudotuberculosis from river water was done using direct KOH and HeLa cell treatments (3, 5, 6). These strains were isolated from the area shown in Fig. 1. Serotypes Ib and 4b showed a similar distribution among wild animals, river water and humans. Strains of serotypes 2b, 2c, 3, 4a, 6 and 10 were rarely isolated from humans, river water and wild animals; therefore, distribution of these strains among the three sources could not be assessed. Serotype Ib strains were isolated from river water and wildlife animals in a limited area of the Chugoku Mountainous District, an area in which serotype 1b strains were prevalent among humans. Serotype 4b strains were widely isolated from river water in the Shimane Peninsula and the Chugoku Mountainous District, areas in which serotype 4b strains were predominantly isolated from humans and wild animals. REAP. REAP was done using the enzyme BamHI (9).
Results REAP. REAP of Y. pseudotuberculosis strains using the enzyme BamHI is shown in Table 2. REAP patterns of Y. pseudotuberculosis revealed 16 (A to P) distinct patterns (9). The predominant serotypes Ib and 4b were classified into 6 and 7 patterns, respectively (Fig. 2). Serotype 1b strains isolated from humans belonged to four REAP patterns (D, E, F and H), and almost all isolates from river water samples and wildlife animals belonged to REAP pattern D, except for one strain each which belonged to patterns B and H. Serotype 4b strains isolated from humans belonged to four REAP patterns (B, G, Hand K) and almost all isolates from river water samples and wild animals beTable 1. Serotypes of Y. pseudotuberculosis isolated from humans, river water and wild animals in Shimane Prefecture No. of isolates of serotype: Source Patients a River water Well water Wild miceb Raccoon dogs Hares b Deerb Marten b Wild duckb Total a b
Total
Ib
43 65 1 9 29 1 6 1 2
13 16 1 1 3 1
157
36
Data from reference 7. Data from reference 4.
2b
3
4a
4b
1
2 2
1
28 45 7 24
1
6
1
10
1
6 1 1
1 1
5
1
112
1
1
500
H. Fukushima and M. Gomyoda
Chugoku Mountain District
10km Fig. 1. Regional distribution of Y. pseudotuberculosis among wildlife animals, river water and humans in the eastern Shimane Prefecture. Symbols: A, serotype 1b; 0, serotype 2b; ~, serotype 3; 0, serotype 4a; e, serotype 4b; 0, serotype 6; -k, serotype 10.
Total
Patients River water Well water Wild mice Raccoon dogs Hares Deer Marten Wild duck
Source
1
157
6 1 2
43 65 1 9 29
Total
1
1
B
29
1
8 15 1 1 3
D
1
1
E
1b
2
2
F
3
2 1
H
1
1
H
2b
4
1
1 2
B
3
1
1
D
1
1
L
4a
33
1 3
52
1
2
11
3
7 5
12
G
25
D
6 15
B
4b
4
6
4
8
2
K
H
No. of strains (with indicated REAP pattern by BamHI) of serotype:
Table 2. REAP patterns of Y. pseudotuberculosis isolates in Shimane Prefecture
12
4
3
5
L
1
1
B
6
1
1
D
10
tTl
~
'"0:.
....
V> 0
0 i:I
8.
i:I ~
'"0;'
-
0-
;:
"
...
~ <::so:.
0
;: \:l...
"<:;
~.
;i.
~
0
....,
~
0"
o·
(1)
a
0.:
'"0
502
H. Fukushima and M. Gomyoda
Serotype 1b
o
Fig. 2. REAP patterns of BamHI digestion of plasmid DNA within serotypes 1b and 4b of Y. pseudotuberculosis. Lane MW, lambda DNA digested with HindlIl as a molecular marker. The strains of serotype 1b are represented as follows. Lanes: B,RD43; D,Pa1994; E,Pa3492; F,Pa9198; H, Pa12116; and 0, 510-1. The strains of serotype 4b are represented as follows. Lanes: B, Pa3422; D, Wla643; G, Pa3450, H, 87-11, K, 143; L, W134; and M, 81-286.
longed to REAP patterns Band G. Several isolates from raccoon dogs belonged to REAP patterns D and K. REAP pattern L was observed in twelve strains isolated from river water and wildlife animals but not from humans. Serotypes 2b, 3, 4a, 6 and 10 were previously classified as belonging to 5, 4, or 2, or a single different REAP patterns, respectively (9), and in this study, the isolates fell into one or two REAP patterns. Almost all isolates from humans, river water samples and wild animals belonged to serotype 1b REAP pattern D and serotype 4b REAP patterns B, G and L.
Regional distribution of REAP patterns The regional distribution of predominant REAP patterns of serotypes 1b and 4b was closely related among wildlife animals, river water and humans (Fig. 3). Sertoype Ib REAP pattern D was frequently observed among isolates from river water and wild animals in the limited area of the Chugoku Mountainous District, an area in which serotype Ib REAP pattern D was observed among human isolates. The distribution of serotype 4b REAP pattern B was mainly observed among isolates from humans, river water and wild animals on the southern border of the epidemic area of serotype Ib REAP pattern D in the Chugoku Mountainous area. The distribution of serotype 4b
Epidemiology of Yersinia pseudotuberculosis Infection
503
Chugoku Mountain District
••
10km
Fig. 3. Regional distribution of REAP patterns of Y. pseudotuberculosis serotypes Ib and 4b among wildlife animals, river water and humans in eastern Shimane Prefecture. Symbols: e, Humans; A, River and well water; 0, Wild mouse; 1cr, Raccoon dog; D, Deer; !c" Marten; ., Hare; <), Wild duck.
REAP pattern G was mainly observed among isolates from humans, river water and wildlife animals in Shimane Peninsula and some strains were found on the western border of the epidemic area of serotype Ib REAP pattern D in the Chugoku Mountainous area. Serotype 4b REAP pattern L was observed among isolates from river water and deer in the western part of the Shimane Peninsula but not from humans.
Discussion An effective and solid barrier against the spreading of certain subtypes of Y. pseudotuberculosis by a predominant type of Y. pseudotuberculosis among wildlife animals was suggested by findings that certain subtypes were prevalent among humans and wildlife animals in a limited area. The predominant type strains are widely prevalent among humans and wildlife animals surrounding the area in which these subtypes are prevalent among wildlife animals (Figs. 1 and 3). The possibility of this phenomenon was noted in experimentally induced infections with pathogenic Yersinia in mice (1, 12, 15) and wild mice (2). Wild mice convalescing from infection with Y. pseudotuberculosis are protected upon subsequent challenge with homologeneous and heterogeneous serotypes of Y. pseudotuberculosis (2). This protection is commonly 34
Zbl. B,kt. 282/4
504
H. Fukushima and M. Gomyoda
observed in a subsequent challenge with virulent Yersinia organisms, such as Y. pestis, Y. pseudotuberculosis and pathogenic Y. enterocolitica, of mice convalescing from infection with Y. pseudotuberculosis or pathogenic Y. enterocolitica (1, 12, 15). This acquired resistance to virulent Yersinia organisms is due to a cross-immunity (1), and factors linked to the protection against re-infection with virulent Yersinia organisms may be V and W antigens (15). This phenomenon may dominate the distribution of species and serotype of virulent Yersinia organisms in nature. Alonso et al. (1) pointed out that Y. enterocolitica, demonstrated to be widely spread among mammalian populations in various parts of the world, might act as a silent but efficient barrier against Y. pestis from natural foci. In the present study, this hypothesis was given support. Almost all isolates from humans and wildlife animals belonged to serotype 1b REAP pattern D and serotype 4b REAP patterns B, G and L, and the most predominant type was serotype 4b REAP pattern G. The majority of serotype 1 b REAP pattern D strains was prevalent among humans and wildlife animals in a limited area of Chugoku Mountain district (Fig. 3). In much the same area with serotype 1b REAP pattern D strains, serotype 4b REAP pattern B strains were prevalent. However, the majority of serotype 4 b REAP pattern G strains were prevalent in Shimane Peninsula, west of the area in which the strains of serotype 1b REAP pattern D and serotype 4b REAP pattern B were prevalent. Both strains of serotype 1b REAP pattern D and serotype 4b REAP pattern B were rarely isolated in Shimane Peninsula in which strains of serotype 4b REAP pattern G were prevalent. These findings suggest that Y. pseudotuberculosis serotype 4b REAP pattern G, which was widely spread among wildlife animals in the examined area, might act as an efficient barrier against other types of Y. pseudotuberculosis. Y. pseudotuberculosis is an important causative agent of zoonosis. Infection with Y. pseudotuberculosis is acquired by direct or indirect contact with animals (11). Unchlorinated drinking water from wells, springs and streams in mountainous areas of Japan is considered to have caused many sporadic and epidemic infections (9, 10, 13). However, the actual circumstances of contamination of environmental substances such as river water and soil remained unknown because an effective method for selective isolation of Y. pseudotuberculosis had not been developed. Isolation of Yersinia pseudotuberculosis in river water was done using direct KOH and HeLa cell treatments (3,5,7). Details on the distribution of Y. pseudotuberculosis in water from rivers in the eastern part of Shimane Prefecture, Japan, an area in which Y. pseudotuberculosis serotype 1b and 4b strains are prevalent among wild animals and humans, were first obtained using direct KOH and HeLa cell treatments. Thus, a detailed epidemiological link among humans, wildlife animals and environmental substances such as water can be examined by serotyping and REAP. The role of unchlorinated drinking water as a route of human infection was suggested by the following observations. Patients, wildlife animals and river water strains had the same cultural, biological, pathogenic and molecular characteristics and the same epidemiological aspects. The majority of water strains belonged to serotypes 1band 4b, types most prevalent among wild animals and found most frequently in patients with gastroenteritis due to Y. pseudotuberculosis. The distribution of each serotype in river water was the same as that in humans and wild animals. Namely, serotype 1b strains were mainly isolated from river water in the limited area of Chugoku Mountain district where serotype 1b strains were prevalent among humans and wildlife animals, although the serotype 4b strains were widely distributed in· river water and in humans and wild animals, regardless of the area (Fig. 1).
Epidemiology of Yersinia pseudotuberculosis Infection
505
In the present work, REAP of the Y. pseudotuberculosis strains isolated from humans, wildlife animals and river water in the eastern part of Shimane Prefecture demonstrated for the first time the close link between human Y. pseudotuberculosis infections, harbouring of Y. pseudotuberculosis by wildlife animals and contamination of environmental substances with Y. pseudotuberculosis. The distribution of serotype and REAP patterns of the isolates from river water was much the same as those of the isolates from humans and wildlife animals (Table 2 and Fig. 3). Serotype 4b REAP pattern L strains were frequently isolated from water samples obtained from rivers in a limited area of the western part of Shimane Peninsula in which this type is prevalent in deer; patients with this type have not been identified in this area. These findings strongly suggest that environmental substances such as water and soil are contaminated with Y. pseudotuberculosis excreted from wildlife animals infected with this species.
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