APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1978, p. 995-1002 0099-2240/78/0035-0995$02.00/0 Copyright X 1978 American Society for Microbiology
Vol. 35, No. 6
Printed in U.S.A.
Distribution of Ribonucleic Acid Coliphages in South and East Asia K. FURUSE,'* T. SAKURAI,2 A. HIRASHIMA,' M. KATSUKI,' A. ANDO,' AND I. WATANABE' Department of Molecular Biology, School of Medicine, Keio University, Shinanomachi, Shinjuka-ku, Tokyo 160,1 and Department ofMicrobiology, Yakult Institute for Microbiological Research, 1796, Yaho, Kunitachi-shi, Tokyo 186,2 Japan
Received for publication 22 December 1977
We investigated the distribution of ribonucleic acid (RNA) coliphages in the Philippines, Singapore, Indonesia, India, and Thailand by collecting sewage samples from domestic drainage in November 1976. Of the 221 samples collected from domestic drainage, 50 contained RNA phages (52 strains). By serological analysis, 46 of the 52 strains were found to belong to group III. It can thus be said that the most prevalent RNA phages in Southeast Asia (at least, in the Philippines, Singapore, and Indonesia) were group III phages. Investigations of sewage samples collected from domestic drainage in Japan indicate that the most prevalent RNA phages in mainland Japan (north of Kyushu) are group II phages, whereas group III phages are predominant in the southem part of Japan (south of Amamiohshima Island). We therefore propose a borderline between Kyushu and Amamiohshima Island for the geographical distribution of RNA coliphages in the domestic drainage of South and East Asia. Moreover, one strain (ID2) was inactivated to some extent with the antisera of four groups of RNA phages. This is thought to be significant from the evolutionary viewpoint. We have isolated many ribonucleic acid (RNA) coliphages from sewage samples collected in several countries and classified them into four groups (I through IV) according to several biological and physicochemical criteria such as serological properties (15, 16, 18), ultraviolet light sensitivity (8), buoyant density in CsCl (14), sedimentation constant of their RNAs (unpublished data), and template specificity of the RNA to their replicases (12). Moreover, recent isolation of serological intermediates (phages JP34 and JP500) between groups I and II and another (phage MX1) between groups III and IV (6, 7), together with several other observations (1, 12, 13, 17), suggest that groups I and II can be incorporated into a large group (A) and groups III and IV into another large group (B). Concerning the ecology of bacteriophages, we have made extensive efforts to determine the geographical distribution of RNA phages in sewage, because we believe this to constitute one of their natural habitats. We collected numerous sewage samples from several countries such as Taiwan (1970) (10), Brazil (1971) (11), Japan (1972 to 1977) (2, 5, 7), and Central and South America (1975) (6). Analysis of these samples revealed several interesting features in the pattern of distribution of RNA phages, as follows. (i) Few RNA phages were isolated from sewage samples collected in the countries of Central and South America. (ii) In Asian countries, many
kinds of RNA phages were isolated from the same source. (iii) In Japan, group II RNA phages were predominant on the mainland, whereas most RNA phages isolated from the southwest islands of Japan belonged to group III. These findings led us to undertake a more intensive survey of the Asian area in an attempt to elucidate the distributional pattern of RNA phages in relation to that of Japan. In this paper, we report the distribution of RNA coliphages in five Asian countries (the Philippines, Singapore, Indonesia, India, and Thailand). We also search for new types of RNA phages (i.e., RNA phages not belonging to any of the four known groups or having common characteristics within them) to clarify the relationships among the four known groups of RNA phages.
MATERIALS AND METHODS Media. The PG medium (used for collection of sewage samples, isolation of RNA phages, and dilution of phage and antiphage sera) and PGYC medium (used for the preparation of crude phage lysate) were as described previously (1). Bacterial strains. Escherichia coli K-12S A/A(F+), Q13(ribonuclease I-, Hfr), and W3110(F-) (abbreviated simply to A/A, Q13, and W3110, respectively, hereafter) were used as host strains for the isolation and preparation of RNA phages. Antiphage sera. Antiphage sera of groups I (MS2, BO1, JP501), II (GA, BZ13, TH1, KU1, JP34), III (Qf, VK, ST, TW18, MX1), and IV (SP, Fl, TW19, TW28) 995
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FURUSE ET AL.
APPL. ENVIRON. MICROBIOL.
grouping of newly isolated RNA phages. Collection of sewage samples. (i) Basic details. The countries, cities, number of samples collected, dates of collection, and base laboratories used for preparation of the original phage samples are listed in Table 1. (ii) Collection of sewage samples and preparation of original phage samples. The materials used for the isolation of RNA phages were limited to sewage samples from domestic drainage and raw sewage from sewage treatment plants. We have found that such materials may contain various types of coliphages (7, 10, 11). Approximately 1 g of sewage sample was taken from domestic drainage and suspended in 5 ml of PG medium by using an attached spoon in a small, sterilized plastic tube. Samples (5 to 10 ml) of raw sewage from the sewage treatment plants were also collected into vacant, sterilized plastic tubes. The samples were then stored in a refrigerator or at room temperature for 2 to 5 days, treated with 0.5 ml of chloroform to kill bacteria, and centrifuged to remove bacterial debris and certain other precipitates. The supernatant fractions (original phage samples) so obtained were transferred to small glass tubes, sealed with an air-tight rubber stopper, and transported back to Japan under normal temperature for examination. (iii) Records of collection. The sample numbers,
were used for the serological
place, source of material, environmental conditions, dates of collection, pH, and temperature of the sewage were recorded. When circumstances permitted, photographs were also taken to illustrate the general environImental conditions. Isolation of RNA phages. Each 0.1 ml of original phage sample was plated on E. coli strains A/A(F+), Q13(Hfr), and W3110(F-), and single plaques on the individual plates were picked up, suspended in 2 ml of PG, and treated with 0.5 ml of chloroform. After centrifugation, the supematant fraction was used for further analysis. In the present study, 3 to 10 plaques were usually picked up per plate. These phage stocks were subjected to spot tests on E. coli strains A/A, Q13, W3110, and A/A including ribonuclease (100 ug per plate). Phages which lysed strains A/A and/or Q13 but did not lyse strains W3110 and A/A (with ribonuclease) were picked up, purified several times by the single-plaque isolation method, and stocked in a cold room (4°0) as RNA phages. Grouping and subgrouping by the serological method. As shown in Table 3, newly isolated RNA phages were subjected to spot tests on A/A or Q13 with (K = 1 per plate) or without antiserum of six standard phages (MS2[I], JP34[II], GA[II], Q/B[III], VK[III], and SP[IV]). After this preliminary classification by the spot test, each RNA phage was subjected to further analysis for subgrouping by the plating
TABLE 1. Details of countries, cities, treatment plants, number of samples, and dates of collection Country
City
Treatment plant/base laboratory
No. of samples
Date
Philippines
Manila
Makati Sewage Treatment Plant
1
Nov. 3, 1976
Singapore
Singapore
Tanglin Sewage Pumping Station
1
Nov. 6, 1976
India
Delhi Delhi Delhi Agra
North Side Sewage Purification Plant Kilokali Water Supply and Treatment Okhla Sewage Treatment Plant Agra Sewage Pumping House Total for treatment plants
1 1 1 1 6
Nov. 23, Nov. 23, Nov. 23, Nov. 25,
Philippines
Manila
Institute of Hygiene, University of the Philippines
Quezon Total
1976 1976 1976 1976
38
Nov. 2-3, 1976
23 61
Nov. 3, 1976
Singapore
Singapore
Department of Pathology
29
Nov. 6-7, 1976
Indonesia
Jakarta
Biomedical Research Centre, National Institute of Health Research and Development
20
Nov. 11, 1976
14 34
Nov. 13, 1976
14
Nov. 20-21, 1976
34 11 59
Nov. 23-24, 1976 Nov. 25, 1976
38
Nov. 30-Dec. 1, 1976
Yogjakarta Total India
Madras
National Institute of Communicable Diseases
Delhi Agra Total Thailand
Bangkok
Faculty of Tropical Medicine, Mahidol University
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RNA PHAGE DISTRIBUTION IN SOUTH AND EAST ASIA
997
TABLE 2. Serological relationships among four groups of RNA phages Antiserum Phage
I MS2
II GA
III
QfB
IV SP
1 x 0.-4b 0.7 1.0 1.3 Group I (a)a MS2 1 x 1O-4 0.6 1.3 1.3 Group II (a) GA 1 x 10-5 1.1 1.0 0.9 Group III (a) Q8 1.0 1.2 1.2 8 x 10-4 Group IV (a) SP (a), One subgroup of group I. b The degree of inactivation by an antiserum was obtained from [PFU on serum plate (K = 1/plate) per PFU on serum-free plate]. a
method (7) on E. coli strain A/A with (4 = 1 per plate) or without antiserum. The degree of inactivation (plaque-forming units [PFU] on serum plate per PFU on serum-free plate) was measured, and the values for the newly isolated phages were compared with those of standard ones (Table 2). The RNA phages thus obtained were designated serially as PP1 through PP31 for phages from the Philippines, SP1 through SP12 for those from Singapore, IN1 through IN7 for those from Indonesia, ID1 and ID2 for those from India, and TL1 and TL2 for those from Thailand according to the sample number for each country, respectively.
RESULTS Frequency of appearance of coliphages and RNA phages in the samples. A sample which contained one or more coliphages (RNA phages) per 0.1 ml of original phage sample was judged to be a coliphage(+) [RNA phage(+)] sample. As shown in Table 3, coliphages were detected in all sewage samples from the sewage treatment plants of three countries and in almost all sewage samples from the domestic drainage of the five countries. The amounts of total coliphages in these sewage samples were fairly high, ranging from 10 to 107 PFU/ml (many showing 102 to 105 PFU/ml). RNA phages were isolated at high frequency from samples from the Philippines (48%), Singapore (35%), and Indonesia (21%). This result was similar to that reported for other Asian countries: Taiwan (28%) (10) and Japan (20 to 70%) (5, 7, 9). The sewage samples from India and Thailand, however, had few RNA phages (3.4 and 5.3%, respectively), a result which is consistent with data for Brazil (5%) (11) and Mexico (2%) (6). The relative amounts of RNA phages in the sewage samples from the Philippines, Singapore, and Indonesia were fairly high, occupying from 10 to 90% of the total coliphages. The corresponding values for the samples from India and Thailand were also similar to those for the above three countries even though the actual number of RNA phage(+) samples was very few. No significant differences in sewage pH (most values
were between 5 and 7), sewage temperature (20 to 30°C), volume of sewage water, or apparent environmental conditions were detected among the domestic drainage in the five countries. Serological grouping and geographical distribution of RNA phages. When two or more RNA phages isolated from the same original sample showed similar inactivation patterns to each other, they were considered as the same strain. (The majority of samples conformed to this case.) The results of serological grouping by the spot test and of subgrouping by the plating method are shown in Tables 3 and 4, respectively. All of the RNA phages tested were inactivated by one of the six standard antisera except for one strain, ID2. We thus classified the newly isolated 54 strains according to the four known groups of RNA phages. By comparing the inactivation patterns of each phage by the antisera of several subgroups of RNA phages with those of the standard ones, group I RNA phage PP25 was further classified into subgroup (a) of group I, and group II RNA phages PP3, SP5, IN7, and TL2 were classified into subgroups (c), (a), (d), and (d) of group II, respectively. Group III RNA phages isolated in the Philippines (16 out of 29 strains), Singapore (all 11 strains), Indonesia (all 6 strains), and Thailand (1 strain) were classified into subgroup (a). Nine other of the Philippines strains were classified into subgroup (b), and the remainder of the Philippines phages (PP1, -5, -10, and -20) were classified into a new subgroup (g). Group III RNA phage ID1 was neutralized well by an antiserum of TW18, and the pattern of neutralization by other antisera was similar to that of TW18. This phage was thus assigned to subgroup (d) of group III. Because group IV RNA phage ID2 was neutralized well by the antisera of TW19 and TW28 and was also sensitive to the antisera of groups I, II, and III to some extent, it was classified into a new subgroup (f) of group IV (Table 4). As shown in Table 3, most of the RNA phages isolated from the Philippines, Singapore, and
998
APPL. ENVIRON. MICROBIOL.
FURUSE ET AL.
TABLE 3. Frequencies of isolation of total coliphages and RNA phages in collected materials Group RNA phage Coliphage
(+)a______b_Group)
city
(treatment plant)
No. of strains'
No. of
No. of samples
samples
I
III
IV
0
1
100
1
100
1
0
0
1
1
100
1
100
1
0
0
1
0
1
100
0
0
0
0
0
0
0
1
100
0
0
0
0
0
0
0
1
100
0
0
0
0
0
0
0
1
100
0
0
0
0
0
0
0
6
100
2
33.3
2
0
0
2
0
Manila Quezon Total
38 23 61
100 100 100
21 8 29
55.3 34.8 47.5
21 9 30
0 1 1
1 0 1
20 8 28
0 0 0
Singapore
20
69.0
10
34.5
11
0
1
10
0
Jakarta Yogjakarta Total
17 13 30
85.0 92.9 88.2
3 4 7
15.0 28.6 20.6
3 4 7
0 0 0
0 1 1
3 3 6
0 0 0
Madras Delhi Agra Total
14 33 11 58
100 97.1 100 98.3
1 0 1 2
7.1 0 9.1 3.4
1 0 1 2
0 0 0 0
0 0 0 0
1 0 0 1
0 0 1 1
Bangkok
34
89.5
2
5.3
2
0
1
1
0
Manila (Makati Sewage Treatment Plant) Singapore (Tanglin Sewage Pumping Station) Delhi (North Side Sewage Purification Plant) Delhi (Kilokali Water Supply and Treatment) Delhi (Okhla Sewage Treatment Plant) Agra (Agra Sewage Pumping House) Total of treatment plants
1 22.6 52 1 4 46 50 203 91.9 Grand total a Number of samples containing one or more coliphages per 0.1 ml of original phage sample. b Number of samples containing one or more RNA phages per 0.1 ml of original phage sample. 'Number of RNA phages exhibiting different serological properties in the same original phage sample (see text). TABLE 4. Serological grouping of RNA phages isolated in Southeast Asia Group
Phage
Subgroup
No. of strains
I
(a)
PP25
1
II
(a)
SP5 PP3 IN7, TL2
1 1 2
(c) (d) III
(a)
(b) (d) (g) IV
(f)
PP4, 6, 7, 8, 11, 12, 15, 18, 19, 23, 24, 26, 27, 28, 30, 31
SP1, 2, 3, 4, 6, 7,.8, 9, 10, 11, 12, IN1, 2, 3, 4, 5, 6, TL1 PP2, 9, 13, 14, 16, 17, 21, 22, 29
34
ID1 PP1, 5, 10, 20
9 1 4
ID2
1
Indonesia could be assigned to group III: phages belonging to groups I, II, or IV were noticeably few. It is thus considered that a borderline can be drawn between Amamiohshima Island and
Kyushu with regard to the geographical distribution of RNA phages in South and East Asia (Fig. 1; Table 5). New RNA phage. At the initial stage of
VOL. 35, 1978
999
RNA PHAGE DISTRIBUTION IN SOUTH AND EAST ASIA
1.
I
:
0
_~
I 1.
1.
1.
Delhi--
&-Agra
ritlgdd,m 1.
-
lrirovotjina 1.
Jakawr
FIG. 1. Distribution of RNA coliphages in south and east Asia. The data refer to Tables 3 and 5.
classification by the spot test, ID2 showed only a small cross-reaction with two antisera, MS2(I) and SP(IV), and even at the second stage of classification by the plating method, it showed only a weak susceptibility to six standard
antisera (MS2[I], JP34[II], GA[II], QJ?[III], VK[III], and SP[IV]). We therefore tested the susceptibility of ID2 to 17 antisera of four groups of RNA phages. As shown in Table 6, ID2 was neutralized weli by the antisera of TW19 and TW28; however, it was also neutralized significantly by those of group I, II, and III phages, although to a lesser extent. We thus tentatively assigned phage ID2 to a new subgroup (fl of group IV. The inactivation patterns of group IV RNA phages [subgroups (a), (b), (c), and (d)] by the same 17 antisera are also shown in Table 6. In
contrast to ID2, these phages were inactivated only by the antisera of group IV, and not by those of groups I, II, and Ill. We have previously isolated serological intermediates between groups I and II (JP34 and JP500) and between groups III and IV (MX1). However, we did not find any intermediate phages among groups I, II, III, and IV. We therefore believe the present isolate (ID2) to be the first to share common characteristics among groups I, II, III, and IV. A detailed characterization of ID2 in addition to its serological character is now in progress. DISCUSSION Concerning the geographical distribution of RNA phages, it is of considerable interest to compare the present data with those previously
1000
APPL. ENVIRON. MICROBIOL.
FURUSE ET AL.
TABLE 5. Distributional pattern of RNA phages in Taiwan and Japan yr samples Survey Survey samples No. of
a_______ No. of
Group
RNA phage (+)a
Coliphage Country
Group_____
No. of
Reference
Taiwan
1970
121
samples 107
88.5
No. of samples 34
Japan proper
1972-1976
736
505
68.6
373
50.7
433
2
331
100
13
6
46.2
5
38.5
5
0
5
0
1973, 1975
57
47
82.5
30
52.6
32
0
6
26
0 8; unpub-
1973, 1975
62
39
62.9
25
40.3
25
0
15
10
1973
20
17
85.0
10
50.0
12
0
2
10
0 8; unpublished data 0 8
1973
18
15
83.3
11
61.1
11
0
11
0
93
74
79.6
33
35.5
38
0
21
17
0 8; unpub-
47
43
91.5
30
63.8
37
1
23
13
lished data 0 8; unpublished data
54
72.0
20
26.7
20
0
2
18
0
261
92.9
163
58.0
178
4
20
154
0
41
87.2
28
59.6
28
0
0
28
5
50.0
5
0
0
5
(area)
%
I
II
III
IV
8
22
2
strainsa 28.1
38
6
12
0 6,7; unpub-
lished data
North (Rishiri 1975 Island) South Ogasawara Island Hachijojima Island Miyakejima Island Niijima Island West Iki Island
0 8
lished data
1973, 1975, 1977 Tsushima Is- 1973, 1977 land
Southwest 75 1975 Amamioshima Island 281 Okinawa 1973, 1974 Mainland 47 Ishigakijima 1974 Island 10 Iriomotejima 1974 Island For explanation, see Table 3.
10
100
obtained in East Asia (1970 to 1977) (Table 5). Tables 3 and 5 clearly show that in Japan proper (Hokkaido, Honshu, Shikoku, and Kyushu) and in the islands in the seas adjacent to Japan
(Rishiri, Hachijojima, Miyakejima, Niijima, Iki, and Tsushima Islands), the most prevalent RNA phages are those of group II (group II/group III, 3:1), whereas in the Southwest islands of Japan (Amamiohshima Island, the mainland of Okinawa, and Ishigakijima and Iriomotejima Islands) and in Taiwan, the Philippines, Singapore, and Indonesia, the most prevalent RNA phages are those of group III. Moreover, some tendency exists for the ratio of group III to group II to increase according to separation from Japan proper toward the southern islands. In this respect, it is important for us to determine the distributional pattern of RNA phages in the center of the Asian continent. Dhillon et al. have revealed that sewage samples collected from both rural and urban areas of Hong Kong included many RNA phages. These were classified into groups I (MS2-related
0 8
Unpublished data
2; unpublished data 0 Unpublished data 0 Unpublished data
phages) and III (Qfl-related phages). They also showed that the number of samples containing MS2-related phages was almost the same as the number containing QJ-related phages, although the density of the former exceeded that of the latter (3, 4). Thus, from the viewpoint of the geographical distribution of RNA phages, the sewage of Hong Kong appears to have certain features in contrast with those of other Asian countries. However, it will be necessary to compare the results obtained by several procedures on the same sewage samples. At the present time, it can be said that in Japan, group I RNA phages have been isolated exclusively from the feces of cows and pigs and from the raw sewage of certain restricted sewage treatment plants (unpublished data). Although little is known about what constitutes the ideal conditions for isolation of RNA phages from their natural habitats, we have tentatively employed rich media (peptone-glucose), a fixed incubation temperature of 370C, and three strains of E. coli K-12 as host bacteria
RNA PHAGE DISTRIBUTION IN SOUTH AND EAST ASIA
VOL. 35, 1978
1001
TABLE 6. Serological properties of the new RNA phage ID2a Phage Group IV
Antiserum
(a) SP
(b)
Group I (a) MS2 (b) BO1 (c) JP501
New RNA phage ID2
FI
(c) TW19
(d) TW28
1.la 1.3 1.1
0.8 0.8 0.9
1.0 1.0 0.9
1.0 0.9 0.7
0.4 0.8 0.7
Group II (a) GA (b) BZ13 (c) TH1 (d) KUl (e) JP34
1.1 1.1 1.1 1.1 1.0
1.1 1.3 0.9 0.6 0.8
1.0 1.0 0.9 1.0 1.0
1.0 0.9 0.9 0.8 0.8
0.5 0.4 0.2 0.2 0.2
Group III (a) Qfi (b) VK (c) ST (d) TW18 (f) MXL
1.1 0.6 0.8 0.9 0.1
0.9 1.1 1.0 0.9 0.8
1.1 1.0 1.0 1.0
1.0 0.8 0.8 0.8 0.6
0.7 0.5 0.4 0.3 0.2
0.2 1.1 2 x 10-3 7 x 1i-5
0.6 0.7
Group IV 2 x 10-4 (a) SP 0.7 (b) Fl 0.3 (c) TWl9 0.6 (d) TW28 For explanation, see Table 2.
0.1 5 x 10-4 0.2 0.1
0.7 0.2 0.9 6 x 1i-5 2 x 10-4
1 X 10-2 7 x 1O-3
a
for the isolation, propagation, and titration of the phages. We have checked the influence of the temperature in transportation of the phage samples and observed no significant difference between normal conditions and refrigerated ones in the isolation frequency of RNA phages at least within several days. Under these conditions, we were in fact able to isolate RNA phages from sewage samples at a relatively high frequency, and the RNA phages we have tested so far (two to three strains of each group) have formed plaques on F+ strains of E. coli B and C with a fairly high plating efficiency comparable to that of K. We therefore believe our isolation procedure to cover a considerable fraction of the RNA coliphages to be isolated from the sewage samples. Our data appear to reflect more strongly the influence of the global physical condition such as geographical position or general climate surrounding- the domestic drainage, because they were derived exclusively from an analysis of sewage samples from domestic drainage and there is some evidence to suggest that group II RNA phages are the most temperature-sensitive ones and can propagate at the lowest temperature among the known four groups of RNA phages in in vitro experiments (unpublished
data). In spite of the current limitations in materials and methods, the time is ripe to accumulate data of this kind, because our knowledge of the distribution of coliphages in their natural habitats, especially from the ecological viewpoint, remains very limited. We are unable to explain the significance of coincidences such that the ID1 and ID2 isolated in India display serological affinities with the TW18 (a rare phage in group III) and TW28 (a rare phage in group IV), respectively, isolated in Taiwan. ACKNOWLEDGMENTS We wish to express our sincere appreciation to the governments of the Philippines, Singapore, Indonesia (particularly the LIPI), India, and Thailand (particularly the NRC) for permission to carry out research in their respective countries. We are also very much indebted to the following individuals whose institutions are listed in Table 1 for their kind support and collaboration throughout our research: Benjamin D. Cabrera, Veronica Chan, Sng Ewe Hui, Moses Yu, Lim Ah Loo, J. Sulianti Saroso, Iskak Koiman, B. L. Wattal, S. N. Ray, Ratna Sarkar, Chamlong Harinasuta, Savanat Tharavanij, and T. Akamatsu and Y. Sasaki (Yakult [Thailand] Co., Ltd). Kind assistance in sample collection was given by Soetresno Eram (Laboratory Kesehatan), M. P. Mehrotra (TB Demonstration and Training Centre), Raphiphat Kasemsook (Bureau of Public Health), and members of the six sewage treatment plants listed in Table 1. We wish to express our deep gratitude
1002
APPL. ENVIRON. MICROBIOL.
FURUSE ET AL.
to M. Sasa (National Institute for Environmental Studies) for introducing us to the suitable organizations (listed in Table 1) in each country and for providing valuable advice and assistance in our research project. Thanks are also due to the Tokyo Embassies of the five countries visited, the Ministry of Foreign Affairs of Japan, the Ministry of Education, Science and Culture of Japan, members of the Japanese Embassies in Manila, Singapore, Jakarta, Madras, Delhi, and Bangkok for their kind advice and cooperation. This study was aided principally by the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan, but thanks are also due to Yakult Honsha Co., Ltd. (Hisami Matsuzono) for additional financial support.
LITERATURE CITED 1. Ando, A., K. Furuse, T. Miyake, T. Shiba, and I. Watanabe. 1976. Three complementation subgroups in group IV RNA phage SP. Virology 74:64-72. 2. Aoi, T., T. Sakurai, and I. Watanabe. 1974. Isolation and grouping of RNA phages. VI. A survey in the Okinawa Island. J. Keio Med. Soc. 51:337-349. 3. Dhillon, E. K. S., and T. S. Dhillon. 1974. Synthesis of indicator strains and density of ribonucleic acid-containing coliphages in sewage. Appl. Microbiol. 27:640-649. 4. Dhillon, T. S., E. K. S. Dhillon, H. C. Chau, W. K. Li, and A. H. C. Tsang. 1976. Studies on bacteriophage distribution: virulent and temperate bacteriophage content of mammalian feces. Appl. Environ. Microbiol. 32:68-74. 5. Furuse, K., A. Ando, and I. Watanabe. 1975. Isolation and grouping of RNA phages. V. A survey in the islands in the adjacent seas of Japan. J. Keio Med. Soc.
52:259-263. 6. Furuse, K., A. Ando, and L. Watanabe. 1975. Isolation and grouping of RNA phages. VII. A survey in Peru, Bolivia, Mexico, Kuwait, France, Australia and the United States of America. J. Keio Med. Soc.
52:355-361. 7. Furuse, K., T. Aoi, T. Shiba, T. Sakurai, T. Miyake, and I. Watanabe. 1973. Isolation and grouping of RNA phages. IV. A survey in Japan. J. Keio Med. Soc.
50:363-376. 8. Furuse, K., T. Sakurai, and I. Watanabe. 1967. The effects of ultraviolet irradiation on various RNA phages. Virus 17:159-164. 9. Furuse, K., and I. Watanabe. 1973. Alteration in the distributional pattern of RNA phages. I. Patterns in summer and in winter. J. Keio Med. Soc. 50:437-443. 10. Miyake, T., K. Furuse, T. Shiba, T. Aoi, T. Sakurai, and I. Watanabe. 1971. Isolation and grouping of RNA phages in Taiwan. J. Keio Med. Soc. 48:25-34. 11. Miyake, T., K. Furuse, T. Shiba, T. Aoi, T. Sakurai, and I. Watanabe. 1973. Isolation and grouping of RNA phages. II. A survey in Brazil. J. Keio Med. Soc. 50:353-362. 12. Miyake, T., I. Haruna, T. Shiba, Y. H. Itoh, K. Yamane, and I. Watanabe. 1971. Grouping of RNA phages based on the template specificity of their RNA replicases. Proc. Natl. Acad. Sci. U.S.A. 68:2022-2024. 13. Miyake, T., and T. Shiba. 1970. Formation of hybrid particles in RNA phages. I. Hybrid particles between RNA phages SP and Fl. Virology 43:675-684. 14. Nishihara, T., and I. Watanabe. 1969. Discrete buoyant density distribution among RNA phages. Virology 39:360-362. 15. Sakurai, T., T. Miyake, T. Shiba, and I. Watanabe. 1968. Isolation of a possible fourth group of RNA phages. Jpn. J. Microbiol. 12:544-546. 16. Sakurai, T., I. Watanabe, and T. Ohno. 1967. Isolation and serological grouping of RNA phages. Virus 17:165-171. 17. Shiba, T. 1974. In vitro assembly of virus particles. II. Formation of hybrid particles between various RNA phages in vitro. J. Keio Med. Soc. 51:305-314. 18. Watanabe, I., T. Miyake, T. Sakurai, T. Shiba, and T. Ohno. 1967. Isolation and grouping of RNA phages. Proc. Jpn. Acad. 43:204-209.
Vol. 35, No. 6
Printed in U.S.A.
Distribution of Ribonucleic Acid Coliphages in South and East Asia K. FURUSE,'* T. SAKURAI,2 A. HIRASHIMA,' M. KATSUKI,' A. ANDO,' AND I. WATANABE' Department of Molecular Biology, School of Medicine, Keio University, Shinanomachi, Shinjuka-ku, Tokyo 160,1 and Department ofMicrobiology, Yakult Institute for Microbiological Research, 1796, Yaho, Kunitachi-shi, Tokyo 186,2 Japan
Received for publication 22 December 1977
We investigated the distribution of ribonucleic acid (RNA) coliphages in the Philippines, Singapore, Indonesia, India, and Thailand by collecting sewage samples from domestic drainage in November 1976. Of the 221 samples collected from domestic drainage, 50 contained RNA phages (52 strains). By serological analysis, 46 of the 52 strains were found to belong to group III. It can thus be said that the most prevalent RNA phages in Southeast Asia (at least, in the Philippines, Singapore, and Indonesia) were group III phages. Investigations of sewage samples collected from domestic drainage in Japan indicate that the most prevalent RNA phages in mainland Japan (north of Kyushu) are group II phages, whereas group III phages are predominant in the southem part of Japan (south of Amamiohshima Island). We therefore propose a borderline between Kyushu and Amamiohshima Island for the geographical distribution of RNA coliphages in the domestic drainage of South and East Asia. Moreover, one strain (ID2) was inactivated to some extent with the antisera of four groups of RNA phages. This is thought to be significant from the evolutionary viewpoint. We have isolated many ribonucleic acid (RNA) coliphages from sewage samples collected in several countries and classified them into four groups (I through IV) according to several biological and physicochemical criteria such as serological properties (15, 16, 18), ultraviolet light sensitivity (8), buoyant density in CsCl (14), sedimentation constant of their RNAs (unpublished data), and template specificity of the RNA to their replicases (12). Moreover, recent isolation of serological intermediates (phages JP34 and JP500) between groups I and II and another (phage MX1) between groups III and IV (6, 7), together with several other observations (1, 12, 13, 17), suggest that groups I and II can be incorporated into a large group (A) and groups III and IV into another large group (B). Concerning the ecology of bacteriophages, we have made extensive efforts to determine the geographical distribution of RNA phages in sewage, because we believe this to constitute one of their natural habitats. We collected numerous sewage samples from several countries such as Taiwan (1970) (10), Brazil (1971) (11), Japan (1972 to 1977) (2, 5, 7), and Central and South America (1975) (6). Analysis of these samples revealed several interesting features in the pattern of distribution of RNA phages, as follows. (i) Few RNA phages were isolated from sewage samples collected in the countries of Central and South America. (ii) In Asian countries, many
kinds of RNA phages were isolated from the same source. (iii) In Japan, group II RNA phages were predominant on the mainland, whereas most RNA phages isolated from the southwest islands of Japan belonged to group III. These findings led us to undertake a more intensive survey of the Asian area in an attempt to elucidate the distributional pattern of RNA phages in relation to that of Japan. In this paper, we report the distribution of RNA coliphages in five Asian countries (the Philippines, Singapore, Indonesia, India, and Thailand). We also search for new types of RNA phages (i.e., RNA phages not belonging to any of the four known groups or having common characteristics within them) to clarify the relationships among the four known groups of RNA phages.
MATERIALS AND METHODS Media. The PG medium (used for collection of sewage samples, isolation of RNA phages, and dilution of phage and antiphage sera) and PGYC medium (used for the preparation of crude phage lysate) were as described previously (1). Bacterial strains. Escherichia coli K-12S A/A(F+), Q13(ribonuclease I-, Hfr), and W3110(F-) (abbreviated simply to A/A, Q13, and W3110, respectively, hereafter) were used as host strains for the isolation and preparation of RNA phages. Antiphage sera. Antiphage sera of groups I (MS2, BO1, JP501), II (GA, BZ13, TH1, KU1, JP34), III (Qf, VK, ST, TW18, MX1), and IV (SP, Fl, TW19, TW28) 995
996
FURUSE ET AL.
APPL. ENVIRON. MICROBIOL.
grouping of newly isolated RNA phages. Collection of sewage samples. (i) Basic details. The countries, cities, number of samples collected, dates of collection, and base laboratories used for preparation of the original phage samples are listed in Table 1. (ii) Collection of sewage samples and preparation of original phage samples. The materials used for the isolation of RNA phages were limited to sewage samples from domestic drainage and raw sewage from sewage treatment plants. We have found that such materials may contain various types of coliphages (7, 10, 11). Approximately 1 g of sewage sample was taken from domestic drainage and suspended in 5 ml of PG medium by using an attached spoon in a small, sterilized plastic tube. Samples (5 to 10 ml) of raw sewage from the sewage treatment plants were also collected into vacant, sterilized plastic tubes. The samples were then stored in a refrigerator or at room temperature for 2 to 5 days, treated with 0.5 ml of chloroform to kill bacteria, and centrifuged to remove bacterial debris and certain other precipitates. The supernatant fractions (original phage samples) so obtained were transferred to small glass tubes, sealed with an air-tight rubber stopper, and transported back to Japan under normal temperature for examination. (iii) Records of collection. The sample numbers,
were used for the serological
place, source of material, environmental conditions, dates of collection, pH, and temperature of the sewage were recorded. When circumstances permitted, photographs were also taken to illustrate the general environImental conditions. Isolation of RNA phages. Each 0.1 ml of original phage sample was plated on E. coli strains A/A(F+), Q13(Hfr), and W3110(F-), and single plaques on the individual plates were picked up, suspended in 2 ml of PG, and treated with 0.5 ml of chloroform. After centrifugation, the supematant fraction was used for further analysis. In the present study, 3 to 10 plaques were usually picked up per plate. These phage stocks were subjected to spot tests on E. coli strains A/A, Q13, W3110, and A/A including ribonuclease (100 ug per plate). Phages which lysed strains A/A and/or Q13 but did not lyse strains W3110 and A/A (with ribonuclease) were picked up, purified several times by the single-plaque isolation method, and stocked in a cold room (4°0) as RNA phages. Grouping and subgrouping by the serological method. As shown in Table 3, newly isolated RNA phages were subjected to spot tests on A/A or Q13 with (K = 1 per plate) or without antiserum of six standard phages (MS2[I], JP34[II], GA[II], Q/B[III], VK[III], and SP[IV]). After this preliminary classification by the spot test, each RNA phage was subjected to further analysis for subgrouping by the plating
TABLE 1. Details of countries, cities, treatment plants, number of samples, and dates of collection Country
City
Treatment plant/base laboratory
No. of samples
Date
Philippines
Manila
Makati Sewage Treatment Plant
1
Nov. 3, 1976
Singapore
Singapore
Tanglin Sewage Pumping Station
1
Nov. 6, 1976
India
Delhi Delhi Delhi Agra
North Side Sewage Purification Plant Kilokali Water Supply and Treatment Okhla Sewage Treatment Plant Agra Sewage Pumping House Total for treatment plants
1 1 1 1 6
Nov. 23, Nov. 23, Nov. 23, Nov. 25,
Philippines
Manila
Institute of Hygiene, University of the Philippines
Quezon Total
1976 1976 1976 1976
38
Nov. 2-3, 1976
23 61
Nov. 3, 1976
Singapore
Singapore
Department of Pathology
29
Nov. 6-7, 1976
Indonesia
Jakarta
Biomedical Research Centre, National Institute of Health Research and Development
20
Nov. 11, 1976
14 34
Nov. 13, 1976
14
Nov. 20-21, 1976
34 11 59
Nov. 23-24, 1976 Nov. 25, 1976
38
Nov. 30-Dec. 1, 1976
Yogjakarta Total India
Madras
National Institute of Communicable Diseases
Delhi Agra Total Thailand
Bangkok
Faculty of Tropical Medicine, Mahidol University
VOL. 35, 1978
RNA PHAGE DISTRIBUTION IN SOUTH AND EAST ASIA
997
TABLE 2. Serological relationships among four groups of RNA phages Antiserum Phage
I MS2
II GA
III
QfB
IV SP
1 x 0.-4b 0.7 1.0 1.3 Group I (a)a MS2 1 x 1O-4 0.6 1.3 1.3 Group II (a) GA 1 x 10-5 1.1 1.0 0.9 Group III (a) Q8 1.0 1.2 1.2 8 x 10-4 Group IV (a) SP (a), One subgroup of group I. b The degree of inactivation by an antiserum was obtained from [PFU on serum plate (K = 1/plate) per PFU on serum-free plate]. a
method (7) on E. coli strain A/A with (4 = 1 per plate) or without antiserum. The degree of inactivation (plaque-forming units [PFU] on serum plate per PFU on serum-free plate) was measured, and the values for the newly isolated phages were compared with those of standard ones (Table 2). The RNA phages thus obtained were designated serially as PP1 through PP31 for phages from the Philippines, SP1 through SP12 for those from Singapore, IN1 through IN7 for those from Indonesia, ID1 and ID2 for those from India, and TL1 and TL2 for those from Thailand according to the sample number for each country, respectively.
RESULTS Frequency of appearance of coliphages and RNA phages in the samples. A sample which contained one or more coliphages (RNA phages) per 0.1 ml of original phage sample was judged to be a coliphage(+) [RNA phage(+)] sample. As shown in Table 3, coliphages were detected in all sewage samples from the sewage treatment plants of three countries and in almost all sewage samples from the domestic drainage of the five countries. The amounts of total coliphages in these sewage samples were fairly high, ranging from 10 to 107 PFU/ml (many showing 102 to 105 PFU/ml). RNA phages were isolated at high frequency from samples from the Philippines (48%), Singapore (35%), and Indonesia (21%). This result was similar to that reported for other Asian countries: Taiwan (28%) (10) and Japan (20 to 70%) (5, 7, 9). The sewage samples from India and Thailand, however, had few RNA phages (3.4 and 5.3%, respectively), a result which is consistent with data for Brazil (5%) (11) and Mexico (2%) (6). The relative amounts of RNA phages in the sewage samples from the Philippines, Singapore, and Indonesia were fairly high, occupying from 10 to 90% of the total coliphages. The corresponding values for the samples from India and Thailand were also similar to those for the above three countries even though the actual number of RNA phage(+) samples was very few. No significant differences in sewage pH (most values
were between 5 and 7), sewage temperature (20 to 30°C), volume of sewage water, or apparent environmental conditions were detected among the domestic drainage in the five countries. Serological grouping and geographical distribution of RNA phages. When two or more RNA phages isolated from the same original sample showed similar inactivation patterns to each other, they were considered as the same strain. (The majority of samples conformed to this case.) The results of serological grouping by the spot test and of subgrouping by the plating method are shown in Tables 3 and 4, respectively. All of the RNA phages tested were inactivated by one of the six standard antisera except for one strain, ID2. We thus classified the newly isolated 54 strains according to the four known groups of RNA phages. By comparing the inactivation patterns of each phage by the antisera of several subgroups of RNA phages with those of the standard ones, group I RNA phage PP25 was further classified into subgroup (a) of group I, and group II RNA phages PP3, SP5, IN7, and TL2 were classified into subgroups (c), (a), (d), and (d) of group II, respectively. Group III RNA phages isolated in the Philippines (16 out of 29 strains), Singapore (all 11 strains), Indonesia (all 6 strains), and Thailand (1 strain) were classified into subgroup (a). Nine other of the Philippines strains were classified into subgroup (b), and the remainder of the Philippines phages (PP1, -5, -10, and -20) were classified into a new subgroup (g). Group III RNA phage ID1 was neutralized well by an antiserum of TW18, and the pattern of neutralization by other antisera was similar to that of TW18. This phage was thus assigned to subgroup (d) of group III. Because group IV RNA phage ID2 was neutralized well by the antisera of TW19 and TW28 and was also sensitive to the antisera of groups I, II, and III to some extent, it was classified into a new subgroup (f) of group IV (Table 4). As shown in Table 3, most of the RNA phages isolated from the Philippines, Singapore, and
998
APPL. ENVIRON. MICROBIOL.
FURUSE ET AL.
TABLE 3. Frequencies of isolation of total coliphages and RNA phages in collected materials Group RNA phage Coliphage
(+)a______b_Group)
city
(treatment plant)
No. of strains'
No. of
No. of samples
samples
I
III
IV
0
1
100
1
100
1
0
0
1
1
100
1
100
1
0
0
1
0
1
100
0
0
0
0
0
0
0
1
100
0
0
0
0
0
0
0
1
100
0
0
0
0
0
0
0
1
100
0
0
0
0
0
0
0
6
100
2
33.3
2
0
0
2
0
Manila Quezon Total
38 23 61
100 100 100
21 8 29
55.3 34.8 47.5
21 9 30
0 1 1
1 0 1
20 8 28
0 0 0
Singapore
20
69.0
10
34.5
11
0
1
10
0
Jakarta Yogjakarta Total
17 13 30
85.0 92.9 88.2
3 4 7
15.0 28.6 20.6
3 4 7
0 0 0
0 1 1
3 3 6
0 0 0
Madras Delhi Agra Total
14 33 11 58
100 97.1 100 98.3
1 0 1 2
7.1 0 9.1 3.4
1 0 1 2
0 0 0 0
0 0 0 0
1 0 0 1
0 0 1 1
Bangkok
34
89.5
2
5.3
2
0
1
1
0
Manila (Makati Sewage Treatment Plant) Singapore (Tanglin Sewage Pumping Station) Delhi (North Side Sewage Purification Plant) Delhi (Kilokali Water Supply and Treatment) Delhi (Okhla Sewage Treatment Plant) Agra (Agra Sewage Pumping House) Total of treatment plants
1 22.6 52 1 4 46 50 203 91.9 Grand total a Number of samples containing one or more coliphages per 0.1 ml of original phage sample. b Number of samples containing one or more RNA phages per 0.1 ml of original phage sample. 'Number of RNA phages exhibiting different serological properties in the same original phage sample (see text). TABLE 4. Serological grouping of RNA phages isolated in Southeast Asia Group
Phage
Subgroup
No. of strains
I
(a)
PP25
1
II
(a)
SP5 PP3 IN7, TL2
1 1 2
(c) (d) III
(a)
(b) (d) (g) IV
(f)
PP4, 6, 7, 8, 11, 12, 15, 18, 19, 23, 24, 26, 27, 28, 30, 31
SP1, 2, 3, 4, 6, 7,.8, 9, 10, 11, 12, IN1, 2, 3, 4, 5, 6, TL1 PP2, 9, 13, 14, 16, 17, 21, 22, 29
34
ID1 PP1, 5, 10, 20
9 1 4
ID2
1
Indonesia could be assigned to group III: phages belonging to groups I, II, or IV were noticeably few. It is thus considered that a borderline can be drawn between Amamiohshima Island and
Kyushu with regard to the geographical distribution of RNA phages in South and East Asia (Fig. 1; Table 5). New RNA phage. At the initial stage of
VOL. 35, 1978
999
RNA PHAGE DISTRIBUTION IN SOUTH AND EAST ASIA
1.
I
:
0
_~
I 1.
1.
1.
Delhi--
&-Agra
ritlgdd,m 1.
-
lrirovotjina 1.
Jakawr
FIG. 1. Distribution of RNA coliphages in south and east Asia. The data refer to Tables 3 and 5.
classification by the spot test, ID2 showed only a small cross-reaction with two antisera, MS2(I) and SP(IV), and even at the second stage of classification by the plating method, it showed only a weak susceptibility to six standard
antisera (MS2[I], JP34[II], GA[II], QJ?[III], VK[III], and SP[IV]). We therefore tested the susceptibility of ID2 to 17 antisera of four groups of RNA phages. As shown in Table 6, ID2 was neutralized weli by the antisera of TW19 and TW28; however, it was also neutralized significantly by those of group I, II, and III phages, although to a lesser extent. We thus tentatively assigned phage ID2 to a new subgroup (fl of group IV. The inactivation patterns of group IV RNA phages [subgroups (a), (b), (c), and (d)] by the same 17 antisera are also shown in Table 6. In
contrast to ID2, these phages were inactivated only by the antisera of group IV, and not by those of groups I, II, and Ill. We have previously isolated serological intermediates between groups I and II (JP34 and JP500) and between groups III and IV (MX1). However, we did not find any intermediate phages among groups I, II, III, and IV. We therefore believe the present isolate (ID2) to be the first to share common characteristics among groups I, II, III, and IV. A detailed characterization of ID2 in addition to its serological character is now in progress. DISCUSSION Concerning the geographical distribution of RNA phages, it is of considerable interest to compare the present data with those previously
1000
APPL. ENVIRON. MICROBIOL.
FURUSE ET AL.
TABLE 5. Distributional pattern of RNA phages in Taiwan and Japan yr samples Survey Survey samples No. of
a_______ No. of
Group
RNA phage (+)a
Coliphage Country
Group_____
No. of
Reference
Taiwan
1970
121
samples 107
88.5
No. of samples 34
Japan proper
1972-1976
736
505
68.6
373
50.7
433
2
331
100
13
6
46.2
5
38.5
5
0
5
0
1973, 1975
57
47
82.5
30
52.6
32
0
6
26
0 8; unpub-
1973, 1975
62
39
62.9
25
40.3
25
0
15
10
1973
20
17
85.0
10
50.0
12
0
2
10
0 8; unpublished data 0 8
1973
18
15
83.3
11
61.1
11
0
11
0
93
74
79.6
33
35.5
38
0
21
17
0 8; unpub-
47
43
91.5
30
63.8
37
1
23
13
lished data 0 8; unpublished data
54
72.0
20
26.7
20
0
2
18
0
261
92.9
163
58.0
178
4
20
154
0
41
87.2
28
59.6
28
0
0
28
5
50.0
5
0
0
5
(area)
%
I
II
III
IV
8
22
2
strainsa 28.1
38
6
12
0 6,7; unpub-
lished data
North (Rishiri 1975 Island) South Ogasawara Island Hachijojima Island Miyakejima Island Niijima Island West Iki Island
0 8
lished data
1973, 1975, 1977 Tsushima Is- 1973, 1977 land
Southwest 75 1975 Amamioshima Island 281 Okinawa 1973, 1974 Mainland 47 Ishigakijima 1974 Island 10 Iriomotejima 1974 Island For explanation, see Table 3.
10
100
obtained in East Asia (1970 to 1977) (Table 5). Tables 3 and 5 clearly show that in Japan proper (Hokkaido, Honshu, Shikoku, and Kyushu) and in the islands in the seas adjacent to Japan
(Rishiri, Hachijojima, Miyakejima, Niijima, Iki, and Tsushima Islands), the most prevalent RNA phages are those of group II (group II/group III, 3:1), whereas in the Southwest islands of Japan (Amamiohshima Island, the mainland of Okinawa, and Ishigakijima and Iriomotejima Islands) and in Taiwan, the Philippines, Singapore, and Indonesia, the most prevalent RNA phages are those of group III. Moreover, some tendency exists for the ratio of group III to group II to increase according to separation from Japan proper toward the southern islands. In this respect, it is important for us to determine the distributional pattern of RNA phages in the center of the Asian continent. Dhillon et al. have revealed that sewage samples collected from both rural and urban areas of Hong Kong included many RNA phages. These were classified into groups I (MS2-related
0 8
Unpublished data
2; unpublished data 0 Unpublished data 0 Unpublished data
phages) and III (Qfl-related phages). They also showed that the number of samples containing MS2-related phages was almost the same as the number containing QJ-related phages, although the density of the former exceeded that of the latter (3, 4). Thus, from the viewpoint of the geographical distribution of RNA phages, the sewage of Hong Kong appears to have certain features in contrast with those of other Asian countries. However, it will be necessary to compare the results obtained by several procedures on the same sewage samples. At the present time, it can be said that in Japan, group I RNA phages have been isolated exclusively from the feces of cows and pigs and from the raw sewage of certain restricted sewage treatment plants (unpublished data). Although little is known about what constitutes the ideal conditions for isolation of RNA phages from their natural habitats, we have tentatively employed rich media (peptone-glucose), a fixed incubation temperature of 370C, and three strains of E. coli K-12 as host bacteria
RNA PHAGE DISTRIBUTION IN SOUTH AND EAST ASIA
VOL. 35, 1978
1001
TABLE 6. Serological properties of the new RNA phage ID2a Phage Group IV
Antiserum
(a) SP
(b)
Group I (a) MS2 (b) BO1 (c) JP501
New RNA phage ID2
FI
(c) TW19
(d) TW28
1.la 1.3 1.1
0.8 0.8 0.9
1.0 1.0 0.9
1.0 0.9 0.7
0.4 0.8 0.7
Group II (a) GA (b) BZ13 (c) TH1 (d) KUl (e) JP34
1.1 1.1 1.1 1.1 1.0
1.1 1.3 0.9 0.6 0.8
1.0 1.0 0.9 1.0 1.0
1.0 0.9 0.9 0.8 0.8
0.5 0.4 0.2 0.2 0.2
Group III (a) Qfi (b) VK (c) ST (d) TW18 (f) MXL
1.1 0.6 0.8 0.9 0.1
0.9 1.1 1.0 0.9 0.8
1.1 1.0 1.0 1.0
1.0 0.8 0.8 0.8 0.6
0.7 0.5 0.4 0.3 0.2
0.2 1.1 2 x 10-3 7 x 1i-5
0.6 0.7
Group IV 2 x 10-4 (a) SP 0.7 (b) Fl 0.3 (c) TWl9 0.6 (d) TW28 For explanation, see Table 2.
0.1 5 x 10-4 0.2 0.1
0.7 0.2 0.9 6 x 1i-5 2 x 10-4
1 X 10-2 7 x 1O-3
a
for the isolation, propagation, and titration of the phages. We have checked the influence of the temperature in transportation of the phage samples and observed no significant difference between normal conditions and refrigerated ones in the isolation frequency of RNA phages at least within several days. Under these conditions, we were in fact able to isolate RNA phages from sewage samples at a relatively high frequency, and the RNA phages we have tested so far (two to three strains of each group) have formed plaques on F+ strains of E. coli B and C with a fairly high plating efficiency comparable to that of K. We therefore believe our isolation procedure to cover a considerable fraction of the RNA coliphages to be isolated from the sewage samples. Our data appear to reflect more strongly the influence of the global physical condition such as geographical position or general climate surrounding- the domestic drainage, because they were derived exclusively from an analysis of sewage samples from domestic drainage and there is some evidence to suggest that group II RNA phages are the most temperature-sensitive ones and can propagate at the lowest temperature among the known four groups of RNA phages in in vitro experiments (unpublished
data). In spite of the current limitations in materials and methods, the time is ripe to accumulate data of this kind, because our knowledge of the distribution of coliphages in their natural habitats, especially from the ecological viewpoint, remains very limited. We are unable to explain the significance of coincidences such that the ID1 and ID2 isolated in India display serological affinities with the TW18 (a rare phage in group III) and TW28 (a rare phage in group IV), respectively, isolated in Taiwan. ACKNOWLEDGMENTS We wish to express our sincere appreciation to the governments of the Philippines, Singapore, Indonesia (particularly the LIPI), India, and Thailand (particularly the NRC) for permission to carry out research in their respective countries. We are also very much indebted to the following individuals whose institutions are listed in Table 1 for their kind support and collaboration throughout our research: Benjamin D. Cabrera, Veronica Chan, Sng Ewe Hui, Moses Yu, Lim Ah Loo, J. Sulianti Saroso, Iskak Koiman, B. L. Wattal, S. N. Ray, Ratna Sarkar, Chamlong Harinasuta, Savanat Tharavanij, and T. Akamatsu and Y. Sasaki (Yakult [Thailand] Co., Ltd). Kind assistance in sample collection was given by Soetresno Eram (Laboratory Kesehatan), M. P. Mehrotra (TB Demonstration and Training Centre), Raphiphat Kasemsook (Bureau of Public Health), and members of the six sewage treatment plants listed in Table 1. We wish to express our deep gratitude
1002
APPL. ENVIRON. MICROBIOL.
FURUSE ET AL.
to M. Sasa (National Institute for Environmental Studies) for introducing us to the suitable organizations (listed in Table 1) in each country and for providing valuable advice and assistance in our research project. Thanks are also due to the Tokyo Embassies of the five countries visited, the Ministry of Foreign Affairs of Japan, the Ministry of Education, Science and Culture of Japan, members of the Japanese Embassies in Manila, Singapore, Jakarta, Madras, Delhi, and Bangkok for their kind advice and cooperation. This study was aided principally by the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan, but thanks are also due to Yakult Honsha Co., Ltd. (Hisami Matsuzono) for additional financial support.
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