Isolation of Yersinia pestis of unusual protein content obtained from Central Java.

Notes Isolation of Yersinia pestis of Unusual Protein Content Obtained from Central Java* by BRUCE W. HUDSON 1 & MARTIN I. GOLDENBERG 2 While classification of isolates of Yersinia (Pasteu- rella) pestis into specific subgroups is of little clinical importance, a number of plague investigators have been impressed with the need for such a classification (Girard, 1954). The best known categorization is that of Devignat (1951) in which plague organisms are divided into 3 subgroups or varieties-namely, antiqua, orientalis, and mediaevalis-on the basis of glycerol and nitrate fermentation. The value of this classification has been pointed out by Pollitzer (1960) who stated that the finding of 2 distinct varieties, as in Kenya, leads to an assumption that there exist 2 independent reservoirs of infection. Other attempts to classify Y. pestis strains have been made using various immunological character- istics (Dodin, 1963; Ransom et al., 1955). In addi- tion, promising results have been reported through the use of biochemical tests to delineate geographi- cally distributed variants of Y. pestis (Martinevskij, 1968). A different approach to the evaluation of Y. pestis variants has been taken in the Ecological Investiga- tions Laboratories. During the last decade, gel- electrophoretic techniques have been developed to a high degree of reproducibility. In addition to their clinical application for the diagnosis of various disease states, these techniques have been used in studies of the taxonomic classification of various species and subspecies of animals (Johnson & Wicks, 1964; Rasmussen & Koehn, 1966; Bongardt et al., 1968). Because of the relative ease with which these techniques can be applied, and their potential utility in the definition of geographic variants of Y. pestis, the techniques have been applied to antigens of a number of Y. pestis strains isolated from various major plague areas. The resulting observations, reported here, suggest that there may be significant * From the Ecological Investigations Laboratories, Center for Disease Control, US Department of Health, Education, and Welfare, P.O. Box 551, Fort Collins, Colo., USA. I Chief, Immunobiology Unit, Zoonoses Section. 2 Chief, Microbiology Unit, Zoonoses Section. demonstrable differences between plague strains found in different geographical areas. Materials and methods The discontinuous acrylamide gel-electrophoresis method of Davis and Ornstein (Davis, 1964) was used in this investigation. Protein patterns were examined after staining by use of the Chromoscan.3 4 Altogether, 10 selected strains of Y. pestis were inoculated into white mice. After 24 hours the strains were recovered from the mice by culturing the dissected spleens on blood agar plates, sub- culturing the resulting growth in trypticase soy broth for 24 hours at 28°C, then using this broth culture to inoculate Roux bottles containing trypticase soy agar. After incubation at 28°C for 3 days, the mate- rial was harvested in 0.85 % sodium chloride solution, using glass beads to remove growth, and killed by adding it to 3-4 volumes of cold (-20°C) acetone. After remaining at room temperature (20°C) over- night, the precipitate was washed twice in ace- tone, then dried under vacuum. The acetone-dried bacteria were placed in 0.1 M borate (pH 7.0) buffer (1: 20 w/v), disrupted by sonic disintegration for 1 minute, frozen at - 20°C, thawed, and centrifuged to clarify the extract. The clear supernatant liquid obtained by this procedure was useful for a period of 1-2 weeks when kept at 40C. The strains used, and their geographical origins, are listed in the accompanying table. The North American, Bolivian, and Nepalese strains were iso- lated by staff of the Center for Disease Control, Fort Collins, Colo., USA. The Kolar 3 strain from Mysore State, India, and strain PKR-133 from Iran, were obtained from Dr M. Bahmanyar of the Institut Pasteur de l'Iran, Teheran. Strain 195/P is the Haffkine Institute's vaccine strain, which has been 3Supplied by Joyce, Loebl and Co., Ltd., Gateshead, England. ' The use of trade names is for identification only, and does not constitute endorsement by the Public Health Service or the US Department of Health, Education, and Welfare. 2611 -917 NOTES GEOGRAPHICAL ORIGIN AND EPIDEMIOLOGICAL INFORMATION FOR 10 STRAINS OF YERSINIA PESTIS SUBJECTED TO DISC ELECTROPHORESIS ANALYSIS Strain designation Origin Epidemiological data 1. BAS 2400 San Mateo County, Calif., USA Microtus californicus fleas, 1966 2. 68-1-109 Denver, Colo., USA Sciurius niger, 1968 3. 68-32-19 N. Mex., USA Cynomys fleas, 1968 4. Aguilar Bolivia Man, 1965 5. 195/P India Virulent laboratory strain used for vaccine production 6. Kolar 3 Kolar, Mysore State, India Man, 1963 7. Ramgiri (Rhamagiri) Nepal Man, 1967 8. PKR-133 Kurdistan, Iran Rodent fleas 9. Muradi Bojolali, Central Java, Indonesia Man, 1968 10. Java 1241 Bojolali, Central Java, Indonesia Man, 1968 DENSITOMETRIC TRACINGSa OF ACRYLAMIDE GEL ELECTROPHORESIS PATTERNS OF 10 STRAINS OF YERSINIA PESTIS 0 J ,' a The numbers correspond to the numbered strains listed in the accompanying table. (.D t-ON 918 YERSINIA PESTIS FROM JAVA WITH UNUSUAL PROTEIN CONTENT 919 maintained in this laboratory for several decades. The Java 1241 strain was isolated by Indonesian Government personnel working at the Area Health Laboratory, Jogjakarta. The Muradi strain was isolated by the combined efforts of staff of the Center for Disease Control and Indonesian Government personnel. Results and discussion The results of disc electrophoresis analyses of the strains studied are shown in the accompanying figure. The gross differences in protein patterns of the Javanese strains of Y. pestis from those of the Asian and American strains used for comparison are imme- diately evident. Immunological work is in progress to identify the protein component or components responsible for the marked differences, but it is not yet complete. There is no evidence that the components contribut- ing to these patterns are different immunologically or chemically from those already known to be common to most strains of Y. pestis (Lawton et al., 1960). Plague was first imported into Java at the port of Surabaja in 1910; from there it spread and became endemic in the inland mountain areas. The disease was first noted in Central Java in the 1920s and was known to persist there until 1959. No plague was reported in Java from 1959 to 1967. The two isolates exhibiting unusual protein patterns were obtained during a plague outbreak that occurred in the Bojolali Regency of Central Java from August 1967 to April 1968. The first cases were reported from Sepandan, a ward in the village of Selo, and from Suradadi, in the village of Tarubatang. These were the same wards from which plague was first reported in the last previously known outbreak in 1959 (Baltazard & Bahmanyar, 1960). The iso- lated occurrence of plague during the 1967-68 out- break, coupled with epidemiological and geogra- phical similarities to the 1959 outbreak, leads us to suspect that an entrenched source of rodent plague exists in that locality. The demonstrable difference between the 1968 Java plague strains and other recent strains from Asia and from America also supports this hypothesis. The significance of the patterns seen with these strains must be determined by the examination of additional Javanese plague strains, temporally and spatially remote from the 1968 outbreak. REFERENCES Baltazard, M. & Bahmanyar, M. (1960) Bull. Wld Hlth Org., 23, 217 Bongardt, H., Richens, V. B. & Howard, W. E. (1968) J. Mammal., 49, 544 Davis, B. J. (1964) Ann. N. Y. Acad. Sci., 121, 404 Devignat, R. (1951) Bull. Wld Hlth Org., 4, 247 Dodin, A. (1963) Ann. Inst. Pasteur, 105, 1098 Girard, G. (1954) Maroc. med., 33, 1016 Johnson, M. L. & Wicks, M. (1964) Serum-protein electro- phoresis in mammals: significance in the higher taxo- nonmic categories. In: Leone, C. A., ed., Taxonomic biochemistry and serology, New York, Ronald Press, pp. 681-694 Lawton, W. D., Fukui, G. M. & Surgalla, M. J. (1960) J. Immunol., 84, 475 Martinevskij, I. L. (1968) Proprietes biologiques et gene- tiques des souches depeste et depseudotuberculose isolees en divers points du globe. In: Regamey, R. H., ed., Proceedings of the International Symposium on Pseudo- tuberculosis, Paris, 1967, Basel, Karger, pp. 265-274 Pollitzer, R. (1960) Bull. Wld Hlth Org., 23, 313 Ransom, J. P., Quan, S. F., Hoggan, M. D. & Omi, G. (1955) Proc. Soc. exp. Biol. Med., 88, 173 Rasmussen, D. I. & Koehn, R. K. (1966) Genetics, 54, 1353 Transliteration from Cyrillic characters The "International System for the Trans- literation of Cyrillic Characters ", set out in Recommendation ISO/R9-1954 (E) of the Inter- national Organization for Standardization, is normally used in the Bulletin ofthe World Health Organization for personal names, titles of publi- cations, etc. However, papers accepted for publication may contain names transliterated differently, and if the original Cyrillic spelling is not recognizable inconsistencies may occur. For convenience the transliteration from Russian according to ISO/R9 is given below: Translitteration des Caracteres cyrilliques Le # Systeme international pour la translitte- ration des caracteres cyrilliques> presente dans la Recommandation ISO/R9-1954 (F) de l'Orga- nisation internationale de Normalisation est g6n6ralement utilise dans le Bulletin de l'Organi- sation mondiale de la Sante pour les noms de personnes, les titres de publications, etc. Cepen- dant des articles acceptes pour publication peuvent contenir des noms translitteres diff&e remment et si l'orthographe cyrillique originale n'est pas reconnaissable un manque d'unifor- mit6 peut s'ensuivre. A toutes fins utiles, la translitteration du russe selon la recommandation ISO/R9 est indiquee ci-apres: Le e cyrillique ne doit 6tre translitt6r6 Trans- Trans- Cyrillic fitratom Cyrillic fitratom character fruiman Examples and remarks character fruian Examples and remarks Caractere Trans- Exemples et observations Caractere Trans- Exemples et observations du russe du russe A, a a ARpec = Adres Y, y u .YTPo = Utro 6, 6 b Ba6a = Baba (),I4 f ()H3HKa = Fizika B, B v Bbi = Vy X, x h XHMH4eCK<Hf = Himiceskij r, r g rnaBa = Glava L, i c [leHTpanbHbtli -- Central'nyj FoJIoBa = Golova -, ' | 1|lacbi = Casy d JAa = Da W, uj s IlKona = Skola E, e(e)1 e (e) Eule = Esce LU, u sc LUeKa = keka >1, NK z M<ypHaJi = Zurnal (medial, or" In modern Russian, where 3, 3 z 3ne3Jra = Zvezda mEdiaI) "ou" sometimes replaces medial b, transliteration is still ".I'l, H i IIJ1H = Iii ~b, 'b En russe moderne, oiu le ' rem- fl, u J -bi H, -on-i i o place quelquefois le b medial, la -,u, ~ J~ *J translitt6ration reste ". K, K k 1(aK = Kak (final) (Not JI, n 1 lo6HTb Ljubit' trans-literated. M, M m MyK = Muz Non trans- H, H n HHKHH1 = Niznij littere.) 0, 0 0 06LuecTBo = Obscestvo bl, bi y EblbI = Byl [n, n p nepBbli = Pervyj b, b 'orl 'ou' ManeHbKHu = Malen'kij P, p Pbi6a Ryba 3, 3 e 3To = Eto C, c s CecTpa = Sestra 10, io ju IOCHbIMi = Juznyj T, T t TOBapJiuW = Tovarik 1, 51 ja 5l1iio = Jajco I Cyrillic e to be transliterated by e only when the diacritical appears in the original. par e que loraque la diacritique apparalt dans l'original.