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Geographical distribution of neotropical Leishmania of the subgenus Leishmania analysed by ribosomal oligonucleotide probes
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Geographical distribution of neotropical Leishmania of the subgenus Leishmania analysed by ribosomal oligonucleotide probes S. R. B. Uliana’, E. Ishikawa’, V. A. Stempliuk’, A. de Souza’, J. J. Shawl** and L. M. Floeter’ Departamento de Parasitologia, Institute de Winter’ Ci&ias Biokdicas, Universidade de Sco Paulo, SZo Paulo, Brazil; ‘Institute Evandro Chagas, Be&n, Par& Brazil Leishmania, SSU rDNA, identification, vectors, reservoirs, Central America, South America
Keywords:
In the Americas, Leishmania (Leishmania) amazonensis is one of the most widely distributed species associated with both cutaneous leishmaniasis and anergic diffuse cutaneous leishmaniasis (ADCL) (SHAW & LAINSON, 1987). Another neotropical species of this subgenus, classically associated with chiclero’s ulcer and also ADCL is L. (L.) mexicana. The latter is considered to be confined to Central and North America. However, in the same area, other leishmania belonging to the subgenus Leishmania, such as L. (L.) venezuelensis and L. (L.) pifanoi, have been described from cases of cutaneous leishganiasis, as well as some isolates presumpively identified as L. CL.1 mexicana (GRIMALDI et al.. 1989). The major l&o’wn reservdirs of these Lkshmakia species are small sylvatic rodents. The proven vectors so far all belong to theflaviscutellata series but the species varies regionally. The main vector of L. (L.) amazonensis is Lutzomyiaflaviscutellata and that of L. (L.) mexicana is Lu. olmeca. Sand flies of other groups are, however, suspected vectors in places such as Texas, USA, and the Dominican Republic (YOUNG & DUNCAN, 1994). The understanding of the eco-epidemiology of this important group of Leishmania is hindered by the limited number of available markers for identification of isolates from hitherto unstudied areas. The description of DNA sequences useful in both identification and taxonomy is opening new windows in studies on leishmaniasis (GRIMALDI & TESH, 1993). In a previous paper we described a coupled PCRhybridization assay, where a set of oligo&cleotides, derived from the small subunit ribosomal RNA (SSU rRNA) sequence, provided the means for distinguiihing 4 groups of leishmania: (i) L. (L.) amazonensis, (ii) L. (L.) mexicana, (iii) L. (L.) chagasi, and (iv) all named species ofthe subgenus Viannia (ULIANA et aZ., 1994). At that time, 2 strains of L. (L.) mexicana did not follow the hybridization pattern of the reference strain, having profiles similar to L. (L.) amazonensis. In this paper, we examined a greater number of L. (L.) mexicana and L. (L.) amazonensis strains, isolated from broadly distributed geographical areas, as well as some reference strains of Leishmania species. Tissue samples from experimentally infected hamsters and mammal reservoirs captured in the Amazon region were also tested. The Leishmania strains used are listed in Tables 1 and 2. The parasites are from the Leishmaniu Bank of the Instituto Evandro Chagas in BelCm (Pa& Brazil) and were typed with a panel of 24 monoclonal antibodies Address for correspondence: Dr Lucile M. Floeter-Winter, Departamento de Parasitologia, Institute de Ciencias Biomedicas,Universidade de SBoPaula, Av. Prof. Lineu Prestes, 1374 SHoPaulo - SP - 05508-900, Brazil; fax +55 11 8187329, e-mail [email protected]
AND HYGIENE
(2000) 94,261-264
using the methods described by SHAW et al. (1989). Organisms were cultured in biphasic Blood Agar base medium (Difco B45; Difco Laboratories, Detroit, Mich., USA) according to the method of WALTON et al. ( 1977). DNA was purified from cultured parasites or tissue samples as described previously (ULIANA et al., 1994). SSU rDNA amplification was performed on all isolates as previously described, using primers Sl/S4 or S12/S4. This last pair of oligonucleotides amplifies a 539-nt fragment corresponding to the 3’ end of Leishmania SSU rDNA (ULIANA et al., 1994). Slot blots, labelling of oligonucleotides and hybridization were performed as previously described (ULIANA et al., 1994). The amplified product was slot blotted and hybridized with the probes S8, S9, SlO and S16. Results for the reference strains are shown on Table 1. The L. (L.) amazonensis reference strain (M2269) hybridized only with S8, the L. (L.) mexicana reference strain (M7369) was positive only with S9. Two strains of L. (L.) major included in the study showed hybridization with S8, the same pattern observed for L. amazonensis. The L. (L.) infanturn hybridization profile was the same as L. (L.) chagasi and L. (L.) donovani, being positive for S9 and S16. The reference strains not previously tested, L. (Viannia) Zainsoni and L. (V.) shawi, of the Vianniu subgenus hybridized with S 10. A strain of Enabtypanum schaudini hybridized with the S 10 probe. The common reactivity between Viannia species and Enobypanum means that it is impossible to use the SlO probe for identifying sylvatic reservoir hosts and vectors. It is interesting that both have a peripylarian development yet antigenically Endotypanum is closer to L. (L.) amazonensis (SHAW, 1994). The profiies obiained ‘for strains of the mexicana comnlex are shown on Table 2. All strains of L. CL.) mexdana hybridized only with the probe S9. The monoclonal profile for all these strains confirmed the identification & L. (L.) mexicana. The strains of L. (L.I amazonensis confirmed the positivity with S8 for ihis species, as was observed for the strains of L. (L.) aristidesi and L. (L.) garnhami tested. A strain of L. (L.) pifanoi showed hybridization with S9, similar to the pattern observed for L. (L.) mexicana strains. Six new isolates were also tested. Of these, 3 were isolated in Minas Gerais State. Brazil. 2 were from the Dominican Republic and 1 f&m Bohvia. The strains isolated in Minas Gerais had a monoclonal reactivity similar to L. (L.) amazonensis reference stains and were positive with S8 probe, corresponding to the pattern observed for L. (L.) amazonensis and L. major. The 2 strains from the Dominican Republic were not tested for monoclonal antibodies but shbwed positive hybridization with S9. comnatible with L. CL.) mexicana strains. Lastly, an isolate fEom Bolivia rea&ei with S8. The hybridization patterns of DNA obtained from biopsies of animals experimentally infected with Leishmania reference strains agreed completely with the monoclonal profiles and previously described oligo patterns (Table 3). Animals infected with species of the subgenus Viunnia, including L. (V.) Zainsoni, L. (V.) shawi and L. ( V.) panamensis hybridized only with S 10. L. (L.) amazonensis hamster samples were positive with S8 and the L. (L.) chugusihamster tissue hybridized only with the S9 and S16 probes. A biopsy of a clean control hamster was negative for all probes. The results obtained with hamster tissue from experimental infections suggested that this technique could be applied to tissues of sylvatic caught animals. Table 4 shows the results of testing samples collected from 5 wild rodents. Of the 3 Proechi6ys gugannensis, 2 were positive with S8. For 1 ofthese animals we tested liver, spleen and skin samples and had positive results with liver and skin. All tissues of the Echimvs were nositive with S8. The ~001 of Nectomys liver and spleen was positive with bo& s8 and S 10. This is an interesting result and suggests mixed
262
S. R. B. ULIANA
Table 1. Hybridization
of probes SS, S9, SlO and S16 with DNA of the International
Reference
ETAL.
strains
Slot blot hybridization pattern’ IEC No.”
ICNb
Species
M7369 Ml1302 M2269 Ml3330 Ml3331 M8408 M6426 M5725
MNYClBZl1962lti379 MHOM/TN/1980/IPT1 MHOM/BR/1973/M2269 MHOM/IN/DD8’ MRHO/SU/1959/l’ MCEB/BR/1984/M8408 MHOM/BR/198 l/M6426 MCHO/BR/1979/M5725
L. (L.) mexicana L. (L.) infanturn L. (L.) amazonensis L. (L.) major L. (L.) major L. (V.) shawi L. (V.) Zainsoni Endotypanum schaudini
S8 + + + -
S9
SlO
+ + -
+ + +
“Institute Evandro Chagas Number. bIntemational Code Number. ‘Slot blot hybridization pattern: S8, L. (L.) amazonensis;SlO, subgenus fiunnia; S9 L. (L.) S16, L. (L.) chug&. *Reactivity pattern with monoclonal antibody panel. ‘Strain received as L. (L.) donovani but typed as L. (L.) major.
Table 2. Hybridization mexicana complex
of probes
S16 MAb* + -
chug&,
SS, S9; SlO and S16 with DNA of strains
L. L. L. L. L. L. L. E.
(L.) mexicana (L.) infantum (L.) amazonensis (L.) major (L.) major (V.) shawi (V.) Zuinsoni schaudini
L. (15.) mexicana,
considered
L. (L.)
as belonging
crqpica;
to the
Slot blot hybridization pattemc IEC No.”
ICNb
Species
M4517 M7369 M7326 M6036 M6037 M7088 M2269 Ml1613 M8488 Ml2275 M6331 Ml3333 M8489 Ml 1766 Ml 1764 Ml 1763
MNYC/BZ/1962lM379’ MNYC/BR/1962/M379 MHOM/BZ/1982/BEL2 1 MHOM/D0/0000/1137 MHOM/D0/0000/1l39 MHOM/VE/1976/ESTHER-RAMOS MHOM/BR/1973lM2269 MHOM/BR/1998/M11613 MHOWO/OO/J07 180 MPHI/BR/199/M12275 MORY/PA/1968/GML3 MHOM./VE/1976/JAP78 MHOM/BO/OOOO/EV09479 MHOM/BR/1986/BH129 MHOM/BR/1986/BH135 MHOM/BR/1986/BH122
L. CL.) mexicana L. (L.) mexicana L. (L.) mexicana (Dominican Rep.) (Dominican Rep.) L. (L.) pifanoi L. (L.) amazonensis L. (L.) amazonensis L. (L.) amazonensis L. (L.) amazonensis L. (L.) aristidesi L. (L.) garnhami L. (L.) sp. majoplike major-like major-like ,
S8
S9
SlO MAb* L. (L.) mexicana L. (L.) mexicana L. (L.) mexicana mexicana-like mexicana-like L. (L.) pifanoi L. (L.) amazonensis L. (L.) amazonensis L. (L.) amazonensis L. (L.) amazonensis L. (L.) amazonensis L. (L.) garnhami amazonensis-like major-like major-like major-like
I
-
+ + +
-
+
-
-
; + + + +
-
-
“-*For the key, seeTable 1. ‘Isolate received from another laboratory that was later discovered to be the reference strain MNYC/BR/1962/M379.
Table 3. Hybridization hamsters
of probes
SS, S9, SlO and S16 with DNA from biopsies
of experimentally
infected
Slot blot hybridization pattemc IEC No.” or Field No. Ml4732 M6426 Ml4855 M4037 PH8 Ml4733 M6445 M2682
ICNb
Species or host
MHOM/BR/1994/M14732 MHOM/BR/1981/M6426 MHOM/BR/1994/M14855 MHOM/l’A/1967/BOYTON IFLA/BR/1968/PH8 MHOM/BR/1994/M14733 MCER/BR/1981/M6445 MHOM/BlU1974/PP75
“dFor the key, see Table
1.
guyanensis complex L. (V.) lainsoni L. (V.) shawi L. (V.) panamfmsis L. (L.) amazonensis L. (L.) amazonensis L. (L.) chagasi L. (L.) chugusi Negative hamster
S8
S9
-
-
+ +
-
-
: -
SlO S16 MAb*
+ + + + -
+ + -
guyanensis complex L. (V.) Zuinsoni L. (V.) shawi L. (V.) panamensis L. (L.) amazonensis L. (L.) amazonensis L. (L.) chug& L. (L.) chug&
263
DNA TYPING OF LEISHMANIA
Table 4. Hybridization Amapa States, Brazil
of probes
S8, S9, SlO and S16 with DNA from wild animals
captured
Slot blot hybridization Field No. Al A2 07 07 07
i: 06
A3
Host (tissue) Proechimys guyannensis (spleen) Proechimys guyannensis (liver) Proechimys guyannensis (liver) Proechimys guyannensis (skin) Proechimys guyannensis (spleen) Echimys sp. (liver) Echimys sp. (skin) Echimys sp. (spleen) Nectomys squamipes (pool liver and spleen)
in Parh and
pattern”
S8
s9
+ +
-
SlO -
S16 -
-
-
-
+
-
-t
+ + + +
-
Al, Proechimys guyannensis, adult, male, from Melgado region, Pari State.
A2, Pmechimysguyannensis,adult, male, from Ipixuna region, Parl State. Animals 06 (Echimys sp.) and 07 (Proechimys guyannenesis) from Abaett, Maracacupumirim River. A3, Nectomys squamipes, adult, female, from Amapi State. “See the footnote to Table 1.
Figure. Diagramatic representation of distribution areasfor the Leishmania species rested in this study correlated to their reactivity with the rDNA oligonucleotides. The map represents Central and part of South America; S8 and S9 are 2 probes used in this study.
infection with parasites of 2 different subgenera. A similar infection of 2 parasites of 2 different subgenera had been previously reported in P. guyannensis (LAINSON etal., 1981). The geographical origin of strains is shown in the Figure. Strain distribution follows a pattern where different areas can be correlated with S9/S8 positivity. Reactivity with S9 probe prevails in Central American isolates while S8-positive samples are placed from the north of Colombia/Venezuela to the south of the continent. The geographical distribution of different vectors and vertebrate host species represents the most likely boundaries or barriers in the distribution ofthe parasite. Thus it is necessary to relate their geographical distribution to those of parasite populations. As Leishmania do not reproduce by sexual processes, an isolated population is considered to be a clone with genetically identical individuals (TIBAYRENC & AYALA, 1991). The overall lack of genetic recombination, except in certain as yet undefined circumstances, and continual natural mutation may work as an efficient method for speciation
(CIBULSKIS, 1986). Geographical isolation may lead to the expansion of genetically distinct parasite clones. Many Leishmania species were described and named according to the geographical distribution of the initial isolates. Initially, the description of new species was based mainly on phenotypical characters, i.e., morphology, pathology and development in the vector. The clinical forms most characteristic in each area have been taken as being typical of a particular species. Subsequently, characterization of strains using isoenzyme profiles (LE BLANCQ et al., 1987) and reactivity with monoclonal antibodies (GRIMALDI et al., 1987) represented major advances in the understanding of the ecoepidemiology of Leishmania. The next step was the application of DNA-based methodology, which led to more precise inferences about the relationships between different species. In this paper, we have analysed isolates from widely distributed geographical areas of Latin America and shown that the rDNA sequence correlates with monoclonal antibody profiles, strengthening the usefulness of rDNA typing. Identification based on DNA eliminates the need for isolation and culture of the parasite, small tissue samples being adequate for testing. Positive results obtained with material from wild animals and biopsies of experimentally infected hamsters attest the field applicability of the technique. A doubt on the usefulness of the method was previously raised by discrepancies of the profiles of L. (L.) mexicana stocks. One stock of L. (L.) mexicana, BEL21, had previously reacted with the S8 probe (ULIANA et al., 1994). To solve this problem we obtained another line of the reference strain of L. (L.) me&ma, whose identity had been confirmed with monoclonal antibodies. This time it reacted with the S9 probe. From this, we conclude that the first stock of BEL21 had been mixed with another strain that reacts with S8. The usefulness of typing isolates to species groups from the field is clear as well as the use of the method as a tool for epidemiological studies. First, the application of rDNA typing to the detection of infected reservoirs and vectors will give a lead to more detailed studies even if the parasites have not been isolated. Secondly, the identification of parasites, even to a species group, will, in certain situations, contribute to a better understanding of the different pathologies. Acknowledgements This work received financial support from the Funda@o de
Amparo g Pesquisa do Estado de SHo Paulo (FAPESP) and CNPq. We also thank Cassiano P. Nunes for assistance with drawing.
264
S. R. B. ULIANA ETAL.
References Cibulskis, R. E. (1986). Mutation and recombination in the Trypanosomatidae. In: Letihmania Taxonomie et phylogenbe Applications kcot$idimiologiques Colloquelnternational, Rioux, J. A. (editor). Montepellier: ORSTOM, pp. 297-303. Grimaldi, G. Jr & Tesh, R. B. (1993). Leishmaniases of the New World: current concepts and implications for future research. Clinical and Microbiology Reviews, 6,230-250. Grimaldi, G. Jr, David, J. R. & McMahon-Pratt, D. (1987). Identification and distribution of New World Leishmaniu species characterized by serodeme analysis using monoclonal antibodies. AmericanJoumalof TropicaZMedicineandHygime, 36,270-287. Grimaldi, G. Jr, Tesh, R. B. & McMahon-Pratt, D. (1989). A review of the geographic distribution and epidemiology of leishmaniasis in the New World. American Journal of Tropical Medicine and Hygiene, 41, 687-725. Lainson, R., Shaw, J. J., Ready, I’. D., Miles, M. A. & P6voa, M. (198 1). Leishmaniasis in Brazil: XVI. Isolation and identification of Leishmania species from sandflies, wild mammals and man in north Par& State, with particular reference to L. bruziliensis guyunensis causative agent of “pian-bois”. Transactions of the Royal Society of Tropkzl Medicine and Hygiene, 75, 530-536. Le Blancq, S. M., Lanham, S. M. & Evans, D. A. (1987). Comparative isoenzyme profiles of Old and New World Leishmania. In: Letihmaniases in Biology and Medicine, Vol. 1, Peters, W. & Killick-Kendrick, R. (editors). London: Academic Press, pp. 543-550. Shaw, J. J. (1994). Taxonomy of the genus Leishmania: present and future trends and their implications. Mem&ius do Znstiwto Oswald0 Cruz, 89,47 l-478.
Shaw, J. J. & Lainson, R. (1987). Recent advances in studies of the ecology and epidemiology of New World Leishmania. In: TheLkshmaniases in Biology andMedicine, Vol. 1, Peters, W. & KiyKigiendrick, R. (editors). London: Academic Press, pp. Shaw, J. J., ishikawa, E. A. Y. & Lainson, R. (1989). A rapid and sensitive method for the identification of Leishmania with monoclonal antibodies using fluorescein-labelled avidin. Transactions of the Royal Society of Tropical Medicine and Hygiene, 83,783-784. Tibayrenc, M. & Ayala, F. J. (1991). Towards a populationgenetics of microorganisms-the clonal theory of parasitic protozoa. Parasitology Today, 7, 228-232. Uliana, S. R. B., Nelson, K., Beverley, S. M., Camargo, E. P. & Floeter-Winter, L. M. (1994). Discrimination amongst Leashmania by polymerase chain reaction and hybridization with small subunit ribosomal DNA derived oligonucleotides. Journal of Eukayotik Microbiology, 41,324-330. Walton,B. C., Shaw, J. J. &Lainson, R. (1977). Observationson the in v&o cultivation of Leishmania braziliensis. Journal of Parasitology, 63, 1118- 1119. Young, D. G. &Duncan, M. A. (1994). Guide to the identification and geographic distribution of Lutzomyia sand flies in Mexico, the West Indies, Central and South America (Diptera: Psychodidae). Memoirs of the American Entomological Institute no. 54. Gainsville, Florida: Associated Publishers, American Entomological Institute.
Received November
19 October 1999
1999;
accepted for
publication
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9
OF THE ROYAL
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I
1 Short Report
)
SOCIETY
OF TROPICAL
MEDICINE
Geographical distribution of neotropical Leishmania of the subgenus Leishmania analysed by ribosomal oligonucleotide probes S. R. B. Uliana’, E. Ishikawa’, V. A. Stempliuk’, A. de Souza’, J. J. Shawl** and L. M. Floeter’ Departamento de Parasitologia, Institute de Winter’ Ci&ias Biokdicas, Universidade de Sco Paulo, SZo Paulo, Brazil; ‘Institute Evandro Chagas, Be&n, Par& Brazil Leishmania, SSU rDNA, identification, vectors, reservoirs, Central America, South America
Keywords:
In the Americas, Leishmania (Leishmania) amazonensis is one of the most widely distributed species associated with both cutaneous leishmaniasis and anergic diffuse cutaneous leishmaniasis (ADCL) (SHAW & LAINSON, 1987). Another neotropical species of this subgenus, classically associated with chiclero’s ulcer and also ADCL is L. (L.) mexicana. The latter is considered to be confined to Central and North America. However, in the same area, other leishmania belonging to the subgenus Leishmania, such as L. (L.) venezuelensis and L. (L.) pifanoi, have been described from cases of cutaneous leishganiasis, as well as some isolates presumpively identified as L. CL.1 mexicana (GRIMALDI et al.. 1989). The major l&o’wn reservdirs of these Lkshmakia species are small sylvatic rodents. The proven vectors so far all belong to theflaviscutellata series but the species varies regionally. The main vector of L. (L.) amazonensis is Lutzomyiaflaviscutellata and that of L. (L.) mexicana is Lu. olmeca. Sand flies of other groups are, however, suspected vectors in places such as Texas, USA, and the Dominican Republic (YOUNG & DUNCAN, 1994). The understanding of the eco-epidemiology of this important group of Leishmania is hindered by the limited number of available markers for identification of isolates from hitherto unstudied areas. The description of DNA sequences useful in both identification and taxonomy is opening new windows in studies on leishmaniasis (GRIMALDI & TESH, 1993). In a previous paper we described a coupled PCRhybridization assay, where a set of oligo&cleotides, derived from the small subunit ribosomal RNA (SSU rRNA) sequence, provided the means for distinguiihing 4 groups of leishmania: (i) L. (L.) amazonensis, (ii) L. (L.) mexicana, (iii) L. (L.) chagasi, and (iv) all named species ofthe subgenus Viannia (ULIANA et aZ., 1994). At that time, 2 strains of L. (L.) mexicana did not follow the hybridization pattern of the reference strain, having profiles similar to L. (L.) amazonensis. In this paper, we examined a greater number of L. (L.) mexicana and L. (L.) amazonensis strains, isolated from broadly distributed geographical areas, as well as some reference strains of Leishmania species. Tissue samples from experimentally infected hamsters and mammal reservoirs captured in the Amazon region were also tested. The Leishmania strains used are listed in Tables 1 and 2. The parasites are from the Leishmaniu Bank of the Instituto Evandro Chagas in BelCm (Pa& Brazil) and were typed with a panel of 24 monoclonal antibodies Address for correspondence: Dr Lucile M. Floeter-Winter, Departamento de Parasitologia, Institute de Ciencias Biomedicas,Universidade de SBoPaula, Av. Prof. Lineu Prestes, 1374 SHoPaulo - SP - 05508-900, Brazil; fax +55 11 8187329, e-mail [email protected]
AND HYGIENE
(2000) 94,261-264
using the methods described by SHAW et al. (1989). Organisms were cultured in biphasic Blood Agar base medium (Difco B45; Difco Laboratories, Detroit, Mich., USA) according to the method of WALTON et al. ( 1977). DNA was purified from cultured parasites or tissue samples as described previously (ULIANA et al., 1994). SSU rDNA amplification was performed on all isolates as previously described, using primers Sl/S4 or S12/S4. This last pair of oligonucleotides amplifies a 539-nt fragment corresponding to the 3’ end of Leishmania SSU rDNA (ULIANA et al., 1994). Slot blots, labelling of oligonucleotides and hybridization were performed as previously described (ULIANA et al., 1994). The amplified product was slot blotted and hybridized with the probes S8, S9, SlO and S16. Results for the reference strains are shown on Table 1. The L. (L.) amazonensis reference strain (M2269) hybridized only with S8, the L. (L.) mexicana reference strain (M7369) was positive only with S9. Two strains of L. (L.) major included in the study showed hybridization with S8, the same pattern observed for L. amazonensis. The L. (L.) infanturn hybridization profile was the same as L. (L.) chagasi and L. (L.) donovani, being positive for S9 and S16. The reference strains not previously tested, L. (Viannia) Zainsoni and L. (V.) shawi, of the Vianniu subgenus hybridized with S 10. A strain of Enabtypanum schaudini hybridized with the S 10 probe. The common reactivity between Viannia species and Enobypanum means that it is impossible to use the SlO probe for identifying sylvatic reservoir hosts and vectors. It is interesting that both have a peripylarian development yet antigenically Endotypanum is closer to L. (L.) amazonensis (SHAW, 1994). The profiies obiained ‘for strains of the mexicana comnlex are shown on Table 2. All strains of L. CL.) mexdana hybridized only with the probe S9. The monoclonal profile for all these strains confirmed the identification & L. (L.) mexicana. The strains of L. (L.I amazonensis confirmed the positivity with S8 for ihis species, as was observed for the strains of L. (L.) aristidesi and L. (L.) garnhami tested. A strain of L. (L.) pifanoi showed hybridization with S9, similar to the pattern observed for L. (L.) mexicana strains. Six new isolates were also tested. Of these, 3 were isolated in Minas Gerais State. Brazil. 2 were from the Dominican Republic and 1 f&m Bohvia. The strains isolated in Minas Gerais had a monoclonal reactivity similar to L. (L.) amazonensis reference stains and were positive with S8 probe, corresponding to the pattern observed for L. (L.) amazonensis and L. major. The 2 strains from the Dominican Republic were not tested for monoclonal antibodies but shbwed positive hybridization with S9. comnatible with L. CL.) mexicana strains. Lastly, an isolate fEom Bolivia rea&ei with S8. The hybridization patterns of DNA obtained from biopsies of animals experimentally infected with Leishmania reference strains agreed completely with the monoclonal profiles and previously described oligo patterns (Table 3). Animals infected with species of the subgenus Viunnia, including L. (V.) Zainsoni, L. (V.) shawi and L. ( V.) panamensis hybridized only with S 10. L. (L.) amazonensis hamster samples were positive with S8 and the L. (L.) chugusihamster tissue hybridized only with the S9 and S16 probes. A biopsy of a clean control hamster was negative for all probes. The results obtained with hamster tissue from experimental infections suggested that this technique could be applied to tissues of sylvatic caught animals. Table 4 shows the results of testing samples collected from 5 wild rodents. Of the 3 Proechi6ys gugannensis, 2 were positive with S8. For 1 ofthese animals we tested liver, spleen and skin samples and had positive results with liver and skin. All tissues of the Echimvs were nositive with S8. The ~001 of Nectomys liver and spleen was positive with bo& s8 and S 10. This is an interesting result and suggests mixed
262
S. R. B. ULIANA
Table 1. Hybridization
of probes SS, S9, SlO and S16 with DNA of the International
Reference
ETAL.
strains
Slot blot hybridization pattern’ IEC No.”
ICNb
Species
M7369 Ml1302 M2269 Ml3330 Ml3331 M8408 M6426 M5725
MNYClBZl1962lti379 MHOM/TN/1980/IPT1 MHOM/BR/1973/M2269 MHOM/IN/DD8’ MRHO/SU/1959/l’ MCEB/BR/1984/M8408 MHOM/BR/198 l/M6426 MCHO/BR/1979/M5725
L. (L.) mexicana L. (L.) infanturn L. (L.) amazonensis L. (L.) major L. (L.) major L. (V.) shawi L. (V.) Zainsoni Endotypanum schaudini
S8 + + + -
S9
SlO
+ + -
+ + +
“Institute Evandro Chagas Number. bIntemational Code Number. ‘Slot blot hybridization pattern: S8, L. (L.) amazonensis;SlO, subgenus fiunnia; S9 L. (L.) S16, L. (L.) chug&. *Reactivity pattern with monoclonal antibody panel. ‘Strain received as L. (L.) donovani but typed as L. (L.) major.
Table 2. Hybridization mexicana complex
of probes
S16 MAb* + -
chug&,
SS, S9; SlO and S16 with DNA of strains
L. L. L. L. L. L. L. E.
(L.) mexicana (L.) infantum (L.) amazonensis (L.) major (L.) major (V.) shawi (V.) Zuinsoni schaudini
L. (15.) mexicana,
considered
L. (L.)
as belonging
crqpica;
to the
Slot blot hybridization pattemc IEC No.”
ICNb
Species
M4517 M7369 M7326 M6036 M6037 M7088 M2269 Ml1613 M8488 Ml2275 M6331 Ml3333 M8489 Ml 1766 Ml 1764 Ml 1763
MNYC/BZ/1962lM379’ MNYC/BR/1962/M379 MHOM/BZ/1982/BEL2 1 MHOM/D0/0000/1137 MHOM/D0/0000/1l39 MHOM/VE/1976/ESTHER-RAMOS MHOM/BR/1973lM2269 MHOM/BR/1998/M11613 MHOWO/OO/J07 180 MPHI/BR/199/M12275 MORY/PA/1968/GML3 MHOM./VE/1976/JAP78 MHOM/BO/OOOO/EV09479 MHOM/BR/1986/BH129 MHOM/BR/1986/BH135 MHOM/BR/1986/BH122
L. CL.) mexicana L. (L.) mexicana L. (L.) mexicana (Dominican Rep.) (Dominican Rep.) L. (L.) pifanoi L. (L.) amazonensis L. (L.) amazonensis L. (L.) amazonensis L. (L.) amazonensis L. (L.) aristidesi L. (L.) garnhami L. (L.) sp. majoplike major-like major-like ,
S8
S9
SlO MAb* L. (L.) mexicana L. (L.) mexicana L. (L.) mexicana mexicana-like mexicana-like L. (L.) pifanoi L. (L.) amazonensis L. (L.) amazonensis L. (L.) amazonensis L. (L.) amazonensis L. (L.) amazonensis L. (L.) garnhami amazonensis-like major-like major-like major-like
I
-
+ + +
-
+
-
-
; + + + +
-
-
“-*For the key, seeTable 1. ‘Isolate received from another laboratory that was later discovered to be the reference strain MNYC/BR/1962/M379.
Table 3. Hybridization hamsters
of probes
SS, S9, SlO and S16 with DNA from biopsies
of experimentally
infected
Slot blot hybridization pattemc IEC No.” or Field No. Ml4732 M6426 Ml4855 M4037 PH8 Ml4733 M6445 M2682
ICNb
Species or host
MHOM/BR/1994/M14732 MHOM/BR/1981/M6426 MHOM/BR/1994/M14855 MHOM/l’A/1967/BOYTON IFLA/BR/1968/PH8 MHOM/BR/1994/M14733 MCER/BR/1981/M6445 MHOM/BlU1974/PP75
“dFor the key, see Table
1.
guyanensis complex L. (V.) lainsoni L. (V.) shawi L. (V.) panamfmsis L. (L.) amazonensis L. (L.) amazonensis L. (L.) chagasi L. (L.) chugusi Negative hamster
S8
S9
-
-
+ +
-
-
: -
SlO S16 MAb*
+ + + + -
+ + -
guyanensis complex L. (V.) Zuinsoni L. (V.) shawi L. (V.) panamensis L. (L.) amazonensis L. (L.) amazonensis L. (L.) chug& L. (L.) chug&
263
DNA TYPING OF LEISHMANIA
Table 4. Hybridization Amapa States, Brazil
of probes
S8, S9, SlO and S16 with DNA from wild animals
captured
Slot blot hybridization Field No. Al A2 07 07 07
i: 06
A3
Host (tissue) Proechimys guyannensis (spleen) Proechimys guyannensis (liver) Proechimys guyannensis (liver) Proechimys guyannensis (skin) Proechimys guyannensis (spleen) Echimys sp. (liver) Echimys sp. (skin) Echimys sp. (spleen) Nectomys squamipes (pool liver and spleen)
in Parh and
pattern”
S8
s9
+ +
-
SlO -
S16 -
-
-
-
+
-
-t
+ + + +
-
Al, Proechimys guyannensis, adult, male, from Melgado region, Pari State.
A2, Pmechimysguyannensis,adult, male, from Ipixuna region, Parl State. Animals 06 (Echimys sp.) and 07 (Proechimys guyannenesis) from Abaett, Maracacupumirim River. A3, Nectomys squamipes, adult, female, from Amapi State. “See the footnote to Table 1.
Figure. Diagramatic representation of distribution areasfor the Leishmania species rested in this study correlated to their reactivity with the rDNA oligonucleotides. The map represents Central and part of South America; S8 and S9 are 2 probes used in this study.
infection with parasites of 2 different subgenera. A similar infection of 2 parasites of 2 different subgenera had been previously reported in P. guyannensis (LAINSON etal., 1981). The geographical origin of strains is shown in the Figure. Strain distribution follows a pattern where different areas can be correlated with S9/S8 positivity. Reactivity with S9 probe prevails in Central American isolates while S8-positive samples are placed from the north of Colombia/Venezuela to the south of the continent. The geographical distribution of different vectors and vertebrate host species represents the most likely boundaries or barriers in the distribution ofthe parasite. Thus it is necessary to relate their geographical distribution to those of parasite populations. As Leishmania do not reproduce by sexual processes, an isolated population is considered to be a clone with genetically identical individuals (TIBAYRENC & AYALA, 1991). The overall lack of genetic recombination, except in certain as yet undefined circumstances, and continual natural mutation may work as an efficient method for speciation
(CIBULSKIS, 1986). Geographical isolation may lead to the expansion of genetically distinct parasite clones. Many Leishmania species were described and named according to the geographical distribution of the initial isolates. Initially, the description of new species was based mainly on phenotypical characters, i.e., morphology, pathology and development in the vector. The clinical forms most characteristic in each area have been taken as being typical of a particular species. Subsequently, characterization of strains using isoenzyme profiles (LE BLANCQ et al., 1987) and reactivity with monoclonal antibodies (GRIMALDI et al., 1987) represented major advances in the understanding of the ecoepidemiology of Leishmania. The next step was the application of DNA-based methodology, which led to more precise inferences about the relationships between different species. In this paper, we have analysed isolates from widely distributed geographical areas of Latin America and shown that the rDNA sequence correlates with monoclonal antibody profiles, strengthening the usefulness of rDNA typing. Identification based on DNA eliminates the need for isolation and culture of the parasite, small tissue samples being adequate for testing. Positive results obtained with material from wild animals and biopsies of experimentally infected hamsters attest the field applicability of the technique. A doubt on the usefulness of the method was previously raised by discrepancies of the profiles of L. (L.) mexicana stocks. One stock of L. (L.) mexicana, BEL21, had previously reacted with the S8 probe (ULIANA et al., 1994). To solve this problem we obtained another line of the reference strain of L. (L.) me&ma, whose identity had been confirmed with monoclonal antibodies. This time it reacted with the S9 probe. From this, we conclude that the first stock of BEL21 had been mixed with another strain that reacts with S8. The usefulness of typing isolates to species groups from the field is clear as well as the use of the method as a tool for epidemiological studies. First, the application of rDNA typing to the detection of infected reservoirs and vectors will give a lead to more detailed studies even if the parasites have not been isolated. Secondly, the identification of parasites, even to a species group, will, in certain situations, contribute to a better understanding of the different pathologies. Acknowledgements This work received financial support from the Funda@o de
Amparo g Pesquisa do Estado de SHo Paulo (FAPESP) and CNPq. We also thank Cassiano P. Nunes for assistance with drawing.
264
S. R. B. ULIANA ETAL.
References Cibulskis, R. E. (1986). Mutation and recombination in the Trypanosomatidae. In: Letihmania Taxonomie et phylogenbe Applications kcot$idimiologiques Colloquelnternational, Rioux, J. A. (editor). Montepellier: ORSTOM, pp. 297-303. Grimaldi, G. Jr & Tesh, R. B. (1993). Leishmaniases of the New World: current concepts and implications for future research. Clinical and Microbiology Reviews, 6,230-250. Grimaldi, G. Jr, David, J. R. & McMahon-Pratt, D. (1987). Identification and distribution of New World Leishmaniu species characterized by serodeme analysis using monoclonal antibodies. AmericanJoumalof TropicaZMedicineandHygime, 36,270-287. Grimaldi, G. Jr, Tesh, R. B. & McMahon-Pratt, D. (1989). A review of the geographic distribution and epidemiology of leishmaniasis in the New World. American Journal of Tropical Medicine and Hygiene, 41, 687-725. Lainson, R., Shaw, J. J., Ready, I’. D., Miles, M. A. & P6voa, M. (198 1). Leishmaniasis in Brazil: XVI. Isolation and identification of Leishmania species from sandflies, wild mammals and man in north Par& State, with particular reference to L. bruziliensis guyunensis causative agent of “pian-bois”. Transactions of the Royal Society of Tropkzl Medicine and Hygiene, 75, 530-536. Le Blancq, S. M., Lanham, S. M. & Evans, D. A. (1987). Comparative isoenzyme profiles of Old and New World Leishmania. In: Letihmaniases in Biology and Medicine, Vol. 1, Peters, W. & Killick-Kendrick, R. (editors). London: Academic Press, pp. 543-550. Shaw, J. J. (1994). Taxonomy of the genus Leishmania: present and future trends and their implications. Mem&ius do Znstiwto Oswald0 Cruz, 89,47 l-478.
Shaw, J. J. & Lainson, R. (1987). Recent advances in studies of the ecology and epidemiology of New World Leishmania. In: TheLkshmaniases in Biology andMedicine, Vol. 1, Peters, W. & KiyKigiendrick, R. (editors). London: Academic Press, pp. Shaw, J. J., ishikawa, E. A. Y. & Lainson, R. (1989). A rapid and sensitive method for the identification of Leishmania with monoclonal antibodies using fluorescein-labelled avidin. Transactions of the Royal Society of Tropical Medicine and Hygiene, 83,783-784. Tibayrenc, M. & Ayala, F. J. (1991). Towards a populationgenetics of microorganisms-the clonal theory of parasitic protozoa. Parasitology Today, 7, 228-232. Uliana, S. R. B., Nelson, K., Beverley, S. M., Camargo, E. P. & Floeter-Winter, L. M. (1994). Discrimination amongst Leashmania by polymerase chain reaction and hybridization with small subunit ribosomal DNA derived oligonucleotides. Journal of Eukayotik Microbiology, 41,324-330. Walton,B. C., Shaw, J. J. &Lainson, R. (1977). Observationson the in v&o cultivation of Leishmania braziliensis. Journal of Parasitology, 63, 1118- 1119. Young, D. G. &Duncan, M. A. (1994). Guide to the identification and geographic distribution of Lutzomyia sand flies in Mexico, the West Indies, Central and South America (Diptera: Psychodidae). Memoirs of the American Entomological Institute no. 54. Gainsville, Florida: Associated Publishers, American Entomological Institute.
Received November
19 October 1999
1999;
accepted for
publication
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