RESULTS AND DISCUSSION
preliminary results on the reactivity of monoclonal antibodies derived for selected
species complexes of Leishmania (McMahon-Pratt et al. 1982, 1985, 1986, Jaffe &
McMahon-Pratt 1983, Jaffe et al. 1984, Shaw et al. 1986, Pan & McMahon-Pratt 1988,
Hanham et al. 1990) were confirmed in our recent studies using a large sample (1,500 Leishmania
stocks, isolated from humans and a variety of mammalian and sandfly hosts) from
different localities in the New World. Included are results of our work as well as data
from other recently published studies (Grimaldi et al. 1987, 1989, 1991, Aguilar et al.
1989, Barral et al. 1991, Darce et al. 1991, Falqueto et al. 1991, Hashiguchi et al. 1991,
Kreutzer et al. 1991, Ponce et al. 1991, Bonfante-Garrido et al. 1992).
the reactivity of the monoclonal antibodies did not show any variation related to time in
culture, culture media, or parasite virulence (Grimaldi et al. 1987). However, variation
in the sensitivity of the test may occur due to the type of screening assay used (i.e.,
immunofluorescence, RIA or the ELISA technique). As an exemple, the L. donovani
group-specific epitope recognized by monoclonal antibody D2, using RIA (Jaffe et al. 1984,
Grimaldi et al. 1987) or ELISA, is weakly detected by IFA. However, as indicated in
previous studies (McMahon-Pratt et al. 1986, Grimaldi et al. 1987, 1991, Barral et al.
1991), independent of the origin of Leishmania stock (i.e., host species involved
or the clinical state of the infection) or of geographic area of isolation, some of the
monoclonal antibodies showed a high and consistent qualitative specificity at a species
level. In these analyses (see Table), the following monoclonals
were the most specific: D2 (LXXVIII, 2E5-A8) for L. (L.) chagasi; B11 (VII-5G3-F3)
for L. (V.) panamensis; B18 (XIV-2A5-A10) for L. (V.) braziliensis; M3
(IX-5H9-C10) for L. (L.) amazonensis; and V1 (CLXXVI-3C11-F14) for L. (L.)
venezuelensis. However, significant differences between the reactivity patterns with
specific monoclonal antibodies could be observed among stocks from certain species
complexes of Leishmania from distinct endemic areas. These differences can be
related with strain variation in the level of expression of certain antigenic
determinants, as recognized by some of the monoclonal antibodies. For instance, Venezuelan
isolates of L. (V.) braziliensis showed a distinct profile (Bonfante-Garrido et al.
1992) when they were compared with the same parasite species which circulates in Bolivia,
Brazil or Colombia (Barral et al. 1991, Grimaldi et al. 1991, Grimaldi &
McMahon-Pratt, unpublished data). Also, the species-specific epitope recognized by
monoclonal antibody B19 (XLIV-5A2-B9) (Grimaldi et al. 1987) could not be detected in some
variant strains of L. (V.) guyanensis (Grimaldi et al. 1991), indicating that they
had lost the epitope. In addition, some L. (V.) braziliensis isolates from the
Brazilian Amazon Region (Grimaldi et al. 1991), as well as other variant strains of this
parasite from Bolivia and Peru (Grimaldi & McMahon-Pratt, unpublished data) did not
react with the specific monoclonal antibody B16 (XIII-3E6-B11) that identify this species
(Shaw et al. 1986, McMahon-Pratt et al. 1986, Grimaldi et al. 1987, Barral et al. 1991).
Furthermore, although naturally occurring hybrid parasites (L. (V.) braziliensis x L.
(V.) guyanensis) from Venezuela reacted with the specific monoclonal antibodies (B16
and B18) for L. (V.) braziliensis (Bonfante-Garrido et al. 1992, Grimaldi &
McMahon-Pratt, unpublished data), conflicting results were obtained when the L. (V.)
braziliensis x L. (V.) panamensis hybrids from Nicaragua were analyzed by the
same method (Darce et al. 1991). Indeed, these monoclonals as well as the L. (V.)
panamensis-specific monoclonal antibody (B11) did not react with the later hybrid
isolates, indicating that they had lost the epitopes specific to both the parental species
(Momen et al. 1993). On the other hand, numerical analysis of the enzymic profiles of L.
(L.) venezuelensis isolates showed that this species was phenetically closely related
to the WHO L. (L.) mexicana reference strain. However, these strains did not react
with any of the monoclonal antibodies group-specific for L. mexicana complex
parasites, other than the species-specific (V1) monoclonal antibody (Bonfante-Garrido et
aforementioned, not all known species of Leishmania are recognized by a
distinct/specific monoclonal antibody (Grimaldi et al. 1987, 1991, 1992, Kreutzer et al.
1991). However, the patterns observed with the less specific monoclonals (as defined by
either qualitative or quantitative reactions with the expressed antigens) are indicative
of these species. For example, monoclonal antibodies B3 (VI-4D10-D12) and B12
(XIII-3H6-A12) were found to be useful in the confirmation of L. (V.) braziliensis
from Argentina, Bolivia, Brazil, Colombia, Nicaragua and Peru (Grimaldi et al. 1987,
Barral et al. 1991, Grimaldi & McMahon-Pratt, unpublished data) or L. (V.)
panamensis from Colombia, Costa Rica, Ecuador, Honduras and Nicaragua (Grimaldi et
al., 1987 Grimaldi & McMahon-Pratt, unpublished data). In contrast, these monoclonals
were not reactive in RIA tests with stocks of L. (V.) guyanensis, L. (V.) shawi, L.
(V.) lainsoni, L. (V.) colombiensis or L. (V.) equatorensis regardless of
geographic origin (Grimaldi et al., 1991, 1992, Kreutzer et al. 1991, Bonfante-Garrido et
al. 1992, Grimaldi & McMahon-Pratt, unpublished data). In addition, although B4 (VI-2
A5-A4) crossreacted with L. (V.) panamensis, L. (V.) colombiensis and L.
(V.) equatorensis (Kreutzer et al. 1991, Grimaldi et al. 1992), the former species
could be easily distinguished using in conjunction the more specific monoclonal antibody
(B11). Moreover, certain of the quantitative antigenic variations occurring betwen groups
or species often exceed that detected within each of these taxonomic groups (Grimaldi et
are other unusual features about leishmaniases of the New World. Although American
cutaneous leishmaniasis is usually caused by parasite species belonging to the L.
braziliensis or L. mexicana complex (Lainson & Shaw 1987, Grimaldi et al.
1989), a few cases of the disease from Brazil (Momen et al. 1985) and Ecuador (Hashiguchi
et al. 1991) have been associated with a parasite similar to the Old World L. (L.)
major. Interestingly, these L. major-like parasites cross-reacted with several
monoclonal antibodies (T1, XLVI-5B8-A8; T2, XLVI-4H12-C2; T3, XLVI-5A5-D4; T4,
LXVIII-1A4-G1; and T8, LXVII-3E12-F8) (Momen et al. 1985, Hashiguchi et al. 1991) produced
against members of the L. major or L. tropica complex (Jaffe &
McMahon-Pratt 1983). In addition, our experience would indicate the existence of a number
of other leishmanial parasites circulating in the Americas [e.g., L. (V.) colombiensis;
L. (L.) equatorensis; and L. (L.) venezuelensis] that also cross-reacted with the L.
major species-specific monoclonals (Kreutzer et al. 1991, Bonfante-Garrido et al.
1992, Grimaldi et al. 1992). Work is now in progress to better define the phylogenetic
relationship between these parasites and Old World L. (L.) major strains. Whatever
the explanation for the existence of these L. major related parasites, the results
point to caution for all researchers working with New World Leishmania isolates. We
recommend that when classifying these parasites, reference strains of Old World species as
well as the L. major-specific monoclonals (e.g., the monoclonal T1, XIX-2D8-D7) be
included for comparison.
monoclonal antibodies (D-2, B-4, B-5, B-7, B-16, B-19, M-3, M-7, P-9, T-9) analyzed in
this study were selected by the WHO Workshops and recommended for general use in the
identification of Leishmania species. The analyses of strains brought by the
participants of the Cali Workshop also pointed strongly to the need for the incorporation
of additional monoclonals (e.g., B-3, B-11, B-12, B-18, V-1) in an expanded crosspanel. We
should mention that a free "Monoclonal antibody kit" for
diagnosis/identification of Leishmania species, consisting of lyophilized aliquots
(100 µl) of the monoclonal antibodies (titers 10-4 to 10-6) will be
available soon; as part of the kit, a description of methods (immunofluores-cence and the
ELISA technique) will also be provided. Requests for the kit will be made to Dr F
Modabber, World Health Organization, Geneva, Switzerland. A formal request form indicating
the potential application and resources available for analyses will be requested by WHO.
conclusion, problems related to the differentiation and identification of some leishmanial
parasites were encountered using serodeme analysis with specific monoclonal antibodies, as
well as when those samples were analyzed by isoenzyme characterization (Grimaldi et al.
1987, 1989, 1991). Some of these isolates represent additional new species (Grimaldi et
al. 1991, 1992, Kreutzer et al. 1991) or hybrid parasites (Darce et al. 1991,
Bonfante-Garrido et al. 1992) and further investigation with new monoclonal antibodies is
recommended in these situations. A comparison of the discriminatory ability of the two
typing methods for Leishmania using Simpson's index of diversity showed that
serodeme analysis is more discriminating, even though the zymodeme analysis produced more
groups (Cupolillo et al. 1993). The continual discovery of new leishmanial species in
tropical America is, in part, a reflection of the increasingly sophisticated methods for
parasite differentiation. However, it also indicates that there has been a much greater
evolutionary divergence among this parasite group in the New World, compared to the Old
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