MOLECULAR IDENTIFICATION OF NAEGLERIA SPP by Ns92M02

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									              MOLECULAR IDENTIFICATION OF NAEGLERIA SPP.
                         ISOLATED IN MEXICO
                              1                      1                                  2
        Fernando Lares Villa , Eunice Guzmán Fierros , and Johan F. De Jonckheere
     1. Department of Agronomic and Veterinary Sciences, Technological Institute of Sonora.
            5 de febrero 818 Sur, Cd. Obregón, Sonora, 85000, Mexico. flares@itson.mx
               2. Scientific Institute of Public Health, B-1050 Brussels, Belgium.

                   Mode: Poster Thematic: Environment and Public Health
                          Key words: amoebae, Naegleria, isolation

Introduction. There have been several            one from the Distrito Federal, only yielded
cases        of       primary       amoebic      isolates at 20ºC incubation. Seven
meningoencephalitis (PAM) caused by              Naegleria spp. Were identified, N.
Naegleria fowleri in Mexico. Identifications     lovaniensis, N. tihangensis, N. americana,
of the genus Naegleria were mainly on            N. clarki, N. pagei, N. indonesiensis, and N.
morphological      basis   but    sometimes      byersi. The strain of N. indonesiensis differs
isoenzymes were used. In Mexico, two high        by one substitution in the ITS2 sequence
temperature tolerant Naegleria australiensis     from one of the type strain, while the
and Naegleria lovaniensis could be               sequences of the N. clarki isolate is totally
identified by this method amongst the            identical to those of the type strain. One of
isolates. However, several Naegleria             the two isolates of N. pagei differs a bit in
isolates of these studies could not be           the ITS2 sequence. In strains of the new N.
identified. Strains of N. fowleri have also      lovaniensis isolates is unique as it is the
been isolated from the environment and           first time a one bp deletion is observed in
PAM cases in Mexico (1).                         the ITS2 of the species.
Objective. To identify as much different         Conclusion. We have attempted to find as
Naegleria species as possible from different     much different strains as possible which
sites in Sonora and from other five states of    belong to Naegleria. While only N. fowleri,
Mexico.                                          N. lovaniensis and N. australiensis had
Material and Methods. The sampling area          been identified in Mexico with certainty
covered the states of Sonora, Sinaloa,           before, we are adding, six other Naegleria
Jalisco, Michoacán, Estado de México, and        spp. to the list. This study is only a small
Distrito Federal. Water bodies sampled           survey and more elaborate studies are
were ponds, rivers, lakes, and a water           needed to know the dispersal and
reservoir. The samples were kept for a           biodiversity of Naegleria spp. in Mexico.
week at room temperature, before been            References.
processed in triplicate and incubated at         1.     Lares-Villa F., De Jonckheere J. F.,
20ºC, 37ºC and 42ºC, respectively. Only          Visvesvara G. S., Rechi-Iruretagayena A.,
isolates that showed typical Naegleria           Ferreira-Guerrero E., Fernandez-Quintnilla G.
morphology were further investigated.            and Ruiz-Matus C. 1993. Five cases of primary
                                                 amoebic encephalitis in Mexically, B.C. México:
Flagellate formation was tested in distilled     study of the isolates. J. Clin Microbiol, 31, 685-
water at room temperature. DNA was               688.
isolated from pelleted trophozoites using        2. Hugo, E. R., Stewart, V. J., Gast, R. J.,
the UNSET method (2). The ITS1, 5.8S and         Byers, T. J. 1992. Purification of the amoeba
ITS2 rDNA were PCR-amplified using an            mtDNA using the UNSET procedure. In Soldo A.
ITS forward primer and an ITS reverse            T. and Lee J. J. (Ed.) Protocols in protozoolgy,
primer, corresponding to the 3’ end of the       Allen Press, Lawrence, Kansas, p. D-7.1-2.
SSU rDNA and the 5’ end of the LSU rDNA,         3. De Jonckheere, J. F. 2004. Molecular
respectively (3). The PCR product was            definition and the ubiquity of species in the
                                                 genus Naegleria. Protist 155, 89-103.
sequenced with the amplification primers         4. De Jonckheere, J. F. 1998. Sequence
without cloning with a Beckman CEQ2000           variation in the ribosomal internal transcribed
sequencer using the CEQ Dye Terminator           spacer, including 5.8S, of Naegleria spp. Protist
Cycle Sequencing Kit. Total ITS1, 5.8S and       149, 221-228.
ITS2 sequences of the Naegleria isolates
were aligned to those of published
sequences of different Naegleria spp. (4).
Results and Discussion. Except one
sample from Michoacán all samples yielded
amoebae. Two samples, one from Jalisco,

								
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