MOLECULAR IDENTIFICATION OF NAEGLERIA SPP.
ISOLATED IN MEXICO
Fernando Lares Villa1, Eunice Guzmán Fierros1, and Johan F. De Jonckheere2
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
cases
of
primary
amoebic
meningoencephalitis (PAM) caused by
Naegleria fowleri in Mexico. Identifications
of the genus Naegleria were mainly on
morphological
basis
but
sometimes
isoenzymes were used. In Mexico, two high
temperature tolerant Naegleria australiensis
and Naegleria lovaniensis could be
identified by this method amongst the
isolates. However, several Naegleria
isolates of these studies could not be
identified. Strains of N. fowleri have also
been isolated from the environment and
PAM cases in Mexico (1).
Objective. To identify as much different
Naegleria species as possible from different
sites in Sonora and from other five states of
Mexico.
Material and Methods. The sampling area
covered the states of Sonora, Sinaloa,
Jalisco, Michoacán, Estado de México, and
Distrito Federal. Water bodies sampled
were ponds, rivers, lakes, and a water
reservoir. The samples were kept for a
week at room temperature, before been
processed in triplicate and incubated at
20ºC, 37ºC and 42ºC, respectively. Only
isolates that showed typical Naegleria
morphology were further investigated.
Flagellate formation was tested in distilled
water at room temperature. DNA was
isolated from pelleted trophozoites using
the UNSET method (2). The ITS1, 5.8S and
ITS2 rDNA were PCR-amplified using an
ITS forward primer and an ITS reverse
primer, corresponding to the 3’ end of the
SSU rDNA and the 5’ end of the LSU rDNA,
respectively (3). The PCR product was
sequenced with the amplification primers
without cloning with a Beckman CEQ2000
sequencer using the CEQ Dye Terminator
Cycle Sequencing Kit. Total ITS1, 5.8S and
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,
one from the Distrito Federal, only yielded
isolates at 20ºC incubation. Seven
Naegleria spp. Were identified, N.
lovaniensis, N. tihangensis, N. americana,
N. clarki, N. pagei, N. indonesiensis, and N.
byersi. The strain of N. indonesiensis differs
by one substitution in the ITS2 sequence
from one of the type strain, while the
sequences of the N. clarki isolate is totally
identical to those of the type strain. One of
the two isolates of N. pagei differs a bit in
the ITS2 sequence. In strains of the new N.
lovaniensis isolates is unique as it is the
first time a one bp deletion is observed in
the ITS2 of the species.
Conclusion. We have attempted to find as
much different strains as possible which
belong to Naegleria. While only N. fowleri,
N. lovaniensis and N. australiensis had
been identified in Mexico with certainty
before, we are adding, six other Naegleria
spp. to the list. This study is only a small
survey and more elaborate studies are
needed to know the dispersal and
biodiversity of Naegleria spp. in Mexico.
References.
1.
Lares-Villa F., De Jonckheere J. F.,
Visvesvara G. S., Rechi-Iruretagayena A.,
Ferreira-Guerrero E., Fernandez-Quintnilla G.
and Ruiz-Matus C. 1993. Five cases of primary
amoebic encephalitis in Mexically, B.C. México:
study of the isolates. J. Clin Microbiol, 31, 685688.
2. Hugo, E. R., Stewart, V. J., Gast, R. J.,
Byers, T. J. 1992. Purification of the amoeba
mtDNA using the UNSET procedure. In Soldo A.
T. and Lee J. J. (Ed.) Protocols in protozoolgy,
Allen Press, Lawrence, Kansas, p. D-7.1-2.
3. De Jonckheere, J. F. 2004. Molecular
definition and the ubiquity of species in the
genus Naegleria. Protist 155, 89-103.
4. De Jonckheere, J. F. 1998. Sequence
variation in the ribosomal internal transcribed
spacer, including 5.8S, of Naegleria spp. Protist
149, 221-228.