(Updated November 2023)
Balamuthia mandrillaris was first identified in 1990, isolated from a pregnant mandrill (Papio sphinx) that died of meningoencephalitis (Visvesvara et al., 1990, J. Clin. Microbiol. 28: 2750; Visvesvara, Schuster, and Martinez, 1993, J. Eukaryot. Microbiol. 40: 504). Since its original identification, a number of cases of fatal encephalitis attributable to B. mandrillaris have been reported in humans. The environmental niche for this amoebae is still being investigated, in part through the availability of molecular sequences developed in our lab.
[Trophozoite of Balamuthia mandrillaris – By G. Vishvesvara – CDC]
The first DNA sequence identifying Balamuthia mandrillaris was published by our lab as part of the expansion of sequence types in Acanthamoeba (Stothard et al. 1998; Journal of Eukaryotic Microbiology). This was a sequence of the nuclear ribosomal small subunit (18S) rRNA gene of the original Balamuthia mandrillaris isolate (CDC:V039) provided by our close collaborator Govinda Vishvesvara of the Centers for Disease Control and Prevention (CDC). Results from this phylogenetic analysis suggested that B. mandrillaris might be the closest known relative of Acanthamoeba within the Amoebozoa and the Acanthamoebidae. At the present time, this is still the case, although members of the genus Protacanthamoeba show about the same level of sequence divergence for the 18S rRNA gene and the mitochondrial cytochrome oxidase subunit I gene from Acanthamoeba.
Subsequently we have been involved with a number of collaborators in developing and using molecular tools to identify putative isolates of B. mandrillaris. Collaborators have included labs at the CDC (Govinda Vishvesvara and colleagues), the California Department of Public Health (Fred Schuster, and Thelma Dunnebacke) and colleagues at the Instituto Technologica de Sonora in Ciudad Obregon, Mexico (Fernando Lares-Villa and Luis Fernando Lares).
MOLECULAR STUDIES OF VARIATION IN B. MANDRILLARIS
The primary structure of the Balamuthia mandrillaris 18S rRNA gene sequence was very different than that found in members of Acanthamoeba. As mentioned, the first 18S rRNA sequence from Balamuthia was obtained as part of the study published as Stothard et al., 1998 (Journal of Eukaryotic Microbiology). The sequence for B. mandrillaris that we obtained using universal eukaryotic 18SrRNA primers was 1972 bases in length. This is about 150 bases longer than a “typical” eukaryotic 18S rRNA gene, such as we had observed in studies of Hartmanella (now Vermamoeba) vermiformis. It was about 300 bases shorter than the sequences of isolates of Acanthamoeba. The Balamuthia 18S rRNA sequence does not share the extra hypervariable regions of the gene that are found in Acanthamoeba.
Our studies showed that genetic variation in the sequence of the nuclear 18S rRNA gene was very low. This prompted us to investigate whether other genes might be able to provide more differentiation among isolates of Balamuthia.
In 2003, we determined the sequence of a major portion of the mitochondrial 16S-like small subunit rRNA from Balamuthia mandrillaris (Booton et al. 2003. J. Clin. Microbiol. 41: 453-455). The length of segment determined for this gene was 1109 bases in length, but homologous to a slightly longer region in the Acanthamoeba mitochondrial 16S-like rRNA gene, which had a length of 1154 bases. The equivalent region in the Vermamoeba (Hartmanella) vermiformis mitochondrial 16S-like rRNA sequence is 1110 bases. We subsequently showed that in B. mandrillaris the mitochondrial small ribosomal (16S-like) rRNA gene contains more information about differences between isolates than is contained within the nuclear 18S rRNA gene. As a consequence, most of the subsequent studies on genetic variability in B. mandrillaris have focused on the mitochondrial rRNA gene as a target.
We have also obtained the sequence of the mitochondrial cytochrome oxidase subunit I (COX-I or COI) gene from Balamuthia mandrillarus (Monica Crary, Ph.D. thesis, 2012). Some comparisons among isolates for COI have begun, given the recent availability of complete mitochondrial genome sequences from several isolates.
Our collaborators have also been very active in identifying procedures to culture B. mandrillaris from both soil and more recently from water (Lares, et al, Experimental Parasitology, 2014).
SEQUENCES IN THE DNA DATABASES FOR BALAMUTHIA
Information on variation between isolates for both the nuclear and mitochondrial small ribosomal subunit rRNA is given on another page of this site. Also provided on that page is information concerning genome studies of Balamuthia mandrillaris. Genome studies include WGS analysis of several isolates and complete mitochondrial genome information for an expanded number of isolates.
QUESTION: IS BALAMUTHIA REPRESENTED BY ONLY A SINGLE SPECIES?
Until recently, Balamuthia was represented by only a single species, B. mandrillaris. This changed in 2022, when a second species of Balamuthia. B. spinosa sp. n. was described (Lotonin et al. 2022). The 18S rRNA sequence from B. spinosa shows ~7% sequence divergence from the homologous sequences of B. mandrillaris.
In addition, the DNA databases suggest that additional “species” of Balamuthia may exist that have yet to be recognized. At least three 18S rRNA sequences from environmental surveys have been deposited that show ~7% divergence from both B. mandrillaris and B. spinosa sp. nov. (as well as from each other). These are: FN867196, MK499380 and MK499381. Are these unrecognized species level taxa within Balamuthia.
To put the 7% differences between different “Balamuthia” in context, the 18S rRNA gene of the Neff strain of Acanthamoeba terricola differs from various Group I Acanthamoeba (A. astronyxis, A. comanodi, A. tubiashi or A. byersi) by more than 18% . The sequence divergence of either B. mandrillaris or B. spinosa sp. nov. from Acanthamoeba is ~20-21%.