J. Eukaryot. Microbiol., 54(1), 2007 pp. 38–41r 2006 The Author(s)Journal compilation r 2006 by the International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2006.00140.x
Analysis of the b-Tubulin Genes from Enterocytozoon bieneusi Isolates from a
DONNA E. AKIYOSHI,a LOUIS M. WEISS,b XIAOCHUAN FENG,a BRYONY A. P. WILLIAMS,c PATRICK J. KEELING,c
aDivision of Infectious Diseases, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts 01536, and
bDepartment of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA, and
cCanadian Institute for Advanced Research, Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada
Enterocytozoon bieneusi is the most common and clinically significant microsporidium associated with chronic diarrhea
and wasting in immunocompromised humans. Albendazole, which is effective against several helminths, protozoa, and microsporidia, isrelatively ineffective against infections due to E. bieneusi. A likely explanation for the observed clinical resistance to albendazole wasdiscovered from sequence analysis of the E. bieneusi b-tubulin from isolates from an infected human and a naturally infected rhesusmacaque. The b-tubulin of E. bieneusi has a substitution at Glu198, which is one of six amino acids reported to be associated with benz-imidazole sensitivity.
Key Words. Albendazole resistance, beta-tubulin gene, microsporidia.
THE microsporidia represent a large and diverse group of positive for Enterocytozoon bieneusi. Spores were purified from
obligate intracellular eukaryotic parasites and to date, 1,200
the human and rhesus macaque stool samples (Sheoran et al. 2005;
species have been identified. Of these, Enterocytozoon bieneusi is
Zhang et al. 2005), and were confirmed to be E. bieneusi by elec-
the most clinically significant species associated with AIDS-re-
tron microscopy and sequencing of the internal transcribed spacer
lated human microsporidiosis (reviewed in Cali 1991; Curry and
(ITS) of the small subunit rRNA gene. The spores isolated from
Canning 1993; Didier et al. 2004; Keeling and Fast 2002; Mathis,
the human patient and rhesus macaque will be referred to as the
Weber, and Deplazes 2005; Wittner 1999), with symptoms includ-
H206 isolate and M231 isolate, respectively.
ing chronic diarrhea, wasting, and malabsorption. Enterocytozoon
Cloning and sequencing of the b-tubulin genes. The b-tubulin
bieneusi has also been identified in immunocompetent patients
gene from the M231 isolate was first isolated from a whole genome
(Albrecht and Sobottka 1997; Gainzarain et al. 1998; Sandfort et
amplified (Molecular Staging Inc., New Haven, CT) library cloned
al. 1994), in individuals receiving immunosuppressive therapy
into the pHCSmart-Kan vector (Lucigen Corp., Middleton, WI).
(Guerard et al. 1999; Rabodonirina et al. 1996), and in macaques,
The E. bieneusi b-tubulin genes from the H206 and M231 isolates
both immunocompetent and those infected with simian immuno-
were then amplified from purified genomic DNA (1 ng; DNeasy
deficiency virus (SIV) (Green et al. 2004; Mansfield et al. 1997).
Tissue Kit; Qiagen Inc., Valencia, CA) using primers, MEb btub-I
Microtubules, which are formed by polymerization of the
(50-AACGGGCAGCTGAGTAGTTTAAGTGATT-30) and MEb
a- and b-tubulin subunits, are a major component of the mito-
tic spindle. The benzimidazoles have been found to prevent
using the Expand High Fidelity PCR System (Roche Diagnostics
both the polymerization of the tubulin subunits by binding to the
Corp., Indianapolis, IN). The PCR products were cloned into
b-tubulin subunit, thus preventing elongation of the microtubules,
pCR4TOPO (Invitrogen Corp., Carlsbad, CA) and used to trans-
and depolymerization of the two subunits. The benzimidazoles
form E. coli TOP10 cells. Inserts from four independent clones
are toxic to fungi and helminths (Davidse 1986; Lacey 1988), and
were double-strand sequenced for each isolate.
have been used to effectively treat both microsporidiosis due toEncephalitozoon spp. (De Groote et al. 1995; Katiyar and Edlind
1997; Molina et al. 1998; Ridoux and Drancourt 1998), and someprotozoan infections including Giardia intestinalis (Katiyar et al.
Analysis of the b-tubulin of Enterocytozoon bieneusi. The
1994; Lemee et al. 2000). However, these benzimidazole deriv-
E. bieneusi b-tubulin gene was first identified in the M231 amp-
atives, including albendazole, are relatively ineffective against E.
lified genomic library. One clone, with an open reading frame
bieneusi infections (Blanshard et al. 1992; Conteas et al. 2000;
of 1,314 bp, had significant similarity (E-value 5 e À 177) to other
b-tubulin sequences in the NCBI database using the BLASTp
In this communication, we report the analysis of the b-tubulin
program (Altschul et al. 1990; McGinnis and Madden 2004). PCR
gene from E. bieneusi isolated from an HIV-infected human and
primers, Meb btub-I and MEb-btub-J, were designed to amplify
an SIV-infected rhesus macaque (Macaca mulatta). The sequence
the b-tubulin gene from purified spores isolated from a human
data provide an explanation for the reported resistance of E.
patient (H206) and a rhesus macaque (M231). These genes were
compared using the ClustalW algorithm (Thompson, Higgins, andGibson 1994). Both genes were 1,314 bp and were identical ex-cept for five synonymous transitions (99.62% sequence identity).
The A-T content was 55.4% and 55.6% for the M231 and H206 b-
Parasite strains. An HIV-positive adult patient admitted to the
tubulin genes, respectively. Analysis of the 50—and 30-flanking
Mulago Hospital in Kampala, Uganda, and an SIV-infected rhesus
regions revealed no obvious canonical elements, such as promoter
macaque (Macaca mulatta), housed at the New England Regional
elements or polyadenylation sequences.
Primate Research Center (Southborough, MA), were found to be
The E. bieneusi b-tubulin gene encoded a 438-amino acid
polypeptide with a molecular weight of 49,116 daltons. A highdegree of sequence identity (68%–73%) was observed between
Corresponding Author: D. Akiyoshi, Division of Infectious Diseases,
the E. bieneusi b-tubulin and b-tubulins from the family Enceph-
Tufts Cummings School of Veterinary Medicine, North Grafton, Mas-sachusetts 01536—Telephone number: 508-839-7935; FAX number:
alitozoonidae, Antonospora locustae, Trachipleistophora homin-
508-839-7911; e-mail: [email protected]
is, Saccharomyces cerevisiae, and Homo sapiens. However, the
AKIYOSHI ET AL.—b-TUBULIN GENES FROM E. BIENEUSI
+ E. bieneusi
MREIIHVQAGQCGNQIGTKFWEEISREHGIDENGKKCGCDGDDGWCDETNRISVYYNQSSSNKYVPRAVLVDLEPGTMEAIRNHPMGNIFRPDNFIFGQS 100E. cuniculi
MREIIHLQTGQCGNQVGCKFWETISGEHGIDQTGRYVGTSDN-----QLERINVYYNEASSKKYVPRAVLIDLEPGTMDAVRQGPFGELFRPDNFVFGQS 95E. hellem
MREIIHLQTGQCGNQVGCKFWETISGEHGIDQTGKYVGTSDN-----QLERVNVYYNEASSKKYVPRAVLIDLEPGTMDAVRQGPFGDLFRPDNFVFGQS 95E. intestinalis
REIIHIQTGQCGNQIGAKFWETISGEHGVDPSGRYVGTSDL-----QIERINVYYNEASGKKYVPRAVLIDLEPGTMDSVRAGPFGELFRPDNFVFGQS 94N. locustae
VGSKFWEVISEEHGINNEGHFVGHSSN-----QLERINVYYNEASSSKYVPRAVLIDLEPGTMDSVRAGPLGRLFRPDNFIFGQS 80T. hominis
IGTKFWEVISEEHGINNLGQYTGTKDN-----QLDRISVYYNESSTKQYVPRAVLVDLEPGTMDTLRSGPLGSLFRPDNYVFGQS 80S. cerevisiae
MREIIHISAGQYGNQIGAAFWETICGEHGLDFNGTYHGHDDI-----QKERLNVYFNEASSGKWVPRSINVDLEPWTIDAVRNSAIGNLFRPDNYIFGQS 95H. sapiens
MREIVHIQAGQCGNQIGAKFWEVISDEHGIDPTGSYHGDSDL-----QLERINVYYNEAAGNKYVPRAILVDLEPGTMDSVRSGPFGQIFRPDNFVFGQS 95 .*. *** *. ***. * * . *. **.*. . ***. .**** *.* * .*****.****
+ + E. bieneusi
GAGNNWAKGHYTEGAELCEQVLECIRKEAEKTDCLQGFQLTHSLGGGTGSGMGTLLVSKIKEEFPDRMLATFSVVPSPKVSDTVVEPYNATLSFHQLVEN 200E. cuniculi
GAGNNWAKGHYTEGAELIDSVMDVVRKEAESSDCLQGFQITHSLGGGTGAGMGTLLLSKIREDFPDRMICTFSVVPSPKVSDTVVEPYNATLSIHQLVEN 195E. hellem
GAGNNWAKGHYTEGAELIDSVMDVVRKEAESSDCLQGFQITHSLGGGTGAGMGTLLLSKIREDFPDRMICTFSVVPSPKVSDTVVEPYNATLSIHQLVEN 195E. intestinalis
GAGNNWAKGHYTEGAELIDAVMDVVRKEAESCDCLQGFQITHSLGGGTGAGMGTLLIAKIREDFPDRMICTFSVVPSPKVSDTVVEPYNATLSIHQLVEN 194N. locustae
GAGNNWAKGHYTEGAELIDSVLDVVRKEAESSDCLQGFQFTHSLGGGTGAGMGTLLISKIREEYPDRMMCTFSVVPSPKVSDTVVEPYNATLSIHQLVEN 180T. hominis
GAGNNWAKGHYTEGAELIENVMDCVRREAEKSNCLQGFQITHSLGGGTGAGMGTLLISKIREEFPDRMMCTFSVVPSPKVSDTVVEPYNATLSIHQLVEN 180S. cerevisiae
SAGNVWAKGHYTEGAELVDSVMDVIRREAEGCDSLQGFQITHSLGGGTGSGMGTLLFSKIKEELPDRMMATFSVLPSPKTSDTVVEPYNATLSVHQLVEH 195H. sapiens
*** ************ . *. .*.*.* . .***** *********.****** .**.*. ***. ****.**** ************* *****.
+ + + E. bieneusi
ANQTFCIDNDALYEICTKTLKLKNPSYNDLNSLVSKVMSGITTCLRFPGQLNSDLRKLAVNMIPFPRLHFFCVGYAPLCSEASTQYRNITVSDLTAQLFD 300E. cuniculi
ADETFCIDNEALYDICFRTLKLNNPGYGDLNHLVSLVMSGVTTCLRFPGQLNADLRKLAVNMIPFPRLHFFVAGFAPLIAIGTQKFKTYSVSELTQQMFD 295E. hellem
ADETFCIDNEALYDICFRTLKMSNPGYGDLNHLVSLVMSGVTTCLRFPGQLNADLRKLAVNMIPFPRLHFFVVGSAPLIAIGTQKFKTYSVSELTQQMFD 295E. intestinalis
ADEVFCIDNEALYDICFRTLKLSNPGYGDLNHLVSLVMSGVTSCLRFPGQLNADLRKLAVNMIPFPRLHFFLVGFAPLTAVGSQKFKTYSVSELTQQMFD 294N. locustae
ADETFCIDNEALYDICFRTLKLSTPGYGELNRLVSLVMSGVTTCLRFPGQLNADLRKLAVNMVPFPRLHFFIVGFAPLIAQGTSQYRTYSVSELTSQMFD 280T. hominis
ADETFCIDNEALYNICFNILKLKNPGYADLNRLVSLVMSGVTTCLRFPGQLNADLRKLAVNMIPFPRLHFFMIGFAPLIAEGMASYRSYSVSELTQQMFD 280S. cerevisiae
SDETFCIDNEALYDICQRTLKLNQPSYGDLNNLVSSVMSGVTTSLRYPGQLNSDLRKLAVNLVPFPRLHFFMVGYAPLTAIGSQSFRSLTVPELTQQMFD 295H. sapiens
. . .****.*** ** **. * * .** *** ***.*.**.*****.********.******** * *** . . .* .** *.**
SKNMMTACNPRDGRYLTAAVYFRGKMSMKEVDEQMNLMQTRTMDSFVEWIPNNVQTAVCSVPPKDVEMSATFIGNTTSIQEIFKRVGEQFSSMFKRKAFL 400E. cuniculi
SKNMMTACDPRKGRYLTVAAMFRGKISMKDVDEQMSMVQSKNSSLFVEWIPSNVKTAVCDIAPTGLEMSATFVGNTTSIQELFKRISDQFTVMFRRKAFL 395E. hellem
SKNMMTACDPRKGRYLTVAAMFRGKISMKDVDEQMSMVQSKNSTLFVEWIPSNVKTAVCDIAPTGLEMSATFVGNTTSIQELFKRISDQFTVMFRRKAFL 395E. intestinalis
SKNMMTASDPRKGRYLTAAAMFRGRISTKDVDEQMSMVQSKNSSYFVEWIPSNIKVAVCDIAPTGLEMSATFVGNSTSIQELFKRVSDQFTVMFRRKAFL 394N. locustae
SKNMMAASDPRHGRYLTVAAVFRGKISMKDVDEQMLQVQTRNSAHFVEWIPNNVKTAVCDIPPSGLDMSATFIGNSTSIQELFKRISDQFSVMFRRKAFL 380T. hominis
SKNMMAASDPKHGRYLTVATIFRGNISMKDVDEQLHNIQSRNASNFVEWIPNNVKTAVCDIPPSTLDMSATFIGNTTAIQELFKRIAEQFQLMFRRKAFL 380S. cerevisiae
AKNMMAAADPRNGRYLTVAAFFRGKVSVKEVEDEMHKVQSKNSDYFVEWIPNNVQTAVCSVAPQGLDMAATFIANSTSIQELFKRVGDQFSAMFKRKAFL 395H. sapiens
.****.*. *. ***** * *** .* *.*. .* . ****** *. *** . * . *.***. *.*.***.***. .** **.*****
HWYTGEGMDEAEFTEAEANLQDLLSEYQQYRDSGVGGYN 439E. cuniculi
HWYTGEGMDEMEFSEAESNMNDLLSEYQQYQDATIEDAEEFLVN 439E. hellem
HWYTGEGMDEMEFSEAESNMNDLLSEYQQYQDATVEDAEEFLVN 439E. intestinalis
HWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATVEDAEEFLVN 438N. locustae
HWYTGEG 387T. hominis
HWYTQEG 387S. cerevisiae
HWYTSEGMDELEFSEAESNMNDLVSEYQQYQEATVEDDEEVDENGDFGAPQNQDEPITENFE 457H. sapiens
ClustalW alignment of the b-tubulin amino acid sequences from Enterocytozoon bieneusi, Encephalitozoon cuniculi (GenBank Accession
no. NP_597591), Encephalitozoon hellem (GenBank Accession no. AAB12034), Encephalitozoon intestinalis (GenBank Accession no. AAN78302),Nosema locustae (GenBank accession no. AAN35161), Trachipleistophora hominis (GenBank Accession no. AAF31660), Saccharomyces cerevisiae(GenBank Accession no. CAA24603), and Homo sapiens (GenBank Accession no. T08726). Positions with identical residues in all eight sequences areindicated by asterisks (below). Conserved changes are indicated by a dot (below). The b-tubulin sequence from E. bieneusi had an apparent five aminoacid insertion (boxed), which was not found in other fungal, microsporidial or mammalian sequences. Changes in the six amino acids (His6, Phe167,Glu198, Phe200, Arg241; numbering based on S. cerevisiae), which are associated with benzimidazole resistance, are indicated by a cross (1) above.
Glu198, which has a high association with benzimidazole sensitivity, is changed to a glutamine in E. bieneusi. Nucleotide sequences of the b-tubulin genesare available in the GenBank database under Accession numbers, DQ242639 (human) and DQ242640 (rhesus macaque).
E. bieneusi b-tubulin has near its amino terminus an additional
Phe200, which confer resistance to benzimidazole (Katiyar et al.
five residues (DGWCD) that are unlikely to represent an intron
1994; Kwa, Veenstra, and Roos 1994). With respect to Ala165, the
because of the absence of a canonical GT-AG splice boundary, as
b-tubulins of the Encephalitozoonidae have five of the six resi-
well as, an AT-AC boundary (Fig. 1).
dues, but with Ala165 changed to a Cys165. Yet, they are highly
The most significant finding was a molecular explanation for
sensitive to albendazole (Cruz, Bartlett, and Edlind 1994; Katiyar
the observed clinical resistance of E. bieneusi to benzimidazoles.
et al. 1994). This residue is also a cysteine in Giardia lamblia
The E. bieneusi b-tubulin has only five of the six amino acids that
(Kirk-Mason, Turner, and Chakrabory 1988) and Cryptococcus
have been reported to be associated with benzimidazole activity
neoformans (Li et al. 1996), which are also sensitive to albenda-
(His6, Phe167, Glu198, Phe200 or Arg241; numbering based on the
zole. Contrary to this, Ala165 is replaced by a glycine in thiaben-
S. cerevisiae sequence, Fig. 1) (Jung, Wilder, and Oakley 1992;
dazole-resistant strains of Aspergillus nidulans. Of these six
Ohrbach, Porro, and Yanofsky 1986; Thomas, Neff, and Botstein
amino acids, His6 and Arg241, are not highly predictive of benz-
1985). The one residue difference occurred at position 198 in
imidazole sensitivity because these two residues are conserved in
which glutamine was substituted for glutamic acid.
both benzimidazole-resistant and benzimidazole-sensitive organ-
Organisms resistant to benzimidazole lack one or both of
isms. Phe167 is also not highly predictive of benzimidazole sensi-
Glu198 or Phe200. Both E. bieneusi and Entamoeba histolytica
tivity because the Trichomonas vaginalis b-tubulins have tyrosine
have changes at Glu198, and both are relatively resistant to alben-
instead of phenylalanine at this position (Katiyar and Edlind
dazole. Cryptosporidium parvum and Acanthamoeba polyphaga
1997), yet the organism is sensitive to the benzimidazoles. Over-
have changes at both Glu198 and Phe200, and both are resistant to
all, the data for these six amino acids show that substitutions of
benzimidazole. The helminths, fungi, and humans have changes at
either Glu198 or Phe200 are highly associated with benzimidazole
J. EUKARYOT. MICROBIOL., VOL. 54, NO. 1, JANUARY– FEBRUARY 2007
sensitivity, and changes of one or both of these amino acids results
of disseminated Encephalitozoon cuniculi in a patient with AIDS:
in resistance to benzimidazole. The E. bieneusi b-tubulin data are
successful therapy. J. Infect. Dis., 171:1375–1378.
Didier, E. S., Stovall, M. E., Green, L. C., Brindley, P. J., Sestak, K. &
The microsporidia have been placed within the fungal clade
Didier, P. J. 2004. Epidemiology of microsporidiosis: sources and
based on phylogenetic analyses of the a- and b-tubulin genes
modes of transmission. Vet. Parasitol., 126:145–166.
Dieterich, D., Lew, E., Kotler, D., Poles, M. & Orenstein, J. 1994.
(Edlind et al. 1996; Keeling and Doolittle 1996), sequencing of
Treatment with albendazole for intestinal disease due to Enterocytozo-
the E. cuniculi genome (Katinka et al. 2001; Vivares et al. 2002),
on bieneusi in patients with AIDS. J. Infect. Dis., 169:178–183.
the existence of relic mitochondrial genes in the nuclear genome,
Edlind, T. D., Li, J., Visvesvara, G. S., Vodkin, M. H., McLaughlin, G. L.
and the presence of mitosomes and Golgi-like membranes. In ad-
& Katiyar, S. K. 1996. Phylogenetic analysis of b-tubulin sequences
dition, morphological and life cycle data are consistent with their
from amitochondrial protozoa. Mol. Phylogenet. Evol., 5:359–367.
placement within the fungal clade (reviewed in Germot, Philippe,
Gainzarain, J. C., Canut, A., Lozano, M., Labora, A., Carreras, F., Fenoy,
and Le Guyander 1997; Keeling and Doolittle 1996). We attempt-
S., Navajas, R., Pieniazek, N. J., da Silva, A. J. & del Aguila, C. 1998.
ed to align the E. bieneusi b-tubulin to 67 other b-tubulin se-
Detection of Enterocytozoon bieneusi in two human immunodeficiency
quences representative of the major eukaryotic groups, and then
virus-negative patients with chronic diarrhea by polymerase chainreaction in duodenal biopsy specimens and review. Clin. Infect. Dis.,
use maximum likelihood methods (TREE-PUZZLE 5.2 and
PHYML; Guindon and Gascuel 2003; Schmidt et al. 2002) to de-
Germot, A., Philippe, H. & Le Guyader, H. 1997. Evidence for loss of
termine the phylogenetic position of E. bieneusi. Unfortunately,
mitochondria in Microsporidia from a mitochondrial-type HSP70 in
the E. bieneusi b-tubulin sequence was found to be highly diver-
Nosema locustae. Mol. Biochem. Parasitol., 87:159–168.
gent and its position within the tree was not well resolved (data
Green, L. C., Didier, P. J., Bowers, L. C. & Didier, E. S. 2004. Natural and
experimental infection of immunocompromised rhesus macaques
While E. bieneusi is clinically the most significant human
(Macaca mulatta) with the microsporidian Enterocytozoon bieneusi
microsporidium, it is also the least understood with respect to
genotype D. Microbes Infect., 6:996–1002.
its biology and epidemiology. Greater than 50 genotypes have
Guerard, A., Rabodonirina, M., Cotte, M. L., Liguory, O., Piens, M. A.,
Daoud, S., Picot, S. & Touraine, J. L. 1999. Intestinal microsporidiosis
been identified using the ITS sequence (Mathias et al. 2005; Sulai-
occurring in two renal transplant recipients treated with mycophenolate
man et al. 2004). A recent study characterized E. bieneusi isolates
mofetil. Transplantation, 68:699–707.
from cattle and found some genotypes were host-adapted while
Guindon, S. & Gascuel, O. 2003. A simple, fast, and accurate algorithm
others were identical to the human genotype K, suggesting that
to estimate large phylogenies by maximum likelihood. Syst. Biol., 52:
these isolates might have the potential to infect humans (Sulaiman
et al. 2004). Additional independent genetic markers are needed to
Jung, M. K., Wilder, I. B. & Oakley, B. R. 1992. Amino acid alterations in
provide more genotyping tools, which would also aid in clarifying
the benA (b-tubulin) of Aspergillus nidulans that confer benomyl
the genetic structure of E. bieneusi. The determination of the se-
resistance. Cell Motil. Cytoskeleton, 22:170–174.
quence of the b-tubulin gene provides the first such independent
Katinka, M. D., Duprat, S., Cornillot, E., Me´te´nier, G., Thomarat, F.,
marker for studies on the heterogeneity of E. bieneusi populations.
Prensier, G., Bare, V., Peyretaillade, E., Brottier, P., Wincker, P.,Delbac, F., El Alaoui, H., Peyret, P., Saurin, W., Gouy, M., Weissen-
But more importantly, the significant finding of this study was the
bach, J. & Vivares, C. P. 2001. Genome sequence and gene compaction
correlation between the observed clinical resistance of E. bieneusi
of the eukaryote parasite Encephalitozoon cuniculi. Nature, 414:
to benzimidazoles and its b-tubulin sequence.
Katiyar, S. K. & Edlind, T. D. 1997. In vitro susceptibilities of the AIDS-
associated microsporidian Encephalitozoon intestinalis to albendazole,
its sulfoxide metabolite, and 12 additional benzimidazole derivatives.
This work was supported by National Institutes of Health grants
Antimicrob. Agents Chemother., 41:2729–2732.
R21 AI52792 and R21 AI064118. LMW was supported by RO1
Katiyar, S. K., Gordon, V. R., McLaughlin, G. L. & Edlind, T. D. 1994.
Antiprotozoal activities of benzimidazoles and correlations with
AI31788. The authors thank Joel Hanawalt for technical assistance.
b-tubulin sequence. Antimicrob. Agents Chemother., 38:2086–2090.
Keeling, P. J. & Doolittle, W. F. 1996. Alpha-tubulin from early diverging
eukaryotic lineages and the evolution of the tubulin family. Mol.
Albrecht, H. & Sobottka, I. 1997. Enterocytozoon bieneusi infection in
Keeling, P. J. & Fast, N. M. 2002. Microsporidia: biology and evolution
patients who are not infected with human immunodeficiency virus. Clin.
of highly reduced intracellular parasites. Ann. Rev. Microbiol., 56:
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. 1990.
Kirk-Mason, K. E., Turner, M. J. & Chakrabory, P. R. 1988. Cloning and
Basic local alignment search tool. J. Mol. Biol., 215:403–410.
sequence of b-tubulin cDNA from Giardia lamblia. Nucleic Acids Res.,
Blanshard, C., Ellis, D. S., Tovey, D. G., Dowell, S. & Gazzard, B. G.
1992. Treatment of intestinal microsporidiosis with albendazole in
Kwa, M. S., Veenstra, J. G. & Roos, M. H. 1994. Benzimidazole resistance
patients with AIDS. AIDS, 6:311–313.
in Haemonchus contortus is correlated with a conserved mutation at
Cali, A. 1991. General microsporidian features and recent findings on
amino acid 200 in beta-tubulin isotype 1. Mol. Biochem. Parasitol.,
AIDS isolates. J. Protozool., 38:625–630.
Conteas, C. N., Berlin, O. G., Ash, L. R. & Pruthi, J. S. 2000. Therapy for
Lacey, E. 1988. The role of the cytoskeletal protein, tubulin, in the mode
human gastrointestinal microsporidiosis. Am. J. Trop. Med. Hyg.,
of action and mechanism of drug resistance to benzimidazoles. Int.
Cruz, M. C., Bartlett, M. S. & Edlind, T. D. 1994. In vitro susceptibility of
Lemee, V., Zaharia, I., Nevez, G., Rabodonirina, M., Brasseur, P., Ballet,
the opportunistic fungus Cryptococcus neoformans to anthelmintic
J. J. & Favennec, L. 2000. Metronidazole and albendazole susceptibility
benzimidazoles. Antimicrob. Agents Chemother., 38:378–380.
of 11 clinical isolates of Giardia duodenalis from France. J. Antimicrob.
Curry, A. & Canning, E. U. 1993. Human microsporidiosis. J. Infect.,
Li, J., Katiyar, S. K., Hamelin, A., Visvesvara, G. S. & Edlind, T. D. 1996.
Davidse, L. C. 1986. Benzimidazole fungicides: mechanism of action and
Tubulin genes from AIDS-associated microsporidia and implications
biological impact. Ann. Rev. Phytopathol., 24:43–65.
for phylogeny and benzimidazole sensitivity. Mol. Biochem. Parasitol.,
De Groote, M. A., Visvesvara, G., Wilson, M. L., Pieniazek, N. J.,
Siemenda, S. B., deSilva, A. J., Leitch, G. J., Bryan, R. T. &
Mansfield, K. G., Carville, A., Shvetz, D., MacKey, J., Tzipori, S. &
Reves, R. 1995. Polymerase chain reaction and culture confirmation
Lackner, A. A. 1997. Identification of an Enterocytozoon bieneusi-like
AKIYOSHI ET AL.—b-TUBULIN GENES FROM E. BIENEUSI
microsporidian parasite in simian-immunodeficiency-virus-inoculated
Schmidt, H. A., Strimmer, K., Vingron, M. & von Haeseler, A. 2002.
macaques with hepatobiliary disease. Am. J. Pathol., 150:1395–1405.
TREE-PUZZLE: maximum-likelihood phylogenetic analysis using
Mathias, A., Weber, R. & Deplazes, P. 2005. Zoonotic potential of the
quartets and parallel computing. Bioinformatics, 18:502–504.
microsporidia. Clin. Microbiol. Rev., 18:423–445.
Sheoran, A. S., Feng, X., Kitaka, S., Green, L., Pearson, C., Didier, E. S.,
McGinnis, S. & Madden, T. L. 2004. BLAST: at the core of a powerful
Chapman, S., Tumwine, J. K. & Tzipori, S. 2005. Purification of En-
and diverse set of sequence analysis tools. Nucleic Acids Res., 32:
terocytozoon bieneusi from stools and production of specific antibodies.
Molina, J. M., Chastang, C., Goguel, J., Michiels, J. F., Sarfati, C.,
Sulaiman, I. M., Fayer, R., Yang, C., Santin, M., Matos, O. & Xiao, L.
Desportes-Livage, I., Horton, J., Derouin, F. & Modai, J. 1998.
2004. Molecular characterization of Enterocytozoon bieneusi in cattle
Albendazole for treatment and prophylaxis of microsporidiosis due to
indicates that only some isolates have zoonotic potential. Parasitol.
Encephalitozoon intestinalis in patients with AIDS. J. Infect. Dis.,
Thomas, J. H., Neff, N. F. & Botstein, D. 1985. Isolation and character-
Orbach, M. J., Porro, E. B. & Yanofsky, C. 1986. Cloning and character-
ization of mutations in the b-tubulin gene of Saccharomyces cerevisiae.
ization of the gene for b-tubulin from a benomyl-resistant mutant of
Neurospora crassa and its use as dominant selectable marker. Mol. Cell.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. 1994. CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment
Rabodonirina, M., Bertocchi, M., Desportes-Livage, I., Cotte, L., Levrey,
through sequence weighting, position-specific gap penalties and weight
H., Piens, M. A., Monneret, G., Celard, M., Mornex, J. F. & Mojon, M.
matrix choice. Nucleic Acids Res., 22:4673–4680.
1996. Enterocytozoon bieneusi as a cause of chronic diarrhea in a heart-
Vivare`s, C. P., Gouy, M., Thomarat, F. & Me´te´nier, G. 2002. Functional
lung transplant recipient who was seronegative for human immunodefi-
and evolutionary analysis of a eukaryotic parasitic genome. Curr. Opin.
ciency virus. Clin. Infect. Dis., 23:114–117.
Ridoux, O. & Drancourt, M. 1998. In vitro susceptibilities of the micro-
Wittner, M. (ed.). 1999. The Microsporidia and Microsporidiosis. ASM
sporidia Encephalitozoon cuniculi, Encephalitozoon hellem, and En-
cephalitozoon intestinalis to albendazole and its sulfoxide and sulfone
Zhang, Q., Singh, I., Sheoran, A., Feng, X., Nunnari, J., Carville, A. &
metabolites. Antimicrob. Agents Chemother., 42:3301–3303.
Tzipori, S. 2005. Production and characterization of monoclonal anti-
Sandfort, J., Hannemann, A., Gelderblom, H., Stark, K., Owen, R. L. &
bodies against Enterocytozoon bieneusi purified from rhesus macaques.
Ruf, B. 1994. Enterocytozoon bieneusi infection in an immunocompe-
tent patient who had acute diarrhea and who was not infected with thehuman immunodeficiency virus. Clin. Infect. Dis., 19:514–516.
Received: 01/09/05, 06/13/06; accepted: 06/25/06
SEDRAH SELECTIONS PARSHAS BO 5772 BS”D Ch. 7, v. 17: “Hi’nei onochi ma’keh” – Behold I will smite – Many commentators explain the appropriateness of smiting the Egyptians with specifically the ten plagues, as recorded in our and the following parshios. The Kli Yokor offers: 1) The Nile was smitten in response to Paroh’s saying that the Nile was his and he created himself (
The current issue and full text archive of this journal is available atwww.emeraldinsight.com/0828-8666.htmMarkfield Institute of Higher Education, Markfield, UK, andCentre for Islamic Banking, Finance and Management,University of Brunei Darussalam, Bander Seri Begawan, BruneiAbstractPurpose – As per Islamic business ethics, corporate social responsibility (CSR) of the businessorganizations