Cryptosporidium Research
Division of Biology
Kansas State
University
Updated: 15 November 2004
Recent advances in the ability to cultivate Cryptosporidium parvum
in cell culture have allowed for the development of a 96-well ELISA for
rapid screening of pharmaceuticals. This system is also useful for
studying receptor/ligand interactions. Research on its development
was supported almost entirely by Pfizer Animal Health after an NIH study
section decided that development of such an ELISA was technically
unfeasible. Since that time, our laboratory has tested
over 1,500 pharmaceuticals against the parasite using the system. Most
recently, the laboratory received an R21 grant from NIH to develop better
methods of cultivating the parasite both in vitro and in ovo.
Eggleston, M.T., M. Tilley, and S.J. Upton. 1994. Enhanced development of
Cryptosporidium parvum in vitro by removal of oocyst toxins from
infected cell monolayers. Journal of the Helminthological Society of
Washington 61(1): 118-121.
Upton, S.J., M. Tilley, M.V. Nesterenko, and D.B. Brillhart. 1994. A simple and
reliable method of producing in vitro infections of Cryptosporidium
parvum (Apicomplexa). FEMS Microbiology Letters 118(1-2): 45-50.
Upton, S.J., M. Tilley, and D.B. Brillhart. 1994. Comparative development of
Cryptosporidium parvum (Apicomplexa) in 11 continuous host cell lines.
FEMS Microbiology Letters 118(3): 233-236.
Upton, S.J., M. Tilley, and D.B. Brillhart. 1994. Comparative development of
Cryptosporidium parvum in MDBK and HCT-8 cells under select
atmospheres. Biomedical Letters 49(196): 265-271.
Upton, S.J., M. Tilley, and D.B. Brillhart. 1995. Effects of select medium
supplements on in vitro development of Cryptosporidium parvum in HCT-8
cells. Journal of Clinical Microbiology 33(2): 371-375.
Woods, K.M., M.V. Nesterenko, and S.J. Upton. 1995. Development of a
microtitre ELISA to quantify development of Cryptosporidium parvum
in vitro. FEMS Microbiology Letters 128(1): 89-93.
Nesterenko, M.V., K.M.Woods, and S.J. Upton. 1996. Effects of manganese
salts on the AIDS-related pathogen, Cryptosporidium parvum in vitro and
in vivo. Biological Trace Element Research 56: 243-253.
Woods, K.M., M.V. Nesterenko, and S.J. Upton. 1996. Efficacy of 101
antimicrobials and other agents on development of Cryptosporidium parvum
in vitro. Annals of Tropical Medicine and Parasitology 90(6): 603-615.
Upton, S.J. 1997. In vitro cultivation of Cryptosporidium. In:
Cryptosporidiosis of Man and Animals, 2nd ed. Chapter 8. (Fayer, R.,
ed.). CRC Press, Boca Raton. pp. 181-207.
Woods, K.M. and S.J. Upton. 1998. Efficacy of select antivirals against
Cryptosporidium parvum in vitro. FEMS Microbiology Letters 168(1): 59-63.
Lindsay, D.S., K.M. Woods, S.J. Upton, and B.L. Blagburn. 2000. Activity
of decoquinate against Cryptosporidium parvum in cell cultures and
neonatal mice. Vet. Parasitol. 89: 307-311.
Kayser, O., W.R. Waters, K.M. Woods, S.J. Upton, J.S. Keithly, and A.F.
Kiderlen. 2001. Evaluation of in vitro activity of aurones and related
compounds against Cryptosporidium parvum. Planta Med. 67: 722-725.
Kayser, O., W.R. Waters, K.M. Woods, S.J. Upton, J.S. Keithly, H. Laatsch,
and A.F. Kiderlen. 2002. Evaluation of in vitro and in vivo activity of
benzindazole-4,9-quinones against Cryptosporidium parvum. J. Antimicrob.
Chemother. 50(6): 975-980.
In the six years, the parasitology laboratory at Kansas State
University has generated complete or partial sequences for over 120 genes
from Cryptosporidium parvum. Both
genomic and cDNA libraries are used in this research. Additional areas of
research and expertise
include pulse field mapping of genes to chromosomes, isolation of
developmentally regulated genes, and sequencing of extrachromosomal
elements. We now know, for instance, that the genome of C.
parvum consists of 8 chromosomes with few introns, 2 dsRNA strands of
viral origin, and a total of
about 10.4 mbp.
Khramtsov, N.V., M. Tilley, D.S. Blunt, B.A. Montelone, and S.J. Upton. 1995.
Cloning and analysis of a Cryptosporidium parvum gene encoding a
protein with homology to cytoplasmic form Hsp70. Journal of Eukaryotic
Microbiology 42(4): 416-422.
Khramtsov, N.V., D.S. Blunt, B.A. Montelone, and S.J. Upton. 1996. The
putative acetyl-CoA synthetase gene of Cryptosporidium parvum and a new
conserved protein motif in acetyl-CoA synthetases. Journal of
Parasitology 82(3): 423-427.
Blunt, D.S., B.A. Montelone, S.J. Upton, and N.V. Khramtsov. 1996.
Sequence of the parasitic protozoan, Cryptosporidium parvum, putative
protein disulfide isomerase-encoding DNA. Gene 181: 221-223.
Blunt, D.S., N.V. Khramtsov, S.J. Upton, and B.A. Montelone. 1997.
Molecular karyotype analysis of Cryptosporidium parvum: evidence for
eight chromosomes and a low-molecular size molecule. Clinical and
Diagnostic Laboratory Immunology 4(1): 11-13.
Khramtsov, N.V., B. Oppert, B.A. Montelone, and S.J. Upton. 1997.
Sequencing, analysis and expression in Escherichia coli of a gene
encoding a 15 kDa Cryptosporidium parvum protein. Biochemical and
Biophysical Research Communications 230: 164-166.
Khramtsov, N.V., K.M. Woods, M.V. Nesterenko, C.C. Dykstra, and S.J.
Upton. 1997. Virus-like double-stranded RNAs in the pathogenic
protozoan, Cryptosporidium parvum. Molecular Microbiology 26(2): 289-300.
LeBlancq, S.M., N.V. Khramtsov, F. Zamani, S.J. Upton, and T.W. Wu. 1997.
Ribosomal RNA gene organization in Cryptosporidium parvum. Molecular and
Biochemical Parasitology 90: 463-478.
Khramtsov, N.V. and S.J. Upton. 1998. High-temperature inducible cell-free
transcription and replication of double-stranded RNAs within the parasitic
protozoan Cryptosporidium parvum. Virology 245(2): 331-337.
Woods, K.M., M. Tilley, A. Iseli, S.J. Upton, B.A. Montelone, and
N.V. Khramtsov. 1999. Sequence of the gene encoding hsp90e from
Cryptosporidium parvum. DNA Sequence 10(4-5): 339-342.
Zhu, G., M.J. Marchewka, K.M. Woods, S.J. Upton and
J. S. Keithly. 2000. Molecular analysis of a type I fatty acid synthase in
Cryptosporidium parvum. Mol. Biochem. Parasitol. 102(2): 253-260.
Khramtsov, N.V., P.A. Chung, C.C. Dykstra, J.K. Griffiths, U.N. Morgan,
M.J. Arrowood, and S.J. Upton. 2000. Presence of dsRNAs in human and
calf isolates of Cryptosporidium parvum. J. Parasitol. 86(2): 275-282.
Khramtsov, N.V. and S.J. Upton. 2000. Association of RNA polymerase
complexes of the parasitic protozoan Cryptosporidium parvum with
virus-like particles: heterogeneous system. J. Virol. 74(13): 5788-5795.
Morgan, U.M., Xiao, L., Monis, P., Sulaiman, I., Pavlasek, I., Blagburn,
B., Olson, M., Upton, S.J., Khramtsov, N.V., Lal, A., Elliot, A., and
Thompson, R.C.A. 2000. Molecular and phylogenetic analysis of
Cryptosporidium muris from various hosts. Parasitology 120: 457-464.
Chung, P.A., J. Kasper, N.V. Khramtsov, and S.J. Upton. 2000. Cloning and
molecular characterization of a gene encoding a Cryptosporidium
parvum putative 20s proteasone B1-type subunit. DNA Sequence
11(3-4): 309-314.
Khramtsov, N.V. and Upton, S.J. 2003. dsRNAs of Cryptosporidium. J.
Parasitol. 89 (suppl.): 165s-168s.
Rochelle, P.A., Fallar, D., Marshall, M.M., Montelone, B.A., Upton, S.J.,
and Woods, K. 2004. Irreversible UV inactivation of Cryptosporidium spp.
despite the presence of UV repair genes. J. Euk. Microbiol. 51(5):
553-562.
Rochelle, P.A., Mofidi, A.A., Marshall, M.M., Upton, S.J., Montelone,
B.A., Woods, K., and Di Giovanni, G. 2004. An investigation of UV
disinfection and repair in Cryptosporidium parvum. AWWA Research
Foundation, Denver, CO. 83 pp.
Rochelle, P.A., Upton, S.J., Montelone, B.A., and Woods, K. 2005. The
response of Cryptosporidium parvum to UV light. Trends Parasitol. 21: (in
press).
At least a dozen surface proteins/glycoproteins are known to occur
on the surface of Cryptosporidium sporozoites. Research at Kansas State
University is actively involved in determining function and kinetics of
these surface molecules, especially those interacting with the host cell. Thus far, specific
functions have been assigned to five C.
parvum surface molecules, and either
monoclonal antibodies or polyclonal antiserum have been made to
each. Additional areas of biochemical research on this parasite include
collaborations on lipid composition and polyamine biosynthetic pathways.
Tilley, M. and S.J. Upton. 1990. Electrophoretic characterization of
Cryptosporidium parvum (KSU-1 isolate) (Apicomplexa: Cryptosporidiidae).
Canadian Journal of Zoology 68(7): 1513-1519.
Tilley, M., R. Fayer, A. Guidry, S.J. Upton, and B.L. Blagburn. 1990.
Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae) oocyst and
sporozoite antigens recognized by bovine colostral antibodies.
Infection and Immunity 58(9): 2966-2971.
Tilley, M., S.J. Upton, and P.S. Freed. 1990. A comparative study on the
biology of Cryptosporidium serpentis and Cryptosporidium parvum
(Apicomplexa: Cryptosporidiidae). Journal of Zoo and Wildlife Medicine
21(4): 463-467.
Tilley, M., S.J. Upton, R. Fayer, J.R. Barta, C.E. Chrisp, P.S. Freed, B.L.
Blagburn, B.C. Anderson, and S.M. Barnard. 1991. Identification of a 15
kilodalton surface glycoprotein on sporozoites of Cryptosporidium
parvum. Infection and Immunity 59(3): 1002-1007.
Fayer, R., M. Tilley, S.J. Upton, A.J. Guidry, D.W. Thayer, M. Hildreth, and J.
Thomson. 1991. Production and preparation of hyperimmune bovine
colostrum for passive immunotherapy of cryptosporidiosis. Journal of
Protozoology 38(6): 38s-39s.
Tilley, M. and S.J. Upton. 1991. Sporozoites and merozoites of
Cryptosporidium parvum share a common epitope recognized by a monoclonal
antibody and two-dimensional electrophoresis. Journal of
Protozoology 38(6): 48s-49s.
Tilley, M., S.J. Upton, and C.E. Chrisp. 1991. A comparative study on the
biology of Cryptosporidium sp. from guinea pigs and Cryptosporidium parvum
(Apicomplexa). Canadian Journal of Microbiology 37(12): 949-952.
Tilley, M., M.T. Eggleston, and S.J. Upton. 1993. Multiple oral inoculations
with Cryptosporidium parvum as a means of immunization for production of
monoclonal antibodies. FEMS Microbiology Letters 113(10): 235-240.
Mitschler, R.R., R. Welti, and S.J. Upton. 1994. A comparative study of lipid
compositions of Cryptosporidium parvum (Apicomplexa) and Madin-Darby
bovine kidney cells. Journal of Eukaryotic Microbiology 41(1): 8-12.
Tilley, M. and S.J. Upton. 1994. Both CP15 and CP25 are left as trails behind
gliding sporozoites of Cryptosporidium parvum (Apicomplexa). FEMS
Microbiology Letters 120(3): 275-279.
Nesterenko, M.V., M. Tilley, and S.J. Upton. 1995. A metallo-dependent
cysteine proteinase of Cryptosporidium parvum associated with the
surface of sporozoites. Microbios 83(2): 77-88.
Yarlett, N., M.P. Martinez, G. Zhu, J.S. Keithly, K. Woods, and S.J.
Upton. 1996. Cryptosporidium parvum: polyamine biosynthesis from
agamatine. Journal of Eukaryotic Microbiology 43(5): 73s.
Nesterenko, M.V., K.M. Woods, and S.J. Upton. 1997. Effects of manganese
salts on the AIDS-related pathogen, Cryptosporidium parvum in vitro and
in vivo. Biological Trace Element Research 56(3): 243-253.
Tilley, M. and Upton, S.J. 1997. Biochemistry of Cryptosporidium. In:
Cryptosporidiosis of Man and Animals, 2nd ed. Chapter 7. (Fayer, R.,
ed.). CRC Press, Boca Raton. pp. 163-180.
Keithly, J.S., G.Zhu, S.J. Upton, K.M. Woods, M.P. Martinez, and N.
Yarlett. 1997. Polyamine biosynthesis in Cryptosporidium parvum and its
implications for chemotherapy. Mol. Biochem. Parasitol. 88: 35-42.
Nesterenko, M.V., K.M. Woods, and S.J. Upton. 1999. Receptor/ligand
interactions between Cryptosporidium parvum and the surface of the host
cell. Biochimica et Biophysica Acta 1454(2): 165-173.
In addition to the species of Cryptosporidium infecting humans, isolates
from wildlife, Zoo and captive specimens are also studied. We hope that, in
time, comparing species and isolates both at the structural and molecular
levels will aid in determining whether a sample of oocysts collected from
surface waters has the potential to infect humans. In
collaboration with Auburn University, we obtained a 3-year EPA grant to
collect various isolates and species from the environment,
make gDNA libraries of each, and deposit these libraries in the
American Type Culture Collection. These libraries will prove
useful to various investigators who wish to develop species and isolate
specific molecular probes.
Current, W.L., S.J. Upton, and T.B. Haynes. 1986. The life cycle of
Cryptosporidium baileyi n. sp. (Apicomplexa, Cryptosporidiidae) infecting
chickens. Journal of Protozoology 33(2): 289-296.
Upton, S.J. and Barnard, S.M. 1987. Two new species of coccidia (Apicomplexa:
Eimeriidae) from Madagascar gekkonids. Journal of Protozoology 34(4):
452-454.
Upton, S.J., M.E. Tilley, G.L. Marchin, and L.R. Fina. 1988. Efficacy of a
pentaiodide resin disinfectant on Cryptosporidium parvum (Apicomplexa:
Cryptosporidiidae) oocysts in vitro. Journal of Parasitology 74(4):
719-721.
Upton, S.J., C.T., McAllister, P.S. Freed, and S.M. Barnard. 1989.
Cryptosporidium spp. in wild and captive reptiles. Journal of Wildlife
Diseases 25(1): 20-30.
Upton, S.J., Freed, P.S., Burdick, D.A., and McAllister, C.T. 1990. Seven new
species of coccidia (Apicomplexa: Eimeriorina) from reptiles in
Madagascar. Canadian Journal of Zoology 68(11): 2368-2375.
Tilley, M., S.J. Upton, B.L. Blagburn, and B.C. Anderson. 1990. Identification
of outer oocyst wall proteins of three species of Cryptosporidium
(Apicomplexa: Cryptosporidiidae) by 125I surface labeling. Infection and
Immunity 58(1): 252-253.
Tilley, M., S.J. Upton, and P.S. Freed. 1990. A comparative study on the
biology of Cryptosporidium serpentis and Cryptosporidium parvum
(Apicomplexa: Cryptosporidiidae). Journal of Zoo and Wildlife Medicine
21(4): 463-467.
Upton, S.J. 1990. Chapter 10. Cryptosporidiosis in lower vertebrates. In:
Cryptosporidiosis in man and animals. Dubey, J.P., Speer, C.A., and
Fayer, R., eds. CRC Press, Boca Raton. pp. 149-156.
Todd, S.C., M.S. Phillips, G.L. Marchin, and S.J. Upton. 1991. Cryptosporidium
and Giardia in surface waters in and around Manhattan, Kansas.
Transactions of the Kansas Academy of Sciences 94(3-4): 101-106.
Tilley, M., S.J. Upton, and C.E. Chrisp. 1991. A comparative study on the
biology of Cryptosporidium sp. from guinea pigs and Cryptosporidium parvum
(Apicomplexa). Canadian Journal of Microbiology 37(12): 949-952.
Lindsay, D.S., S.J. Upton, D.S. Owens, U.M. Morgan, J.R. Mead, and
B.L. Blagburn. 2000. Cryptosporidium andersoni n. sp. (Apicomplexa:
Cryptosporiidae) from cattle, Bos taurus. J. Euk. Microbiol. 47(1):91-95.
Fayer, R., U.M. Morgan, and S.J. Upton. 2000. Epidemiology of
Cryptosporidium: transmission, detection and
identification. Int. J. Parasitol.
30(12-13): 1305-1322.
Duszynski, D.W. and S.J.Upton. 2001. The common coccidia of wild mammals:
Cyclospora, Eimeria, Isospora (Eimeriidae) and Cryptosporidium
(Cryptosporidiidae). In, Parasitic Diseases of Wild Mammals, 2nd edition.
W.M. Samuel and A. Kocan, eds. Wildlife Disease Association, Iowa State
University Press, Ames, Iowa. 720 pp.
Palmer, C.J., Xiao, L., Terashima, A., Guerra, H., Gotuzzo, E., Saldias,
G., Bonilla, J.A., Zhou, L., Lindquist, A., and Upton, S.J. 2003.
Cryptosporidium muris, a rodent pathogen recovered from a human in Peru.
Emerg. Inf. Dis. 9: 1174-1176.
Xiao, L., Fayer, R., Ryan, U., and Upton, S.J. 2004. Cryptosporidium
taxonomy: recent advances and implications for public health. Clin.
Microbiol. Rev. 17(1): 72-97.
In addition to more traditional types of basic research, one important area
of cryptosporidial research in the Division of Biology has been on the
development of novel techniques and assays useful for studying
Cryptosporidium spp. These areas of research are typically high risk/high
failure, and tend to be supported more from private sources rather than
federal funding agencies. However, work on these techniques
has allowed for the development of the microtiter ELISA, a rapid silver
stain method useful for detecting proteins in SDS-PAGE in under 30
minutes, a rapid (3 minute) sporozoite purification
technique, and a rapid
sporozoite biotin labeling procedure.
Nesterenko, M.V. and S.J. Upton. 1994. A simple modification of Blum's silver
stain allows for 30 minute detection of proteins in polyacrylamide
gels. Journal of Biochemical and Biophysical Methods 28(2): 239-242.
Woods, K.M., M.V. Nesterenko, and S.J. Upton. 1995. Development of a
microtitre ELISA to quantify development of Cryptosporidium parvum
in vitro. FEMS Microbiology Letters 128(1): 89-93.
Upton, S.J. and H.H. Gillock. 1996. Infection dynamics of Cryptosporidium
parvum in ICR outbred suckling mice. Folia Parasitologica (Praha)
43: 101-106.
Nesterenko, M.V. and S.J. Upton. 1996. A rapid microcentrifuge procedure for
purification of Cryptosporidium sporozoites. Journal of
Microbiological Methods 25(1): 87-89.
Nesterenko, M.V., K.M. Woods, and S.J. Upton. 1997. Effective
nonradioactive method of surface labeling Cryptosporidium parvum
sporozoites. Acta Tropica 65(1): 53-57.
For more information concerning Cryptosporidium research in the Division of
Biology at Kansas State University, contact:
Nomarski interference contrast photomicrograph
of CsCl gradient purified oocysts of
Cryptosporidium parvum. Each
oocyst measures about 5.2 x 4.6 micrometers and contains four infective
sporozoites. Based on these measurements, then the total area covered by
an individual oocyst should be about 18.787 square micrometers and the
individual volume about 57.613 cubic
micrometers. Considering that the specific gravity of oocysts has been
calculated at 1.109 using a refractometer, then about
15,651,314,725 oocysts should make a metric gram!
The parasitology laboratory in the Division of Biology, Kansas State
University has been studying the basic and applied biology of
Cryptosporidium spp. and other coccidia since
1986. Over 200 papers in refereed journals have originated from
the laboratory since that time. In 1991, emphasis was
placed on developing a program where expertise from multiple areas could
be used collectively to problem solve. Post-doctoral
fellows, graduate students, and undergraduates from around the world, with
expertise as diverse as molecular biology, biochemistry, immunology, and in
vitro cultivation, were selected for their abilities to work
together and problem solve. None of these individuals were specialists in
coccidian biology, but all received secondary training in that field and
were allowed to utilize their previous backgrounds to enhance upon
established techniques and dogma. Currently, four individuals are
actively engaged in research with expertise in molecular biology,
immunology, taxonomy, and in vitro cultivation.
In vitro cultivation/pharmaceutical screening
Upton, S.J., M. Tilley, R.R. Mitschler, and B.S. Oppert. 1991.
Incorporation of
exogenous uracil by Cryptosporidium parvum in vitro. Journal of
Clinical Microbiology 29(5): 1062-1065.
Molecular Biology
Jenkins, M., R. Fayer, M. Tilley, and S.J. Upton. 1993. Cloning and expression
of a cDNA encoding epitopes shared by 15-kDa and 60-kilodalton proteins
of Cryptosporidium parvum sporozoites. Infection and Immunity 61(6):
2377-2382.
Biochemistry
Tilley, M., S.J. Upton, B.L. Blagburn, and B.C. Anderson. 1990. Identification
of outer oocyst wall proteins of three species of Cryptosporidium
(Apicomplexa: Cryptosporidiidae) by 125I surface labeling. Infection and
Immunity 58(1): 252-253.
Wildlife and Zoo parasitology
Upton, S.J. and Current, W.L. 1985. The species of Cryptosporidium
(Apicomplexa: Cryptosporidiidae) infecting mammals. Journal of
Parasitology 71(5): 625-629.
Techniques
Tilley, M., M.T. Eggleston, and S.J. Upton. 1993. Multiple oral inoculations
with Cryptosporidium parvum as a means of immunization for production of
monoclonal antibodies. FEMS Microbiology Letters 113(10): 235-240.
Division of Biology, Ackert Hall
Kansas State University
Manhattan, KS 66506
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