![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Appl Environ Microbiol, June 1998, p. 2262-2265, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Randomly Amplified Polymorphic DNA PCR Analysis of
Bovine Cryptosporidium parvum Strains Isolated from the
Watershed of the Red River of the North
Kevin V.
Shianna,
Russell
Rytter, and
Jonathan G.
Spanier*
Department of Microbiology and Immunology,
University of North Dakota School of Medicine, Grand Forks, North
Dakota 58202
Received 15 December 1997/Accepted 19 March 1998
 |
ABSTRACT |
Cryptosporidium parvum is a protozoan parasite that
causes the disease cryptosporidiosis in a variety of mammals, including neonatal calves and humans. Millions of oocysts are shed during acute
cryptosporidiosis, and zoonotic transmission is inferred, though not
proven, to be a general phenomenon. Very little is known about the
degree of strain variation exhibited by bovine and human isolates,
though such knowledge would enable the amount of bovine-to-human
transmission to be more precisely analyzed. This research was initiated
to determine whether variations exist among bovine strains isolated
from a localized geographic area, the watershed of the Red River of the
North. Sixteen strains were isolated and compared to each other and to
two human and two calf strains from Australia by randomly amplified
polymorphic DNA PCR. A statistical analysis of the data indicated that
the isolates belonged to four different groups of strains.
| |
TEXT |
Cryptosporidium parvum
(phylum Apicomplexa) is the causative agent of cryptosporidiosis, a
disease that results in a severe, yet self-limiting, diarrhea in
immunocompetent humans but a chronic, severely debilitating and
sometimes fatal infection in immunocompromised humans (15).
Cryptosporidiosis is also a major cause of death in neonatal calves and
is responsible for annual economic losses amounting to millions of
dollars (9). Although there have been some encouraging pilot
studies, C. parvum is not currently treatable with
antimicrobial drugs, nor is an effective vaccine available (6, 7,
15).
Millions of oocysts, the infectious form of the parasite, are shed from
infected animals into the environment during acute cryptosporidiosis
and often continue to be shed for days, weeks, or even months after the
acute disease has resolved (15, 20). Disease outbreaks often
involve water (1), and zoonotic transmission from cattle is
inferred. However, except in job-related infections of farmworkers and
veterinarians (17), the extent to which cattle act as a
reservoir of human disease is unknown; in fact, initial strain typing
studies suggest that there are bovine- and human-specific strains
(4, 5, 14, 16). However, Morgan et al. (14) determined that 2 of the 14 human isolates they examined were more
closely related to the 8 isolates from cattle than to the other human
isolates. While it is clear that C. parvum is multiclonal, little is known about strain variability, differences in virulence, and
the degree of cross-infectivity between bovine- and human-specific strains.
In this study, we examined the clonality of bovine strains using
randomly amplified polymorphic DNA (RAPD) PCR and demonstrated that
even oocysts isolated from the relatively localized geographic area of
Minnesota and North Dakota are multiclonal. Furthermore, we confirmed
that RAPD PCR is a very sensitive monitor of the genetic diversity of
Cryptosporidium and an especially useful tool for typing
clinical isolates when only limited amounts of material are available.
For all of these reasons, we suggest that RAPD PCR is a feasible method
of testing the hypothesis that bovine strains of C. parvum
are responsible for a significant proportion of human cases of
cryptosporidiosis.
Oocysts were purified from fecal samples (provided by Neil Dyer, State
Veterinary Diagnostic Laboratory, North Dakota State University, Fargo)
by sequential sucrose and CsCl gradients and subsequently washed as
described previously (2, 10). DNA was released from purified
oocysts during six freeze-thaw cycles. One cycle consisted of 2 min in
a dry ice-ethanol bath followed by 2 min in a 98°C dry-heating block.
DNA Dipstick (Stratagene, La Jolla, Calif.) was used to quantitate the
DNA according to the manufacturer's instructions, and the DNA was
stored at 
20°C until used as a PCR template. After DNA Dipstick
quantitation, PCR was performed on similar amounts of DNA from all
samples by using the primers described by Laxer et al. (11).
In addition to detection of isolates positive for
Cryptosporidium (see below), PCR product intensities were
compared to each other. Whereas the DNA Dipstick measured the DNA
concentration, PCR amplification with the primers described by Laxer et
al. measured the amplifiability of each DNA sample. Since the oocyst
DNA was liberated only from the oocyst and was not purified from oocyst
debris, we felt that the relative amplifiability was an important
empirical measurement, especially for the subsequent RAPD reactions,
but in fact, DNA Dipstick quantitation (0.2 to 5.0 ng/PCR) and
amplifiability correlated well for most samples. PCRs were carried out
in a buffer containing 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 2 mM
MgCl2, and a 0.2 mM concentration of each nucleotide in a
final volume of 100 µl. Each reaction mixture also contained 2.5 U of
Taq polymerase, the primer(s), and the template. The primer
sequences and concentrations are shown in Table
1. Sequences within primer 1 and primer 2 were amplified for 35 cycles at 94°C for 1 min, 56°C for 2 min, and 72°C for 3 min followed by a final cycle at 72°C for 9 min.
Products of PCR with RAPD primers R2817 and (GAA)5 were
generated during 35 cycles at 94°C for 1 min, 37°C for 1 min, and
72°C for 2 min followed by a final cycle at 72°C for 9 min. RAPD
primer 1344 was used under conditions identical to those for the other
RAPD primers except that the annealing temperature was 42°C. All
reactions were carried out in a model 110S Tempcycler II thermocycler
(Coy). Agarose gel electrophoresis, photography, Southern transfers,
and hybridizations were performed under standard conditions
(19).
Preliminary characterizations.
In order to determine if the
isolated oocysts were indeed C. parvum, PCR was performed
with the primers described by Laxer et al. (11). These
primers specifically amplify a 452-bp sequence from C. parvum, but not from other Cryptosporidium species,
other parasites, or bacteria (18). All of the amplified
samples produced this band (data not shown), indicating that they all
contained C. parvum DNA. Primers specific for
Escherichia coli and Prevotella (Bacteroides) ruminicola were used to amplify
Cryptosporidium DNA since these organisms are common ruminal
and intestinal inhabitants and would be likely contaminants if bacteria
were present in the oocyst samples in our study (23). When
the E. coli primers were used to amplify C. parvum DNA samples, no products were detected. However, when the
Prevotella (ribosomal DNA) primers were used, a 389-bp
fragment was amplified in four samples (strains 2674, 2894, 2896, and
3296), indicating that they were contaminated with P. ruminicola. In order to determine if the amount of contaminating Prevotella DNA was enough to generate
Prevotella-specific amplification products, a pure culture
of Prevotella (ATCC 19188) was amplified by each RAPD primer
used to radioactively probe the Cryptosporidium RAPD
products from the same primers. Results of even the P. ruminicola-contaminated reaction mixtures showed that there were
no Prevotella-specific RAPD products (21).
RAPD PCR.
RAPD PCR was used to determine the relatedness of
the different Cryptosporidium isolates. Initially, we tried
the four primers that had been successfully used by Morgan et al.
(14): R2817, R2936, GACA, and (GAA)5. Only R2817
and (GAA)5 resulted in reproducible DNA patterns in our
hands. About two dozen additional primers were tested and found to be
unsuitable either because they produced no PCR products or because they
produced products even in the absence of a template (21).
Two suitable primers (1344 and 1640) were eventually found. Ultimately,
primer 1344 was used in conjunction with primers (GAA)5 and
R2817 for our study.
The RAPD PCR analysis included 20 different isolates: 16 from the upper
Midwest in the United States and 4 from Australia. Purified oocysts of
human strains 49-H and 53-H and calf strains 248-C and 8700-C were
provided by Una Morgan, School of Veterinarian Studies, Murdoch
University, Murdoch, Australia. RAPD PCRs were carried out at up to
three template concentrations 25-fold different from one another for
each DNA sample and showed little product variation.
A total of 81 bands were scored: 30 from primer (GAA)5, 34 from primer R2817, and 17 from primer 1344. Similarity coefficients were determined by using the correlation-principal component analysis option in Microsoft Excel/xlSTAT (Table
2). Spearman's correlation coefficients
were determined, and dendrograms were made by utilizing the
hierarchical ascending clustering function (cluster analysis 2, or
Ward's clustering technique). When the data for the three primers were
combined, four different groups were seen (Fig.
1): (i) two human strains from Australia,
(ii) most (13 of 16) of the strains isolated during this study (strains
from North Dakota or Minnesota [ND/MN strains]), (iii) two Australian
calf strains and one ND/MN strain (385), and (iv) two ND/MN strains
(2896 and 3296) (Table 3).
This study is the first to compare C. parvum isolates from
within a specific geographic region. With limited starting material, and neither the means nor the desire to cycle oocysts through animals,
we were able to perform our analysis using the small amount of DNA
required to perform RAPD PCR. Our observation that most of the isolates
we studied belong to one major group was expected. Finding three
strains (385, 2896, and 3296) that are quite different from each other
and from the major group is somewhat surprising, but these results are
similar to the results of Morgan et al. (14), who grouped
one human isolate by itself and another with the bovine isolates they
studied. However, those strains were isolated from samples from across
the entire continent of Australia, while ours were derived from a more
localized geographic area. Three possible explanations for our results
are that (i) multiple, different strains have been brought into this
geographic region, (ii) strains already present have continued to
evolve, and (iii) DNA contamination produced apparent strain
differences. While we cannot disprove the first possibility, examples
of the second are well documented. Microevolution (also referred to as substrain shuffling) is commonly seen in recurrent Candida
albicans vaginal infections (12) and is also
acknowledged to occur during persistent infection with
methicillin-resistant Staphylococcus aureus (13).
A few of the strains we examined were contaminated with enough
Prevotella DNA to generate a ribosomal DNA PCR product; even
though the amount of contaminating DNA was insufficient to generate any
RAPD product, we cannot completely rule out the third possibility
that
the strain differences we observed resulted from bacterial
contamination. Thus, the heterogeneity of these
Cryptosporidium strains needs to be corroborated by an
additional taxonomic technique, such as ribotyping.
It is currently acknowledged that there are human and bovine strain
types of C. parvum (5, 14). Whether or not these strain types are cross-infective in vivo, or how strain variation relates to disease transmission, is unknown. Having a reliable and
comprehensive strain typing system would help address these issues and
might ultimately establish whether shedding of oocysts by cattle and
oocyst migration into surface waters during seasonal runoffs constitute
a significant source of human disease outbreaks, such as those
chronicled in Milwaukee, Wis., and Las Vegas, Nev.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the Grand Forks Water
Treatment Plant, Grand Forks, N. Dak.
| |
FOOTNOTES |
*
Corresponding author. Present address: 143 Smith Ridge
Rd., South Salem, NY 10590. Phone: (914) 533-5373. Fax: (914) 694-3754. E-mail: Spanier{at}cyburban.com.
Present address: Department of Microbiology, North Carolina State
University, Raleigh, NC 27695.
| |
REFERENCES |
| 1.
|
Addiss, D. G.,
M. J. Arrowood,
M. E. Bartlett,
D. G. Colley,
D. D. Juranek, and J. E. Kaplan.
1995.
Assessing the public health threat associated with waterborne cryptosporidiosis: report of a workshop.
Morbid. Mortal. Weekly Rep.
44:1-16[Medline].
|
| 2.
|
Arrowood, M. J., and C. R. Sterling.
1987.
Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients.
J. Parasitol.
73:314-319[Medline].
|
| 3.
|
Avgu tin, G.,
F. Wright, and H. J. Flint.
1994.
Genetic diversity and phylogenetic relationships among strains of Prevotella (Bacteroides) ruminicola from the rumen.
Int. J. Syst. Bacteriol.
44:246-255[Abstract/Free Full Text].
|
| 4.
|
Bonnin, A.,
M. N. Fourmaux,
J. F. Dubremetz,
R. G. Nelson,
P. Gobet,
G. Harly,
M. Buisson,
D. Puygauthiertoubas,
F. Gabrielpospisil,
M. Naciri, and P. Camerlynck.
1996.
Genotyping human and bovine isolates of Cryptosporidium parvum by polymerase chain reaction restriction fragment length polymorphism analysis of a repetitive DNA sequence.
FEMS Microbiol. Lett.
137:207-211[Medline].
|
| 5.
|
Carraway, M.,
S. Tzipori, and G. Widmer.
1997.
A new restriction fragment length polymorphism from Cryptosporidium parvum identifies genetically heterogeneous parasite populations and genotypic changes following transmission from bovine to human hosts.
Infect. Immun.
65:3958-3960[Abstract].
|
| 6.
|
Harp, J. A., and J. P. Goff.
1995.
Protection of calves with a vaccine against Cryptosporidium parvum.
J. Parasitol.
81:54-57[Medline].
|
| 7.
|
Hicks, P.,
R. J. Zwiener,
J. Squires, and V. Savell.
1996.
Azithromycin therapy for Cryptosporidium parvum infection in four children infected with human immunodeficiency virus.
J. Pediatr.
129:297-300[Medline].
|
| 8.
|
Hill, T. M.,
M. L. Tecklenburg,
A. J. Pelletier, and P. L. Kuempel.
1989.
tus, the trans-acting gene required for termination of DNA replication in Escherichia coli, encodes a DNA-binding protein.
Proc. Natl. Acad. Sci. USA
86:1593-1597[Abstract/Free Full Text].
|
| 9.
|
House, J. A.
1978.
Economic impact of rotavirus and other neonatal disease agents of animals.
J. Am. Vet. Med. Assoc.
173:573-576[Medline].
|
| 10.
|
Kilani, R. T., and L. Sekla.
1987.
Purification of Cryptosporidium oocysts and sporozoites by cesium chloride and Percoll gradients.
Am. J. Trop. Med. Hyg.
36:505-508.
|
| 11.
|
Laxer, M. A.,
B. K. Timblin, and R. J. Patel.
1991.
DNA sequences for the specific detection of Cryptosporidium parvum by the polymerase chain reaction.
Am. J. Trop. Med. Hyg.
45:688-694.
|
| 12.
|
Lockhart, S. R.,
B. D. Reed,
C. L. Pierson, and D. R. Soll.
1996.
Most frequent scenario for recurrent Candida vaginitis is strain maintenance with "substrain shuffling": demonstration by sequential DNA fingerprinting with probes Ca3, C1, and CARE2.
J. Clin. Microbiol.
34:767-777[Abstract].
|
| 13.
|
Maslow, J. N.,
S. Brecher,
J. Gunn,
A. Durbin,
M. A. Barlow, and R. D. Arbeit.
1995.
Variation and persistence of methicillin-resistant Staphylococcus aureus strains among individual patients over extended periods of time.
Eur. J. Clin. Microbiol. Infect. Dis.
14:282-290[Medline].
|
| 14.
|
Morgan, U. M.,
C. C. Constantine,
P. O'Donoghue,
B. P. Meloni,
P. A. O'Brien, and R. C. Thompson.
1995.
Molecular characterization of Cryptosporidium isolates from humans and other animals using random amplified polymorphic DNA analysis.
Am. J. Trop. Med. Hyg.
52:559-564.
|
| 15.
|
O'Donoghue, P. J.
1995.
Cryptosporidium and cryptosporidiosis in man and animals.
Int. J. Parasitol.
25:139-195[Medline].
|
| 16.
|
Ortega, Y. R.,
R. R. Sheehy,
V. A. Cama,
K. K. Oishi, and C. R. Sterling.
1991.
Restriction fragment length polymorphism analysis of Cryptosporidium parvum isolates of bovine and human origin.
J. Protozool.
38:40s-41s.
|
| 17.
|
Pohjola, S.,
H. Oksanen,
L. Jokipii, and A. M. Jokipii.
1986.
Outbreak of cryptosporidiosis among veterinary students.
Scand. J. Infect. Dis.
18:173-178[Medline].
|
| 18.
|
Rochelle, P. A.,
R. De Leon,
M. H. Stewart, and R. L. Wolfe.
1997.
Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water.
Appl. Environ. Microbiol.
63:106-114[Abstract].
|
| 19.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
In
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 20.
|
Scott, C. A.,
H. V. Smith, and H. A. Gibbs.
1994.
Excretion of Cryptosporidium parvum oocysts by a herd of beef suckler cows.
Vet. Rec.
134:172[Medline].
|
| 21.
|
Shianna, K. V.
1997.
In
M.S. thesis.
University of North Dakota, Grand Forks.
|
| 22.
|
Skokotas, A.,
H. Hiasa,
K. Marians,
L. O'Donnell, and T. Hill.
1995.
Mutations in the Escherichia coli Tus protein define a domain positioned close to the DNA in the Tus-Ter complex.
J. Biol. Chem.
270:30941-30948[Abstract/Free Full Text].
|
| 23.
|
Van Gylswyk, N. O.
1990.
Enumeration and presumptive identification of some functional groups of bacteria in the rumen of dairy cows fed grass silage-based diets.
FEMS Microbiol. Ecol.
73:243-254.
|
Appl Environ Microbiol, June 1998, p. 2262-2265, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Straub, T. M., Daly, D. S., Wunshel, S., Rochelle, P. A., DeLeon, R., Chandler, D. P.
(2002). Genotyping Cryptosporidium parvum with an hsp70 Single-Nucleotide Polymorphism Microarray. Appl. Environ. Microbiol.
68: 1817-1826
[Abstract]
[Full Text]
-
Simmering, R., Kleessen, B., Blaut, M.
(1999). Quantification of the Flavonoid-Degrading Bacterium Eubacterium ramulus in Human Fecal Samples with a Species-Specific Oligonucleotide Hybridization Probe. Appl. Environ. Microbiol.
65: 3705-3709
[Abstract]
[Full Text]
Top
Abstract
Text
