[Emerging Infectious Diseases]  
[Volume 4 No. 4 /October-December 1998]


Dispatches

Differentiating Human from Animal Isolates of Cryptosporidium parvum

Irshad M. Sulaiman, Lihua Xiao, Chunfu Yang, Lilian Escalante, Anne Moore,
Charles B. Beard, Michael J. Arrowood, and Altaf A. Lal
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

------------------------------------------------------------------------
      We analyzed 92 Cryptosporidium parvum isolates from humans and
      animals by a polymerase chain reaction/restriction fragment
      length polymorphism method based on the thrombospondin-related
      anonymous protein 2 gene sequence. Used as a molecular marker,
      this method can differentiate between the two genotypes of C.
      parvum and elucidate the transmission of infection to humans.

Cryptosporidium parasites cause infection in humans and other vertebrates,
including mammals, birds, reptiles, and fish. More than 20 species of
Cryptosporidium have been reported, of which six are considered valid
species on the basis of oocyst morphologic features and site of infection
(1,2). Cryptosporidium parvum, the species that infects humans and most
mammals, has a monoxenous life cycle in which all stages of asexual and
sexual development occur within one host. The parasite generates large
numbers of viable oocysts in feces. Cross-infection studies in various
mammalian systems have indicated zoonotic transmission to humans (1,3). C.
parvum has caused waterborne outbreaks of cryptosporidiosis and (in AIDS
patients) life-threatening diarrhea for which no effective treatment exists
(4). A waterborne outbreak of cryptosporidiosis in Milwaukee, Wisconsin, in
1993 affected more than 400,000 people (5).

Molecular characterization techniques used to detect intraspecific
variations in C. parvum include isozyme profiles (6); random amplified
polymorphic DNA (RAPD) analyses (7); nucleotide sequence studies of the 18S
rRNA (8,9) and DHFR gene (10); and polymerase chain reaction-restriction
fragment length polymorphism (PCR-RFLP) analysis of the undefined
repetitive sequence (11), polythreonine motifs, and oocyst wall protein
(12,13). Two distinct genotypes of C. parvum parasites have been detected
in humans.

In a previous article, we identified several mutations in the gene
thrombospondin-related anonymous protein 2 of C. parvum (TRAP-C2) that
differentiate between anthroponotic and zoonotic infection in humans (14).
Our objective in the present study was to develop a simple, rapid protocol
that can be used as a diagnostic tool to differentiate between the two
genotypes of C. parvum and elucidate the transmission of infection in
humans. We analyzed 92 C. parvum isolates from humans, calves, deer, dogs,
and monkeys and found that this new PCR/RFLP method based on the TRAP-C2
gene sequence can be used as a molecular marker to differentiate between
the two genotypes of C. parvum.

Analytic Approach

Isolates

We summarized data for 92 isolates of C. parvum, 50 from human and 42 from
animal sources (Tables 1, 2). Twenty-one of the 50 human isolates were from
AIDS patients; the rest were primarily from cryptosporidiosis outbreak
case-patients. Seven of the human isolates came from a previous TRAP-C2
sequencing study (14), but because of the lack of DNA, other isolates we
used in the previous study were not used in this study. Fecal samples were
stored at 4 C in 2.5% potassium dichromate before oocysts were isolated.
Oocysts were purified from fecal samples by first using the discontinuous
density sucrose gradient centrifugation and then the Percoll gradient
centrifugation (15,16).

 Table 1. Cryptosporidium parvum human isolates, restriction pattern, and
 sequence type

 ------------------------------------------------------------------------

                                                 Restric- Se-
                                                 tion     quence
 Source                      Isolate    Host     pattern  type
 ------------------------------------------------------------------------
 1993 Milwaukee              HM3     Hum(sup a)  Hum      Hum(sup b)     

 1993 Milwaukee              HM5        Hum      Hum      Hum

 1993 Milwaukee              HM7        Hum      Hum      ND(sup c)     

 1995 Florida                HFL1       Hum      Hum      Hum(sup b)     

 1995 Florida                HFL5       Hum      Hum      Hum(sup b)     

 1995 Florida                HFL6       Hum      Hum      ND

 1995 Atlanta                HGA1       Hum      Hum      Hum(sup b)     

 1995 Atlanta                HGA4       Hum      Hum      Hum

 1995 Atlanta                HGA5       Hum      Hum      ND

 1995 Atlanta                HGA6       Hum      Hum      ND

 1996 Canada                 HCAN9      Hum    Bov(sup d) Bov(sup b)     

 1995 Canada                 NC30       Hum      Hum      ND

 1994 Nevada                 HCNV2      Hum      Hum      ND

 1994 Nevada                 HCNV4      Hum      Hum      ND

 1997 Pennsylvania           PA41       Hum      Bov      Bov(sup b)     

 1997 Pennsylvania           PA46       Hum      Bov      Bov(sup b)     

 1997 HIV-Guatemala          HGMO7      Hum      Hum      Hum

 1997 HIV-Guatemala          HGMO8      Hum      Hum      ND

 1997 HIV-Guatemala          HGMO9      Hum      Hum      Hum

 1997 HIV-Guatemala          HGMO10     Hum      Hum      Hum

 1997 Minnesota              HMOB1      Hum      Bov      Hum

 1997 Minnesota              HMOB3      Hum      Bov      Bov

 1997 Minnesota              HMOB4      Hum      Bov      Bov

 1997 Minnesota              HMOB5      Hum      Bov      Bov

 1997 HIV-New Orleans        HNO2       Hum      Hum      Hum

 1997 HIV-New Orleans        HNO3       Hum      Hum      Hum

 1997 HIV-New Orleans        HNO4       Hum      Hum      ND

 1997 HIV-New Orleans        HNO5       Hum      Bov      Bov

 1997 HIV-New Orleans        HNO6       Hum      Hum      Hum

 1997 HIV-New Orleans        HNO7       Hum      Hum      Hum

 1997 HIV-New Orleans        HNO8       Hum      Hum      Hum

 1997 HIV-New Orleans        HNO10      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO11      Hum      Bov      ND

 1997 HIV-New Orleans        HNO12      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO13      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO14      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO15      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO16      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO17      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO18      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO19      Hum      Hum      ND

 1997 India                  HIND4      Hum      Hum      Hum

 1997 India                  HIND5      Hum      Hum      ND

 1998 Washington State       HWA1       Hum      Hum      Hum

 1998 Washington State       HWA2       Hum      Hum      ND

 1998 Washington State       HWA3       Hum      Hum      ND

 1998 Washington State       HWA4       Hum      Hum      ND

 1998 Washington State       HWA5       Hum      Hum      Hum

 1998 Washington State       HWA6       Hum      Hum      Hum

 1998 Washington State       HWA7       Hum      Hum      Hum

 ------------------------------------------------------------------------
 (sup a)Hum=human
 (sup b)Sequencing data reported earlier (14)
 (sup c)ND= Not done.
 (sup d)Bov=bovine.

 ------------------------------------------------------------------------

 Table 2. Cryptosporidium parvum bovine isolates, restriction
 pattern, and sequence type

 ---------------------------------------------------------------
                                          Restric-   Se-
                                          tion       quence
 Source                Isolate   Host     pattern    type
 ---------------------------------------------------------------
 1996 Alabama          AAL35     Calf   Bov(sup a)   Bov
 1996 Georgia          AGA43     Calf     Bov        Bov
 1996 Georgia          AGA44   Mon(sup b) Bov        Bov
 1997 Georgia          AGA75     Calf     Bov        Bov
 1996 Idaho            AID21     Calf     Bov        Bov
 1996 Kansas           AKA19     Calf     Bov        Bov
 1996 Maryland         AMD36     Calf     Bov        Bov
 1996 Maryland         AMD38     Deer     Bov        Bov
 1996 Massachusetts    AMA61-    Calf     Bov        Bov
                       GCH1
 1997 Iowa             AIO62     Calf     Bov        Bov
 1996 Ohio             AOH6      Calf     Bov        Bov
 1996 Ohio             AOH7      Calf     Bov        Bov
 1996 Ohio             AOH8      Calf     Bov        Bov
 1996 Ohio             AOH9      Calf     Bov        Bov
 1996 Ohio             AOH10     Calf     Bov        Bov
 1996 Ohio             AOH11     Calf     Bov        Bov
 1996 Ohio             AOH12     Calf     Bov        Bov
 1996 Ohio             AOH13     Calf     Bov        Bov
 1996 Ohio             AOH14     Calf     Bov        Bov
 1996 Ohio             AOH15     Calf     Bov        Bov
 1996 Ohio             AOH16     Calf     Bov        Bov
 1996 Ohio             AOH17     Calf     Bov        Bov
 1997 Ohio             AOH45     Calf     Bov        Bov
 1997 Ohio             AOH47     Calf     Bov        Bov
 1997 Ohio             AOH48     Calf     Bov        Bov
 1997 Ohio             AOH49     Calf     Bov        Bov
 1997 Ohio             AOH50     Calf     Bov        Bov
 1997 Ohio             AOH52     Calf     Bov        Bov
 1997 Ohio             AOH53     Calf     Bov        Bov
 1997 Ohio             AOH54     Calf     Bov        Bov
 1997 Ohio             AOH55     Calf     Bov        Bov
 1997 Ohio             AOH56     Calf     Bov        Bov
 1997 Ohio             AOH57     Calf     Bov        Bov
 1997 Ohio             AOH58     Calf     Bov        Bov
 1997 Ohio             AOH59     Calf     Bov        Bov
 1997 Ohio             AOH107    Dog      Bov        Bov
 1996 Oklahoma         AOK3      Beef     Bov        Bov
                                 cattle
 1996 Oklahoma         AOK29     Calf     Bov        Bov
 1997 Pennsylvania     APE89     Calf     Bov        Bov
 1996 Utah             AUT37     Calf     Bov        Bov
 1996 Washington       AWA5      Beef     Bov        Bov
                                 cattle
 1997 West Virginia    AWV65     Calf     Bov        Bov
 ---------------------------------------------------------------
 (sup a)Bov=bovine.
 (sup b)Mon=monkey.

Extraction of Genomic DNA and PCR Amplification

We followed the protocol of Kim et al. in isolating the total genomic DNA
from the purified oocyst (17). A 369 base pair (bp) fragment of the TRAP-C2
gene of C. parvum was amplified by using a forward (cua cua cua cua CAT ATT
CCC TGT CCC TTG AG) and a reverse (cau cau cau cau TGG ACA ACC CAA ATG CAG
AC) primer (lower case represents nucleotide used for cloning); these
primers correspond to positions 848-867 (positive strand) and 1,180-1,199
(negative strand) of the GenBank sequence X77586, respectively. The PCR
reaction consisted of 50 ng genomic DNA, 200 mM of each dNTP (Perkin Elmer,
Foster City, CA), 40 ng of primer, 1X PCR buffer, and 0.5 units of Taq
polymerase (GIBCO BRL, Frederick, MD) in a total volume of 100 ml. DNA
amplification was carried out for 35 cycles, each consisting of denaturing
(94 C, 45 sec), annealing (48 C, 45 sec), and elongating (72 C, 60 sec),
with an initial hot start at 94 C for 5 min in a Perkin Elmer Gene Amp PCR
9600 thermocycler. An additional cycle of 7 min at 72 C was done for final
extension. Each experiment used three negative controls (reaction mixtures
without Taq polymerase, primers, or template DNA) and a positive control.

DNA Sequencing and Analysis

PCR products were purified by the Wizard PCR Preps DNA purification system
(Promega, Madison, WI) and cloned by the CLONEAMP pAMP1 System for Rapid
Cloning of Amplification Products (GIBCO BRL, Frederick, MD) according to
the manufacturer's protocol. DNA sequencing of recombinant clones that had
the correct size insert was carried out on an ABI 377 Automated Sequencer
by the dRhodomine Terminator Cycle Sequencing Kit (Perkin Elmer-Applied
Biosystems).

RFLP

To develop an RFLP technique for differentiating between the two genotypes
of C. parvum, the TRAP-C2 sequences were aligned and mapped for restriction
enzyme sites by the Genetics Computer Group program (18). Enzymes with
predicted exclusive cutting in each genotype were used in RFLP development
and analysis. For RFLP analysis, 10 ml of amplification products was
digested in a 30-ml reaction mix consisting of 10 units of BfaI (New
England BioLabs, Beverly, MA), BsetEI (Boehringer Mannheim, Indianapolis,
IN), Eco571 (MBI Fermentas, Gariciuno, Vilnius, Lithuania), HaeIII (New
England BioLabs), HphI (New England BioLabs), MaeIII (Boehringer Mannheim,
Germany), NruI (New England BioLabs), PacI (New England BioLabs), or Tsp45I
(New England BioLabs), and 3 ml of respective restriction buffer for 1 hr,
under conditions recommended by the supplier. The digested products were
fractionated on 2.0% agarose gel and visualized by ethidium bromide
staining.

Findings

Sequence Analysis of Human and Bovine Isolates

 Table 3. Human and bovine Cryptosporidium
 parvum isolates based on multiple alignment(sup a)

 -----------------------------------------------
     Position        Human           Bovine
      (nt)          genotype        genotype
 -----------------------------------------------
       51              G                A
       78              C                T
       100             T                G
       147             C                T
       280           T or C             C
 -----------------------------------------------
 (sup a)Representative sequences have been 
 deposited in the GenBank, with accession 
 numbers AF082521 to AF082524.

Two genotypes of C. parvum exist in humans,as shown by the primary sequence
of the TRAP-C2 gene (14). Nucleotide sequences differed at five positions
between most human and bovine isolates. To confirm and extend this
observation, we sequenced additional human and bovine isolates, as well as
isolates from dogs, deer, and monkeys. We obtained 42 additional sequences
of the TRAP-C2 gene from animal sources and 27 additional sequences of the
TRAP-C2 gene from human sources; results of DNA sequencing confirmed that
C. parvum is highly conserved at the TRAP-C2 locus. All animal isolates,
including those from nonbovine animals, showed bovine genotype
characteristics (Table 2). Differences between the two genotypes are shown
in Table 3. Of the additional 23 human isolates showing human genotype
pattern, four isolates (HGMO7, HGMO9, HGMO10, and HNO18) showed "C" at the
fifth place, whereas the rest showed "T".

PCR-RFLP Method To Discriminate between Human and Bovine Genotype Isolates


 [Figure.] Human- and bovine-specific restriction enzymes
 showed distinct banding pattern for genotypes of
 Cryptosporidium parvum isolates. The different lanes
 represent the TRAP-C2 PCR-amplified products belonging
 to AGA43, AMD36, AOH6, HM3, and HM5 isolates of C.
 parvum, respectively, after digestion with HaeIII
 (Lanes 1-5, human-specific marker) and BstE II (Lanes
 7-11, bovine-specific marker) restriction enzymes and
 agarose gel electrophoresis. Lane 6 is the 100 bp
 marker. Samples AGA43, AMD36 and AOH6 are bovine
 (bovine genotype) and samples HM3 and HM5 are human
 (human genotype). Human- or bovine-specific restriction
 enzyme markers can cut only the TRAP-C2 amplified
 product of the respective genotype of C. parvum
 isolates.

To avoid expensive and lengthy DNA sequencing when determining the genotype
of C. parvum isolates, we developed a simpler, quicker methodPCR
amplification of the TRAP-C2 gene followed by RFLP. Restriction enzyme
mapping on the aligned sequences of both genotypes showed five
human-genotypespecific (HaeI, HaeIII, NruI, PacI, and ThaI) and six
bovine-genotypespecific (BfaI, BsetEI, Eco571, HphI, MaeIII, and Tsp45I)
restriction enzymes. All human-genotype and bovine-genotypespecific
restriction enzymes except HaeI and ThaI were tested for the TRAPC-2
PCR-amplified products of genomic DNA of C. parvum. After restriction and
gel electrophoresis, the resulting bands were the size predicted by the
mapping analysis (Figure). Digestion of PCR products with these enzymes
resulted in a distinct band pattern for the human genotype and bovine
genotype isolates. In all cases, the DNA sequencing and PCR-RFLP mapping
data matched.

Using PCR-RFLP in Outbreak Investigations

We validated the PCR-RFLP technique by using isolates from outbreaks and
sporadic cases of human cryptosporidiosis. Human genotype characteristics
were evident in all samples from HIV-infected patients from Guatemala and
most patients with sporadic clinical cases, as well as samples from the
following outbreaks: Milwaukee (1993), Florida (1995), Atlanta (1995),
Canada (1995), Nevada (1994), and Washington (1998). Of the 17 samples from
HIV-infected patients in New Orleans, two demonstrated bovine-genotype
pattern, while the rest were similar to human genotype. However,
bovine-genotype characteristics were evident in the human isolates from
outbreaks in British Columbia, Canada (1996), Minnesota (1997), and
Pennsylvania (1997).

Conclusions

We examined a large number of C. parvum isolates (92) from human and animal
sources from patients in outbreak and nonoutbreak settings to determine the
two transmission routes of the parasite in humans. Molecular markers were
generated by restriction digestion of PCR-amplified TRAP-C2 products with
one of the 12 enzymes to differentiate the two genotypes of C. parvum. The
results based on TRAP-C2 gene PCR-RFLP showed that this method could also
be used in future cryptosporidiosis outbreak investigations.

Results of our characterization of outbreak and nonoutbreak cases of human
cryptosporidiosis indicate that anthroponotic organisms account for most
cases. We find a large number of human genotype parasites in sporadic cases
and in HIV-infected patients. Most cryptosporidiosis outbreaks examined are
caused by anthroponotic (human genotype) parasites. Our results suggest
similar epidemiologic features of cryptosporidiosis in HIV-infected persons
from New Orleans and Guatemala because both were infected with human
genotype parasites.

The results of this study confirm the polymorphic nature of C. parvum. As
we showed in a previous study, two alleles of the TRAP-C2 gene exist, each
representing a distinct genotype of C. parvum with different transmission
cycles in humans. The simple PCR-RFLP technique we developed can
effectively differentiate between these two genotypes and transmission
cycles and can be used as a tool in outbreak investigations of
cryptosporidiosis. Information generated from these investigations will be
useful not only in identifying the sources of contamination but also in
controlling the disease.

Acknowledgments

      We thank Barbara Herwaldt, Corinne S. L. Ong, P. Vijyalakshmi, Bruce
Anderson, William Shulaw, A. Morse, and J. Inungu for providing
Cryptosporidium isolates.

      This work was supported in part by funding from Environmental
Protection Agency (DW 75937984-01-1).

      Dr. Sulaiman is a postdoctoral research associate in the Molecular
Vaccine Section, Division of Parasitic Diseases, National Center for
Infectious Diseases, CDC. For the last 7 years, he has focused on the
genetic polymorphism of various organisms. He is now conducting molecular
typing of Cryptosporidium to understand the transmission routes of the
parasite.

      Address for correspondence: Altaf A. Lal, Division of Parasitic
Diseases, National Center for Infectious Diseases, Centers for Disease
Control and Prevention, Mail Stop F-12, 4770 Buford Highway, Atlanta, GA
30341-3717, USA; fax: 770-488-4454; e-mail: aal1@cdc.gov.

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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

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