t

J. Parasitol., 90(4), 2004, pp. 769-773

American Society of Parasitologists 2004

IN PIGS
CRYPTOSPORIDIIDAE)
SUIS N. SP. (APICOMPLEXA:
CRYPTOSPORIDIUM
(SUS SCROFA)
U. M. Ryan, P. Monis*, H. L. Enemarkt, 1. Sulaimants B. Samarasinghe, C. Read, R. Buddle, 1. Robertson, L. Zhout,
R. C. A. Thompson, and L Xiaot
Divisionof HealthSciences, MurdochUniversity,MurdochDrive,Murdoch,Perth,WA6150, Australia.e-mail:unaryan@2central.murdoch.edu.au
ABSTRACT:

Molecular

and biological

characteristics

are structurally
indistinguishable
described.
Oocysts
4.9-4.4
,um (mean
lack sporocysts,
and measure
50).

Cryptosporidium

analyses
at the
from all known

suis

is not

transmissible

RNA,
18S ribosomal
and genotypes
species

of a new

species

from those
= 4.6 ,um) x
to nude

heat shock protein

of Cryptosporidium,

mice
70,

and

from

of Cryptosporidium

of Cryptosporidium
4.0-4.3
,uwm (mean
is poorly

the feces

of pigs

(Sus

scrofa)

infectious

for

cattle.

Molecular

and

phylogenetic

C. suis to be genetically
and actin gene loci demonstrate
suis.
and thus is named as Cryptosporidium

At present, 13 species of Cryptosporidiumare regardedas

valid on the basis of differences in genetics, oocyst morphology, and site of infection: C. muris in rodents;C. andersoniin
cattle;C. parvumin ruminantsand humans;C. wrairi in guinea
pigs; C. hominis in humans;C. meleagridis, C. baileyi, and C.
galli in birds; C. serpentis and C. saurophilumin snakes and
lizards;C. molnariin fish; C.felis in cats; and C. canis in dogs
(Fayeret al., 2000, 2001; Alvarez-Pelliteroand Sitja-Bobadilla,
2002; Morgan-Ryanet al., 2002; Ryan, Xiao et al., 2003).
Traditionally,taxonomicclassificationfor coccidia and other
protozoahas been based on phenotypiccharacterssuch as morphological features and host specificity (Fayer et al., 2000).
However, morphology has been shown to be an unreliable
means of delineating species within Cryptosporidium(Fall et
al., 2003). This is in part because of the fact that oocysts of
Cryptosporidiumspp. are among the smallestexogenous stages
of the apicomplexansand also because of the lack of distinguishing morphologicalcharacters,as there are only 2 characters that can be analyzed (length by width and shape index).
Cryptosporidiumparvum is the most widely studied species,
and whereas no morphologicaldifference has been identified,
thereis now strongevidence thatthereare numerousgenetically
distinctgenotypes withinthe C. parvumgroup,which are likely
to be cryptic species (Xiao et al., 1999; Morgan-Ryanet al.,
2002; Xiao, Sulaimanet al., 2002; Xiao et al., 2003).
Recent genetic and biological characterizationstudies have
identified2 distinctapparentlyhost-adaptedgenotypesof Cryptosporidiumin pigs, i.e., the Cryptosporidiumpig genotype I
and pig genotype II (Morganet al., 1998; Pereiraet al., 1998;
Morgan et al., 1999; Sulaimanet al., 2000, 2002; Enemarket
al., 2003; Ryan, Samarasingheet al., 2003). In the present
study, we presentevidence thatpig genotype I is in fact a valid
species and propose the name Cryptosporidiumsuis (pig genotype I).

is

parvum;
they are passed
fully sporulated,
= 4.2 ,uwm); length to width ratio 1.1 (n =
distinct

MATERIALSAND METHODS
Sources of parasite isolates
fecal samples
Isolates were obtainedfrom Cryptosporidium-positive
from 3- to 5-wk-old pigs (see TableI).
Deoxyribonucleic acid extraction, polymerase chain reaction, and
sequence analyses
Oocystswere purified,anddeoxyribonucleicacid (DNA) was extracted as describedpreviously (Xiao et al., 1999). Fragmentsof the 18S
ribosomalRNA (rRNA)( 830 bp), heat shock protein(HSP)70 ( 325
bp), and actin ( 1,095 bp) genes were amplifiedby polymerasechain
reaction(PCR) as describedpreviously (Xiao et al., 1999; Morganet
al., 2001; Sulaimanet al., 2002). PCR productswere purifiedusing
Qiagen spin columns (Qiagen, Valencia, California)or Wizard PCR
Prep Kit (Promega,Madison,Wisconsin)and sequencedin both directions on an ABI377 or ABI3100 Autosequencerusing an ABI BigDyec3
TerminatorCycle Sequencingkit (AppliedBiosystems,FosterCity,California) accordingto the manufacturer'sinstructions.Each isolate was
sequencedat least twice with independentPCR products.
Phylogenetic analyses
Nucleotide sequences obtainedfrom this study were aligned against
each other and against those obtained previously using Clustal X
(Thompsonet al., 1997); (sequence alignmentscan be obtainedfrom
the authorsupon request).Plasmodiumfalciparumwas used as an outgroupfor HSP 70 (GenBankM19753), 18S rRNA (GenbankM19172),
and actin (GenbankM19146) analyses.Distance-basedanalysesof 18S
rRNA, HSP 70, and actin sequenceswere performedusing MEGAversion 2.1 (Kumaret al., 2001). Neighborjoining (NJ) trees were constructedon the basis of genetic distancescalculatedwith the TamuraNei model.
Parsimonyand maximum-likelihood(ML) analyseswere used to validate the phylogeneticrelationshipinferredby the NJ analyses. Parsimony analyses were performedby PAUP* (version 4.0b2), using the
heuristicsearchoption. The following settings were used for heuristic
parsimonyanalysis:all characterswere treatedas unorderedwith equal
weight, and gaps were treatedas missing; startingtrees obtainedby
stepwise addition;additionsequence = simple; branchswappingalgorithm = TBR. For HSP 70 sequences, ML analysis (performedby
PAUP* [version4.0b21)using an heuristicsearchwas conductedusing
the following settings:HKY85model settingswith 2 substitutiontypes;

Received 30 July 2003; revised 29 October2003; accepted 18 November2003.

* MicrobiologyUnit, AustralianWaterQualityCentre,HodgsonRoad,
Bolivar,SA 5110, Australia.
t Section for Parasitology,Danish VeterinaryInstitute,27, Bulowsvej,
DK-1790 CopenhagenV, Denmark.
:tDivision of ParasiticDiseases, NationalCenterfor InfectiousDiseases, Centersfor Disease Controland Prevention,PublicHealthServices, U.S. Departmentof Healthand HumanServices, Atlanta,Georgia
30341.
769

used in this study.

TABLE
I. Isolates of Cryptosporidium
Isolate code
SP1
WAP1
WAP5
WAP8
WAP12

Age of host (wk)

3-4
3-4
3-4
3-4
5

Geographicorigin
Switzerland
WesternAustralia
WesternAustralia
WesternAustralia
WesternAustralia

770

THE JOURNAL OF PARASITOLOGY,VOL. 90, NO. 4, AUGUST 2004

C.

pa7vum-C

FIGURE1.

C.

genotype

oocysts of C. parvumand C. suis n. sp. Bar = 5 ,uwm.

Nomarskiinterferencemicroscopyof Cryptosporidium

-Cattlegenotype
C.parvum- C. hominis
notype
57, 55

- Mousegenotype
1S
pialgenotype
51,ns

C. suis
5 1, ns

82, ns

ratio estimatedby ML; empiricalbase frequentransition-transversion

cies used; starting branch lengths obtained using Rogers-Swofford
method;branch-lengthoptimizationby l-dimensionalNewton-Raphson
with pass limit = 20; startingtrees obtainedby stepwise addition;addition sequence = as-is; branchswappingalgorithm= TBR. Bootstrap
analysesfor distance-basedand parsimonymethodswere conductedusing 1,000 replicatesto assess the reliabilityof inferredtree topologies.
Bootstrapswere conductedfor ML analysis using 1,000 replicatesfor
the HSP 70 analysisand 108 replicates(reducedbecauseof computing
and time constraints)for the actin analysis.
Nucleotide sequence accession numbers
The nucleotidesequencesof the 18S ribosomalDNA (rDNA), HSP
70, and actin gene sequences of Cryptosporidiumspp. isolates have
been deposited in GenBankunder the accession numbersAF108861,
AF221533, and AF382344.
DESCRIPTION

Cryptosporidium suis (pig genotype 1)

65, ns

(Fig 1)
C. fe

rgenotyg

lis

peII

-C. serpentis
C. galli
4

SC.

C. muris

andersoni
Plasmodium

suis

0.05 nucleotidesubstitutions
/ site

FIGURE2. Evolutionary relationships of Cryptc vsporidiumisolates

inferred by NJ analysis of Tamura-Nei distances callculatedfrom pairwise comparisons of the 18S rDNA sequences. Pezrcentagebootstrap
support (>50%) from 1,000 replicate samples (analytzed by NJ and parsimony methods, respectively) is indicated at the le ft of the supported
node. ns = node not supported by method.

Description: Oocysts are excreted already sporulated.They

measure4.9-4.4 Fm (mean = 4.6 Fm) X 4.0-4.3 Fm (mean
= 4.2 Fm); length to width ratio 1.1 (n = 50).
Typehosts: Pigs (Sus scrofa).
Other hosts: There has been 1 report of this species in humans (Xiao, Bern et al., 2002).
Type locality: Perth,WesternAustralia.
Other localities. Cosmopolitan.
Location in host: Epithelial cells of the small and large intestine (Enemarket al., 2003).
Prepatent period: 4.8 days (range 2-9) (Enemark et al.,
2003).
Patent period: 12.6 days (range 9-15) (Enemark et al.,
2003).
Sporulationtime: Oocysts are excreted fully sporulated.
Material deposited: A phototype of sporulatedoocysts and
Genbankaccession numbershave been depositedin the United
States NationalParasiteCollection (USNPC), Beltsville, Maryland. USNPC 094038.
Etymology:This species is named Cryptosporidiumsuis as it
appearsto be adaptedto this host.

RYANETAL.-CRYPTOSPORIDIUM
SUISIN PIGS

87, ns, ns-C.

84. 73 55
75,ns,nst
80. 51, ns
93, 54, ns

+
70- 54857

/
RR RJ

nc

parvum

C pa^Ug

771

-Cattlegenotype
-Mouse

genotype

C hominis
LC.wrairi

Ferretgenotype
C.meleagridis
Marsupial
genotype

100, 100, 100

Pig genotype II

P. falciparum
l

0.05 nucleotidesubstitutions
/ site

Evolutionaryrelationshipsof Cryptosporidium
isolates inferredby NJ analysis of Tamura-Neidistancescalculatedfrom pairwise
comparisonsof the hsp-70 DNA sequences.Percentagebootstrapsupport(>50%) from 1,000 replicatesamples(analyzedby NJ, parsimony,and
ML methods,respectively)is indicatedat the left of the supportednode. ns = node not supportedby method.
FIGURE3.

Remarks

Previousstudieshave shown thatthis species is not infectious

for mice (Morganet al., 1999) and is poorly infectiousfor cattle
(Enemarket al., 2003). Experimentalinfections indicated that
C. suis is adaptedto porcine hosts as demonstratedby the absence of clinical signs despite the excretionof high numbersof
oocysts (Enemarket al., 2003). This is supportedby another
recent study of C. suis in Yorkshire-Landrace
piglets on a pig
farm northeastof Calgary,Canada.In this study, oocyst excretion was not associatedwith diarrhea(Guselle et al., 2003).
The 18S rDNA gene sequenceswere obtainedfrom 5 C. suis
isolates (TableI). These sequences were comparedwith Cryptosporidium sequence information obtained from GenBank.
Both distance-basedand parsimonyanalyses demonstratedthat
the C. suis isolates formed a cohesive genetic group that was
distinctfrom all other species or genotypes of Cryptosporidium
(Fig. 2, NJ tree illustrated).The position of C. suis was poorly
resolved by either forms of analysis. Both methodsof analysis
placed C. suis within a clustercontainingthe variousgenotypes
of C. parvum, C. hominis, C. meleagridis, C. wrairi, C. felis,
C. canis, and C. saurophilum.However, the exact position of
C. suis varied dependingon the method of analysis, and there
was generally poor bootstrapsupportfor much of the tree topology, with the exception of the cluster forming C. suis and
the cluster comprising C. serpentis, C. galli, C. muris, and C.
andersoni.
Partialsequences of the CryptosporidiumHSP 70 gene were

obtainedfrom4 C. suis isolates (TableI). These sequenceswere

comparedwith Cryptosporidium
sequenceinformationobtained
from GenBank. The phylogenetic relationshipsof the isolates
could be better resolved compared with the results obtained
using the 18S rDNA sequence data. All methods of analyses
strongly supportedthe placementof the C. suis isolates into a
single unique cluster, which was in agreement with the 18S
rDNA analysis (Fig. 3, NJ tree illustrated).In addition,the analysis suggested some substructuringwithin C. suis, with isolates
WP8 and SP1 forming a subgroup.In contrast with the 18S
rDNA tree, the HSP 70 analysis placed C. suis externalto the
clustercontainingthe variousgenotypes of C. parvum,C. hominis, C. meleagridis,C. wrairi, C.felis, and C. canis. This placement was supportedby all methods of analysis and received
high bootstrapsupport(87-94%, dependingon the method of
analysis).
Partialactin gene sequence informationwas obtainedfrom 3
C. suis isolates (TableI). These sequenceswere comparedwith
sequences from a range of Cryptosporidiumspecies and genotypes obtainedfrom GenBank.Phylogeneticanalysis was consistent with the 18S rDNA and HSP 70 sequenceanalyses,with
the C. suis isolates forming a distinct cluster.NJ and ML analyses of the actin data (Fig. 4) were consistent with the 18S
rDNA tree (Fig. 2), placing C. suis externalto the clustercontaining C. hominis, C. wrairi, C. meleagridis, and the cattle,
mouse, ferret,and marsupialgenotypes.Heuristicanalysisidentified 6 equally most parsimonious trees (not shown), 4 of
which placed C. suis in the same position as the NJ tree.

772

THEJOURNAL
OF PARASITOLOGY,
VOL.90, NO. 4, AUGUST2004

61, 58, 59-C.

10O,10O,100

parvum - mouse genotype

L-C.

99 96.

hominis
parvum-Cattle genotype

C. wrairi
Ferret genotype

100, 91, 91

C. meleagridis

90,ns,62

Marsupial genotype

SP1
, 95

100,looWAPI
69,ns,nSWAP5

C. suis

C. saurophilum

. canls

100,100, 100

PiggenotypeII

C. baileyi

C.galli

|
100, 100, 10

serpentis
C. andersoni
C. muris

C.

82, 8
89, 96, 98

0.05 nucleotidesubstitutions
/ site

FIGURE4. Evolutionaryrelationshipsof Cryptosporidium

isolates
inferredby NJ analysis of Tamura-Neidistancescalculatedfrom pairwise comparisonsof the actin DNA sequences. Percentagebootstrap
support(>50%) from 1,000 replicatesamples(analyzedby NJ andparsimony methods,respectively)or 108 replicates(ML analysis) is indicated at the left of the supportednode. ns = node not supportedby
method.

DISCUSSION
Because of the difficulties associated with the taxonomy of
Cryptosporidiumspp., several guidelines have been developed
as an aid in establishingnew species in this genus (Xiao et al.,
2003). When naming new species of Cryptosporidium,
4 basic
requirementsshould be fulfilled. First,morphometricstudiesof
the parasite'soocysts must be undertaken.Second, the parasite
must be characterizedgenetically.Third,there must be a demonstrationof naturaland, if feasible, some experimentalhost
specificity.Finally, the study must be done in compliancewith
the InternationalCode of Zoological Nomenclature.Thus, the
taxonomicdescriptionsshould constitutevalid publishedwork,
and the morphologicaldescriptionitself and appropriatephotographsshould be deposited in a recognized museum, i.e., at
the USNPC in Beltsville, Maryland(Xiao et al., 2003).
In the present study, C. suis was shown to be morphologically identical to the C. parvum 'cattle' genotype. However,
phylogenetic analyses confirm the validity of C. suis at 3 independentloci. The HSP 70 data appearto give the best-resolved phylogeny for Cryptosporidiumoverall, as determined
by bootstrapanalysis.The topology of the HSP 70 tree is largely supportedby the actin and 18S rDNA trees, with the main
exception being the placementof C. suis. Both actin and HSP
70 datasetsprovide strongsupportfor the clusteringof C. hominis, C. wrairi, C. meleagridis,and the cattle, mouse, ferret,and
marsupialgenotypes.

For the 18S locus, C. suis sharedonly 97% similaritywith

the C. parvumcattle genotype and C. hominis.This is less than
the similaritybetween C. meleagridisand the C. parvumcattle
genotype and C. hominis (98.6%) and between the C. parvum
'cattle' genotype and C. hominis (99.42%). Cryptosporidium
suis does not appearto be closely relatedto the novel pig genotype II because they sharedonly 93% similarityat the 18S
locus.
There have been few studies that have examined the infectivity and pathogenicityof C. suis because the majorityof previous studies either did not genotype the porcine isolates used
or used oocysts from calves (Tziporiet al., 1981, 1982; Heine
et al., 1984; Ayeni et al., 1985).
A recent study provides evidence that C. suis is adaptedto
a porcine host and causes either mild or no clinical signs in
pigs (Enemarket al., 2003). The study reportedthe prepatent
period for C. suis to be 4.8 days (range 2-9), comparedwith
3.5 days (range 2-5) for the C. parvum cattle genotype (Enemarket al., 2003). Pereiraet al. (2002) reportedthe prepatent
period for the cattle genotype in gnotobiotic piglets to be 5.6
days. However,it is difficult to compareresults from different
studies because differentC. parvumcattle genotype isolates of
Cryptosporidiumwere used in differenthosts (cattle vs. pigs).
It has been shown that differentisolates of the same genotype
can exhibit very differentpathogenicityand infectivity in the
same host (Okhuysenet al., 1999). In the study by Enemarket
al. (2003), there were distinct differences in pathogenicitybetween C. suis and the C. parvum cattle genotype in pigs. For
example, no mortalitywas seen in piglets infected with C. suis,
whereasinfection with the C. parvumcattle genotype was fatal
in 1 of 6 piglets. The severityand durationof diarrheawas also
less in pigs infectedwith C. suis (1.2 days durationvs. 3.5 days
for the C. parvum cattle genotype). In addition,the maximum
oocysts output was not detected until 12 days postinfection
(dpi) in C. suis-infected pigs comparedwith 3.5 days for C.
parvumcattle genotype-infectedpigs (Enemarket al., 2003).
Guselle et al. (2003) examinedoocyst sheddingin pigs (n =
33) in a hog farm northeastof Calgary and reportedthe mean
age of initial oocyst detectionwas 45.2 days afterweaning,with
the mean durationof infection being 28.7 days (Guselle et al.,
2003). Genetic sequences(HSP 70) were obtainedfor 10 of the
33 isolates and all were C. suis (pig genotype I). The study
reportedthat the mean numberof oocysts shed was low and
thattherewas no associationbetween diarrheaand oocyst shedding (Guselle et al., 2003).
Unlike the C. parvumcattle genotype, C. suis is not infective
for nude mice (Morgan et al., 1999) and is poorly infectious
for cattle (Enemarket al., 2003). In the latterstudy, 2 attempts
were made to infect calves with C. suis; 1 resulted in no detectable oocyst production.In the second, there were very low
numbersof oocysts detected at 15 dpi but no clinical signs in
the second attempt(Enemarket al., 2002). Anotherinvestigation revealed that squirrelshave a separategenotype, which is
very similarto C. suis in 18S-PCR-restrictionfragmentlength
polymorphismanalysis (Atwill et al., 2001); however,sequence
informationis requiredto confirmthis observation.
The genetic and biological datadiscussed above indicatethat
the differencesbetween C. suis and other Cryptosporidium
spp.
are comparablewith those between establishedspecies. Therefore, C. suis should be consideredvalid species.

RYANETAL.-CRYPTOSPORIDIUM
SUISIN PIGS

This

project

opment

ACKNOWLEDGMENT
funded
by the Australian Pig Research

was

Corporation

(now

Australian

Pork

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