1104 THE JOURNAL OF PARASITOLOGY, VOL. 92, NO.
5, OCTOBER 2006
Ultrastructure of Babesia WA1 (Apicomplexa: Piroplasma) During Infection of
Erythrocytes in a Hamster Model
W. Braga, J. Venasco, L. Willard, and M. H. Moro*, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine/Kansas
State University, 1800 Denison Avenue, Manhattan, Kansas 66506-5606; *To whom correspondence should be addressed.
e-mail: mmoro@vet.ksu.edu
ABSTRACT: Babesia Washington-1 (WA1) is a newly identified intra-
erythrocyte infectious agent of human babesiosis in the western United
States. The purpose of the present study is to describe the ultrastructural
changes in affected erythrocytes during the infectious process in a sus-
ceptible animal model, the golden Syrian hamster. Two, 1-mo-old fe-
male hamsters were inoculated intraperitoneally (i.p.) with 1.8 ⫻ 109
Babesia WA1-infected erythrocytes originally isolated from a human
case and serially passaged in hamsters. Saphenous vein blood samples
(20 ␮l) were collected at 0, 24, 36, 48, 60, 72, 84, and 96 hr postin-
oculation (PI). Parasitemia was determined at each time interval by
quick staining of blood smears showing 0, 2.5, 5, 10, 12.5, 22.5, 70,
and almost 100% parasitemic erythrocytes at the corresponding PI time
interval, respectively. Animals showed weakness and dehydration 72 hr
PI inoculation, and were killed by 96 hr PI. Selected blood samples
from 0, 24, 48, 72, and 96 hr were fixed in cacodylate buffer, dehydrated
in ethanol gradients, resin embedded, and then thin sectioned and
stained with uranyl acetate and lead citrate for transmission electron
microscopy or gold-coated for scanning electron microscopy (SEM).
Shape and surface membrane changes in erythrocytes were demonstrat-
ed by SEM and were more evident at 72 and 96 hr PI. Infected eryth-
rocytes underwent changes in shape 24 hr PI, from few protrusions to
several perforations, some of them resembling a ‘‘swiss cheese’’ ap-
pearance 96 hr PI. Several erythrocytes had irregular surface mem-
branes and Babesia WA1 organisms were seen at different stages of
development within erythrocytes, from single trophozoites to several
merozoites (young trophozoites), some of them dividing to form typical
tetrads. In general, Babesia WA1 induced severe morphological changes
in the erythrocytes, and these changes were more evident in almost all
infected cells 96 hr PI.
Babesia Washington-1 (WA1) was first isolated from a human patient
in Washington state and since 1991 has been included with Babesia
microti as the causative agents of human babesiosis in the United States
(Quick et al., 1993). The transmission of WA1 was reported to occur
via blood transfusion, and it is difficult to detect among asymptomatic
blood donors (Kjemtrup et al., 2002). Other possible transmission routes
and the identity of the tick vector and the host reservoir remain un-
known (Quick et al., 1993). In contrast to B. microti, WA1 is extremely
virulent to hamsters (Mesocricetus auratus), producing high mortality
by day 10 postinoculation (PI) (Pruthi et al., 1995). Hamsters infected
with WA1 developed high parasitemia and hemolytic anemia (Dao and
Eberhard, 1996), signs characteristic of acute human babesiosis (Pruthi
et al., 1995; Dao, 1996). Babesia WA1 is morphologically similar to B.
microti but molecularly and antigenically distinct. Moreover, its phy-
logenetic tree is closely related to Babesia gibsoni, a common dog
erythrocyte piroplasm (Walter et al., 2001; Goethert and Telford, 2003).
The ultrastructure of B. microti is well documented in host erythro-
cytes and in ticks (Rudzinska et al., 1976, 1979; Rudzinska and Trager,
1977). Morphological hallmarks of Babesia spp. infection in erythro-
cytes are the presence of vacuole(s) within an organism and the patho-
gnomonic tetrads (‘‘Maltese cross’’), which are 4 daughter merozoites
that remain attached to each other after division (Pantanowitz et al.,
2002). Tetrads are not universal to all Babesia species, but they are
abundant in WA1 compared with B. microti (Healy and Ruebush, 1980).
In the present study, 2 female 1-month-old hamsters were inoculated
intraperitoneally (i.p.) with cryopreserved hamster blood containing 1.8
⫻ 109 WA1-infected erythrocytes. The Babesia WA1 strain was origi-
nally isolated from a blood sample of a human patient from the state
of Washington. Saphenous vein blood samples were obtained at 0, 24,
36, 48, 60, 72, 84, and 96 hr PI. Parasitemias were determined at each
J. Parasitol., 92(5), 2006, pp. 1104–1107
䉷 American Society of Parasitologists 2006
FIGURE 1. (A) Hamster erythrocytes infected with WA1 merozoites
96 hr PI. TEM (bar ⫽ 2 ␮m). (B) Hamster erythrocytes uninfected at
time 0. TEM (bar ⫽ 2 ␮m).
time interval by counting 400–500 stained erythrocytes on blood smears
(Protocol-Hema 3, Fisher, Pittsburgh, Pennsylvania). Fresh hamster
blood (20 ␮l) was fixed in chilled modified-Karnovsky’s-cacodylate fix-
ative (a 50:50 solution of 2% paraformaldehyde and 2.5% glutaralde-
hyde in 0.1 M cacodylate buffer), washed in fresh fixative, and postfixed
twice in cold (4 C) 1% osmium tetroxide-cacodylate-buffered fixative
solution for 1 hr. After washes in cold distilled water, the samples were
dehydrated in a series of increasing concentrations of ethyl alcohol so-
lutions. The tissues were stained for 60 min with uranyl acetate added
to 70% alcohol solution. The specimens were then infiltrated with 3
increasing concentrations of resin in acetone, 2:1, 1:1, and 1:2 (treat-
ment periods of 2–12 hr) ending in 100% resin (treatment period of 8–
12 hr). All samples were then embedded in Epon LX112 (EMS, Fort
Washington, Pennsylvania) embedding medium at 45 C for 24 hr and
then at 60 C for another 24 hr. Embedded tissues were trimmed and
sectioned on an ultramicrotome (Ultracut E-Reichert-Jung, Vienna, Aus-
tria). Ultrathin sections (80–85 nm) were placed on copper grids and
stained with uranyl acetate and lead citrate. Grids were examined by
transmission electron microscopy (TEM) with a Hitachi H-300. For
scanning electron microscopy (SEM), the initial steps were similar to
those described above, but after osmium fixation and washes, samples
were placed on 1% poly-L-lysine coverslips for 2 hr and rinsed again
in distilled water and dehydrated with increasing concentrations of ethyl
alcohol solutions. Samples were then treated twice for 5 min with hex-
amethyldesilazane, mounted in aluminum mounts and sputter coated
with gold (Edwards 150A sputter coater equipment) and viewed in the
Hitachi’s H3010 accessory.
Parasitemia in blood smears showed 0, 2.5, 5, 10, 12.5, 22.5, 70, and
almost 100% parasitemic erythrocytes at 0, 24, 36, 48, 60, 72, 84, and
96 hr PI, respectively. ‘‘Maltese cross’’ structures were observed in
blood smears starting 72 hr PI. Single WA1 trophozoites were observed
in the cytoplasm of some erythrocytes during the first 48 hr, but mul-
tiple-merozoite-infected erythrocytes were observed frequently 72 hr PI
with changes in the normal shape of the erythrocyte (Fig. 1A, B). Mer-
ozoites were amoeboid, uninucleated, vacuolated, and had host cell cy-
toplasm. In contrast, trophozoites were multinucleated, nonvacuolated,
and had a high degree of polymorphism. Externally, almost all eryth-
rocytes revealed surface membrane changes 96 hr PI (Figs. 2, 3). Severe
alterations in the surface membrane of erythrocytes also included mul-

FIGURE 2. Severely affected erythrocytes 96 hr PI showing protru-
sions (white arrows), protrusions inside perforations (black arrows), and
1 erythrocyte with multiple perforations. SEM (bar ⫽1 ␮m).
tiple perforations, giving them a ‘‘swiss cheese’’ appearance (Fig. 2).
TEM studies of these multiple-perforated erythrocytes revealed severe
and multiple invaginations of the erythrocyte plasma membrane with
the presence of many merozoites (Fig. 3). Free parasites were observed
singly or in clusters (Fig. 4). Parasites invading erythrocytes were ob-
served with an invagination of the erythrocyte surface membrane that
was in close contact with the plasma membrane of the organism (Fig.
5A, B), and vacuolization of the parasite cytoplasm (Fig. 5B, C). The
FIGURE 3. Severe distortions in 2 erythrocytes 96 hr PI caused by
multiple invaginations of the cell membrane (black arrows) and the
presence of merozoites in the cytoplasm (white arrows). TEM (HC, host
cytoplasm invaginated in 1 parasite; bar ⫽ 0.5 ␮m).
RESEARCH NOTES 1105
FIGURE 4. (A) Free parasites close to an infected erythrocyte 96 hr
PI. TEM (bar ⫽ 0.5 ␮m). (B) Magnification of A, showing free para-
sites. TEM (bar ⫽ 0.25 ␮m).
marginalization of the merozoite inside the erythrocyte allowed for-
mation of a parasitophorous vacuole, which included both the host cell
membrane and the plasma membrane of the parasite just beside the
space left by the invaginated host cytoplasm (Fig. 5D, E). The presence
of protrusions inside perforations was commonly observed by the SEM
(Fig. 5F). Vacuolization of the host cytoplasm was observed in those
cells in which the merozoites were free inside the cell. Enlarged para-
sites were observed apparently invaginating the host cytoplasm, deform-
ing the surface membrane (Fig. 6A, B) and closing the new vacuole by
approximation of anterior and posterior ends, emitting pseudopods, or
both (Fig. 6A–C). The mature trophozoites originated from 4 new mer-
ozoites by tetrad division, an event observed only after 72 hr PI (Fig.
7A). New rosettelike merozoites were recognized by the single nuclei
and vacuole. After division, the parasites developed apical complexes
characterized by organelle formation of rhoptries and micronemes (Fig.
7B). Damage to the surface host cell membrane in contact with the
anterior end of the merozoite was observed in this process (Fig. 7C).
Under the conditions of the present experiment, WA1 was extremely
virulent to hamsters and produced a very high level of parasitemia, up
to 100%, within 96 hr PI. The SEM study revealed changes in the
FIGURE 5. (A) A parasite attached to an erythrocyte, showing in-
vagination of the cell membrane (arrow) 96 hr PI. TEM (bar ⫽ 1 ␮m).
(B) Merozoite invading an erythrocyte 96 hr PI. TEM (bar ⫽ 0.25 ␮m).
(C) Merozoite (N, nucleus) invading an erythrocyte 96 hr PI. The cell
membrane is invaginated; an electron-dense dark area is observed (ar-
rows). TEM (bar ⫽ 0.5 ␮m). (D) Merozoite invading 1 erythrocyte 96
hr PI. The parasite is located beside the invaginated cytoplasm, and a
vacuole is forming (arrow). TEM (bar ⫽ 0.5 ␮m). (E) Merozoite in-
vading 1 erythrocyte 96 hr PI. Vacuolization of the host cytoplasm (HC)
is observed in 1 parasite beside an invaginated cytoplasm (arrow). An-
other parasite is observed (N, nucleus). TEM (bar ⫽ 0.5 ␮m). (F) A
protrusion (arrow) beside a perforation in an infected erythrocyte 72 hr
PI. SEM (bar ⫽ 1␮m).
1106 THE JOURNAL OF PARASITOLOGY, VOL. 92, NO. 5, OCTOBER 2006
FIGURE 6. (A) Vacuolization of the host cytoplasm (HC) in 2 par-
asites 96 hr PI. The HC is invaginated (arrowhead) by 1 parasite that
‘‘closes’’ (arrow) the forming vacuole. TEM (bar ⫽ 0.5 ␮m). (B) Vac-
uolization of the HC in 1 merozoite 96 hr PI. Pseudopods can be ob-
served (arrows) in the forming vacuole. TEM (bar ⫽ 0.5 ␮m). (C)
Merozoites with vacuolized HC 96 hr PI. Two closed points of the
vacuoles are observed (arrows). N, nucleus. TEM (bar ⫽ 0.5 ␮m).
erythrocyte membrane and shape, including protrusions, perforations,
or both.
Extensive damage to the erythrocyte membrane, characterized as pro-
trusions, inclusions, and perforations, have been reported with B. mi-
croti infection of human erythrocytes (Sun et al., 1983). In the present
study, we observed multiple-perforated erythrocytes with an increased
convex shape. To our knowledge, there are no other reports of babesial
infections that cause this type of damage to erythrocytes. Damage to
erythrocyte membranes seemed to correlate with an increase in the per-
centage of parasitemia. Diffuse and pronounced vascular stasis with
massive sequestration of erythrocytes was the cause of severe anoxia
and damage to vital organs in hamsters infected with WA1 (Dao and
Eberhard, 1996).
Previous reports in mice infected with WA1 described endothelial
cell changes associated with pulmonary edema and respiratory distress
(Hemmer et al., 1999). In fatal cases, some key inflammatory cytokines
such as tumor necrosis factor ␣ may play an important role in the path-
ogenesis associated with WA1 (Hemmer et al., 2000). The differential
expression of certain inflammatory mediators in susceptible and resis-
tant strains of mice could explain the different outcome of WA1 infec-
tion (Moro et al., 1998).
The presence of parasites attached to the erythrocyte membrane and
the process of invasion of erythrocytes apparently did not differ as much
as was described for B. microti (Rudzinska et al., 1976). The invagi-
nation of the plasma membrane of the erythrocyte produces a parasi-
tophorus vacuole that contains the merozoite covered by its own mem-
brane and the host plasma membrane. This event appears to occur im-
mediately after attachment since we observed few parasites in this pro-
cess compared with the high number of parasitized erythrocytes. Inside
the cell, the merozoite lies beside the invaginated space observed as
protrusions coming from the walls of the perforated erythrocyte surface.
In the process of invagination, the cytoplasm of the erythrocyte is se-
verely displaced. Similarly, the perforated cells underwent severe
changes in shape caused by the presence of more than 1 intracellular
parasite.
The vacuoles in the merozoites observed in our study were portions
of host cytoplasm generated during the invasion process as described
(Healy and Ruebush, 1980; Kjemtrup et al., 2002). The vacuoles appear
to be a source of nutrients for the invading merozoite (Rudzinska,
1976), but they disappear after growth and the merozoites develop to
mature trophozoites just before the parasite division starts (Rudzinska,
1976; Rudzinska et al., 1976). We observed that the vacuoles also were
formed by an invagination process inside the cell when the merozoite
lies free in the host cytoplasm. In this case, the anterior and posterior
ends of the parasite develop pseudopodia, forming the vacuole inside.
We also observed that vacuolization of the erythrocyte cytoplasm occurs
when the parasites are closer to the cell membrane, causing distortions
in shape when the invagination process starts. This vacuolization sug-
gests that some distortions in the cell membrane, observed as perfora-
tions by SEM, could be formed by this process.
The division of WA1 trophozoites was in general the same as that in
other apicomplexans (Rudzinska, 1976; Rudzinska et al., 1976), how-
ever, with a greater frequency. Erythrocytes containing multiple para-
sites were frequently seen, with trophozoites in division forming char-
FIGURE 7. (A) A dividing trophozoite forming a tetrad 96 hr PI.
Two merozoites with host cytoplasm (HC) vacuoles are observed. TEM
(bar ⫽ 0.5 ␮m). (B) Trophozoite dividing in 4 merozoites (N, nucleus)
with formation of apical complexes (arrows) 96 hr PI. TEM (bar ⫽ 0.5
␮m). (C) Four merozoites with HC vacuoles and a trophozoite in di-
vision (N) 96 hr PI. TEM (bar ⫽ 0.5 ␮m). A magnification of the
damaged erythrocyte membrane (arrowheads) is detailed inside the box
near the apical complex of the parasite (arrow) (bar ⫽ 0.125 ␮m).
acteristic ‘‘tetrads.’’ Interestingly, some merozoites, still attached to
each other by the posterior end, were closer to the erythrocyte mem-
brane. In these parasites, the apical complex was well formed with the
presence of rhoptries and micronemes, which seem to play an important
role during invasion of B. microti (Rudzinska et al., 1976). These or-
ganelles contain enzymes that facilitate exit from the host cell, and their
position could explain the damage to the cell membrane in contact with
the anterior end of the parasite even causing an evagination that mirrors
its shape.
In conclusion, Babesia WA1 was demonstrated to be extremely vir-
ulent in hamsters, inducing severe ultrastructural changes in the eryth-
rocyte membrane and shape. The parasite showed a high degree of
division, pleomorphism, and invasiveness, with almost all erythrocytes
parasitized 96 hr PI.
We are indebted to S. Schul for technical support and to D. Mosier
and J. Green for comments and discussion. This work was supported
in part by National Institutes of Health grant P20RR016443-04.
LITERATURE CITED
DAO, A. H. 1976. Human babesiosis. Comprehensive Therapy 22: 713–
718.
———, AND M. L. EBERHARD. 1996. Pathology of acute fatal babesiosis
in hamsters experimentally infected with the WA-1 strain of Ba-
besia. Laboratory Investigation 74: 853–859.
GOETHERT, H. K., AND S. R. TELFORD, III. 2003. What is Babesia mi-
croti? Parasitology 127: 301–309.
HEALY, G. R., AND T. K. RUEBUSH, II. 1980. Morphology of Babesia
microti in human blood smears. American Journal of Clinical Pa-
thology 73: 107–109.
HEMMER, R. M., D. A. FERRICK, AND P. A. CONRAD. 2000. Up-regulation
of tumor necrosis factor-alpha and interferon-gamma expression in
the spleen and lungs of mice infected with the human Babesia
isolate WA1. Parasitology Research 86: 121–128.
———, E. J. WOZNIAK, L. J. LOWENSTINE, C. G. PLOPPER, V. WONG,
AND P. A. CONRAD. 1999. Endothelial cell changes are associated
with pulmonary edema and respiratory distress in mice infected
with the WA1 human Babesia parasite. Journal of Parasitology 85:
479–489.
KJEMTRUP, A. M., B. LEE, C. L. FRITZ, C. E. EVANS, M. CHEVERNAK,
AND P. A. CONRAD. 2002. Investigation of transfusion transmission
of a WA1-type babesial parasite to a premature infant in California.
Transfusion 42: 1482–1487.
MORO, M. H., C. S. DAVID, J. M. MAGERA, P. J. WETTSTEIN, S. W.
BARTHOLD, AND D. H. PERSING. 1998. Differential effects of infec-
tion with a Babesia-like piroplasm, WA1, in inbred mice. Infection
and Immunity 66: 492–498.
PANTANOWITZ, L., S. AUFRANC III, R. MONAHAN-EARLEY, A. DVORAK,
AND S. R. TELFORD, III. 2002. Morphologic hallmarks of Babesia.
Transfusion 42: 1389.
PRUTHI, R. K., W. F. MARSHALL, J. C. WILTSIE, AND D. H. PERSING. 1995.
Human babesiosis. Mayo Clinic Proceedings 70: 853–862.
QUICK, R. E., B. L. HERWALDT, J. W. THOMFORD, M. E. GARNETT, M.
L. EBERHARD, M. WILSON, D. H. SPACH, J. W. DICKERSON, S. R.

TELFORD, K. R. STEINGART, R. POLLOCK, D. H. PERSING, J. M. KO-
BAYASHI, D. D. JURANEK, AND P. A. CONRAD. 1993. Babesiosis in
Washington state, a new species of Babesia? Annals of Internal
Medicine 119: 284–290.
RUDZINSKA, M. A. 1976. Ultrastructure of intraerythrocytic Babesia mi-
croti with emphasis on the feeding mechanism. Journal of Proto-
zoology 23: 224–233.
———, AND W. TRAGER. 1977. Formation of merozoites in intraeryth-
rocytic Babesia microti. Canadian Journal of Zoology 55: 928–938.
———, ———, S. J. LEWENGRUB, AND E. GUBERT. 1976. An electron
Prevalence of Toxoplasma gondii in Rats (Rattus norvegicus) in Grenada, West Indies
J. P. Dubey, M. I. Bhaiyat*, C. N. L. Macpherson†, C. de Allie*, A Chikweto*, O. C. H. Kwok, and R. N. Sharma*, United States
Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory,
Building 1001, Beltsville, Maryland 20705-2350; *Department of Paraclinical Studies, School of Veterinary Medicine, St. George’s University,
Grenada, West Indies; †Windward Island Research and Education Foundation, True Blue Campus, St. George’s University, Grenada, West
Indies. e-mail: jdubey@anri.barc.usda.gov
ABSTRACT: Cats are important in the natural epidemiology of Toxo-
plasma gondii, because they are the only hosts that can excrete envi-
ronmentally resistant oocysts. Cats are infected with T. gondii via pre-
dation on infected birds and rodents. During 2005, 238 rats (Rattus
norvegicus) were trapped in Grenada, West Indies, and their sera along
with tissue samples from their hearts and brains were examined for T.
gondii infection. Antibodies to T. gondii were assayed by the modified
agglutination test (MAT, titer 1:40 or higher); only 2 (0.8%) of 238 rats
were found to be infected. Brains and hearts of all rats were bioassayed
in mice. Toxoplasma gondii was isolated from the brain and the heart
of only 1 rat, which had a MAT titer of 1:320. All of 5 mice inoculated
with the heart tissue, and the 5 mice inoculated with the brain tissue of
the infected rat remained asymptomatic, even though tissue cysts were
found in their brains. Genetically, the isolates of T. gondii from the
heart and the brain were identical and had genotype III by using the
SAG1, SAG2, SAG3, BTUB, and GRA6 gene markers. These data
indicate that rats are not important in the natural history of T. gondii
in Grenada.
Toxoplasma gondii infections are widely prevalent in human beings
and animals worldwide (Dubey and Beattie, 1988). Humans become
infected postnatally by ingesting tissue cysts from undercooked meat,
consuming food or drink contaminated with oocysts, or by accidentally
ingesting oocysts from the environment. Cats are important in the nat-
ural life cycle of T. gondii because they are the only hosts that can
directly spread T. gondii in the environment. Thus, cats can ‘recycle’
and amplify the infection by releasing millions of infective oocysts into
the environment. In turn, cats are considered to become infected with
T. gondii by ingesting tissues of infected animals, most likely rodents
and birds (Ruiz and Frenkel, 1980).
Grenada is located in the eastern Caribbean and is most southern of
the Windward Islands. It has an area of approximately 344 km2 with a
population of 90,000. Our study on the epidemiology of T. gondii in-
fections in Grenada began in 1995 with a serological survey of T. gondii
in pregnant women and cats. Antibodies to T. gondii were found in
57% of 534 pregnant women and in 35% of 40 cats (Asthana et al.,
2006). Epidemiological data suggested that the ingestion of food or
water contaminated with oocysts was an important mode of transmis-
sion of T. gondii to women (Asthana et al., 2006). More recently, the
prevalence of T. gondii in 102 free-range chickens (Gallus domesticus)
from different areas of Grenada was determined, and serves as a good
indicator of T. gondii oocyst contamination, because free-range chick-
ens feed from the ground. Antibodies to T. gondii were found in 43 of
102 (42%) chickens at a serum dilution of 1:20, and viable T. gondii
was isolated from tissues of 35 of 43 (80%)-seropositive chickens (Dub-
ey et al., 2005). The objective of the present study was to determine
RESEARCH NOTES 1107
microscopic study of Babesia microti invading erythrocytes. Cell
Tissue Research 169: 323–334.
SUN, T., M. J. TENENBAUM, J. GREENSPAN, S. TEICHBERRG, R. T. WANG,
T. DEGNAN, AND M. H. KAPLAN. 1983. Morphologic and clinical
observations in human infection with Babesia microti. The Journal
of Infectious Diseases 148: 239–248.
WALTER, S., H. MEHLHORN, E. ZWEYGARTH, AND E. SCHEIN. 2001. Elec-
tron microscopic investigations on stages of dog piroplasm cultured
in vitro: Asian isolates of Babesia gibsoni and strains of B. canis
from France and Hungary. Parasitology Research 88: 32–37.
J. Parasitol., 92(5), 2006, pp. 1107–1108
䉷 American Society of Parasitologists 2006
the prevalence and genotype of T. gondii in rats (Rattus norvegicus) in
Grenada.
From April to December 2005, 238 rats (119 females, 119 males)
were caught using rattraps set in different locations in the 6 parishes of
Grenada. The rats were transported to the Pathology Laboratory at the
School of Veterinary Medicine, St. George’s University, and then anes-
thetized and killed with an overdose of ether in a closed chamber. The
thoracic cavity was opened, and blood was collected from the heart for
serological studies after which the rats were necropsied. Fresh samples
of heart and brain were taken for T. gondii isolation. All samples were
immediately shipped on ice to the United States Department of Agri-
culture, Agricultural Research Service, Animal and Natural Resources
Institute, Animal Parasitic Diseases Laboratory, Beltsville, Maryland,
for isolation and serological analysis. The rats were received in 12
batches of 10, 4, 13, 9, 9, 12, 33, 22, 24, 23, 33, and 46.
Sera from rats were diluted 2-fold starting at 1:5 dilution and assayed
for T. gondii antibodies with the modified agglutination test (MAT)
conducted as described previously (Dubey and Desmonts, 1987).
Tissues of all rats were bioassayed in mice, irrespective of antibody
level. Brains and hearts of 51 individual rats were pooled separately for
each rat, homogenized, digested in pepsin, and inoculated subcutane-
ously (s.c.) into 2–5 mice (Dubey, 1998). Brains and hearts of seroneg-
ative (MAT ⬍ 1:5) rats were pooled in batches of 5–15, and after di-
gestion they were bioassayed in mice. The mice used were Swiss-Web-
ster albino females obtained from Taconic Farms, Germantown, New
York.
The heart and brain of the rat that had a MAT titer of 1:320 were
homogenized separately, not digested in pepsin, and inoculated s.c. into
5 mice each. Tissue impression smears of mice that died were examined
for T. gondii tachyzoites or tissue cysts. Survivors were bled on day 43
postinoculation (PI), and a 1:25 dilution of serum from each mouse was
tested for T. gondii antibodies with the MAT. Mice were killed 48 days
PI, and brain squashes from all mice were examined microscopically
for tissue cysts as described previously (Dubey and Beattie, 1988). Mice
were considered infected with T. gondii when tachyzoites or tissue cysts
were demonstrable microscopically in smears of their tissues. A portion
of the brain with demonstrable T. gondii was frozen for DNA extraction.
Toxoplasma gondii DNA was extracted from the tissues of infected
mice, and strain typing was performed using genetic markers SAG1,
SAG2, SAG3, BTUB, and GRA6 with modifications of methods de-
scribed previously (Howe et al., 1997; Grigg et al, 2001; Khan et al.,
2005) with minor modifications (Dubey et al., 2006). The primers and
enzymes used were indicated by Dubey et al. (2006).
Antibodies to T. gondii were found in sera of 80 of 238 rats in titers
of 1:5 in 62, 1:10 in 11, 1:20 in 5, 1:40 in 1, and 1:320 in 1. Toxoplasma
gondii was isolated from the brain and the heart of the rat that had a
1108 THE JOURNAL OF PARASITOLOGY, VOL. 92, NO. 5, OCTOBER 2006
titer of 1:320. All 10 mice (5 with heart and 5 with brain) inoculated
with rat tissue developed MAT titers of 1:25 or higher. One mouse from
each group of mice inoculated with the heart and brain was killed on
day 49 PI, and tissue cysts were found in their brains; brain tissue from
these 2 mice was saved for DNA extraction and for cryopreservation
in liquid nitrogen. Tissue cysts were found in the brains of the remaining
8 mice that were killed day 139 PI. All of these 10 infected mice
remained asymptomatic throughout the observation period. The isolates
from the heart and the brain were all of genotype of III.
The low prevalence of T. gondii in rats from Grenada is surprising.
Antibodies (MAT 1:40–1:320) were found in only 2 rats, and T. gondii
was isolated from 1 rat. In the present study, rat sera were tested starting
at 1:5 dilution. In pigs, a titer of 1: 25 is considered indicative of per-
sistent T. gondii infection (Dubey, Thulliez et al., 1995). In the present
study, tissues of all rats were bioassayed for T. gondii, irrespective of
their antibody status, because congenitally infected rats can harbor vi-
able T. gondii in the absence of demonstrable antibodies (Dubey et al.,
1997). Dubey and Frenkel (1998) summarized the worldwide preva-
lence of T. gondii in different species of rats and concluded that the
prevalence of viable T. gondii in R. norvegicus was generally low. One
exception to this finding was reported in rats from an endemic area in
Costa Rica where Ruiz and Frenkel (1980) isolated viable T. gondii
from 15 of 120 rats. Dubey, Weigel et al. (1995), in contrast, isolated
T. gondii from only 1 of 107 rats from 47 pig farms in Illinois. Our
current findings from this study suggest that although T. gondii is prev-
alent in cats, women, and free-range chickens in Grenada, rats are not
important in the epidemiology of T. gondii on the island.
We thank C. Su (Department of Microbiology, The University of
Tennessee, Knoxville, Tennessee) for performing the genotyping and S.
K. Shen and K. Hopkins for technical assistance.
LITERATURE CITED
ASTHANA, S. P., C. N. L. MACPHERSON, S. H. WEISS, R. STEPHENS, R.
N. SHARMA, AND J. P. DUBEY. 2006. Seroprevalence of Toxoplasma
gondii in pregnant women and cats in Grenada, West Indies. Jour-
nal of Parasitology 92: 644–645.
DUBEY, J. P. 1998. Refinement of pepsin digestion method for isolation
of Toxoplasma gondii from infected tissues. Veterinary Parasitol-
ogy 74: 75–77.
———, AND C. P. BEATTIE. 1988. Toxoplasmosis of animals and man.
CRC Press, Boca Raton, Florida, 220 p.
———, M. I. BHAIYAT, C. DE ALLIE, C. N. L. MACPHERSON, R. N.
Toxoplasma gondii in Human Astrocytes In Vitro: Interleukin (IL)-12 and IL-10 Do Not
Influence Cystogenesis
C. Estran, M. P. Brenier-Pinchart*, L. Pelletier†, M. F. Cesbron-Delauw, and H. Pelloux, Laboratoire Adaptation et Pathogénie des
Microorganismes, LAPM, UMR 5163 CNRS-UJF, Université J. Fourier, Campus Santé, Grenoble, France; †Laboratoire de Neurosciences
Précliniques, INSERM U318, Université J. Fourier, Grenoble, France; *To whom correspondence should be addressed.
e-mail: mpbrenierpinchart@chu-grenoble.fr
ABSTRACT: Interleukin (IL)-12, IL-10, and interferon (IFN)-␥ are major
cytokines involved in the immune response against Toxoplasma gondii.
Nevertheless, the role of IL-12 and IL-10 in the control of parasite
replication and cytogenesis is not known yet, whereas the importance
of IFN-␥ is documented. Furthermore, it is of paramount importance to
study the interaction between T. gondii and cells from the central ner-
vous system, e.g., astrocytes. In this study, we report that IL-12 and IL-
10 have no effect on penetration, replication, or cystogenesis of the T.
gondii Prugniaud strain in human astrocytes in vitro and do not antag-
onize the role of IFN-␥ on cystogenesis.
Toxoplasma gondii is a universally widespread protozoan. Toxoplas-
mosis is a health care problem, causing congenital toxoplasmosis and
SHARMA, C. SREEKUMAR, M. C. B. VIANNA, S. K. SHEN, O. C. H.
KWOK, K. B. MISKA, D. E. HILL, AND T. LEHMANN. 2005. Isolation,
tissue distribution, and molecular characterization of Toxoplasma
gondii from chickens in Grenada, West Indies. Journal of Parasi-
tology 91: 557–560.
———, AND G. DESMONTS. 1987. Serological responses of equids fed
Toxoplasma gondii oocysts. Equine Veterinary Journal 19: 337–
339.
———, AND J. K. FRENKEL. 1998. Toxoplasmosis of rats: A review,
with considerations of their value as an animal model and their
possible role in epidemiology. Veterinary Parasitology 77: 1–32.
———, A. N. PATITUCCI, C. SU, N. SUNDAR, O. C. H. KWOK, AND S.
K. SHEN. 2006. Characterization of Toxoplasma gondii isolates in
free-range chickens from Chile, South America. Veterinary Para-
sitology. 140 76–82.
———, S. K. SHEN, O. C. H. KWOK, AND P. THULLIEZ. 1997. Toxo-
plasmosis in rats (Rattus norvegicus): Congenital transmission to
first and second generation offspring and isolation of Toxoplasma
gondii from seronegative rats. Parasitology 115: 9–14.
———, P. THULLIEZ, R. M. WEIGEL, C. D. ANDREWS, P. LIND, AND E.
C. POWELL. 1995. Sensitivity and specificity of various serologic
tests for detection of Toxoplasma gondii infection in naturally in-
fected sows. American Journal of Veterinary Research 56: 1030–
1036.
———, R. M. WEIGEL, A. M. SIEGEL, P. THULLIEZ, U. D. KITRON, M.
A. MITCHELL, A. MANNELLI, N. E. MATEUS-PINILLA, S. K. SHEN, O.
C. H. KWOK, AND K. S. TODD. 1995. Sources and reservoirs of
Toxoplasma gondii infection on 47 swine farms in Illinois. Journal
of Parasitology 81: 723–729.
GRIGG, M. E., J. GANATRA, J. C. BOOTHROOYD, AND T. P. MARGOLIS.
2001. Unusual abundance of atypical strains associated with human
ocular toxoplasmosis. Journal of Infectious Diseases 184: 633–639.
HOWE, D. K, S. HONORÉ, F. DEROUIN, AND L. D. SIBLEY. 1997. Deter-
mination of genotypes of Toxoplasma gondii strains isolated from
patients with toxoplasmosis. Journal of Clinical Microbiology 35:
1411–1414.
KHAN, A., C. SU, M. GERMAN, G. A. STORCH, D. B. CLIFFORD, AND L.
D. SIBLEY. 2005. Genotyping of Toxoplasma gondii strains from
immunocompromised patients reveals high prevalence of type I
strains. Journal of Clinical Microbiology 43: 5881–5887.
RUIZ, A., AND J. K. FRENKEL. 1980. Intermediate and transport hosts of
Toxoplasma gondii in Costa Rica. American Journal of Tropical
Medicine and Hygiene 29: 1161–1166.
J. Parasitol., 92(5), 2006, pp. 1108–1110
䉷 American Society of Parasitologists 2006
cerebral reactivation in immunocompromised patients. Infection induces
a strong immune response and immunocompetent cells secrete IFN-␥,
IL-12, and tumor necrosis factor (TNF)-␣ (Ricard et al., 1996; Bliss et
al., 2000). Another cytokine that is important at the early stage is IL-
10, which down-regulates IL-12 and IFN-␥ secretion, and avoids im-
munopathogenicity of the immune response (Wilson et al., 2005). Al-
though infection is controlled, a percentage of parasites survive as dor-
mant cysts, particularly in the host’s central nervous system. Parasites
replicate in human astrocytes, and these cells have been found to sup-
port more replication of T. gondii than other cells in vitro (Halonen et
al., 1996; Fagard et al., 1999). The importance of IL-12 and IL-10
during the immune response is known. More precisely, IL-10 has been
shown to inhibit parasite-killing functions of IFN-␥–activated murine

TABLE 1. Invasion rate and multiplication of Prugniaud strain in pri-
mary human astrocyte cell cultures.
Invasion rate
Multiplication
Infected cells/
Treatment 1,000 cells Tg/1,000 cells
Astrocytes ⫹ Tg 47 ⫾ 18 620 ⫾ 70
Astrocytes ⫹ Tg ⫹ IFN-␥ (500 U/ml) 48 ⫾ 21 112 ⫾ 58*
Astrocytes ⫹ Tg ⫹ IL-12 (25 ng/ml) 53 ⫾ 20 622 ⫾ 91
Astrocytes ⫹ Tg ⫹ IL-10 (100 ng/ml) 46 ⫾ 17 613 ⫾ 58
Human astrocytes (105 cells/ml) were infected with 105 parasites/ml (ratio ⫽ 1).
Cytokines were added at the time of T. gondii (Tg) infection for 3 and 48 hr in
invasion and multiplication assays, respectively. Invasion rate was determined 3
hr postinfection. Moreover, the multiplication rate was counted 48 hr postinfection.
Data are means ⫾ standard deviations from 3 experiments assessed in duplicate.
* Statistically significant difference (P ⬍ 0.05) (Student t-test).
macrophages. Yet, their roles regarding cyst formation of T. gondii in
human astrocytes remain to be investigated (Gazzinelli et al., 1992).
The objective of this study was to investigate the role of IL-12, IL-10,
and IFN-␥ on parasite cystogenesis in human astrocytes in vitro.
The 2 types of cells, i.e., primary human astrocytes and glioblastoma
cell line U373 (ATCC HTB-17), were grown in 24-well plates on glass
coverslips at 37 C in a 5% CO2 atmosphere as described previously
(Brenier-Pinchart et al., 2004). Briefly, glioblastoma cell line U373 MG
was grown in Dulbecco’s modified Eagle’s medium (DMEM), supple-
mented with 1% synthetic serum Ultroser G (Life Technologies, Eragny,
France), 5% L-glutamine, and 5% penicillin-streptomycin, and primary
human astrocytes were grown in DMEM supplemented with 5% fetal
bovine serum (Cambrex Bio Science, Verviers, Belgium), 2% L-gluta-
mine, and 1% penicillin-streptomycin. Primary human astrocytes were
obtained from 1 patient after epileptic surgery at Grenoble Hospital
(Grenoble, France). Neuropathological study demonstrated the absence
of tumor formation. Cells were positive for glial fibrillary acidic protein
and negative for neurofilament or nestin by immunostaining. Toxoplas-
mic serology of this patient was negative. Cells were infected with
Prugniaud strain, a nonvirulent cystogenic strain in the mouse (type II)
(Brenier-Pinchart et al., 2004). Recombinant human cytokines (IL-12,
IL-10, and IFN-␥) were purchased from R&D Systems (Abington, Ox-
ford, U.K.). The cytokines concentrations used were 500 UI/ml for IFN-
␥; 2.5, and 25 ng/ml for IL-12; and 10 and 100 ng/ml for IL-10 (Dau-
bener et al., 1993; Subauste et al., 2000; Del Rio et al., 2004). Invasion
rate and parasite multiplication were observed by stained Diff Quick at
3 and 48 hr postinfection (PI), respectively. At day 6 PI, cystogenesis
was observed using an indirect immunofluorescence assay by using the
cyst specific monoclonal antibody (Ab) CC2 (Gross et al., 1995), and
cysts were counted. Two types of cysts could be detected with this
antibody, i.e., wall-stained cysts and matrix-stained cysts, with the first
type considered as more mature than the second type (early stage of
development) (Ricard et al., 1999). For invasion rate and parasite mul-
tiplication assays, cells were treated by cytokines at the time of T. gondii
infection for 3 and 48 hr, respectively. For cyst formation study, cyto-
kines were added at the time of infection and again with each renewal
culture medium (days 1 and 3). Moreover, in some experiments on
cystogenesis, a pretreatment of cells by IFN-␥ was made 48 hr before
infection, and IFN-␥ was associated with IL-10 or IL-12 treatment per-
formed at the time of infection and at days 1 and 3, when the culture
medium was renewed.
IL-12 and IL-10 did not significantly modify the invasion rate of T.
gondii in primary human astrocyte cell cultures infected with the Prug-
niaud strain (Table I). In our model, IL-12, IL-10, and IFN-␥ did not
have any effect on parasite penetration. Furthermore, the treatment by
IL-12 and IL-10 for 48 hr did not affect replication of the Prugniaud
strain in primary human astrocytes cell cultures (Table I). As expected,
the parasite multiplication in primary human astrocyte cell cultures was
significantly inhibited in the presence of IFN-␥ (Table I) (Oberdorfer et
al., 2003). Invasion and multiplication rates of T. gondii in U373 cell
lines were similar to those obtained in astrocytes (data not shown). As
reported previously, 2 types of cysts could be differentiated, i.e., a ma-
RESEARCH NOTES 1109
FIGURE 1. Cystogenesis in primary human astrocyte cell cultures
after treatment by cytokines for 6 days. Astrocytes (105/ml) were in-
fected with 1.5 ⫻ 104 T. gondii/ml, and cysts were counted by indirect
immunofluorescence with Ab CC2 6 days PI. Results from 3 experi-
ments assessed in duplicate (means ⫾ standard errors). *, statistically
significant difference (P ⬍ 0.05) (Student’s t-test).
trix-stained cysts and a wall-stained cysts with Ab CC2 (Ricard et al.,
1996). In our model, IL-12 and IL-10 did not have an effect on cyst
formation of T. gondii, whereas IFN-␥ inhibited cyst formation (Fig. 1).
Moreover, we have associated a pretreatment of the cells by IFN-␥ (for
48 hr before infection) to the treatment by IL-12 and IL-10. The number
of cysts observed in the presence of IFN-␥ was not modified when IL-
12 and IL-10 were added. Indeed, the total number of cysts in astrocytes
was 149 ⫾ 40 without any treatment and 2 ⫾ 1 when IFN-␥ was present
in cell culture (with or without IL-12 and IL-10) (mean ⫾ SD of 3
experiments). Thus, IL-12 and IL-10 did not antagonize the ability of
IFN-␥ to inhibit cystogenesis of T. gondii in astrocyte cells. As for both
invasion and replication rates, the results obtained with primary human
astrocyte cell cultures were close to those obtained with the U373 cell
line (data not shown).
Recently, Wilson et al. (2005) reported that IL-10 antagonizes the
ability of IFN-␥ to inhibit replication of T. gondii in murine astrocytes
21 hr PI. Our results confirm that IFN-␥ has no effect on penetration
but that it inhibits multiplication and cyst formation of the parasite in
human astrocyte cells. However, the study presented here suggests that
IL-10 does not antagonize IFN-␥ effect on cyst formation in human
astrocytes 6 days PI. In murine astrocytes, IL-10 antagonizes IFN-␥’s
protective effects on T. gondii multiplication (Wilson et al., 2005),
whereas our study shows that this cytokine does not reveal an antago-
nism with IFN-␥ on cyst formation in human astrocytes. In conclusion,
despite the importance of IL-12 and IL-10 in the immune control of T.
gondii infection, these cytokines do not have any effect on the modu-
lation of penetration, replication, and cystogenesis of T. gondii in human
astrocytes cells in this in vitro model.
Special thanks to U. Gross (University of Gottingen, Gottingen, Ger-
many) for kindly providing the antibody CC2, to S. Durville for English
proofreading, and to J. Simon and F. Durand for advice and support.
LITERATURE CITED
BLISS, S. K., B. A. BUTCHER, AND E. Y. DENKERS. 2000. Rapid recruit-
ment of neutrophils containing prestored IL-12 during microbial
infection. Journal of Immunology 165: 4515–4521.
BRENIER-PINCHART, M. P., E. BLANC-GONNET, P. N. MARCHE, F. BERGER,
F. DURAND, P. AMBROISE-THOMAS, AND H. PELLOUX. 2004. Infection
of human astrocytes and glioblastoma cells with Toxoplasma gon-
dii: Monocyte chemotactic protein-1 secretion and chemokine ex-
pression in vitro. Acta Neuropathologica 3: 245–249.
DAUBENER, W., K. PILZ, S. SEGHROUCHNI ZENNATI, T. BILZER, H. G.
FISCHER, AND U. HADDING. 1993. Induction of toxoplasmostasis in
a human glioblastoma by interferon ␥. Journal of Neuroimmunol-
ogy 43: 31–38.
DEL RIO, L., B. A. BUTCHER, S. BENNOUNA, S. HIENY, A. SHER, AND E.
Y. DENKERS. 2004. Toxoplasma gondii triggers myeloid differenti-
ation factor 88-dependent IL-12 and chemokine ligand 2 (monocyte
chemoattractant protein 1) responses using distinct parasite mole-
cules and host receptors. Journal of Immunology 172: 6954–6960.
FAGARD, R., H. VAN TAN, C. CREUZET, AND H. PELLOUX. 1999. Differ-
ential development of Toxoplasma gondii in neural cells. Parasi-
tology Today 15: 504–507.
1110 THE JOURNAL OF PARASITOLOGY, VOL. 92, NO. 5, OCTOBER 2006
GAZZINELLI, R. T., I. P. OSWALD, S. L. JAMES, AND A. SHER. 1992. IL-
10 inhibits parasite killing and nitrogen oxide production by IFN-
␥-activated macrophages. Journal of Immunology 148: 1792–1796.
GROSS, U., H. BORMUTH, C. GAISSMAIER, C. DITTRICH, V. KRENN, W.
BOHNE, AND D. J. FERGUSON. 1995. Monoclonal rat antibodies di-
rected against Toxoplasma gondii suitable for studying tachyzoite-
bradyzoite interconversion in vivo. Clinical and Diagnostic Labo-
ratory Immunology 2: 542–548.
HALONEN, S. K., W. D. LYMAN, AND F. C. CHIU. 1996. Growth and
development of Toxoplasma gondii in human neurons and astro-
cytes. Journal of Neuropathology and Experimental Neurology 55:
1150–1156.
OBERDORFER, C., O. ADAMS, C. R. MACKENZIE, C. J. DE GROOT, AND
W. DAUBENER. 2003. Role of IDO activation in anti-microbial de-
fense in human native astrocytes. Advances in Experimental Med-
icine and Biology 527: 15–26.
Paleoparasitological Records in a Canid Coprolite From Patagonia, Argentina
M. H. Fugassa, G. M. Denegri, N. H. Sardella, A. Araújo*, R. A. Guichón, P. A. Martinez†, M. T. Civalero‡, and C. Aschero§,
Departamento de Biologı́a, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata–CONICET, Argentina; *Escola
Nacional de Saúde Pública—Fundac˛ao Oswaldo Cruz, Brazil; †Departamento de Biologı́a, Facultad de Ciencias Exactas y Naturales,
Universidad Nacional de Mar del Plata; ‡Instituto Nacional de Antropologı́a y Pensamiento Latinoamericano–CONICET, Argentina;
§Universidad Nacional de Tucumán–CONICET, Argentina. e-mail: mfugassa@mdp.edu.ar
ABSTRACT: In this note, organic remains identified as a canid coprolite
were examined. The material was dated at 6,540 ⫾ 110 B.P.; it was
collected in the Perito Moreno National Park, Santa Cruz, Argentina.
Paleoparasitological analysis was performed following standard proce-
dures. Coprolite fragments were rehydrated in a trisodium phosphate
aqueous solution and subjected to spontaneous sedimentation for mi-
croscope analysis. Eggs of nematodes identified as Trichuris sp., Cap-
illaria sp., Uncinaria sp., and an ascaridid (probably Toxascaris sp.) or
spirurids (presumably Physaloptera sp.), plus a cestode (Anoplocephal-
idae), presumably Moniezia sp., were found.
Paleoparasitological analysis was recently applied to bioanthropo-
logical and archaeological studies in southern Patagonia. The studied
FIGURE 1. Coprolite (CCP5, Santa Cruz Province, Argentina, 6,540
⫾ 110 yr B.P.). Bar ⫽ 2 cm.
RICARD, J., H. PELLOUX, A. L. FAVIER, U. GROSS, E. BRAMBILLA, AND P.
AMBROISE-THOMAS. 1999. Toxoplasma gondii: role of the phospha-
tidylcholine-specific phospholipase C during cell invasion and in-
tracellular development. Experimental Parasitology 3: 231–237.
———, ———, S. PATHAK, B. PIPY, AND P. AMBROISE-THOMAS. 1996.
TNF-␣ enhances Toxoplasma gondii cyst formation in human fi-
broblasts through the sphingomyelinase pathway. Cellular Signal-
ing 6: 439–442.
SUBAUSTE, C. S., AND M. WESSENDARP. 2000. Human dendritic cells
discriminate between viable and killed Toxoplasma gondii tachy-
zoites: Dendritic cell activation after infection with viable parasites
results in CD28 and CD40 ligand signaling that controls IL-12-
dependent and -independent T cell production of IFN-␥. Journal of
Immunology 165: 1498–1505.
WILSON, E. H., U. WILLE-REECE, F. DZIERSZINSKI, AND C. A. HUNTER.
2005. A critical role for IL-10 in limiting inflammation during toxo-
plasmic encephalitis. Journal of Neuroimmunology 165: 63–74.
J. Parasitol., 92(5), 2006, pp. 1110–1113
䉷 American Society of Parasitologists 2006
area is characterized by human populations that exhibited various life
style strategies, but mainly the hunter–gatherer type of subsistence (As-
chero, Belleli, and Goni, 1992; Civalero, 1995; Aschero, 1996; Civalero
and Aschero, 2003; Civalero and Franco, 2003).
The first parasites found in human coprolites from Patagonia were
eggs of Enterobius vermicularis and ancylostomids (Zimmerman and
Morila, 1983; Gonc˛alves et al., 2003).
In the present study, a coprolite was found in an archaeological site
in Santa Cruz Province, Argentina, and was examined for parasites. The
coprolite recovered from cave 5 of the archaeological locality known
as Cerro Casa de Piedra (CCP), at 47⬚57⬘S and 72⬚05⬘W, in a Patagonian
forest steppe ecotone in the valley of Burmeister Lake, Perito Moreno
National Park, Santa Cruz, Argentina, was examined. Cerro Casa de
Piedra is a hill, of volcanic origin, with caves and rock shelters facing
to the north. Radiocarbon dates of the cave 5 reached 6,800 yr B.P.
(Aschero, Belleli, Civalero et al., 1992). Estimates of human occupation
periods range from about 6,780 to 6,540 B.P., 5,170 to 4,330 B.P. to
2,740 to 2,550 B.P (Aschero, 1996). The coprolite was collected from
layer 4, with an age of 6,540 ⫾ 110 (Beta 27796). Sediment in this
level contained plant remains associated with a hearth, and abundant
animal remains, including broken bones, cut artifacts of shaved skin
and furs, feces, and feathers associated with lithic materials (Aschero,
1982).
Samples of 0.5 g from the surface and from the interior of the cop-
rolite were rehydrated in a trisodium phosphate aqueous solution (TSP)
following Callen and Cameron (1960) and subjected to spontaneous
sedimentation for microscope analysis (Lutz, 1919). Twenty slides of
each sample were made and examined by light microscopy for parasites.
Rehydrated sediment was preserved at 4 C in 2 ways, i.e., in vials with
TSP and 10% acetic formalin and in vials without fixative. Parasites
were identified by conventional methods (Thienpont et al., 1979) and
photographed.
Coprolite morphology points to a human or canine origin (Fig. 1).
The widest diameter of the coprolite was 25 mm. Reddish hairs, about
30 mm in length, were found. The rehydrated fraction contained a high
percentage of vegetable fibers. Other macroscopical remains, i.e., rodent
hairs and bones, insect fragments, and grass inflorescences, were abun-
dant. Macroscopic and microscopic charcoal fragments also were pres-

FIGURE 2. (a) Moniezia sp. egg showing hexacanth embryo and pir-
iform apparatus. (b) Trichuris sp. egg. (c) Capillaria sp. egg. (d) As-
caridid—possibly Toxascaris sp. or Physaloptera sp. egg. (e) Ancylos-
tomid egg, probably Uncinaria sp. (f ) Adult of Oribatida, Aphelacari-
dae, presumably Aphelacarus sp. Bar ⫽ 10 ␮, except for f (bar ⫽ 100
␮).
ent. The rehydrated fraction without fixative exhibited the typical smell
of fox urine.
Microscopic examination of the sample confirmed the presence of
nematode eggs identified as Trichuris sp., Capillaria sp., and Uncinaria
sp., and an ascaridid (probably Toxascaris sp.) or spirurids (presumably
Physaloptera sp.), plus eggs of a cestode (Anoplocephalidae), presum-
ably Moniezia sp. Up to 14 eggs per slide of the cestode were present.
The latter eggs were quadrangular to triangular, with a yellowish brown
color, and surrounded by a thin membrane containing a hexacanth em-
bryo of 22 ␮m in diameter. In some, hooks and a piriform apparatus
were identified (Fig. 2a). Measurements were 62.5–77.5 ␮m (69.8 ⫾
4.33, n ⫽ 21) by 55.0–70.0 (62.9 ⫾ 4.70; n ⫽ 21). Eggs were identified
as a species of Anoplocephalidae, probably Moniezia sp. Blanchard,
1891.
Twenty Trichuris sp. eggs (Fig. 2b) were found, 50.0–70.0 (62.7 ⫾
4.8, n ⫽ 18) by 26.3–35.0 (30.8 ⫾ 2.0, n ⫽ 18); eggs of Capillaria sp.
(n ⫽ 174) also were recovered, 27.5–85.0 (59.5 ⫾ 7.3, n ⫽ 171) by
20.0–47.5 (30.6 ⫾ 4.9, n ⫽ 171) (Fig. 2c). Thick- and smooth-walled
eggs (n ⫽ 149), 62.5–80.0 (73.0 ⫾ 4.5, n ⫽ 41) by 50.0–72.5 (60.5 ⫾
4.8, n ⫽ 41), were identified as ascaridids, presumably Toxascaris sp.,
RESEARCH NOTES 1111
or Physaloptera sp. (Fig. 2d); numerous thin-walled eggs (n ⫽ 90),
60.0–95.0 (84.8 ⫾ 7.9, n ⫽ 29) by 30.0–68.7 (52.4 ⫾ 6.2, n ⫽ 29), of
an ancylostomid, probably Uncinaria sp., also were found (Fig. 2e).
Numerous acari instars and adults were found in the coprolite, in-
cluding species of Acaridida, Glycyphagidae, Listrophoridae, and prob-
ably Acaridae or Saproglyphidae as well as Prostigmata, Oribatida, and
Aphelacaridae, probably Aphelacarus sp. (Fig. 2f). Each acarine taxon
was represented by at least 1 whole specimen or the remains of an
exoskeleton.
Coprolite morphology is consistent with a human or a canid source
as described by Chame (2003). The fecal mass diameter is typically a
useful diagnostic tool; in this case, it suggests we were dealing with
feces from Pseudalopex culpaeus Molina 1782 (Cornejo Farfán and
Jimenez Milón, 2001), the largest fox inhabiting the area under study.
The reddish hairs found in coprolite did not, however, correspond to
canid fur according to Chehébar and Martı́n (1989). Dog or fox fur
usually occurs in feces when it is accidentally ingested during self-
grooming (Emmons, 1987), but this seems not to be the case for the
fecal material under study here. However, dietary residues found are
consistent with a human diet (Callen and Martı́n, 1969; Faulkner, 1991;
Reinhard et al., 1992). Charcoal, plant fiber, and a diversity of animal
bones, hairs, and grass inflorescences are typically found in human fe-
ces. In contrast, P. culpaeus, a known local canid, is reported to be
omnivorous and consumes fruits, seeds, and even arthropods (Novaro,
1997; Cornejo Farfán and Jimenez Milón, 2001). Charcoal in the sample
explains the association with hearths. The odor emanating from the
rehydrated sample supports the notion that the coprolite came from a
fox. In future studies with coprolites, part of rehydrated sediments
should be processed in the cold and without fixatives so that the smell
can probably be recovered, which would help to clarify the zoological
origin of the sample.
The presence of eggs with a hexacanth embryo and the characteristics
of the eggshell suggest Moniezia sp. as the identity of the cestode
(Thienpont et al., 1979; Denegri, 2001). This cestode, however, belongs
to a group that infects mainly herbivores. If the coprolite came from a
fox, the presence of Moniezia sp. could be explained by coprophagia
or the consumption of an herbivorous animal. Cestodes identified in
different archaeological and paleonthological sites, i.e., species of Tae-
nia, Hymenolepis, Echinococcus, and Diphyllobothrium (Gonçalves et
al., 2003; Matsui et al., 2003; Santoro et al., 2003; Bathurst, 2005) have
frequently been found in human coprolites and sediments. The only
reference of anoplocephalids in archaeological material of which we are
aware is that of an egg of Anoplocephala perfoliata in a piece of cloth
from a medieval tomb (Hidalgo-Arguello et al., 2003). Present anop-
locephalid records in the Patagonian fauna have cited the occurrence of
Moniezia expansa in Lama guanicoe (Beldomenico et al., 2003; C. Ro-
bles, pers. comm.), Moniezia sp. and Thysanosoma actinoides with an
ovine source (Robles and Olaechea, 2001), Moniezia sp. in L. guanicoe
(Navone and Merino, 1989), and Cittotaenia quadrata in Lagidium vis-
cacia (Denegri et al., 2003).
The few Trichuris sp. eggs could not be diagnosed to species. Eggs
measurements do not conform to either T. trichiura or to T. vulpis,
parasites of humans and dogs, respectively (Thienpont et al., 1979).
Species with similar dimensions to the eggs recovered in the present
study, and with wild hosts inhabiting Patagonia and the Andes, are
summarized in Table I. Trichuris sp. eggs could occur due to rodent
consumption; rodent bones and fur were found in coprolite.
Eggs of Capillaria sp., and Uncinaria sp. were not identified to the
species level due to their morphometric diversity, different degree of
conservation, or both. The eggs with thick and smooth walls were not
identified to genus level because the width of the shell resembles Tox-
ascaris sp. or Physaloptera sp. These nematodes are ubiquitous para-
sites of canids (Thienpont et al., 1979; Stein et al., 1994; Ruas et al.,
2003; Valenzuela et al., 2004).
For the identification of hair and bones, we thank Alejandro Cane-
puccia. We acknowledge the reviewers corrections of an early version
of the manuscript. This work was supported by the project ‘‘Ecologı́a
Evolutiva Humana en Patagonia,’’ SECYT–UNMDP 04-09929 and the
‘‘Convenio de Colaboración entre el Instituto Canario de Bioantropol-
ogı́a del OAMC de Tenerife, España y la Facultad de Cs. Sociales de
la UNCPBA, Argentina.’’.
1112 THE JOURNAL OF PARASITOLOGY, VOL. 92, NO. 5, OCTOBER 2006

TABLE I. Species of Trichuris cited for the area under study, eggs measurements and hosts.

Trichuris Egg length ⫻ width

species (␮m) Host Reference

T. tenuis 46–50 ⫻ 28–30 Camelus dromedarius Chandler, 1930

— Lama guanicoe Beldomenico et al., 2003
51.9–79.0 (64.2 ⫾ 5.3) ⫻ L. glama Rickard and Bishop, 1991
28.4–37.1 (31.9 ⫾ 0.9)
L. glama; Vicugna vicugna Cafrune et al., 1999
Trichuris sp. 79.0 ⫻ 32.0 L. guanicoe Beldomenico et al., 2003
Lama sp. Leguı́a et al., 1995
T. bradleyi 57.0–65.0 ⫻ 29.0–34.0 Octodon sp.* Babero et al., 1975
T. fulvis 65.0–72.0 ⫻ 28.0–31.0 Ctenomys sp.* Babero and Murua, 1987
T. robusti 57.0–65.0 ⫻ 29.0–36.0 Ctenomys sp.* Babero and Murua, 1990
T. chilensis 60.0–67.0 ⫻ 32.0–34.0 Akodon sp.* Babero et al., 1976
T. bursacaudata 60.0–70.0 ⫻ 20.0–30.0 Ctenomys sp.* Suriano and Navone, 1994
T. pampeana 50.0–60.0 ⫻ 20.0–30.0 Ctenomys sp.* Suriano and Navone, 1994
T. myocastoris 53.0–60.0 ⫻ 30.0–34.0 Myocaster colpus* Barós et al., 1975
T. dolichotis 75.0 ⫻ 45.0 Dolichotis patagonum* Morini et al., 1955

* Rodents.

LITERATURE CITED and description. Memorias do Instituto Oswaldo Cruz 98(Supple-

ASCHERO, C. A. 1982. Nuevos datos sobre arqueologı́a del Cerro Casa
de Piedra, sitio CCP5. Relaciones 14: 267–284.
———. 1996. El área Rı́o Belgrano-Lago Posadas (Santa Cruz): Prob-
lemas y estado de problemas. In Arqueologı́a sólo Patagonia, J. G.
Otero (ed.). CENPAT, CONICET. Buenos Aires, Argentina, p. 17–
26.
———, C. BELLELI, AND R. A. GOÑI. 1992. Avances en las investiga-
ciones arqueológicos en el Parque Nacional Perito Moreno (prov-
incia de Santa Cruz, Patagonia, Argentina). Cuadernos del Instituto
Nacional de Antropologı́a y Pensamiento Latinoamericano 14:
143–170.
———, ———, M. T. CIVALERO, R. A. GOÑI, G. GURAIEB, AND R.
MOLINARI. 1992. ‘‘Cronologı́a y tecnologı́a en el Parque Nacional
Perito Moreno (PNPM): Continuidad o reemplazos?’’ Arqueologı́a
2: 89–106.
BABERO, B. B., AND R. MURUA. 1987. The helminth fauna of Chile. X.
A new species of whipworm from a Chilean rodent. Transactions
of American Microscopical Society 106: 190–193.
———, AND ———. 1990. A new species of whipworm from South
American hystricomorph rodent. Memorias do Instituto Oswaldo
Cruz 85: 211–213.
———, P. E. CATTAN, AND C. CABELLO. 1975. Trichuris bradleyi sp.
n., a whipworm from Octodon degus in Chile. Journal of Parasi-
tology 61: 1061–1063.
———, ———, AND ———. 1976. A new species of whipworm from
the rodent Akodon longipilis in Chile. Transactions of American
Microscopical Society 95: 232–235.
BARÓS, V., G. MAJUMDAR, AND T. K. MIKAILOV. 1975. Morphology ad
taxonomy of Trichocephalus myocastoris (Enigk, 1933). Folia Par-
asitologica 22: 207–213.
BATHURST, R. R. 2005. Archaeological evidence of intestinal parasites
from coastal shell middens. Journal of Archaeological Science 32:
115–123.
BELDOMENICO, P. M., M. UHART, M. F. BONO, C. MARULL, R. BALDI,
AND J. L. PERALTA. 2003. Internal parasites of free-ranging gua-
nacos from Patagonia. Veterinary Parasitology 118: 71–77.
CAFRUNE, M. M., D. H. AGUIRRE, AND L. G. RICKARD. 1999. Recovery
of Trichuris tenuis Chandler, 1930, from camelids (Lama glama
and Vicugna vicugna) in Argentina. Journal of Parasitology 85:
961–962.
CALLEN, E. O., AND T. W. M. CAMERON. 1960. A prehistoric diet re-
vealed in coprolites. New Scientist 8: 35–40.
CALLEN, E. O., AND P. S. MARTIN. 1969. Plant remains in some copro-
lites from Utah. American Antiguity 34: 329–331.
CHAME, M. 2003. Terrestrial mammal feces: A morphometric summary
ment I): 71–94.
CHANDLER, A. C. 1930. Specific characters in the genus Trichuris, with
a description of a new species, Trichuris tenuis, from a camel.
Journal of Parasitology 16: 198–206.
CHEHEBAR, C., AND S. MARTı́N. 1989. Guı́a para el reconocimiento mi-
croscpico de los pelos de los mamferos de la Patagonia. Doñana,
Acta Vertebrata 16: 247–291.
CIVALERO, M. T. 1995. ‘‘El sitio Casa de Piedra 7: algunos aspectos de
la tecnologı́a lı́tica y las estrategias de movilidad.’’ Cuadernos del
Instituto Nacional de Antropologı́a 16: 283–296.
———, AND C. ASCHERO. 2003. Early occupations at Cerro Casa de
Piedra 7, Santa Cruz Province, Patagonia, Argentina. In Where the
south winds blow, L. Miotti, M. Salemme, and N. Flegenheimer
(eds.). Texas A&M University, College Station, Texas, p. 141–147.
———, AND N. V. FRANCO. 2003. Early human occupations in western
Santa Cruz Province, southernmost South America. Quaternary In-
ternational 109–110: 77–86.
CORNEJO FARFÁN, A., AND P. JIMENEZ MILÓN. 2001. Dieta del zorro an-
dino Pseudalopex culpaeus (Canidae) en el matorral desértico del
sur de Perú. Revista de Ecologı́a Latinoamericana 8: 1–9.
DENEGRI, G. M. 2001. Cestodosis de herbı́voros domósticos de la Re-
pública Argentina de importancia en medicina veterinaria, Editorial
Martı́n, Mar del Plata, Argentina, 111 p.
———, M. C. DOPCHIZ, M. C. ELISSONDO, AND I. BEVERIDGE. 2003.
Viscachataenia n.g., with the redescription of V. quadrata (von
Linstow, 1904) n. comb. (Cestoda: Anoplocephalidae) in Lagidium
viscacia (Rodentia: Chinchillidae) from Argentina. Systematic Par-
asitology 54: 81–88.
EMMONS, L. H. 1987. Comparative feeding ecology of felids in a Neo-
tropical rainforest. Behavioral Ecology and Sociobiology 20: 271–
283.
FAULKNER, C. T. 1991. Prehistoric diet and parasitic infection in Ten-
nessee: Evidence from the analysis of desiccated human paleofeces.
American Antiquity 56: 687–700.
GONC˛ALVES, M. L. C., A. ARAÚJO, AND L. F. FERREIRA. 2003. Human
intestinal parasites in the past: New finding and a review. Memorias
do Instituto Oswaldo Cruz 98(Supplement I): 103–118.
HIDALGO-ARGUELLO, M. R., N. DYEZ-BAÑOS, J. FREGENEDA GRANDES,
AND E. PRADA MARCOS. 2003. Parasitological analysis of Leonese
royalty from collegiate-basilica of St. Isidoro, León (Spain): Hel-
minths, protozoa, and mites. Journal of Parasitology 89: 738–743.
LEGUı́A, P. G., A. E. CASAS, AND J. WHEELER. 1995. Parasitismo en
camélidos prehistóricos. Parasitologı́a al Dı́a 19: 435.
LUTZ, A. 1919. Schistosoma mansoni e a schistosomatose segundo ob-

servacoes feitas no Brasil. Memorias do Instituto Oswaldo Cruz
11: 121–155.
MATSUI, A., M. KANEHARA, AND M. KANEHARA. 2003. Paleoparasitology
in Japan. Discovery of toilet features. Memorias do Instituto Os-
waldo Cruz 98(Supplement I): 127–138.
MORINI, E. G., J. J. BOERO, AND A. RODRIGUEZ. 1955. Parásitos intes-
tinales en el ‘‘mara’’ (Dolichotis patagonum patagonum). Misión
de Estudios de Patologı́a Regional Argentina 26: 83–89.
NAVONE, G., AND M. L. MERINO. 1989. Contribución al conocimiento
de la fauna endoparasitaria de Lama guanicoe Muller, 1776, de
Penı́nsula Mitre, Tierra del Fuego, Argentina. Boletı́n Chileno de
Parasitologı́a 44: 46–51.
NOVARO, A. J. 1997. Pseudalopex culpaeus. Mammalian Species 558:
1–8.
REINHARD, K., P. R. GEIB, M. M. CALLAHAN, AND R. H. HEVLY. 1992.
Discovery of colon contents in a skeletonized burial: Soil sampling
for dietary remains. Journal of Archaeological Science 19: 697–
705.
RICKARD, L. G., AND J. K. BISHOP. 1991. Redescription of Trichuris
tenuis Chandler, 1930, from llamas (Lama glama) in Oregon with
a key to the species of Trichuris present in North American ru-
minants. Journal of Parasitology 77: 70–75.
ROBLES, C., AND F. OLAECHEA. 2001. Salud y enfermedad de las maja-
das. In Ganaderı́a sustentable en la Patagonia Austral, P. Borrelli,
and G. Oliva (eds.). INTA, Santa Cruz, Argentina, p. 225–243.
Host Specificity of Lepeophtheirus crassus (Wilson and Bere) (Copepoda: Caligidae)
Parasitic on the Marlin Sucker Remora osteochir (Cuvier) in the Atlantic Ocean
Ju-shey Ho, Bruce B. Collette*, and Ione Madinabeitia, Department of Biological Sciences, California State University, Long Beach,
California, 90840-3702; *National Marine Fisheries Service Systematics Laboratory, Smithsonian Institution, P.O. Box 37012, National Museum
of Natural History, MRC 0153, Washington, D.C., 20013-7012. e-mail: jsho@csulb.edu
ABSTRACT: Three species of remoras—Remora brachyptera (Lowe),
Remora osteochir (Cuvier), and Remora remora (Linnaeus)—were col-
lected from 4 species of billfishes—Istiophorus platypterus (Shaw),
Makaira nigricans Lacepéde, Tetrapturus albidus Poey, and Tetraptu-
rus pfluegeri Robins and de Sylva—on board a Japanese long-liner
Shoyo Maru during her cruise in 2002 across the Atlantic. However,
only the marlin sucker (R. osteochir) was found to carry a parasitic
copepod, Lepeophtheirus crassus (Wilson and Bere, 1936). Although
12 species of parasitic copepods have been reported from billfishes
around the world ocean, none of them is L. crassus. Thus, L. crassus
is considered a parasite specific to the marlin sucker.
Lepeophtheirus crassus (Wilson and Bere, 1936) was originally re-
ported by Bere (1936) as ‘‘Gloiopotes crassus Wilson and Bere, spec.
nov.’’ from the marlin sucker ‘‘Rhombochirus osteochir’’ attached to
‘‘a billfish (Tetrapturus imperator)’’ caught at Miami, Florida, and an-
other marlin sucker attached to ‘‘a sailfish’’ taken at Ft. Lauderdale,
Florida. Both adults and larval stages of copepods were obtained, with
the former found on the body surface of their host and the latter on the
gill filaments of the same host. The copepod parasite was subsequently
reported from ‘‘Echeneis albesens’’ taken in the Bay of Bengal at
16⬚40⬘N, 92⬚13⬘E by Shiino (1960) from an unidentified remora at-
tached to ‘‘Makaira audex’’ caught in the eastern Pacific at 19⬚34.3⬘S,
92⬚45.4⬘W; from ‘‘Remilegia australis’’ attached to ‘‘Delphinus bairdi’’
caught at 8⬚14⬘N, 84⬚17⬘W by Shiino (1963); and from ‘‘Rhombachirus
osteochir (Cuvier)’’ attached to a shortbill spearfish (Tetrapturus an-
gustirostris Tanaka, 1951) captured in the central Pacific at 21⬚04.5⬘N,
173⬚47.5⬘E by Lewis (1967). Shiino (1960) correctly proposed to trans-
fer the parasite from Gloiopotes to Lepeophtheirus because it lacks a
pair of dorsal aliform plates on the somite of leg 4.
In 2002, 1 of us (B.B.C.) had the opportunity to collect remoras on
RESEARCH NOTES 1113
RUAS, J. L, M. P. SOARES N. A. R. FARIAS, AND J. G. W. BRUM. 2003.
Infecc˛ão por Capillaria hepatica em carnı́voros silvestres (Lycal-
opex gymnocercus e Cerdocyon thous) na região sul do Rio Grande
do Sul. Arquivos do Instituto Biológico 70: 127–130.
SANTORO, C., S. D. VINTON, AND K. REINHARD. 2003. Inca expansion
and parasitism in the Lluta Valley: Preliminary data. Memorias do
Instituto Oswaldo Cruz 98(Supplement I): 161–164.
STEIN, M., D. M. SURIANO, AND A. J. NOVARO. 1994. Nematodes par-
ásitos de Duscycion griseus (Gray, 1837), D. culpaeus (Molina,
1782), y Conepatus chinga (Molina, 1782) (Mamı́fera: Carnı́vora)
en Neuquen, Argentina. Sistemática y Ecologı́a. Boletı́n Chileno
de Parasitologı́a 49: 60–65.
SURIANO, D. M., AND G. T. NAVONE. 1994. Three new species of the
genus Trihuris Roederer, 1761 (Nematoda: Trchuridae) from Cri-
cetidae and Octodontidae rodents in Argentina. Research and Re-
views in Parasitology 54: 39–46.
THIENPONT, D., F. ROCHETTE, AND O. F. J. VANPARIJS. 1979. Diagnóstico
de las helmintiosis por medio del examen coproparasitológico.
Janssen Research Foundation, Beerse, Bélgica, 187 p.
VALENZUELA, G., U. ALARCÓN, AND F. ALVAREZ. 2004. Helmintos par-
ásitos gastrointestinales en zorros Pseudalopex griseus de la Prov-
incia de Última Esperanza Sur de Chile. Actas del XIII Congreso
Chileno de Medicina Veterinaria. http://magallanes.sag.gob.cl/
cong㛮val.pdf.
ZIMMERMAN, M. R., AND R. E. MORILA. 1983. Enterobiasis in pre-Co-
lumbian America. Paleopathology Newsletter 42: 8.
J. Parasitol., 92(5), 2006, pp. 1113–1115
䉷 American Society of Parasitologists 2006
board a Japanese long-liner Shoyo Maru during her cruise across the
Atlantic. Remoras were collected mainly from 4 species of billfishes,
from 2 species of tunas, and 1 species each of swordfish, blue shark,
manta ray, loggerhead turtle, and long-line floats. Each remora removed
from its host was placed immediately in a cloth bag, labeled with the
station number and host, and placed in a bucket of seawater. Later, the
remoras were retrieved for sampling of tissues. Afterwards, the remoras
were replaced in the bags and fixed in 10% formalin. The copepods
were collected at the time of tissue sampling on board Shoyo Maru, in
the laboratory at the Smithsonian Institution, or both places.
It was noted that only those remoras removed from billfishes carried
a species of parasitic copepod. In total, 129 remoras belonging to 3
species were collected from 4 species of billfishes taken during that
cruise: (1) Remora brachyptera (Lowe, 1839) from Istiophorus platyp-
terus (Shaw, 1792), Makaira nigricans Lacepéde, 1802, and Tetrapturus
pfluegeri Robins and de Sylva, 1963; (2) R. osteochir (Cuvier, 1829)
from I. platypterus, M. nigricans, Tetrapturus albidus Poey, 1860, and
T. pfluegeri; and (3) Remora remora (Linnaeus, 1758) from I. platyp-
terus, M. nigricans, and T. albidus (see Table I).
Close examination of the collection revealed that only the marlin
sucker (R. osteochir) carries the parasitic copepod L. crassus. The com-
mon remora (R. remora) seems to be rare on billfish of the Atlantic
Ocean, but not the spearfish remora (R. brachyptera). In total, 21 spear-
fish remoras were collected from billfishes during that cruise, but none
of them carried L. crassus, even those from 4 stations LL 36, LL 37,
LL 38, and LL 39 where both R. osteochir and R. brachyptera were
found (see Table I). Thus, it seems L. crassus is host specific to R.
osteochir in the Atlantic Ocean, but not in the Pacific. Shiino (1963)
reported L. crassus from a different species of remora attached to a
dolphin in the eastern Pacific.
Although 12 species of parasitic copepods have been reported from
1114 THE JOURNAL OF PARASITOLOGY, VOL. 92, NO. 5, OCTOBER 2006

TABLE I. Billfishes taken during the 2002 Cruise of Shoyo Maru in the Atlantic and the remoras that they carried. Only Remora osteochir was
found carrying the parasitic copepod, L. crassus. The number in parentheses indicates the number of billfishes or remoras examined, and the letter
‘‘C⬙ stands for chalimus larva of the parasitic copepod.

Station Locality Billfish Remora sp. L. crassus

LL 1 29⬚57.36⬘N, 50⬚06.30⬘W Makaira nigricans (1) osteochir (1) 1乆

LL 4 34⬚35.35⬘N, 48⬚19.52⬘W Tetrapturus albidus (1) osteochir (2) 1乆
LL 6 34⬚02.18⬘N, 53⬚11.30⬘W T. albidus (1) osteochir (2) 3乆2么2C
LL 9 34⬚03.26⬘N, 57⬚22.42⬘W T. albidus (1) osteochir (2) 3乆2么8C
LL 13 33⬚54.51⬘N, 59⬚43.56⬘W T. albidus (1) osteochir (2) 0
LL 24 36⬚47.41⬘N, 45⬚39.35⬘W T. albidus (1) osteochir (2) 4乆1么
LL 27 33⬚25.44⬘N, 37⬚02.46⬘W Istiophoru platypterus (1) brachyptera (1) 0
LL 29 31⬚49.01⬘N, 27⬚57.57⬘W M. nigricans (2) brachyptera (1) 0
osteochir (1) 1么
LL 30 30⬚13.25⬘N, 28⬚45.31⬘W Tetrapturas pfluegeri (1) osteochir (2) 3乆1么2C
LL 31 7⬚29.35⬘N, 24⬚00.17⬘W M. nigricans (1) osteochir (2) 0
LL 33 4⬚50.09⬘N, 22⬚24.57⬘W I. platypterus (1) osteochir (2) 0
LL 36 8⬚05.27⬘S, 21⬚01.01⬘W I. platypterus (5) brachyptera (4) 0
osteochir (5) 3乆
T. albidus (3) osteochir (4) 2乆1么
remora (1) 0
LL 37 7⬚50.13⬘S, 21⬚09.22⬘W I. platypterus (2) brachyptera (1) 0
osteochir (1) 1乆1么1C
T. albidus (1) osteochir (2) 0
T. pfluegeri (3) osteochir (5) 7乆1么
LL 38 7⬚54.34⬘S, 21⬚14.27⬘W I. platypterus (1) brachyptera (1) 0
T. pfluegeri (6) brachyptera (1) 0
osteochir (12) 34乆8么2C
LL 39 8⬚17.38⬘S, 24⬚28.09⬘W I. platypterus (2) brachyptera (1) 0
osteochir (1) 0
M. nigricans (1) osteochir (2) 1乆2么
T. albidus (1) osteochir (2) 0
T. pfluegeri (1) osteochir (4) 5乆10么1C
LL 40 7⬚40.17⬘S, 25⬚39.06⬘W I. platypterus (2) brachyptera (2) 0
osteochir (1) 0
LL 41 7⬚44.07⬘S, 22⬚17.55⬘W I. platypterus (2) osteochir (2) 0
T. albidus (1) osteochir (2) 0
T. pfluegeri (5) osteochir (4) 7乆6么5C
LL 42 7⬚58.20⬘S, 21⬚03.27⬘W T. pfluegeri (3) brachyptera (2) 0
osteochir (2) 20乆3么
LL 43 8⬚30.38⬘S, 21⬚47.30⬘W M. nigricans (1) brachyptera (1) 0
T. pfluegeri (2) brachyptera (1) 0
osteochir (1) 7乆
LL 44 8⬚26.33⬘S, 21⬚57.35⬘W T. pfluegeri (1) osteochir (1) 9乆2么
LL 45 8⬚15.17⬘S, 22⬚00.18⬘W I. platypterus (1) brachyptera (1) 0
T. pfluegeri (4) brachyptera (2) 0
osteochir (3) 2么
T. albidus (1) osteochir (2) 0
LL 46 8⬚33.36⬘S, 29⬚12.29⬘ W T. pfluegeri (1) osteochir (2) 1乆
LL 47 8⬚39.00⬘S, 29⬚19.12⬘W M. nigricans (1) osteochir (2) 8乆3么2C
T. albidus (1) osteochir (2) 1么
LL 48 8⬚41.24⬘S, 29⬚18.00⬘W I. platypterus (1) remora (1) 0
T. albidus (2) osteochir (5) 1乆1么1C
T. pfluegeri (5) osteochir (8) 5乆
LL 49 9⬚09.36⬘S, 29⬚18.00⬘W T. pfluegeri (1) osteochir (3) 9乆1么1C
LL 50 9⬚30.00⬘S, 29⬚30.36⬘W T. pfluegeri (1) brachyptera (1) 0
T. albidus (1) osteochir (2) 0
LL 51 4⬚16.48⬘N, 41⬚24.36⬘W T. albidus (1) osteochir (2) 0
LL 52 3⬚09.36⬘N, 40⬚19.12⬘W T. albidus (1) osteochir (1) 0
LL 53 3⬚23.24⬘N, 40⬚17.24⬘W I. platypterus (1) osteochir (1) 8乆3么2C
M. nigricans (2) osteochir (2) 0
remora (1) 0
LL 56 14⬚52.12⬘N, 47⬚52.48⬘W M. nigricans (1) osteochir (2) 0

TABLE II. Copepod parasites of billfishes (Istiophoridae).
Order Siphonostomatoida Thorell, 1859
Caligidae Burmeister, 1834
Caligus quadratus Shiino, 1954
Lepeophtheirus eminens Wilson, 1944
Dissonidae Yamaguti, 1963
Dissonus glaber Kurtz, 1924
Euryphoridae Wilson, 1905
Euryphorus brachypterus (Gerstaecker, 1853)
Gloiopotes americanus Cressey, 1967
Gloiopotes huttoni (Thomson, 1889)
Gloiopotes ornatus Wilson, 1905
Pandaridae Milne Edwards, 1840
Pandarus satyrus Dana, 1852
Pennellidae Burmeister, 1834
Lernaeolophus sultanus (Nordmann, 1839)
Pennella biloba (Kirtisinghe, 1933)
Pennella filosa (Linnaeus, 1758)
Pennella makaira Hogans, 1988
billfishes in oceans around the world, none of them is L. crassus (see
Table II). Thus, it is safe to say that L. crassus is a parasite of marlin
sucker and not billfish. As reported by Bere (1936), we have also found
that although adult and juvenile L. crassus attach to the body surface
of R. osteochir, the larval (chalimus) stages were all parasitic on the
Variation in Eimeria Oocyst Count and Species Composition in Weanling Beef Heifers
A. S. Lucas, W. S. Swecker*, G. Scaglia†, D. S. Lindsay, and A. M. Zajac‡, Department of Biomedical Sciences and Pathobiology, Virginia-
Maryland Regional College of Veterinary Medicine, Virginia Tech, Duck Pond Drive, Blacksburg, Virginia 24061-0442; *Department of Large
Animal Clinical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Duck Pond Drive, Blacksburg, Virginia
24061-0442; and †Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, Virginia 24061-0306; ‡To whom correspondence
should be addressed. e-mail: azajac@vt.edu
ABSTRACT: Rectal fecal samples were collected daily on 10 consecu-
tive days in November 2004 from 11 weaned beef heifers to assess
daily variation in fecal oocyst count and species composition. Subse-
quent samples were collected from the same animals on 15 April 2005
and 9 June 2005. Oocyst numbers were determined by the modified
McMaster’s test, and species were identified by examination of oocysts
recovered with the Wisconsin sugar flotation technique. Soil samples
were collected from the heifer pasture on 8 June 2005, and oocysts
were quantified and identified to species. Mean fecal oocyst counts var-
ied little at all sampling dates ranging from 134–377 oocysts/g. Ten
Eimeria spp. were identified in fecal samples collected in November
and April and 11 in June. Eimeria bovis was the most common species
identified at all samplings. Mean species composition showed little var-
iation during the 10-day sampling period in November, remained similar
in April, and varied slightly in June. Twelve Eimeria spp. were iden-
tified in soil samples in proportions similar to those seen in fecal sam-
ples. The results indicate that clinically normal weanling beef heifers
are likely to be infected with a diverse, but relatively stable, community
of Eimeria spp.
Species of Eimeria are gastrointestinal coccidians that infect cattle
worldwide. Reports indicate that calves are infected shortly after birth
and shed relatively high numbers of oocysts during their first year of
life (Fitzgerald, 1962; Ernst et al., 1984, 1987; Diaz de Ramirez et al.,
2001). Most studies reporting species prevalence have found that E.
bovis is the most prevalent species in calves less than 1 yr of age based
RESEARCH NOTES 1115
marlin sucker’s gill filaments. Shiino (1960) provided an excellent re-
description of L. crassus, and our specimens show no significant dis-
crepancy from that redescription.
Permission for B.B.C. to participate in leg 3 of the Shoyo Maru long-
line cruise and to collect remoras was received from Hiroyuki Kinoshita
(Japanese Fisheries Agency) and Hiroaki Okamoto and Kotaro Yokawa
(National Research Institute of Far Seas Fisheries, Shizuoka). In addi-
tion to their own research, remoras were kindly collected on leg 1 of
the cruise by Hiroaki Okamoto, on leg 2 by Lisa Natanson (National
Marine Fisheries Service, Narraganset), and on leg 4 by Maki Ohwada.
Hiroaki Okamoto, Kotaro Yokawa, and Kouichi Hoshino all helped pro-
vide station data for the billfishes collected on the cruise. Completion
of the manuscript of this paper was aided by a grant from the Paramitas
Foundation to J.S.H.
LITERATURE CITED
BERE, R. 1936. Parasitic copepods from Gulf of Mexico fish. The Amer-
ican Midland Naturalist 17: 577–625.
LEWIS, A. G. 1967. Copepod crustaceans parasitic on teleost fishes of
the Hawaiian Islands. Proceedings of the United States National
Museum 121: 1–204.
SHIINO, S. M. 1960. Copepods parasitic on remoras from the Bay of
Bengal. Report of the Faculty of Fisheries, Prefectural University
of Mie 3: 542–552.
———. 1963. Parasitic copepods of the eastern Pacific fishes. 1. Re-
cords of the known species. Report of the Faculty of Fisheries,
Prefectural University of Mie 4: 335–347.
J. Parasitol., 92(5), 2006, pp. 1115–1117
䉷 American Society of Parasitologists 2006
on identification of fecal oocysts (Fitzgerald, 1962; Ernst et al., 1987;
Hasbullah et al., 1990; Diaz de Ramirez et al., 2001). In contrast, Parker
et al. (1984), reported that Eimeria zuernii was the most prevalent oo-
cysts in the feces of freshly weaned beef calves in Australia, and Svenn-
son et al. (1993) found E. alabamensis most prevalent in dairy calves
(4–16 mo of age) just after turnout in Sweden. No reports exist, how-
ever, that describe daily variation in fecal oocyst counts and species
composition in calves. The objective of the present study was to assess
daily variation in fecal oocyst count and species composition in clini-
cally normal weanling beef calves and to determine whether changes
occurred after a period of several months.
Eleven beef heifers (Angus, Charolais, and Hereford breeding), main-
tained at the Virginia Tech Beef Center, Blacksburg, Virginia, were
surveyed for natural coccidia infection. In the previous year, no out-
breaks of coccidiosis had been observed at this facility. Heifers (287 ⫾
19 days of age at the beginning of the study) were sampled for a period
of 10 consecutive days (15 November 2004 to 24 November 2004).
Subsequent samples were collected from 10 of the same heifers 5 mo
later (15 April 2005) and from 9 of the heifers 7 mo later (9 June 2005).
Heifers were weaned in September 2004 and maintained together on a
permanent native grass lot used every year for replacement animals with
free access to water and mineral mix. The heifers were fed daily hay
and corn silage. All heifers remained on the same lot from November
to June at which time they were moved to summer pastures. They were
clinically normal throughout the course of the survey. All fecal samples
were collected per rectum from the cattle at approximately 0930 hr.
1116 THE JOURNAL OF PARASITOLOGY, VOL. 92, NO. 5, OCTOBER 2006

TABLE 1. Prevalence of Eimeria spp. in fecal samples, and mean percentage of oocysts identified per sample for heifers sampled from 15 to 24
November 2004, 15 April 2005, and 9 June 2005.

15–24 November 2004 15 April 2005 9 June 2005

Prevalence Mean oocysts/ Prevalence Mean oocysts/ Prevalence Mean oocysts/
Eimeria spp. (%) sample (%) (%) sample (%) (%) sample (%)

E. bovis 100.0 39.7 100.0 41.5 100.0 27.3

E. zuernii 96.0 13.4 90.0 7.9 89.0 6.9
E. alabamensis 91.0 15.3 90.0 12.2 89.0 5.0
E. auburnensis 91.0 10.2 100.0 5.8 100.0 13.6
E. cylindrica/ellipsoidalis-like 80.0 9.2 100.0 17.5 78.0 3.4
E. canadensis 67.0 7.1 100.0 8.4 100.0 21.8
E. subspherica 46.0 2.0 40.0 1.6 0.0 0.0
E. wyomingensis 18.0 1.7 30.0 1.6 22.0 4.0
E. pelita 34.0 1.1 30.0 0.8 33.0 3.0
E. bukidonensis 13.0 0.4 0.0 0.0 33.0 4.8
E. brasiliensis 0.0 0.0 20.0 2.8 89.0 8.9
E. illinoisensis 0.0 0.0 0.0 0.0 33.0 3.0

Oocyst number in each fecal sample was calculated using the modified
McMaster’s technique with a sensitivity of 25 oocysts/g (OPG) (Whit-
lock, 1948). A modified Wisconsin sugar flotation technique (Coxx and
Todd, 1962) also was performed on each fecal sample, and 50 Eimeria
spp. oocysts per sample were examined at ⫻400 and identified to spe-
cies based on oocyst morphology (Levine and Ivens, 1986). One indi-
vidual (A.L.) carried out all oocyst counts and species identifications.
Because oocysts of E. cylindrica and E. ellipsoidalis could not be dis-
tinguished reliably, all small cylindrical Eimeria spp. oocysts measuring
from 19 to 36 ␮m by 8–18 ␮m were designated E. cylindrica/ellipso-
idalis-like (Levine and Ivens, 1986).
Soil samples (approximately 100 g each) also were collected from
different sites in the replacement heifer lot on 8 June 2005, shortly after
the heifers were removed. All samples were collected from the top 2.5
cm of soil. A general field sample was obtained by collecting approx-
imately 10 g of soil every 10 m in a diagonal across the field. Soil
around the feed bunk and hay ring also was sampled by collecting
approximately 10-g subsamples taken every 2 m from a distance of
approximately 3 m away from and along the length of the feed bunk
and around the entire perimeter of the hay ring. Approximately 10 g of
soil also was collected from beneath 10 fecal pats that were several
days old and selected on a first seen basis while walking a diagonal
across the field. For each area (whole pasture, feed bunk area, hay ring
area, and fecal pats), samples were mixed well and a 20-g subsample
was analyzed using the modified Wisconsin sugar flotation technique
(Coxx and Todd, 1962). Oocysts were identified to species based on
bovine Eimeria spp. oocyst morphological characteristics (Levine and
Ivens, 1986).
Eimeria spp. oocysts were seen in 94% of fecal samples collected
during the 10-day period. Heifer fecal oocyst counts ranged from 0–
FIGURE 1. Mean daily species compositions for heifers sampled 15
November 2005 to 24 November 2005.
700 OPG throughout the sampling period with an overall mean oocyst
count of 192 OPG. Arithmetic mean daily oocyst counts for the heifers
remained similar over the course of the 10-day sampling period, ranging
from 134–377 OPG. Similarly, oocyst counts from individual animals
exhibited little variation over the course of the 10 days, with more
variation in oocyst count seen between individuals within days. Those
animals with relatively high oocyst counts at the start of the study main-
tained higher oocyst counts throughout, whereas those that began with
lower oocyst counts maintained low levels over the sampling period.
These individuals also maintained their relative high or low oocyst
shedding tendency in samples collected in April and June. Ten Eimeria
spp. were recovered from the heifers during this period (Table I). Ei-
meria bovis was the most common species found, present in 100% of
all positive samples. Eimeria zuernii was the second most common
species (96%), followed by E. alabamensis and E. auburnensis (91%),
E. cylindrica/ellipsoidallis-like (80%), and E. canadensis (67%). All
other species were present in fewer than 50% of positive samples.
Mean daily species compositions for the 10-day sampling period are
presented in Figure 1. Eimeria bovis was the most numerous species
identified on all 10 days, with a mean of 40% of the oocysts identified
in each sample being E. bovis. The second most numerous species var-
ied slightly from day to day among E. zuernii, E. alabamensis, and E.
cylindrica/ellipsoidallis-like. Overall, the mean species composition ex-
hibited little daily variation.
Results from the 10 heifers sampled on 15 April 2005 were generally
similar to the results obtained from the 10-day sampling period in No-
vember (Table I). The mean oocyst count (145 OPG) and species com-
position showed little change. Nine of the 10 Eimeria spp. previously
seen were present (E. bukidonensis not seen). Oocysts of E. brasiliensis,
which were not seen the previous November, were seen in 2 of the 10
animals. On 6 June 2005, the mean fecal oocyst count for the 9 heifers
sampled was 173 OPG. Eimeria bovis made up the highest mean per-
centage of oocysts identified per sample, but the mean percentage of E.
canadensis was only slightly lower (Table I). Oocysts of E. canadensis
were first detected on 18 November 2004, and the proportion seemed
to increase from November to the following June. In November, E.
canadensis oocysts made up less than 10% of the oocysts counted in 8
of 11 animals. In June, however, only 1 of the 9 animals had less than
10% E. canadensis and 6 heifers had greater than 20%. No animals had
a lower percentage of E. canadensis in June compared with November.
All soil samples analyzed from the sites around the heifer’s pasture
contained coccidia oocysts. Eighty-four percent of the oocysts identified
were sporulated. The sample collected from the feed bunk had the most
coccidia oocysts (1,532 oocysts in 20 g of soil). Samples analyzed from
the hay ring, beneath fecal pats and from the field contained 284, 232,
and 115 oocysts per 20 g of soil, respectively. The species composition
was similar among all 4 sites. In total, 12 Eimeria spp. were seen in
these samples. The mean percentages of oocysts identified in samples
from all sites are presented in Table II. The percentages of oocysts
 RESEARCH NOTES 1117

TABLE II. Mean percentage of oocysts identified in soil samples from Jones, 1987). Little information is available on levels of oocyst shed-
representative sites on the heifer lot. ding in animals during their first year after weaning. Oocysts counts
were low in all samples, but they may have been higher if samples were
collected at times of stress such as weaning. The results of this study
Eimeria spp. Mean oocysts/sample (%)
suggest that weanling heifers maintain a low level infection with a di-
E. bovis 40.6 verse community of Eimeria spp. Although the pathogenic species (E.
bovis and E. zuernii) predominated, no clinical disease was observed
E. cylindrica/ellipsoidalis-like 15.0
throughout the study. Soil samples from the heifer’s pasture indicate
E. alabamensis 12.1 that there is a diverse community of infective oocysts present in the
E. zuernii 12.0 animal’s environment. The persistent low level infections and continued
E. auburnensis 4.7 reexposure in clinically normal animals suggest that by 9 mo of age,
E. subspherica 4.7 host and parasite have reached a relatively stable equilibrium.
E. canadensis 2.5 We thank the staff of the Virginia Tech Beef Center for coordinating
E. pelita 2.4 animal husbandry. This research was part of a regional initiative, Pas-
E. illinoisensis 2.4 ture-Based Beef Systems for Appalachia, funded in part by USDA–
E. brasiliensis 1.6 ARS.
E. wyomingensis 1.4
E. bukidonensis 0.5 LITERATURE CITED
COXX, D. D., AND A. C. TODD. 1962. Survey of gastrointestinal para-
sitism in Wisconsin dairy cattle. Journal of the American Veteri-
identified in these samples were similar to the percentages reported for nary Medical Association 141: 706–709.
the heifer fecal samples, with E. bovis predominating. Although coc- DIAZ DE RAMIREZ, A., A. HERNANDEZ, A. GARCIA, AND L. N. RAMIREZ-
cidian oocysts of wild mammals and birds may have been present in IGLESIA. 2001. Excretion of oocysts of Eimeria spp. during the first
the soil and could not be differentiated from bovine Eimeria spp., the three months of life in calves from diary farms in western Vene-
similarity in species distribution between manure and soil samples sug- zuela. Revista Cientifica, Facultad de Ciencia Veterinarias, Univ-
gests that most of the recovered oocysts were of bovine origin. ersidad del Zulia 11: 207–212.
There are no published reports describing short term daily variation ERNST, J. V., H. CIORDIA, AND J. A. STUEDEMANN. 1984. Coccidia in
in fecal oocyst count or Eimeria spp. composition in cattle. The results cows and calves on pasture in north Georgia (U.S.A.). Veterinary
of this study, conducted on weanling beef heifers, found little daily Parasitology 15: 213–221.
variation in both fecal oocyst count and species composition over a 10- ———, T. B. STEWART, AND D. R. WHITLOCK. 1987. Quantitative de-
day period. Minimal variation was seen during this period, thus the next termination of coccidian oocysts in beef calves from the coastal
2 samplings were carried out on a single day to assess longer-term plain are of Georgia (U.S.A.). Veterinary Parasitology 23: 1–10.
changes in oocyst count and species composition. Little change was FITZGERALD, P. R. 1962. Coccidia in Hereford calves on summer and
seen in mean fecal oocyst counts from the same heifers sampled ap- winter ranges in feedlots in Utah. Journal of Parasitology 48: 347–
proximately 5 and 7 mo later, although some differences in mean spe- 351.
cies composition was observed. Eimeria brasiliensis and E. illinoisensis HASBULLAH, Y. AKIBA, H. TAKANO, AND K. OGIMOTO. 1990. Seasonal
were not seen in the first sampling, but they were more common and distribution of bovine coccidia in beef cattle herd in the university
numerous in subsequent samples. Likewise, E. canadensis became al- farm. Japanese Journal of Veterinary Science 52: 1175–1179.
most as numerous as E. bovis by June (Table 1). This suggests that LEVINE, N. D., AND V. IVENS. 1986. The coccidian parasites (Protozoa,
there may be host and/or environmental factors that lead to gradual Apicomplexa) of Artiodactyla. Illinois Biological Monographs No.
changes in the dominant Eimeria spp. present in cattle as they age. 55, University of Illinois Press, Urbana, Illinois, 66 p.
Results of this study also indicate that weanling beef heifers are infected PARKER, R. J., K. BOOTHBY, I. POLKINGHORNE, AND R. G. HOLROYD.
with a diverse community of Eimeria spp. This diversity is consistent 1984. Coccidiosis associated with postweaning diarrhoea in beef
with surveys of cattle from around the world (Ernst et al., 1984; Parker calves in a dry tropical region. Australian Veterinary Journal 61:
et al., 1984; Hasbullah et al., 1990; Svensson et al., 1993). 181–183.
Soil samples collected from various locations on the heifer’s lot also ———, AND G. W. JONES. 1987. The development of Eimerian infec-
had a diverse community of Eimeria spp. oocysts. Oocysts of all 12 tions during the first eight months of life in unweaned beef calves
Eimeria spp., identified in fecal samples collected over the course of in a dry tropical region of Australia. Veterinary Parasitology 25:
the whole study, also were seen in similar proportions in these soil 1–7.
samples. This result, together with the fact that the majority (84%) of SVENSSON, C., P. HOOSHMAND-RAD, B. PEHRSON, M. TORNQUIST, AND A.
oocysts recovered from the soil were sporulated, suggests that oocysts UGGLA. 1993. Excretion of Eimeria oocysts in calves during their
in the environment sporulate and remain viable in proportions similar first three weeks after turn-out to pasture. Acta Veterinaria Scan-
to that passed in the feces, at least in the warmer months of the year. dinavia 34: 175–182.
Most published reports dealing with the epidemiology of coccidia WHITLOCK, H. V. 1948. Some modifications of the McMaster helminth
infections in beef cattle focus on cow/calf pairs or freshly weaned calves egg-counting technique apparatus. Journal of the Council for Sci-
(Fitzgerald, 1962; Parker et al., 1984; Ernst et al., 1987; Parker and entific and Industrial Research 21: 177–180.