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1104 THE JOURNAL OF PARASITOLOGY, VOL. 92, NO.

5, OCTOBER 2006

J. Parasitol., 92(5), 2006, pp. 1104–1107


䉷 American Society of Parasitologists 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
FIGURE 1. (A) Hamster erythrocytes infected with WA1 merozoites
microscopy or gold-coated for scanning electron microscopy (SEM).
96 hr PI. TEM (bar ⫽ 2 ␮m). (B) Hamster erythrocytes uninfected at
Shape and surface membrane changes in erythrocytes were demonstrat-
time 0. TEM (bar ⫽ 2 ␮m).
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- time interval by counting 400–500 stained erythrocytes on blood smears
branes and Babesia WA1 organisms were seen at different stages of (Protocol-Hema 3, Fisher, Pittsburgh, Pennsylvania). Fresh hamster
development within erythrocytes, from single trophozoites to several blood (20 ␮l) was fixed in chilled modified-Karnovsky’s-cacodylate fix-
merozoites (young trophozoites), some of them dividing to form typical ative (a 50:50 solution of 2% paraformaldehyde and 2.5% glutaralde-
tetrads. In general, Babesia WA1 induced severe morphological changes hyde in 0.1 M cacodylate buffer), washed in fresh fixative, and postfixed
in the erythrocytes, and these changes were more evident in almost all twice in cold (4 C) 1% osmium tetroxide-cacodylate-buffered fixative
infected cells 96 hr PI. 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
Babesia Washington-1 (WA1) was first isolated from a human patient to 70% alcohol solution. The specimens were then infiltrated with 3
in Washington state and since 1991 has been included with Babesia increasing concentrations of resin in acetone, 2:1, 1:1, and 1:2 (treat-
microti as the causative agents of human babesiosis in the United States ment periods of 2–12 hr) ending in 100% resin (treatment period of 8–
(Quick et al., 1993). The transmission of WA1 was reported to occur 12 hr). All samples were then embedded in Epon LX112 (EMS, Fort
via blood transfusion, and it is difficult to detect among asymptomatic Washington, Pennsylvania) embedding medium at 45 C for 24 hr and
blood donors (Kjemtrup et al., 2002). Other possible transmission routes then at 60 C for another 24 hr. Embedded tissues were trimmed and
and the identity of the tick vector and the host reservoir remain un- sectioned on an ultramicrotome (Ultracut E-Reichert-Jung, Vienna, Aus-
known (Quick et al., 1993). In contrast to B. microti, WA1 is extremely tria). Ultrathin sections (80–85 nm) were placed on copper grids and
virulent to hamsters (Mesocricetus auratus), producing high mortality stained with uranyl acetate and lead citrate. Grids were examined by
by day 10 postinoculation (PI) (Pruthi et al., 1995). Hamsters infected transmission electron microscopy (TEM) with a Hitachi H-300. For
with WA1 developed high parasitemia and hemolytic anemia (Dao and scanning electron microscopy (SEM), the initial steps were similar to
Eberhard, 1996), signs characteristic of acute human babesiosis (Pruthi those described above, but after osmium fixation and washes, samples
et al., 1995; Dao, 1996). Babesia WA1 is morphologically similar to B. were placed on 1% poly-L-lysine coverslips for 2 hr and rinsed again
microti but molecularly and antigenically distinct. Moreover, its phy- in distilled water and dehydrated with increasing concentrations of ethyl
logenetic tree is closely related to Babesia gibsoni, a common dog alcohol solutions. Samples were then treated twice for 5 min with hex-
erythrocyte piroplasm (Walter et al., 2001; Goethert and Telford, 2003). amethyldesilazane, mounted in aluminum mounts and sputter coated
The ultrastructure of B. microti is well documented in host erythro- with gold (Edwards 150A sputter coater equipment) and viewed in the
cytes and in ticks (Rudzinska et al., 1976, 1979; Rudzinska and Trager, Hitachi’s H3010 accessory.
1977). Morphological hallmarks of Babesia spp. infection in erythro- Parasitemia in blood smears showed 0, 2.5, 5, 10, 12.5, 22.5, 70, and
cytes are the presence of vacuole(s) within an organism and the patho- almost 100% parasitemic erythrocytes at 0, 24, 36, 48, 60, 72, 84, and
gnomonic tetrads (‘‘Maltese cross’’), which are 4 daughter merozoites 96 hr PI, respectively. ‘‘Maltese cross’’ structures were observed in
that remain attached to each other after division (Pantanowitz et al., blood smears starting 72 hr PI. Single WA1 trophozoites were observed
2002). Tetrads are not universal to all Babesia species, but they are in the cytoplasm of some erythrocytes during the first 48 hr, but mul-
abundant in WA1 compared with B. microti (Healy and Ruebush, 1980). tiple-merozoite-infected erythrocytes were observed frequently 72 hr PI
In the present study, 2 female 1-month-old hamsters were inoculated with changes in the normal shape of the erythrocyte (Fig. 1A, B). Mer-
intraperitoneally (i.p.) with cryopreserved hamster blood containing 1.8 ozoites were amoeboid, uninucleated, vacuolated, and had host cell cy-
⫻ 109 WA1-infected erythrocytes. The Babesia WA1 strain was origi- toplasm. In contrast, trophozoites were multinucleated, nonvacuolated,
nally isolated from a blood sample of a human patient from the state and had a high degree of polymorphism. Externally, almost all eryth-
of Washington. Saphenous vein blood samples were obtained at 0, 24, rocytes revealed surface membrane changes 96 hr PI (Figs. 2, 3). Severe
36, 48, 60, 72, 84, and 96 hr PI. Parasitemias were determined at each alterations in the surface membrane of erythrocytes also included mul-
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
FIGURE 2. Severely affected erythrocytes 96 hr PI showing protru- space left by the invaginated host cytoplasm (Fig. 5D, E). The presence
sions (white arrows), protrusions inside perforations (black arrows), and of protrusions inside perforations was commonly observed by the SEM
1 erythrocyte with multiple perforations. SEM (bar ⫽1 ␮m). (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-
tiple perforations, giving them a ‘‘swiss cheese’’ appearance (Fig. 2). ing the surface membrane (Fig. 6A, B) and closing the new vacuole by
TEM studies of these multiple-perforated erythrocytes revealed severe approximation of anterior and posterior ends, emitting pseudopods, or
and multiple invaginations of the erythrocyte plasma membrane with both (Fig. 6A–C). The mature trophozoites originated from 4 new mer-
the presence of many merozoites (Fig. 3). Free parasites were observed ozoites by tetrad division, an event observed only after 72 hr PI (Fig.
singly or in clusters (Fig. 4). Parasites invading erythrocytes were ob- 7A). New rosettelike merozoites were recognized by the single nuclei
served with an invagination of the erythrocyte surface membrane that and vacuole. After division, the parasites developed apical complexes
was in close contact with the plasma membrane of the organism (Fig. characterized by organelle formation of rhoptries and micronemes (Fig.
5A, B), and vacuolization of the parasite cytoplasm (Fig. 5B, C). The 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)
FIGURE 3. Severe distortions in 2 erythrocytes 96 hr PI caused by is observed in 1 parasite beside an invaginated cytoplasm (arrow). An-
multiple invaginations of the cell membrane (black arrows) and the other parasite is observed (N, nucleus). TEM (bar ⫽ 0.5 ␮m). (F) A
presence of merozoites in the cytoplasm (white arrows). TEM (HC, host protrusion (arrow) beside a perforation in an infected erythrocyte 72 hr
cytoplasm invaginated in 1 parasite; bar ⫽ 0.5 ␮m). 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- FIGURE 7. (A) A dividing trophozoite forming a tetrad 96 hr PI.
asites 96 hr PI. The HC is invaginated (arrowhead) by 1 parasite that Two merozoites with host cytoplasm (HC) vacuoles are observed. TEM
‘‘closes’’ (arrow) the forming vacuole. TEM (bar ⫽ 0.5 ␮m). (B) Vac- (bar ⫽ 0.5 ␮m). (B) Trophozoite dividing in 4 merozoites (N, nucleus)
uolization of the HC in 1 merozoite 96 hr PI. Pseudopods can be ob- with formation of apical complexes (arrows) 96 hr PI. TEM (bar ⫽ 0.5
served (arrows) in the forming vacuole. TEM (bar ⫽ 0.5 ␮m). (C) ␮m). (C) Four merozoites with HC vacuoles and a trophozoite in di-
Merozoites with vacuolized HC 96 hr PI. Two closed points of the vision (N) 96 hr PI. TEM (bar ⫽ 0.5 ␮m). A magnification of the
vacuoles are observed (arrows). N, nucleus. TEM (bar ⫽ 0.5 ␮m). damaged erythrocyte membrane (arrowheads) is detailed inside the box
near the apical complex of the parasite (arrow) (bar ⫽ 0.125 ␮m).

erythrocyte membrane and shape, including protrusions, perforations, acteristic ‘‘tetrads.’’ Interestingly, some merozoites, still attached to
or both. each other by the posterior end, were closer to the erythrocyte mem-
Extensive damage to the erythrocyte membrane, characterized as pro- brane. In these parasites, the apical complex was well formed with the
trusions, inclusions, and perforations, have been reported with B. mi- presence of rhoptries and micronemes, which seem to play an important
croti infection of human erythrocytes (Sun et al., 1983). In the present role during invasion of B. microti (Rudzinska et al., 1976). These or-
study, we observed multiple-perforated erythrocytes with an increased ganelles contain enzymes that facilitate exit from the host cell, and their
convex shape. To our knowledge, there are no other reports of babesial position could explain the damage to the cell membrane in contact with
infections that cause this type of damage to erythrocytes. Damage to the anterior end of the parasite even causing an evagination that mirrors
erythrocyte membranes seemed to correlate with an increase in the per- its shape.
centage of parasitemia. Diffuse and pronounced vascular stasis with In conclusion, Babesia WA1 was demonstrated to be extremely vir-
massive sequestration of erythrocytes was the cause of severe anoxia ulent in hamsters, inducing severe ultrastructural changes in the eryth-
and damage to vital organs in hamsters infected with WA1 (Dao and rocyte membrane and shape. The parasite showed a high degree of
Eberhard, 1996). division, pleomorphism, and invasiveness, with almost all erythrocytes
Previous reports in mice infected with WA1 described endothelial parasitized 96 hr PI.
cell changes associated with pulmonary edema and respiratory distress We are indebted to S. Schul for technical support and to D. Mosier
(Hemmer et al., 1999). In fatal cases, some key inflammatory cytokines and J. Green for comments and discussion. This work was supported
such as tumor necrosis factor ␣ may play an important role in the path- in part by National Institutes of Health grant P20RR016443-04.
ogenesis associated with WA1 (Hemmer et al., 2000). The differential
expression of certain inflammatory mediators in susceptible and resis-
LITERATURE CITED
tant strains of mice could explain the different outcome of WA1 infec-
tion (Moro et al., 1998). DAO, A. H. 1976. Human babesiosis. Comprehensive Therapy 22: 713–
The presence of parasites attached to the erythrocyte membrane and 718.
the process of invasion of erythrocytes apparently did not differ as much ———, AND M. L. EBERHARD. 1996. Pathology of acute fatal babesiosis
as was described for B. microti (Rudzinska et al., 1976). The invagi- in hamsters experimentally infected with the WA-1 strain of Ba-
nation of the plasma membrane of the erythrocyte produces a parasi- besia. Laboratory Investigation 74: 853–859.
tophorus vacuole that contains the merozoite covered by its own mem- GOETHERT, H. K., AND S. R. TELFORD, III. 2003. What is Babesia mi-
brane and the host plasma membrane. This event appears to occur im- croti? Parasitology 127: 301–309.
mediately after attachment since we observed few parasites in this pro- HEALY, G. R., AND T. K. RUEBUSH, II. 1980. Morphology of Babesia
cess compared with the high number of parasitized erythrocytes. Inside microti in human blood smears. American Journal of Clinical Pa-
the cell, the merozoite lies beside the invaginated space observed as thology 73: 107–109.
protrusions coming from the walls of the perforated erythrocyte surface. HEMMER, R. M., D. A. FERRICK, AND P. A. CONRAD. 2000. Up-regulation
In the process of invagination, the cytoplasm of the erythrocyte is se- of tumor necrosis factor-alpha and interferon-gamma expression in
verely displaced. Similarly, the perforated cells underwent severe the spleen and lungs of mice infected with the human Babesia
changes in shape caused by the presence of more than 1 intracellular isolate WA1. Parasitology Research 86: 121–128.
parasite. ———, E. J. WOZNIAK, L. J. LOWENSTINE, C. G. PLOPPER, V. WONG,
The vacuoles in the merozoites observed in our study were portions AND P. A. CONRAD. 1999. Endothelial cell changes are associated
of host cytoplasm generated during the invasion process as described with pulmonary edema and respiratory distress in mice infected
(Healy and Ruebush, 1980; Kjemtrup et al., 2002). The vacuoles appear with the WA1 human Babesia parasite. Journal of Parasitology 85:
to be a source of nutrients for the invading merozoite (Rudzinska, 479–489.
1976), but they disappear after growth and the merozoites develop to KJEMTRUP, A. M., B. LEE, C. L. FRITZ, C. E. EVANS, M. CHEVERNAK,
mature trophozoites just before the parasite division starts (Rudzinska, AND P. A. CONRAD. 2002. Investigation of transfusion transmission
1976; Rudzinska et al., 1976). We observed that the vacuoles also were of a WA1-type babesial parasite to a premature infant in California.
formed by an invagination process inside the cell when the merozoite Transfusion 42: 1482–1487.
lies free in the host cytoplasm. In this case, the anterior and posterior MORO, M. H., C. S. DAVID, J. M. MAGERA, P. J. WETTSTEIN, S. W.
ends of the parasite develop pseudopodia, forming the vacuole inside. BARTHOLD, AND D. H. PERSING. 1998. Differential effects of infec-
We also observed that vacuolization of the erythrocyte cytoplasm occurs tion with a Babesia-like piroplasm, WA1, in inbred mice. Infection
when the parasites are closer to the cell membrane, causing distortions and Immunity 66: 492–498.
in shape when the invagination process starts. This vacuolization sug- PANTANOWITZ, L., S. AUFRANC III, R. MONAHAN-EARLEY, A. DVORAK,
gests that some distortions in the cell membrane, observed as perfora- AND S. R. TELFORD, III. 2002. Morphologic hallmarks of Babesia.
tions by SEM, could be formed by this process. Transfusion 42: 1389.
The division of WA1 trophozoites was in general the same as that in PRUTHI, R. K., W. F. MARSHALL, J. C. WILTSIE, AND D. H. PERSING. 1995.
other apicomplexans (Rudzinska, 1976; Rudzinska et al., 1976), how- Human babesiosis. Mayo Clinic Proceedings 70: 853–862.
ever, with a greater frequency. Erythrocytes containing multiple para- QUICK, R. E., B. L. HERWALDT, J. W. THOMFORD, M. E. GARNETT, M.
sites were frequently seen, with trophozoites in division forming char- L. EBERHARD, M. WILSON, D. H. SPACH, J. W. DICKERSON, S. R.
RESEARCH NOTES 1107

TELFORD, K. R. STEINGART, R. POLLOCK, D. H. PERSING, J. M. KO- microscopic study of Babesia microti invading erythrocytes. Cell
BAYASHI, D. D. JURANEK, AND P. A. CONRAD. 1993. Babesiosis in Tissue Research 169: 323–334.
Washington state, a new species of Babesia? Annals of Internal SUN, T., M. J. TENENBAUM, J. GREENSPAN, S. TEICHBERRG, R. T. WANG,
Medicine 119: 284–290. T. DEGNAN, AND M. H. KAPLAN. 1983. Morphologic and clinical
RUDZINSKA, M. A. 1976. Ultrastructure of intraerythrocytic Babesia mi- observations in human infection with Babesia microti. The Journal
croti with emphasis on the feeding mechanism. Journal of Proto- of Infectious Diseases 148: 239–248.
zoology 23: 224–233. WALTER, S., H. MEHLHORN, E. ZWEYGARTH, AND E. SCHEIN. 2001. Elec-
———, AND W. TRAGER. 1977. Formation of merozoites in intraeryth- tron microscopic investigations on stages of dog piroplasm cultured
rocytic Babesia microti. Canadian Journal of Zoology 55: 928–938. in vitro: Asian isolates of Babesia gibsoni and strains of B. canis
———, ———, S. J. LEWENGRUB, AND E. GUBERT. 1976. An electron from France and Hungary. Parasitology Research 88: 32–37.

J. Parasitol., 92(5), 2006, pp. 1107–1108


䉷 American Society of Parasitologists 2006

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- the prevalence and genotype of T. gondii in rats (Rattus norvegicus) in
plasma gondii, because they are the only hosts that can excrete envi- Grenada.
ronmentally resistant oocysts. Cats are infected with T. gondii via pre- From April to December 2005, 238 rats (119 females, 119 males)
dation on infected birds and rodents. During 2005, 238 rats (Rattus were caught using rattraps set in different locations in the 6 parishes of
norvegicus) were trapped in Grenada, West Indies, and their sera along Grenada. The rats were transported to the Pathology Laboratory at the
with tissue samples from their hearts and brains were examined for T. School of Veterinary Medicine, St. George’s University, and then anes-
gondii infection. Antibodies to T. gondii were assayed by the modified thetized and killed with an overdose of ether in a closed chamber. The
agglutination test (MAT, titer 1:40 or higher); only 2 (0.8%) of 238 rats thoracic cavity was opened, and blood was collected from the heart for
were found to be infected. Brains and hearts of all rats were bioassayed serological studies after which the rats were necropsied. Fresh samples
in mice. Toxoplasma gondii was isolated from the brain and the heart of heart and brain were taken for T. gondii isolation. All samples were
of only 1 rat, which had a MAT titer of 1:320. All of 5 mice inoculated immediately shipped on ice to the United States Department of Agri-
with the heart tissue, and the 5 mice inoculated with the brain tissue of culture, Agricultural Research Service, Animal and Natural Resources
the infected rat remained asymptomatic, even though tissue cysts were Institute, Animal Parasitic Diseases Laboratory, Beltsville, Maryland,
found in their brains. Genetically, the isolates of T. gondii from the for isolation and serological analysis. The rats were received in 12
heart and the brain were identical and had genotype III by using the batches of 10, 4, 13, 9, 9, 12, 33, 22, 24, 23, 33, and 46.
SAG1, SAG2, SAG3, BTUB, and GRA6 gene markers. These data Sera from rats were diluted 2-fold starting at 1:5 dilution and assayed
indicate that rats are not important in the natural history of T. gondii for T. gondii antibodies with the modified agglutination test (MAT)
in Grenada. conducted as described previously (Dubey and Desmonts, 1987).
Tissues of all rats were bioassayed in mice, irrespective of antibody
Toxoplasma gondii infections are widely prevalent in human beings level. Brains and hearts of 51 individual rats were pooled separately for
and animals worldwide (Dubey and Beattie, 1988). Humans become each rat, homogenized, digested in pepsin, and inoculated subcutane-
infected postnatally by ingesting tissue cysts from undercooked meat, ously (s.c.) into 2–5 mice (Dubey, 1998). Brains and hearts of seroneg-
consuming food or drink contaminated with oocysts, or by accidentally ative (MAT ⬍ 1:5) rats were pooled in batches of 5–15, and after di-
ingesting oocysts from the environment. Cats are important in the nat- gestion they were bioassayed in mice. The mice used were Swiss-Web-
ural life cycle of T. gondii because they are the only hosts that can ster albino females obtained from Taconic Farms, Germantown, New
directly spread T. gondii in the environment. Thus, cats can ‘recycle’ York.
and amplify the infection by releasing millions of infective oocysts into The heart and brain of the rat that had a MAT titer of 1:320 were
the environment. In turn, cats are considered to become infected with homogenized separately, not digested in pepsin, and inoculated s.c. into
T. gondii by ingesting tissues of infected animals, most likely rodents 5 mice each. Tissue impression smears of mice that died were examined
and birds (Ruiz and Frenkel, 1980). for T. gondii tachyzoites or tissue cysts. Survivors were bled on day 43
Grenada is located in the eastern Caribbean and is most southern of postinoculation (PI), and a 1:25 dilution of serum from each mouse was
the Windward Islands. It has an area of approximately 344 km2 with a tested for T. gondii antibodies with the MAT. Mice were killed 48 days
population of 90,000. Our study on the epidemiology of T. gondii in- PI, and brain squashes from all mice were examined microscopically
fections in Grenada began in 1995 with a serological survey of T. gondii for tissue cysts as described previously (Dubey and Beattie, 1988). Mice
in pregnant women and cats. Antibodies to T. gondii were found in were considered infected with T. gondii when tachyzoites or tissue cysts
57% of 534 pregnant women and in 35% of 40 cats (Asthana et al., were demonstrable microscopically in smears of their tissues. A portion
2006). Epidemiological data suggested that the ingestion of food or of the brain with demonstrable T. gondii was frozen for DNA extraction.
water contaminated with oocysts was an important mode of transmis- Toxoplasma gondii DNA was extracted from the tissues of infected
sion of T. gondii to women (Asthana et al., 2006). More recently, the mice, and strain typing was performed using genetic markers SAG1,
prevalence of T. gondii in 102 free-range chickens (Gallus domesticus) SAG2, SAG3, BTUB, and GRA6 with modifications of methods de-
from different areas of Grenada was determined, and serves as a good scribed previously (Howe et al., 1997; Grigg et al, 2001; Khan et al.,
indicator of T. gondii oocyst contamination, because free-range chick- 2005) with minor modifications (Dubey et al., 2006). The primers and
ens feed from the ground. Antibodies to T. gondii were found in 43 of enzymes used were indicated by Dubey et al. (2006).
102 (42%) chickens at a serum dilution of 1:20, and viable T. gondii Antibodies to T. gondii were found in sera of 80 of 238 rats in titers
was isolated from tissues of 35 of 43 (80%)-seropositive chickens (Dub- of 1:5 in 62, 1:10 in 11, 1:20 in 5, 1:40 in 1, and 1:320 in 1. Toxoplasma
ey et al., 2005). The objective of the present study was to determine 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 SHARMA, C. SREEKUMAR, M. C. B. VIANNA, S. K. SHEN, O. C. H.
with rat tissue developed MAT titers of 1:25 or higher. One mouse from KWOK, K. B. MISKA, D. E. HILL, AND T. LEHMANN. 2005. Isolation,
each group of mice inoculated with the heart and brain was killed on tissue distribution, and molecular characterization of Toxoplasma
day 49 PI, and tissue cysts were found in their brains; brain tissue from gondii from chickens in Grenada, West Indies. Journal of Parasi-
these 2 mice was saved for DNA extraction and for cryopreservation tology 91: 557–560.
in liquid nitrogen. Tissue cysts were found in the brains of the remaining ———, AND G. DESMONTS. 1987. Serological responses of equids fed
8 mice that were killed day 139 PI. All of these 10 infected mice Toxoplasma gondii oocysts. Equine Veterinary Journal 19: 337–
remained asymptomatic throughout the observation period. The isolates 339.
from the heart and the brain were all of genotype of III. ———, AND J. K. FRENKEL. 1998. Toxoplasmosis of rats: A review,
The low prevalence of T. gondii in rats from Grenada is surprising. with considerations of their value as an animal model and their
Antibodies (MAT 1:40–1:320) were found in only 2 rats, and T. gondii possible role in epidemiology. Veterinary Parasitology 77: 1–32.
was isolated from 1 rat. In the present study, rat sera were tested starting ———, A. N. PATITUCCI, C. SU, N. SUNDAR, O. C. H. KWOK, AND S.
at 1:5 dilution. In pigs, a titer of 1: 25 is considered indicative of per- K. SHEN. 2006. Characterization of Toxoplasma gondii isolates in
sistent T. gondii infection (Dubey, Thulliez et al., 1995). In the present free-range chickens from Chile, South America. Veterinary Para-
study, tissues of all rats were bioassayed for T. gondii, irrespective of sitology. 140 76–82.
their antibody status, because congenitally infected rats can harbor vi- ———, S. K. SHEN, O. C. H. KWOK, AND P. THULLIEZ. 1997. Toxo-
able T. gondii in the absence of demonstrable antibodies (Dubey et al., plasmosis in rats (Rattus norvegicus): Congenital transmission to
1997). Dubey and Frenkel (1998) summarized the worldwide preva- first and second generation offspring and isolation of Toxoplasma
lence of T. gondii in different species of rats and concluded that the gondii from seronegative rats. Parasitology 115: 9–14.
prevalence of viable T. gondii in R. norvegicus was generally low. One ———, P. THULLIEZ, R. M. WEIGEL, C. D. ANDREWS, P. LIND, AND E.
exception to this finding was reported in rats from an endemic area in C. POWELL. 1995. Sensitivity and specificity of various serologic
Costa Rica where Ruiz and Frenkel (1980) isolated viable T. gondii tests for detection of Toxoplasma gondii infection in naturally in-
from 15 of 120 rats. Dubey, Weigel et al. (1995), in contrast, isolated fected sows. American Journal of Veterinary Research 56: 1030–
T. gondii from only 1 of 107 rats from 47 pig farms in Illinois. Our 1036.
current findings from this study suggest that although T. gondii is prev- ———, R. M. WEIGEL, A. M. SIEGEL, P. THULLIEZ, U. D. KITRON, M.
alent in cats, women, and free-range chickens in Grenada, rats are not A. MITCHELL, A. MANNELLI, N. E. MATEUS-PINILLA, S. K. SHEN, O.
important in the epidemiology of T. gondii on the island. C. H. KWOK, AND K. S. TODD. 1995. Sources and reservoirs of
We thank C. Su (Department of Microbiology, The University of Toxoplasma gondii infection on 47 swine farms in Illinois. Journal
Tennessee, Knoxville, Tennessee) for performing the genotyping and S. of Parasitology 81: 723–729.
K. Shen and K. Hopkins for technical assistance. GRIGG, M. E., J. GANATRA, J. C. BOOTHROOYD, AND T. P. MARGOLIS.
2001. Unusual abundance of atypical strains associated with human
LITERATURE CITED ocular toxoplasmosis. Journal of Infectious Diseases 184: 633–639.
HOWE, D. K, S. HONORÉ, F. DEROUIN, AND L. D. SIBLEY. 1997. Deter-
ASTHANA, S. P., C. N. L. MACPHERSON, S. H. WEISS, R. STEPHENS, R. mination of genotypes of Toxoplasma gondii strains isolated from
N. SHARMA, AND J. P. DUBEY. 2006. Seroprevalence of Toxoplasma patients with toxoplasmosis. Journal of Clinical Microbiology 35:
gondii in pregnant women and cats in Grenada, West Indies. Jour- 1411–1414.
nal of Parasitology 92: 644–645. KHAN, A., C. SU, M. GERMAN, G. A. STORCH, D. B. CLIFFORD, AND L.
DUBEY, J. P. 1998. Refinement of pepsin digestion method for isolation D. SIBLEY. 2005. Genotyping of Toxoplasma gondii strains from
of Toxoplasma gondii from infected tissues. Veterinary Parasitol- immunocompromised patients reveals high prevalence of type I
ogy 74: 75–77. strains. Journal of Clinical Microbiology 43: 5881–5887.
———, AND C. P. BEATTIE. 1988. Toxoplasmosis of animals and man. RUIZ, A., AND J. K. FRENKEL. 1980. Intermediate and transport hosts of
CRC Press, Boca Raton, Florida, 220 p. Toxoplasma gondii in Costa Rica. American Journal of Tropical
———, M. I. BHAIYAT, C. DE ALLIE, C. N. L. MACPHERSON, R. N. Medicine and Hygiene 29: 1161–1166.

J. Parasitol., 92(5), 2006, pp. 1108–1110


䉷 American Society of Parasitologists 2006

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 cerebral reactivation in immunocompromised patients. Infection induces
cytokines involved in the immune response against Toxoplasma gondii. a strong immune response and immunocompetent cells secrete IFN-␥,
Nevertheless, the role of IL-12 and IL-10 in the control of parasite IL-12, and tumor necrosis factor (TNF)-␣ (Ricard et al., 1996; Bliss et
replication and cytogenesis is not known yet, whereas the importance al., 2000). Another cytokine that is important at the early stage is IL-
of IFN-␥ is documented. Furthermore, it is of paramount importance to 10, which down-regulates IL-12 and IFN-␥ secretion, and avoids im-
study the interaction between T. gondii and cells from the central ner- munopathogenicity of the immune response (Wilson et al., 2005). Al-
vous system, e.g., astrocytes. In this study, we report that IL-12 and IL- though infection is controlled, a percentage of parasites survive as dor-
10 have no effect on penetration, replication, or cystogenesis of the T. mant cysts, particularly in the host’s central nervous system. Parasites
gondii Prugniaud strain in human astrocytes in vitro and do not antag- replicate in human astrocytes, and these cells have been found to sup-
onize the role of IFN-␥ on cystogenesis. 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
Toxoplasma gondii is a universally widespread protozoan. Toxoplas- during the immune response is known. More precisely, IL-10 has been
mosis is a health care problem, causing congenital toxoplasmosis and shown to inhibit parasite-killing functions of IFN-␥–activated murine
RESEARCH NOTES 1109

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 FIGURE 1. Cystogenesis in primary human astrocyte cell cultures
Human astrocytes (105 cells/ml) were infected with 105 parasites/ml (ratio ⫽ 1). after treatment by cytokines for 6 days. Astrocytes (105/ml) were in-
Cytokines were added at the time of T. gondii (Tg) infection for 3 and 48 hr in fected with 1.5 ⫻ 104 T. gondii/ml, and cysts were counted by indirect
invasion and multiplication assays, respectively. Invasion rate was determined 3 immunofluorescence with Ab CC2 6 days PI. Results from 3 experi-
hr postinfection. Moreover, the multiplication rate was counted 48 hr postinfection. ments assessed in duplicate (means ⫾ standard errors). *, statistically
Data are means ⫾ standard deviations from 3 experiments assessed in duplicate. significant difference (P ⬍ 0.05) (Student’s t-test).
* Statistically significant difference (P ⬍ 0.05) (Student 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
macrophages. Yet, their roles regarding cyst formation of T. gondii in formation of T. gondii, whereas IFN-␥ inhibited cyst formation (Fig. 1).
human astrocytes remain to be investigated (Gazzinelli et al., 1992). Moreover, we have associated a pretreatment of the cells by IFN-␥ (for
The objective of this study was to investigate the role of IL-12, IL-10, 48 hr before infection) to the treatment by IL-12 and IL-10. The number
and IFN-␥ on parasite cystogenesis in human astrocytes in vitro. of cysts observed in the presence of IFN-␥ was not modified when IL-
The 2 types of cells, i.e., primary human astrocytes and glioblastoma 12 and IL-10 were added. Indeed, the total number of cysts in astrocytes
cell line U373 (ATCC HTB-17), were grown in 24-well plates on glass was 149 ⫾ 40 without any treatment and 2 ⫾ 1 when IFN-␥ was present
coverslips at 37 C in a 5% CO2 atmosphere as described previously in cell culture (with or without IL-12 and IL-10) (mean ⫾ SD of 3
(Brenier-Pinchart et al., 2004). Briefly, glioblastoma cell line U373 MG experiments). Thus, IL-12 and IL-10 did not antagonize the ability of
was grown in Dulbecco’s modified Eagle’s medium (DMEM), supple- IFN-␥ to inhibit cystogenesis of T. gondii in astrocyte cells. As for both
mented with 1% synthetic serum Ultroser G (Life Technologies, Eragny, invasion and replication rates, the results obtained with primary human
France), 5% L-glutamine, and 5% penicillin-streptomycin, and primary astrocyte cell cultures were close to those obtained with the U373 cell
human astrocytes were grown in DMEM supplemented with 5% fetal line (data not shown).
bovine serum (Cambrex Bio Science, Verviers, Belgium), 2% L-gluta- Recently, Wilson et al. (2005) reported that IL-10 antagonizes the
mine, and 1% penicillin-streptomycin. Primary human astrocytes were ability of IFN-␥ to inhibit replication of T. gondii in murine astrocytes
obtained from 1 patient after epileptic surgery at Grenoble Hospital 21 hr PI. Our results confirm that IFN-␥ has no effect on penetration
(Grenoble, France). Neuropathological study demonstrated the absence but that it inhibits multiplication and cyst formation of the parasite in
of tumor formation. Cells were positive for glial fibrillary acidic protein human astrocyte cells. However, the study presented here suggests that
and negative for neurofilament or nestin by immunostaining. Toxoplas- IL-10 does not antagonize IFN-␥ effect on cyst formation in human
mic serology of this patient was negative. Cells were infected with astrocytes 6 days PI. In murine astrocytes, IL-10 antagonizes IFN-␥’s
Prugniaud strain, a nonvirulent cystogenic strain in the mouse (type II) protective effects on T. gondii multiplication (Wilson et al., 2005),
(Brenier-Pinchart et al., 2004). Recombinant human cytokines (IL-12, whereas our study shows that this cytokine does not reveal an antago-
IL-10, and IFN-␥) were purchased from R&D Systems (Abington, Ox- nism with IFN-␥ on cyst formation in human astrocytes. In conclusion,
ford, U.K.). The cytokines concentrations used were 500 UI/ml for IFN- despite the importance of IL-12 and IL-10 in the immune control of T.
␥; 2.5, and 25 ng/ml for IL-12; and 10 and 100 ng/ml for IL-10 (Dau- gondii infection, these cytokines do not have any effect on the modu-
bener et al., 1993; Subauste et al., 2000; Del Rio et al., 2004). Invasion lation of penetration, replication, and cystogenesis of T. gondii in human
rate and parasite multiplication were observed by stained Diff Quick at astrocytes cells in this in vitro model.
3 and 48 hr postinfection (PI), respectively. At day 6 PI, cystogenesis Special thanks to U. Gross (University of Gottingen, Gottingen, Ger-
was observed using an indirect immunofluorescence assay by using the many) for kindly providing the antibody CC2, to S. Durville for English
cyst specific monoclonal antibody (Ab) CC2 (Gross et al., 1995), and proofreading, and to J. Simon and F. Durand for advice and support.
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
LITERATURE CITED
type considered as more mature than the second type (early stage of
development) (Ricard et al., 1999). For invasion rate and parasite mul- BLISS, S. K., B. A. BUTCHER, AND E. Y. DENKERS. 2000. Rapid recruit-
tiplication assays, cells were treated by cytokines at the time of T. gondii ment of neutrophils containing prestored IL-12 during microbial
infection for 3 and 48 hr, respectively. For cyst formation study, cyto- infection. Journal of Immunology 165: 4515–4521.
kines were added at the time of infection and again with each renewal BRENIER-PINCHART, M. P., E. BLANC-GONNET, P. N. MARCHE, F. BERGER,
culture medium (days 1 and 3). Moreover, in some experiments on F. DURAND, P. AMBROISE-THOMAS, AND H. PELLOUX. 2004. Infection
cystogenesis, a pretreatment of cells by IFN-␥ was made 48 hr before of human astrocytes and glioblastoma cells with Toxoplasma gon-
infection, and IFN-␥ was associated with IL-10 or IL-12 treatment per- dii: Monocyte chemotactic protein-1 secretion and chemokine ex-
formed at the time of infection and at days 1 and 3, when the culture pression in vitro. Acta Neuropathologica 3: 245–249.
medium was renewed. DAUBENER, W., K. PILZ, S. SEGHROUCHNI ZENNATI, T. BILZER, H. G.
IL-12 and IL-10 did not significantly modify the invasion rate of T. FISCHER, AND U. HADDING. 1993. Induction of toxoplasmostasis in
gondii in primary human astrocyte cell cultures infected with the Prug- a human glioblastoma by interferon ␥. Journal of Neuroimmunol-
niaud strain (Table I). In our model, IL-12, IL-10, and IFN-␥ did not ogy 43: 31–38.
have any effect on parasite penetration. Furthermore, the treatment by DEL RIO, L., B. A. BUTCHER, S. BENNOUNA, S. HIENY, A. SHER, AND E.
IL-12 and IL-10 for 48 hr did not affect replication of the Prugniaud Y. DENKERS. 2004. Toxoplasma gondii triggers myeloid differenti-
strain in primary human astrocytes cell cultures (Table I). As expected, ation factor 88-dependent IL-12 and chemokine ligand 2 (monocyte
the parasite multiplication in primary human astrocyte cell cultures was chemoattractant protein 1) responses using distinct parasite mole-
significantly inhibited in the presence of IFN-␥ (Table I) (Oberdorfer et cules and host receptors. Journal of Immunology 172: 6954–6960.
al., 2003). Invasion and multiplication rates of T. gondii in U373 cell FAGARD, R., H. VAN TAN, C. CREUZET, AND H. PELLOUX. 1999. Differ-
lines were similar to those obtained in astrocytes (data not shown). As ential development of Toxoplasma gondii in neural cells. Parasi-
reported previously, 2 types of cysts could be differentiated, i.e., a ma- 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- RICARD, J., H. PELLOUX, A. L. FAVIER, U. GROSS, E. BRAMBILLA, AND P.
10 inhibits parasite killing and nitrogen oxide production by IFN- AMBROISE-THOMAS. 1999. Toxoplasma gondii: role of the phospha-
␥-activated macrophages. Journal of Immunology 148: 1792–1796. tidylcholine-specific phospholipase C during cell invasion and in-
GROSS, U., H. BORMUTH, C. GAISSMAIER, C. DITTRICH, V. KRENN, W. tracellular development. Experimental Parasitology 3: 231–237.
BOHNE, AND D. J. FERGUSON. 1995. Monoclonal rat antibodies di- ———, ———, S. PATHAK, B. PIPY, AND P. AMBROISE-THOMAS. 1996.
rected against Toxoplasma gondii suitable for studying tachyzoite- TNF-␣ enhances Toxoplasma gondii cyst formation in human fi-
bradyzoite interconversion in vivo. Clinical and Diagnostic Labo- broblasts through the sphingomyelinase pathway. Cellular Signal-
ratory Immunology 2: 542–548. ing 6: 439–442.
SUBAUSTE, C. S., AND M. WESSENDARP. 2000. Human dendritic cells
HALONEN, S. K., W. D. LYMAN, AND F. C. CHIU. 1996. Growth and
discriminate between viable and killed Toxoplasma gondii tachy-
development of Toxoplasma gondii in human neurons and astro- zoites: Dendritic cell activation after infection with viable parasites
cytes. Journal of Neuropathology and Experimental Neurology 55: results in CD28 and CD40 ligand signaling that controls IL-12-
1150–1156. dependent and -independent T cell production of IFN-␥. Journal of
OBERDORFER, C., O. ADAMS, C. R. MACKENZIE, C. J. DE GROOT, AND Immunology 165: 1498–1505.
W. DAUBENER. 2003. Role of IDO activation in anti-microbial de- WILSON, E. H., U. WILLE-REECE, F. DZIERSZINSKI, AND C. A. HUNTER.
fense in human native astrocytes. Advances in Experimental Med- 2005. A critical role for IL-10 in limiting inflammation during toxo-
icine and Biology 527: 15–26. plasmic encephalitis. Journal of Neuroimmunology 165: 63–74.

J. Parasitol., 92(5), 2006, pp. 1110–1113


䉷 American Society of Parasitologists 2006

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 area is characterized by human populations that exhibited various life
were examined. The material was dated at 6,540 ⫾ 110 B.P.; it was style strategies, but mainly the hunter–gatherer type of subsistence (As-
collected in the Perito Moreno National Park, Santa Cruz, Argentina. chero, Belleli, and Goni, 1992; Civalero, 1995; Aschero, 1996; Civalero
Paleoparasitological analysis was performed following standard proce- and Aschero, 2003; Civalero and Franco, 2003).
dures. Coprolite fragments were rehydrated in a trisodium phosphate The first parasites found in human coprolites from Patagonia were
aqueous solution and subjected to spontaneous sedimentation for mi- eggs of Enterobius vermicularis and ancylostomids (Zimmerman and
croscope analysis. Eggs of nematodes identified as Trichuris sp., Cap- Morila, 1983; Gonc˛alves et al., 2003).
illaria sp., Uncinaria sp., and an ascaridid (probably Toxascaris sp.) or In the present study, a coprolite was found in an archaeological site
spirurids (presumably Physaloptera sp.), plus a cestode (Anoplocephal- in Santa Cruz Province, Argentina, and was examined for parasites. The
idae), presumably Moniezia sp., were found. 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
Paleoparasitological analysis was recently applied to bioanthropo- forest steppe ecotone in the valley of Burmeister Lake, Perito Moreno
logical and archaeological studies in southern Patagonia. The studied 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
FIGURE 1. Coprolite (CCP5, Santa Cruz Province, Argentina, 6,540 hairs and bones, insect fragments, and grass inflorescences, were abun-
⫾ 110 yr B.P.). Bar ⫽ 2 cm. dant. Macroscopic and microscopic charcoal fragments also were pres-
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-
FIGURE 2. (a) Moniezia sp. egg showing hexacanth embryo and pir- locephalid records in the Patagonian fauna have cited the occurrence of
iform apparatus. (b) Trichuris sp. egg. (c) Capillaria sp. egg. (d) As- Moniezia expansa in Lama guanicoe (Beldomenico et al., 2003; C. Ro-
caridid—possibly Toxascaris sp. or Physaloptera sp. egg. (e) Ancylos- bles, pers. comm.), Moniezia sp. and Thysanosoma actinoides with an
tomid egg, probably Uncinaria sp. (f ) Adult of Oribatida, Aphelacari- ovine source (Robles and Olaechea, 2001), Moniezia sp. in L. guanicoe
dae, presumably Aphelacarus sp. Bar ⫽ 10 ␮, except for f (bar ⫽ 100 (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,
ent. The rehydrated fraction without fixative exhibited the typical smell parasites of humans and dogs, respectively (Thienpont et al., 1979).
of fox urine. Species with similar dimensions to the eggs recovered in the present
Microscopic examination of the sample confirmed the presence of
study, and with wild hosts inhabiting Patagonia and the Andes, are
nematode eggs identified as Trichuris sp., Capillaria sp., and Uncinaria
summarized in Table I. Trichuris sp. eggs could occur due to rodent
sp., and an ascaridid (probably Toxascaris sp.) or spirurids (presumably
consumption; rodent bones and fur were found in coprolite.
Physaloptera sp.), plus eggs of a cestode (Anoplocephalidae), presum-
ably Moniezia sp. Up to 14 eggs per slide of the cestode were present. Eggs of Capillaria sp., and Uncinaria sp. were not identified to the
The latter eggs were quadrangular to triangular, with a yellowish brown species level due to their morphometric diversity, different degree of
color, and surrounded by a thin membrane containing a hexacanth em- conservation, or both. The eggs with thick and smooth walls were not
bryo of 22 ␮m in diameter. In some, hooks and a piriform apparatus identified to genus level because the width of the shell resembles Tox-
were identified (Fig. 2a). Measurements were 62.5–77.5 ␮m (69.8 ⫾ ascaris sp. or Physaloptera sp. These nematodes are ubiquitous para-
4.33, n ⫽ 21) by 55.0–70.0 (62.9 ⫾ 4.70; n ⫽ 21). Eggs were identified sites of canids (Thienpont et al., 1979; Stein et al., 1994; Ruas et al.,
as a species of Anoplocephalidae, probably Moniezia sp. Blanchard, 2003; Valenzuela et al., 2004).
1891. For the identification of hair and bones, we thank Alejandro Cane-
Twenty Trichuris sp. eggs (Fig. 2b) were found, 50.0–70.0 (62.7 ⫾ puccia. We acknowledge the reviewers corrections of an early version
4.8, n ⫽ 18) by 26.3–35.0 (30.8 ⫾ 2.0, n ⫽ 18); eggs of Capillaria sp. of the manuscript. This work was supported by the project ‘‘Ecologı́a
(n ⫽ 174) also were recovered, 27.5–85.0 (59.5 ⫾ 7.3, n ⫽ 171) by Evolutiva Humana en Patagonia,’’ SECYT–UNMDP 04-09929 and the
20.0–47.5 (30.6 ⫾ 4.9, n ⫽ 171) (Fig. 2c). Thick- and smooth-walled ‘‘Convenio de Colaboración entre el Instituto Canario de Bioantropol-
eggs (n ⫽ 149), 62.5–80.0 (73.0 ⫾ 4.5, n ⫽ 41) by 50.0–72.5 (60.5 ⫾ ogı́a del OAMC de Tenerife, España y la Facultad de Cs. Sociales de
4.8, n ⫽ 41), were identified as ascaridids, presumably Toxascaris sp., 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.

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———. 1996. El área Rı́o Belgrano-Lago Posadas (Santa Cruz): Prob-
lemas y estado de problemas. In Arqueologı́a sólo Patagonia, J. G. Journal of Parasitology 16: 198–206.
Otero (ed.). CENPAT, CONICET. Buenos Aires, Argentina, p. 17– CHEHEBAR, C., AND S. MARTı́N. 1989. Guı́a para el reconocimiento mi-
26. croscpico de los pelos de los mamferos de la Patagonia. Doñana,
———, C. BELLELI, AND R. A. GOÑI. 1992. Avances en las investiga- Acta Vertebrata 16: 247–291.
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incia de Santa Cruz, Patagonia, Argentina). Cuadernos del Instituto la tecnologı́a lı́tica y las estrategias de movilidad.’’ Cuadernos del
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143–170. ———, AND C. ASCHERO. 2003. Early occupations at Cerro Casa de
———, ———, M. T. CIVALERO, R. A. GOÑI, G. GURAIEB, AND R. Piedra 7, Santa Cruz Province, Patagonia, Argentina. In Where the
MOLINARI. 1992. ‘‘Cronologı́a y tecnologı́a en el Parque Nacional south winds blow, L. Miotti, M. Salemme, and N. Flegenheimer
Perito Moreno (PNPM): Continuidad o reemplazos?’’ Arqueologı́a (eds.). Texas A&M University, College Station, Texas, p. 141–147.
2: 89–106. ———, AND N. V. FRANCO. 2003. Early human occupations in western
BABERO, B. B., AND R. MURUA. 1987. The helminth fauna of Chile. X. Santa Cruz Province, southernmost South America. Quaternary In-
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———, AND ———. 1990. A new species of whipworm from South dino Pseudalopex culpaeus (Canidae) en el matorral desértico del
American hystricomorph rodent. Memorias do Instituto Oswaldo sur de Perú. Revista de Ecologı́a Latinoamericana 8: 1–9.
Cruz 85: 211–213. DENEGRI, G. M. 2001. Cestodosis de herbı́voros domósticos de la Re-
———, P. E. CATTAN, AND C. CABELLO. 1975. Trichuris bradleyi sp. pública Argentina de importancia en medicina veterinaria, Editorial
n., a whipworm from Octodon degus in Chile. Journal of Parasi- Martı́n, Mar del Plata, Argentina, 111 p.
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AND J. L. PERALTA. 2003. Internal parasites of free-ranging gua-
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961–962. AND E. PRADA MARCOS. 2003. Parasitological analysis of Leonese
CALLEN, E. O., AND T. W. M. CAMERON. 1960. A prehistoric diet re- royalty from collegiate-basilica of St. Isidoro, León (Spain): Hel-
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CALLEN, E. O., AND P. S. MARTIN. 1969. Plant remains in some copro- LEGUı́A, P. G., A. E. CASAS, AND J. WHEELER. 1995. Parasitismo en
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servacoes feitas no Brasil. Memorias do Instituto Oswaldo Cruz RUAS, J. L, M. P. SOARES N. A. R. FARIAS, AND J. G. W. BRUM. 2003.
11: 121–155. Infecc˛ão por Capillaria hepatica em carnı́voros silvestres (Lycal-
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MORINI, E. G., J. J. BOERO, AND A. RODRIGUEZ. 1955. Parásitos intes- and parasitism in the Lluta Valley: Preliminary data. Memorias do
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de la fauna endoparasitaria de Lama guanicoe Muller, 1776, de 1782), y Conepatus chinga (Molina, 1782) (Mamı́fera: Carnı́vora)
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cetidae and Octodontidae rodents in Argentina. Research and Re-
REINHARD, K., P. R. GEIB, M. M. CALLAHAN, AND R. H. HEVLY. 1992. views in Parasitology 54: 39–46.
Discovery of colon contents in a skeletonized burial: Soil sampling THIENPONT, D., F. ROCHETTE, AND O. F. J. VANPARIJS. 1979. Diagnóstico
for dietary remains. Journal of Archaeological Science 19: 697– de las helmintiosis por medio del examen coproparasitológico.
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tenuis Chandler, 1930, from llamas (Lama glama) in Oregon with ásitos gastrointestinales en zorros Pseudalopex griseus de la Prov-
a key to the species of Trichuris present in North American ru- incia de Última Esperanza Sur de Chile. Actas del XIII Congreso
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ROBLES, C., AND F. OLAECHEA. 2001. Salud y enfermedad de las maja- cong㛮val.pdf.
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and G. Oliva (eds.). INTA, Santa Cruz, Argentina, p. 225–243. lumbian America. Paleopathology Newsletter 42: 8.

J. Parasitol., 92(5), 2006, pp. 1113–1115


䉷 American Society of Parasitologists 2006

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), board a Japanese long-liner Shoyo Maru during her cruise across the
Remora osteochir (Cuvier), and Remora remora (Linnaeus)—were col- Atlantic. Remoras were collected mainly from 4 species of billfishes,
lected from 4 species of billfishes—Istiophorus platypterus (Shaw), from 2 species of tunas, and 1 species each of swordfish, blue shark,
Makaira nigricans Lacepéde, Tetrapturus albidus Poey, and Tetraptu- manta ray, loggerhead turtle, and long-line floats. Each remora removed
rus pfluegeri Robins and de Sylva—on board a Japanese long-liner from its host was placed immediately in a cloth bag, labeled with the
Shoyo Maru during her cruise in 2002 across the Atlantic. However, station number and host, and placed in a bucket of seawater. Later, the
only the marlin sucker (R. osteochir) was found to carry a parasitic remoras were retrieved for sampling of tissues. Afterwards, the remoras
copepod, Lepeophtheirus crassus (Wilson and Bere, 1936). Although were replaced in the bags and fixed in 10% formalin. The copepods
12 species of parasitic copepods have been reported from billfishes were collected at the time of tissue sampling on board Shoyo Maru, in
around the world ocean, none of them is L. crassus. Thus, L. crassus the laboratory at the Smithsonian Institution, or both places.
is considered a parasite specific to the marlin sucker. It was noted that only those remoras removed from billfishes carried
a species of parasitic copepod. In total, 129 remoras belonging to 3
Lepeophtheirus crassus (Wilson and Bere, 1936) was originally re- species were collected from 4 species of billfishes taken during that
ported by Bere (1936) as ‘‘Gloiopotes crassus Wilson and Bere, spec. cruise: (1) Remora brachyptera (Lowe, 1839) from Istiophorus platyp-
nov.’’ from the marlin sucker ‘‘Rhombochirus osteochir’’ attached to terus (Shaw, 1792), Makaira nigricans Lacepéde, 1802, and Tetrapturus
‘‘a billfish (Tetrapturus imperator)’’ caught at Miami, Florida, and an- pfluegeri Robins and de Sylva, 1963; (2) R. osteochir (Cuvier, 1829)
other marlin sucker attached to ‘‘a sailfish’’ taken at Ft. Lauderdale, from I. platypterus, M. nigricans, Tetrapturus albidus Poey, 1860, and
Florida. Both adults and larval stages of copepods were obtained, with T. pfluegeri; and (3) Remora remora (Linnaeus, 1758) from I. platyp-
the former found on the body surface of their host and the latter on the terus, M. nigricans, and T. albidus (see Table I).
gill filaments of the same host. The copepod parasite was subsequently Close examination of the collection revealed that only the marlin
reported from ‘‘Echeneis albesens’’ taken in the Bay of Bengal at sucker (R. osteochir) carries the parasitic copepod L. crassus. The com-
16⬚40⬘N, 92⬚13⬘E by Shiino (1960) from an unidentified remora at- mon remora (R. remora) seems to be rare on billfish of the Atlantic
tached to ‘‘Makaira audex’’ caught in the eastern Pacific at 19⬚34.3⬘S, Ocean, but not the spearfish remora (R. brachyptera). In total, 21 spear-
92⬚45.4⬘W; from ‘‘Remilegia australis’’ attached to ‘‘Delphinus bairdi’’ fish remoras were collected from billfishes during that cruise, but none
caught at 8⬚14⬘N, 84⬚17⬘W by Shiino (1963); and from ‘‘Rhombachirus of them carried L. crassus, even those from 4 stations LL 36, LL 37,
osteochir (Cuvier)’’ attached to a shortbill spearfish (Tetrapturus an- LL 38, and LL 39 where both R. osteochir and R. brachyptera were
gustirostris Tanaka, 1951) captured in the central Pacific at 21⬚04.5⬘N, found (see Table I). Thus, it seems L. crassus is host specific to R.
173⬚47.5⬘E by Lewis (1967). Shiino (1960) correctly proposed to trans- osteochir in the Atlantic Ocean, but not in the Pacific. Shiino (1963)
fer the parasite from Gloiopotes to Lepeophtheirus because it lacks a reported L. crassus from a different species of remora attached to a
pair of dorsal aliform plates on the somite of leg 4. dolphin in the eastern Pacific.
In 2002, 1 of us (B.B.C.) had the opportunity to collect remoras on 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
RESEARCH NOTES 1115

TABLE II. Copepod parasites of billfishes (Istiophoridae). 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.
Order Siphonostomatoida Thorell, 1859
Permission for B.B.C. to participate in leg 3 of the Shoyo Maru long-
Caligidae Burmeister, 1834
line cruise and to collect remoras was received from Hiroyuki Kinoshita
Caligus quadratus Shiino, 1954 (Japanese Fisheries Agency) and Hiroaki Okamoto and Kotaro Yokawa
Lepeophtheirus eminens Wilson, 1944 (National Research Institute of Far Seas Fisheries, Shizuoka). In addi-
Dissonidae Yamaguti, 1963 tion to their own research, remoras were kindly collected on leg 1 of
Dissonus glaber Kurtz, 1924 the cruise by Hiroaki Okamoto, on leg 2 by Lisa Natanson (National
Euryphoridae Wilson, 1905 Marine Fisheries Service, Narraganset), and on leg 4 by Maki Ohwada.
Euryphorus brachypterus (Gerstaecker, 1853) Hiroaki Okamoto, Kotaro Yokawa, and Kouichi Hoshino all helped pro-
Gloiopotes americanus Cressey, 1967 vide station data for the billfishes collected on the cruise. Completion
Gloiopotes huttoni (Thomson, 1889) of the manuscript of this paper was aided by a grant from the Paramitas
Gloiopotes ornatus Wilson, 1905 Foundation to J.S.H.
Pandaridae Milne Edwards, 1840
Pandarus satyrus Dana, 1852
LITERATURE CITED
Pennellidae Burmeister, 1834
Lernaeolophus sultanus (Nordmann, 1839) BERE, R. 1936. Parasitic copepods from Gulf of Mexico fish. The Amer-
Pennella biloba (Kirtisinghe, 1933) ican Midland Naturalist 17: 577–625.
Pennella filosa (Linnaeus, 1758) LEWIS, A. G. 1967. Copepod crustaceans parasitic on teleost fishes of
Pennella makaira Hogans, 1988 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
billfishes in oceans around the world, none of them is L. crassus (see Bengal. Report of the Faculty of Fisheries, Prefectural University
Table II). Thus, it is safe to say that L. crassus is a parasite of marlin of Mie 3: 542–552.
sucker and not billfish. As reported by Bere (1936), we have also found ———. 1963. Parasitic copepods of the eastern Pacific fishes. 1. Re-
that although adult and juvenile L. crassus attach to the body surface cords of the known species. Report of the Faculty of Fisheries,
of R. osteochir, the larval (chalimus) stages were all parasitic on the Prefectural University of Mie 4: 335–347.

J. Parasitol., 92(5), 2006, pp. 1115–1117


䉷 American Society of Parasitologists 2006

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- on identification of fecal oocysts (Fitzgerald, 1962; Ernst et al., 1987;
tive days in November 2004 from 11 weaned beef heifers to assess Hasbullah et al., 1990; Diaz de Ramirez et al., 2001). In contrast, Parker
daily variation in fecal oocyst count and species composition. Subse- et al. (1984), reported that Eimeria zuernii was the most prevalent oo-
quent samples were collected from the same animals on 15 April 2005 cysts in the feces of freshly weaned beef calves in Australia, and Svenn-
and 9 June 2005. Oocyst numbers were determined by the modified son et al. (1993) found E. alabamensis most prevalent in dairy calves
McMaster’s test, and species were identified by examination of oocysts (4–16 mo of age) just after turnout in Sweden. No reports exist, how-
recovered with the Wisconsin sugar flotation technique. Soil samples ever, that describe daily variation in fecal oocyst counts and species
were collected from the heifer pasture on 8 June 2005, and oocysts composition in calves. The objective of the present study was to assess
were quantified and identified to species. Mean fecal oocyst counts var- daily variation in fecal oocyst count and species composition in clini-
ied little at all sampling dates ranging from 134–377 oocysts/g. Ten cally normal weanling beef calves and to determine whether changes
Eimeria spp. were identified in fecal samples collected in November occurred after a period of several months.
and April and 11 in June. Eimeria bovis was the most common species Eleven beef heifers (Angus, Charolais, and Hereford breeding), main-
identified at all samplings. Mean species composition showed little var- tained at the Virginia Tech Beef Center, Blacksburg, Virginia, were
iation during the 10-day sampling period in November, remained similar surveyed for natural coccidia infection. In the previous year, no out-
in April, and varied slightly in June. Twelve Eimeria spp. were iden- breaks of coccidiosis had been observed at this facility. Heifers (287 ⫾
tified in soil samples in proportions similar to those seen in fecal sam- 19 days of age at the beginning of the study) were sampled for a period
ples. The results indicate that clinically normal weanling beef heifers of 10 consecutive days (15 November 2004 to 24 November 2004).
are likely to be infected with a diverse, but relatively stable, community Subsequent samples were collected from 10 of the same heifers 5 mo
of Eimeria spp. 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
Species of Eimeria are gastrointestinal coccidians that infect cattle permanent native grass lot used every year for replacement animals with
worldwide. Reports indicate that calves are infected shortly after birth free access to water and mineral mix. The heifers were fed daily hay
and shed relatively high numbers of oocysts during their first year of and corn silage. All heifers remained on the same lot from November
life (Fitzgerald, 1962; Ernst et al., 1984, 1987; Diaz de Ramirez et al., to June at which time they were moved to summer pastures. They were
2001). Most studies reporting species prevalence have found that E. clinically normal throughout the course of the survey. All fecal samples
bovis is the most prevalent species in calves less than 1 yr of age based 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 700 OPG throughout the sampling period with an overall mean oocyst
McMaster’s technique with a sensitivity of 25 oocysts/g (OPG) (Whit- count of 192 OPG. Arithmetic mean daily oocyst counts for the heifers
lock, 1948). A modified Wisconsin sugar flotation technique (Coxx and remained similar over the course of the 10-day sampling period, ranging
Todd, 1962) also was performed on each fecal sample, and 50 Eimeria from 134–377 OPG. Similarly, oocyst counts from individual animals
spp. oocysts per sample were examined at ⫻400 and identified to spe- exhibited little variation over the course of the 10 days, with more
cies based on oocyst morphology (Levine and Ivens, 1986). One indi- variation in oocyst count seen between individuals within days. Those
vidual (A.L.) carried out all oocyst counts and species identifications. animals with relatively high oocyst counts at the start of the study main-
Because oocysts of E. cylindrica and E. ellipsoidalis could not be dis- tained higher oocyst counts throughout, whereas those that began with
tinguished reliably, all small cylindrical Eimeria spp. oocysts measuring lower oocyst counts maintained low levels over the sampling period.
from 19 to 36 ␮m by 8–18 ␮m were designated E. cylindrica/ellipso- These individuals also maintained their relative high or low oocyst
idalis-like (Levine and Ivens, 1986). shedding tendency in samples collected in April and June. Ten Eimeria
Soil samples (approximately 100 g each) also were collected from spp. were recovered from the heifers during this period (Table I). Ei-
different sites in the replacement heifer lot on 8 June 2005, shortly after meria bovis was the most common species found, present in 100% of
the heifers were removed. All samples were collected from the top 2.5 all positive samples. Eimeria zuernii was the second most common
cm of soil. A general field sample was obtained by collecting approx- species (96%), followed by E. alabamensis and E. auburnensis (91%),
imately 10 g of soil every 10 m in a diagonal across the field. Soil E. cylindrica/ellipsoidallis-like (80%), and E. canadensis (67%). All
around the feed bunk and hay ring also was sampled by collecting other species were present in fewer than 50% of positive samples.
approximately 10-g subsamples taken every 2 m from a distance of Mean daily species compositions for the 10-day sampling period are
approximately 3 m away from and along the length of the feed bunk presented in Figure 1. Eimeria bovis was the most numerous species
and around the entire perimeter of the hay ring. Approximately 10 g of identified on all 10 days, with a mean of 40% of the oocysts identified
soil also was collected from beneath 10 fecal pats that were several in each sample being E. bovis. The second most numerous species var-
days old and selected on a first seen basis while walking a diagonal ied slightly from day to day among E. zuernii, E. alabamensis, and E.
across the field. For each area (whole pasture, feed bunk area, hay ring cylindrica/ellipsoidallis-like. Overall, the mean species composition ex-
area, and fecal pats), samples were mixed well and a 20-g subsample hibited little daily variation.
was analyzed using the modified Wisconsin sugar flotation technique Results from the 10 heifers sampled on 15 April 2005 were generally
(Coxx and Todd, 1962). Oocysts were identified to species based on similar to the results obtained from the 10-day sampling period in No-
bovine Eimeria spp. oocyst morphological characteristics (Levine and vember (Table I). The mean oocyst count (145 OPG) and species com-
Ivens, 1986). position showed little change. Nine of the 10 Eimeria spp. previously
Eimeria spp. oocysts were seen in 94% of fecal samples collected seen were present (E. bukidonensis not seen). Oocysts of E. brasiliensis,
during the 10-day period. Heifer fecal oocyst counts ranged from 0– 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
FIGURE 1. Mean daily species compositions for heifers sampled 15 these samples. The mean percentages of oocysts identified in samples
November 2005 to 24 November 2005. 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-
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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-
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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.
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seen in mean fecal oocyst counts from the same heifers sampled ap- winter ranges in feedlots in Utah. Journal of Parasitology 48: 347–
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