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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: [email protected] ABSTRACT: Babesia Washington-1 (WA1) is a newly identified intraerythrocyte 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 susceptible animal model, the golden Syrian hamster. Two, 1-mo-old female 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 postinoculation (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 demonstrated by SEM and were more evident at 72 and 96 hr PI. Infected erythrocytes underwent changes in shape 24 hr PI, from few protrusions to several perforations, some of them resembling a ‘‘swiss cheese’’ appearance 96 hr PI. Several erythrocytes had irregular surface membranes 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 unknown (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 phylogenetic 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 erythrocytes and in ticks (Rudzinska et al., 1976, 1979; Rudzinska and Trager, 1977). Morphological hallmarks of Babesia spp. infection in erythrocytes are the presence of vacuole(s) within an organism and the pathognomonic 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 originally 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

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 fixative (a 50:50 solution of 2% paraformaldehyde and 2.5% glutaraldehyde 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 solutions. 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 (treatment 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, Austria). 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 hexamethyldesilazane, 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 multiple-merozoite-infected erythrocytes were observed frequently 72 hr PI with changes in the normal shape of the erythrocyte (Fig. 1A, B). Merozoites were amoeboid, uninucleated, vacuolated, and had host cell cytoplasm. In contrast, trophozoites were multinucleated, nonvacuolated, and had a high degree of polymorphism. Externally, almost all erythrocytes revealed surface membrane changes 96 hr PI (Figs. 2, 3). Severe alterations in the surface membrane of erythrocytes also included mul-

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FIGURE 4. (A) Free parasites close to an infected erythrocyte 96 hr PI. TEM (bar ⫽ 0.5 ␮m). (B) Magnification of A, showing free parasites. TEM (bar ⫽ 0.25 ␮m).

FIGURE 2. Severely affected erythrocytes 96 hr PI showing protrusions (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 observed 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).

marginalization of the merozoite inside the erythrocyte allowed formation 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 parasites were observed apparently invaginating the host cytoplasm, deforming 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 merozoites 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 invagination 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 (arrows). 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 invading 1 erythrocyte 96 hr PI. Vacuolization of the host cytoplasm (HC) is observed in 1 parasite beside an invaginated cytoplasm (arrow). Another 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).

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FIGURE 6. (A) Vacuolization of the host cytoplasm (HC) in 2 parasites 96 hr PI. The HC is invaginated (arrowhead) by 1 parasite that ‘‘closes’’ (arrow) the forming vacuole. TEM (bar ⫽ 0.5 ␮m). (B) Vacuolization of the HC in 1 merozoite 96 hr PI. Pseudopods can be observed (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).

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 division (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).

erythrocyte membrane and shape, including protrusions, perforations, or both. Extensive damage to the erythrocyte membrane, characterized as protrusions, inclusions, and perforations, have been reported with B. microti 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 percentage 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 pathogenesis associated with WA1 (Hemmer et al., 2000). The differential expression of certain inflammatory mediators in susceptible and resistant strains of mice could explain the different outcome of WA1 infection (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 invagination of the plasma membrane of the erythrocyte produces a parasitophorus vacuole that contains the merozoite covered by its own membrane and the host plasma membrane. This event appears to occur immediately after attachment since we observed few parasites in this process 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 severely 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 suggests that some distortions in the cell membrane, observed as perforations 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), however, with a greater frequency. Erythrocytes containing multiple parasites were frequently seen, with trophozoites in division forming char-

acteristic ‘‘tetrads.’’ Interestingly, some merozoites, still attached to each other by the posterior end, were closer to the erythrocyte membrane. 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 organelles 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 virulent in hamsters, inducing severe ultrastructural changes in the erythrocyte 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 Babesia. Laboratory Investigation 74: 853–859. GOETHERT, H. K., AND S. R. TELFORD, III. 2003. What is Babesia microti? 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 Pathology 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 infection 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.

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TELFORD, K. R. STEINGART, R. POLLOCK, D. H. PERSING, J. M. KOBAYASHI, 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 microti with emphasis on the feeding mechanism. Journal of Protozoology 23: 224–233. ———, AND W. TRAGER. 1977. Formation of merozoites in intraerythrocytic Babesia microti. Canadian Journal of Zoology 55: 928–938. ———, ———, S. J. LEWENGRUB, AND E. GUBERT. 1976. An electron

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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. Electron 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.

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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: [email protected] ABSTRACT: Cats are important in the natural epidemiology of Toxoplasma gondii, because they are the only hosts that can excrete environmentally resistant oocysts. Cats are infected with T. gondii via predation 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 natural 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 infections 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 transmission 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 chickens 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 (Dubey et al., 2005). The objective of the present study was to determine

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 anesthetized 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 Agriculture, 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 subcutaneously (s.c.) into 2–5 mice (Dubey, 1998). Brains and hearts of seronegative (MAT ⬍ 1:5) rats were pooled in batches of 5–15, and after digestion they were bioassayed in mice. The mice used were Swiss-Webster 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 described 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

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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 persistent 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 viable T. gondii in the absence of demonstrable antibodies (Dubey et al., 1997). Dubey and Frenkel (1998) summarized the worldwide prevalence 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 prevalent 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. Journal of Parasitology 92: 644–645. DUBEY, J. P. 1998. Refinement of pepsin digestion method for isolation of Toxoplasma gondii from infected tissues. Veterinary Parasitology 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.

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 Parasitology 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 Parasitology. 140 76–82. ———, S. K. SHEN, O. C. H. KWOK, AND P. THULLIEZ. 1997. Toxoplasmosis 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 infected 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. HONORE´, F. DEROUIN, AND L. D. SIBLEY. 1997. Determination 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

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 Pathoge´nie des Microorganismes, LAPM, UMR 5163 CNRS-UJF, Universite´ J. Fourier, Campus Sante´, Grenoble, France; †Laboratoire de Neurosciences Pre´cliniques, INSERM U318, Universite´ J. Fourier, Grenoble, France; *To whom correspondence should be addressed. e-mail: [email protected] 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 nervous system, e.g., astrocytes. In this study, we report that IL-12 and IL10 have no effect on penetration, replication, or cystogenesis of the T. gondii Prugniaud strain in human astrocytes in vitro and do not antagonize the role of IFN-␥ on cystogenesis. Toxoplasma gondii is a universally widespread protozoan. Toxoplasmosis is a health care problem, causing congenital toxoplasmosis and

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 IL10, which down-regulates IL-12 and IFN-␥ secretion, and avoids immunopathogenicity of the immune response (Wilson et al., 2005). Although infection is controlled, a percentage of parasites survive as dormant cysts, particularly in the host’s central nervous system. Parasites replicate in human astrocytes, and these cells have been found to support 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

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TABLE 1. Invasion rate and multiplication of Prugniaud strain in primary human astrocyte cell cultures. Invasion rate Treatment Astrocytes Astrocytes Astrocytes Astrocytes

⫹ ⫹ ⫹ ⫹

Tg Tg ⫹ IFN-␥ (500 U/ml) Tg ⫹ IL-12 (25 ng/ml) Tg ⫹ IL-10 (100 ng/ml)

Multiplication Infected cells/ 1,000 cells Tg/1,000 cells 47 48 53 46

⫾ ⫾ ⫾ ⫾

18 21 20 17

620 112 622 613

⫾ ⫾ ⫾ ⫾

70 58* 91 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), supplemented 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-glutamine, 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. Toxoplasmic 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, Oxford, 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 (Daubener 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 multiplication assays, cells were treated by cytokines at the time of T. gondii infection for 3 and 48 hr, respectively. For cyst formation study, cytokines 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 performed 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 Prugniaud 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-

FIGURE 1. Cystogenesis in primary human astrocyte cell cultures after treatment by cytokines for 6 days. Astrocytes (105/ml) were infected with 1.5 ⫻ 104 T. gondii/ml, and cysts were counted by indirect immunofluorescence with Ab CC2 6 days PI. Results from 3 experiments 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 IL12 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 antagonism 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 modulation 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, Germany) 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 recruitment 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 gondii: Monocyte chemotactic protein-1 secretion and chemokine expression 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 Neuroimmunology 43: 31–38. DEL RIO, L., B. A. BUTCHER, S. BENNOUNA, S. HIENY, A. SHER, AND E. Y. DENKERS. 2004. Toxoplasma gondii triggers myeloid differentiation factor 88-dependent IL-12 and chemokine ligand 2 (monocyte chemoattractant protein 1) responses using distinct parasite molecules and host receptors. Journal of Immunology 172: 6954–6960. FAGARD, R., H. VAN TAN, C. CREUZET, AND H. PELLOUX. 1999. Differential development of Toxoplasma gondii in neural cells. Parasitology Today 15: 504–507.

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GAZZINELLI, R. T., I. P. OSWALD, S. L. JAMES, AND A. SHER. 1992. IL10 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 directed against Toxoplasma gondii suitable for studying tachyzoitebradyzoite interconversion in vivo. Clinical and Diagnostic Laboratory 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 astrocytes. 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 defense in human native astrocytes. Advances in Experimental Medicine and Biology 527: 15–26.

RICARD, J., H. PELLOUX, A. L. FAVIER, U. GROSS, E. BRAMBILLA, AND P. AMBROISE-THOMAS. 1999. Toxoplasma gondii: role of the phosphatidylcholine-specific phospholipase C during cell invasion and intracellular development. Experimental Parasitology 3: 231–237. ———, ———, S. PATHAK, B. PIPY, AND P. AMBROISE-THOMAS. 1996. TNF-␣ enhances Toxoplasma gondii cyst formation in human fibroblasts through the sphingomyelinase pathway. Cellular Signaling 6: 439–442. SUBAUSTE, C. S., AND M. WESSENDARP. 2000. Human dendritic cells discriminate between viable and killed Toxoplasma gondii tachyzoites: Dendritic cell activation after infection with viable parasites results in CD28 and CD40 ligand signaling that controls IL-12dependent 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 toxoplasmic 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. Arau´jo*, R. A. Guicho´n, P. A. Martinez†, M. T. Civalero‡, and C. Aschero§, Departamento de Biologı´a, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata–CONICET, Argentina; *Escola Nacional de Sau´de Pu´blica—Fundac˛ao Oswaldo Cruz, Brazil; †Departamento de Biologı´a, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata; ‡Instituto Nacional de Antropologı´a y Pensamiento Latinoamericano–CONICET, Argentina; §Universidad Nacional de Tucuma´n–CONICET, Argentina. e-mail: [email protected] 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 procedures. Coprolite fragments were rehydrated in a trisodium phosphate aqueous solution and subjected to spontaneous sedimentation for microscope analysis. Eggs of nematodes identified as Trichuris sp., Capillaria sp., Uncinaria sp., and an ascaridid (probably Toxascaris sp.) or spirurids (presumably Physaloptera sp.), plus a cestode (Anoplocephalidae), presumably Moniezia sp., were found.

Paleoparasitological analysis was recently applied to bioanthropological 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.

area is characterized by human populations that exhibited various life style strategies, but mainly the hunter–gatherer type of subsistence (Aschero, 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 coprolite 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 abundant. Macroscopic and microscopic charcoal fragments also were pres-

RESEARCH NOTES

FIGURE 2. (a) Moniezia sp. egg showing hexacanth embryo and piriform apparatus. (b) Trichuris sp. egg. (c) Capillaria sp. egg. (d) Ascaridid—possibly Toxascaris sp. or Physaloptera sp. egg. (e) Ancylostomid egg, probably Uncinaria sp. (f ) Adult of Oribatida, Aphelacaridae, 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), presumably 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 embryo 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.,

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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, including species of Acaridida, Glycyphagidae, Listrophoridae, and probably 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 Farfa´n and Jimenez Milo´n, 2001), the largest fox inhabiting the area under study. The reddish hairs found in coprolite did not, however, correspond to canid fur according to Chehe´bar and Martı´n (1989). Dog or fox fur usually occurs in feces when it is accidentally ingested during selfgrooming (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 Martı´n, 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 feces. In contrast, P. culpaeus, a known local canid, is reported to be omnivorous and consumes fruits, seeds, and even arthropods (Novaro, 1997; Cornejo Farfa´n and Jimenez Milo´n, 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 Taenia, Hymenolepis, Echinococcus, and Diphyllobothrium (Gonc¸alves 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 anoplocephalid records in the Patagonian fauna have cited the occurrence of Moniezia expansa in Lama guanicoe (Beldomenico et al., 2003; C. Robles, 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 viscacia (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 Toxascaris sp. or Physaloptera sp. These nematodes are ubiquitous parasites 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 Canepuccia. We acknowledge the reviewers corrections of an early version of the manuscript. This work was supported by the project ‘‘Ecologı´a Evolutiva Humana en Patagonia,’’ SECYT–UNMDP 04-09929 and the ‘‘Convenio de Colaboracio´n entre el Instituto Canario de Bioantropologı´a del OAMC de Tenerife, Espan˜a y la Facultad de Cs. Sociales de la UNCPBA, Argentina.’’.

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

Egg length ⫻ width (␮m)

T. tenuis

46–50 ⫻ 28–30 — 51.9–79.0 (64.2 ⫾ 5.3) ⫻ 28.4–37.1 (31.9 ⫾ 0.9)

Trichuris sp.

79.0 ⫻ 32.0

T. T. T. T. T. T. T. T.

57.0–65.0 ⫻ 65.0–72.0 ⫻ 57.0–65.0 ⫻ 60.0–67.0 ⫻ 60.0–70.0 ⫻ 50.0–60.0 ⫻ 53.0–60.0 ⫻ 75.0 ⫻ 45.0

bradleyi fulvis robusti chilensis bursacaudata pampeana myocastoris dolichotis

29.0–34.0 28.0–31.0 29.0–36.0 32.0–34.0 20.0–30.0 20.0–30.0 30.0–34.0

Host

Reference

Camelus dromedarius Lama guanicoe L. glama

Chandler, 1930 Beldomenico et al., 2003 Rickard and Bishop, 1991

L. glama; Vicugna vicugna L. guanicoe Lama sp. Octodon sp.* Ctenomys sp.* Ctenomys sp.* Akodon sp.* Ctenomys sp.* Ctenomys sp.* Myocaster colpus* Dolichotis patagonum*

Cafrune et al., 1999 Beldomenico et al., 2003 Leguı´a et al., 1995 Babero et al., 1975 Babero and Murua, 1987 Babero and Murua, 1990 Babero et al., 1976 Suriano and Navone, 1994 Suriano and Navone, 1994 Baro´s et al., 1975 Morini et al., 1955

* Rodents.

LITERATURE CITED ASCHERO, C. A. 1982. Nuevos datos sobre arqueologı´a del Cerro Casa de Piedra, sitio CCP5. Relaciones 14: 267–284. ———. 1996. El a´rea Rı´o Belgrano-Lago Posadas (Santa Cruz): Problemas y estado de problemas. In Arqueologı´a so´lo Patagonia, J. G. Otero (ed.). CENPAT, CONICET. Buenos Aires, Argentina, p. 17– 26. ———, C. BELLELI, AND R. A. GON˜I. 1992. Avances en las investigaciones arqueolo´gicos en el Parque Nacional Perito Moreno (provincia de Santa Cruz, Patagonia, Argentina). Cuadernos del Instituto Nacional de Antropologı´a y Pensamiento Latinoamericano 14: 143–170. ———, ———, M. T. CIVALERO, R. A. GON˜I, G. GURAIEB, AND R. MOLINARI. 1992. ‘‘Cronologı´a y tecnologı´a en el Parque Nacional Perito Moreno (PNPM): Continuidad o reemplazos?’’ Arqueologı´a 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 Parasitology 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. BARO´S, V., G. MAJUMDAR, AND T. K. MIKAILOV. 1975. Morphology ad taxonomy of Trichocephalus myocastoris (Enigk, 1933). Folia Parasitologica 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 guanacos 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 revealed in coprolites. New Scientist 8: 35–40. CALLEN, E. O., AND P. S. MARTIN. 1969. Plant remains in some coprolites from Utah. American Antiguity 34: 329–331. CHAME, M. 2003. Terrestrial mammal feces: A morphometric summary

and description. Memorias do Instituto Oswaldo Cruz 98(Supplement 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. MART´ıN. 1989. Guı´a para el reconocimiento microscpico de los pelos de los mamferos de la Patagonia. Don˜ana, Acta Vertebrata 16: 247–291. CIVALERO, M. T. 1995. ‘‘El sitio Casa de Piedra 7: algunos aspectos de la tecnologı´a lı´tica y las estrategias de movilidad.’’ Cuadernos del Instituto Nacional de Antropologı´a 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 International 109–110: 77–86. CORNEJO FARFA´N, A., AND P. JIMENEZ MILO´N. 2001. Dieta del zorro andino Pseudalopex culpaeus (Canidae) en el matorral dese´rtico del sur de Peru´. Revista de Ecologı´a Latinoamericana 8: 1–9. DENEGRI, G. M. 2001. Cestodosis de herbı´voros domo´sticos de la Repu´blica Argentina de importancia en medicina veterinaria, Editorial Martı´n, 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 Parasitology 54: 81–88. EMMONS, L. H. 1987. Comparative feeding ecology of felids in a Neotropical rainforest. Behavioral Ecology and Sociobiology 20: 271– 283. FAULKNER, C. T. 1991. Prehistoric diet and parasitic infection in Tennessee: Evidence from the analysis of desiccated human paleofeces. American Antiquity 56: 687–700. GONC˛ALVES, M. L. C., A. ARAU´JO, 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-BAN˜OS, J. FREGENEDA GRANDES, AND E. PRADA MARCOS. 2003. Parasitological analysis of Leonese royalty from collegiate-basilica of St. Isidoro, Leo´n (Spain): Helminths, protozoa, and mites. Journal of Parasitology 89: 738–743. LEGU´ıA, P. G., A. E. CASAS, AND J. WHEELER. 1995. Parasitismo en came´lidos prehisto´ricos. Parasitologı´a al Dı´a 19: 435. LUTZ, A. 1919. Schistosoma mansoni e a schistosomatose segundo ob-

RESEARCH NOTES

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 Oswaldo Cruz 98(Supplement I): 127–138. MORINI, E. G., J. J. BOERO, AND A. RODRIGUEZ. 1955. Para´sitos intestinales en el ‘‘mara’’ (Dolichotis patagonum patagonum). Misio´n de Estudios de Patologı´a Regional Argentina 26: 83–89. NAVONE, G., AND M. L. MERINO. 1989. Contribucio´n al conocimiento de la fauna endoparasitaria de Lama guanicoe Muller, 1776, de Penı´nsula Mitre, Tierra del Fuego, Argentina. Boletı´n Chileno de Parasitologı´a 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 ruminants. Journal of Parasitology 77: 70–75. ROBLES, C., AND F. OLAECHEA. 2001. Salud y enfermedad de las majadas. In Ganaderı´a sustentable en la Patagonia Austral, P. Borrelli, and G. Oliva (eds.). INTA, Santa Cruz, Argentina, p. 225–243.

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RUAS, J. L, M. P. SOARES N. A. R. FARIAS, AND J. G. W. BRUM. 2003. Infecc˛a˜o por Capillaria hepatica em carnı´voros silvestres (Lycalopex gymnocercus e Cerdocyon thous) na regia˜o sul do Rio Grande do Sul. Arquivos do Instituto Biolo´gico 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 para´sitos de Duscycion griseus (Gray, 1837), D. culpaeus (Molina, 1782), y Conepatus chinga (Molina, 1782) (Mamı´fera: Carnı´vora) en Neuquen, Argentina. Sistema´tica y Ecologı´a. Boletı´n Chileno de Parasitologı´a 49: 60–65. SURIANO, D. M., AND G. T. NAVONE. 1994. Three new species of the genus Trihuris Roederer, 1761 (Nematoda: Trchuridae) from Cricetidae and Octodontidae rodents in Argentina. Research and Reviews in Parasitology 54: 39–46. THIENPONT, D., F. ROCHETTE, AND O. F. J. VANPARIJS. 1979. Diagno´stico de las helmintiosis por medio del examen coproparasitolo´gico. Janssen Research Foundation, Beerse, Be´lgica, 187 p. VALENZUELA, G., U. ALARCO´N, AND F. ALVAREZ. 2004. Helmintos para´sitos gastrointestinales en zorros Pseudalopex griseus de la Prov´ ltima Esperanza Sur de Chile. Actas del XIII Congreso incia de U Chileno de Medicina Veterinaria. http://magallanes.sag.gob.cl/ cong㛮val.pdf. ZIMMERMAN, M. R., AND R. E. MORILA. 1983. Enterobiasis in pre-Columbian 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: [email protected] ABSTRACT: Three species of remoras—Remora brachyptera (Lowe), Remora osteochir (Cuvier), and Remora remora (Linnaeus)—were collected from 4 species of billfishes—Istiophorus platypterus (Shaw), Makaira nigricans Lacepe´de, Tetrapturus albidus Poey, and Tetrapturus 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 reported 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 another 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 attached 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 angustirostris Tanaka, 1951) captured in the central Pacific at 21⬚04.5⬘N, 173⬚47.5⬘E by Lewis (1967). Shiino (1960) correctly proposed to transfer 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

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 platypterus (Shaw, 1792), Makaira nigricans Lacepe´de, 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. platypterus, 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 common remora (R. remora) seems to be rare on billfish of the Atlantic Ocean, but not the spearfish remora (R. brachyptera). In total, 21 spearfish 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

LL LL LL LL LL LL LL LL

1 4 6 9 13 24 27 29

29⬚57.36⬘N, 34⬚35.35⬘N, 34⬚02.18⬘N, 34⬚03.26⬘N, 33⬚54.51⬘N, 36⬚47.41⬘N, 33⬚25.44⬘N, 31⬚49.01⬘N,

LL LL LL LL

30 31 33 36

30⬚13.25⬘N, 28⬚45.31⬘W 7⬚29.35⬘N, 24⬚00.17⬘W 4⬚50.09⬘N, 22⬚24.57⬘W 8⬚05.27⬘S, 21⬚01.01⬘W

LL 37

50⬚06.30⬘W 48⬚19.52⬘W 53⬚11.30⬘W 57⬚22.42⬘W 59⬚43.56⬘W 45⬚39.35⬘W 37⬚02.46⬘W 27⬚57.57⬘W

7⬚50.13⬘S, 21⬚09.22⬘W

Billfish Makaira nigricans (1) Tetrapturus albidus (1) T. albidus (1) T. albidus (1) T. albidus (1) T. albidus (1) Istiophoru platypterus (1) M. nigricans (2) Tetrapturas pfluegeri (1) M. nigricans (1) I. platypterus (1) I. platypterus (5) osteochir (5) T. albidus (3) I. platypterus (2) T. albidus (1) T. pfluegeri (3) I. platypterus (1) T. pfluegeri (6)

LL 38

7⬚54.34⬘S, 21⬚14.27⬘W

LL 39

8⬚17.38⬘S, 24⬚28.09⬘W

I. platypterus (2)

LL 40

7⬚40.17⬘S, 25⬚39.06⬘W

M. nigricans (1) T. albidus (1) T. pfluegeri (1) I. platypterus (2)

LL 41

7⬚44.07⬘S, 22⬚17.55⬘W

LL 42

7⬚58.20⬘S, 21⬚03.27⬘W

LL 43

8⬚30.38⬘S, 21⬚47.30⬘W

M. nigricans (1) T. pfluegeri (2)

LL 44 LL 45

8⬚26.33⬘S, 21⬚57.35⬘W 8⬚15.17⬘S, 22⬚00.18⬘W

T. pfluegeri (1) I. platypterus (1) T. pfluegeri (4)

LL 46 LL 47

8⬚33.36⬘S, 29⬚12.29⬘ W 8⬚39.00⬘S, 29⬚19.12⬘W

LL 48

8⬚41.24⬘S, 29⬚18.00⬘W

LL 49 LL 50

9⬚09.36⬘S, 29⬚18.00⬘W 9⬚30.00⬘S, 29⬚30.36⬘W

LL 51 LL 52 LL 53

4⬚16.48⬘N, 41⬚24.36⬘W 3⬚09.36⬘N, 40⬚19.12⬘W 3⬚23.24⬘N, 40⬚17.24⬘W

LL 56

14⬚52.12⬘N, 47⬚52.48⬘W

I. platypterus (2) T. albidus (1) T. pfluegeri (5) T. pfluegeri (3)

T. albidus (1) T. pfluegeri (1) M. nigricans (1) T. albidus (1) I. platypterus (1) T. albidus (2) T. pfluegeri (5) T. pfluegeri (1) T. pfluegeri (1) T. albidus (1) T. albidus (1) T. albidus (1) I. platypterus (1) M. nigricans (2) M. nigricans (1)

Remora sp.

L. crassus

osteochir (1) osteochir (2) osteochir (2) osteochir (2) osteochir (2) osteochir (2) brachyptera (1) brachyptera (1) osteochir (1) osteochir (2) osteochir (2) osteochir (2) brachyptera (4) 3乆 osteochir (4) remora (1) brachyptera (1) osteochir (1) osteochir (2) osteochir (5) brachyptera (1) brachyptera (1) osteochir (12) brachyptera (1) osteochir (1) osteochir (2) osteochir (2) osteochir (4) brachyptera (2) osteochir (1) osteochir (2) osteochir (2) osteochir (4) brachyptera (2) osteochir (2) brachyptera (1) brachyptera (1) osteochir (1) osteochir (1) brachyptera (1) brachyptera (2) osteochir (3) osteochir (2) osteochir (2) osteochir (2) osteochir (2) remora (1) osteochir (5) osteochir (8) osteochir (3) brachyptera (1) osteochir (2) osteochir (2) osteochir (1) osteochir (1) osteochir (2) remora (1) osteochir (2)

1乆 1乆 3乆2么2C 3乆2么8C 0 4乆1么 0 0 1么 3乆1么2C 0 0 0 2乆1么 0 0 1乆1么1C 0 7乆1么 0 0 34乆8么2C 0 0 1乆2么 0 5乆10么1C 0 0 0 0 7乆6么5C 0 20乆3么 0 0 7乆 9乆2么 0 0 2么 0 1乆 8乆3么2C 1么 0 1乆1么1C 5乆 9乆1么1C 0 0 0 0 8乆3么2C 0 0 0

RESEARCH NOTES

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

1115

marlin sucker’s gill filaments. Shiino (1960) provided an excellent redescription of L. crassus, and our specimens show no significant discrepancy from that redescription. Permission for B.B.C. to participate in leg 3 of the Shoyo Maru longline 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 addition 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 provide 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 American 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. Records 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

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, VirginiaMaryland 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: [email protected] ABSTRACT: Rectal fecal samples were collected daily on 10 consecutive days in November 2004 from 11 weaned beef heifers to assess daily variation in fecal oocyst count and species composition. Subsequent 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 varied 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 variation during the 10-day sampling period in November, remained similar in April, and varied slightly in June. Twelve Eimeria spp. were identified in soil samples in proportions similar to those seen in fecal samples. 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

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 oocysts in the feces of freshly weaned beef calves in Australia, and Svennson et al. (1993) found E. alabamensis most prevalent in dairy calves (4–16 mo of age) just after turnout in Sweden. No reports exist, however, 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 clinically normal weanling beef calves and to determine whether changes occurred after a period of several months. Eleven beef heifers (Angus, Charolais, and Hereford breeding), maintained at the Virginia Tech Beef Center, Blacksburg, Virginia, were surveyed for natural coccidia infection. In the previous year, no outbreaks 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.

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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 Eimeria spp. E. E. E. E. E. E. E. E. E. E. E. E.

bovis zuernii alabamensis auburnensis cylindrica/ellipsoidalis-like canadensis subspherica wyomingensis pelita bukidonensis brasiliensis illinoisensis

15 April 2005

9 June 2005

Prevalence (%)

Mean oocysts/ sample (%)

Prevalence (%)

Mean oocysts/ sample (%)

Prevalence (%)

Mean oocysts/ sample (%)

100.0 96.0 91.0 91.0 80.0 67.0 46.0 18.0 34.0 13.0 0.0 0.0

39.7 13.4 15.3 10.2 9.2 7.1 2.0 1.7 1.1 0.4 0.0 0.0

100.0 90.0 90.0 100.0 100.0 100.0 40.0 30.0 30.0 0.0 20.0 0.0

41.5 7.9 12.2 5.8 17.5 8.4 1.6 1.6 0.8 0.0 2.8 0.0

100.0 89.0 89.0 100.0 78.0 100.0 0.0 22.0 33.0 33.0 89.0 33.0

27.3 6.9 5.0 13.6 3.4 21.8 0.0 4.0 3.0 4.8 8.9 3.0

Oocyst number in each fecal sample was calculated using the modified McMaster’s technique with a sensitivity of 25 oocysts/g (OPG) (Whitlock, 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 species based on oocyst morphology (Levine and Ivens, 1986). One individual (A.L.) carried out all oocyst counts and species identifications. Because oocysts of E. cylindrica and E. ellipsoidalis could not be distinguished reliably, all small cylindrical Eimeria spp. oocysts measuring from 19 to 36 ␮m by 8–18 ␮m were designated E. cylindrica/ellipsoidalis-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 approximately 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 maintained 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). Eimeria 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 varied slightly from day to day among E. zuernii, E. alabamensis, and E. cylindrica/ellipsoidallis-like. Overall, the mean species composition exhibited 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 November (Table I). The mean oocyst count (145 OPG) and species composition 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 percentage 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

TABLE II. Mean percentage of oocysts identified in soil samples from representative sites on the heifer lot. Eimeria spp. E. E. E. E. E. E. E. E. E. E. E. E.

bovis cylindrica/ellipsoidalis-like alabamensis zuernii auburnensis subspherica canadensis pelita illinoisensis brasiliensis wyomingensis bukidonensis

Mean oocysts/sample (%) 40.6 15.0 12.1 12.0 4.7 4.7 2.5 2.4 2.4 1.6 1.4 0.5

identified in these samples were similar to the percentages reported for the heifer fecal samples, with E. bovis predominating. Although coccidian oocysts of wild mammals and birds may have been present in the soil and could not be differentiated from bovine Eimeria spp., the similarity in species distribution between manure and soil samples suggests that most of the recovered oocysts were of bovine origin. There are no published reports describing short term daily variation in fecal oocyst count or Eimeria spp. composition in cattle. The results of this study, conducted on weanling beef heifers, found little daily variation in both fecal oocyst count and species composition over a 10day period. Minimal variation was seen during this period, thus the next 2 samplings were carried out on a single day to assess longer-term changes in oocyst count and species composition. Little change was seen in mean fecal oocyst counts from the same heifers sampled approximately 5 and 7 mo later, although some differences in mean species composition was observed. Eimeria brasiliensis and E. illinoisensis were not seen in the first sampling, but they were more common and numerous in subsequent samples. Likewise, E. canadensis became almost as numerous as E. bovis by June (Table 1). This suggests that there may be host and/or environmental factors that lead to gradual changes in the dominant Eimeria spp. present in cattle as they age. Results of this study also indicate that weanling beef heifers are infected with a diverse community of Eimeria spp. This diversity is consistent with surveys of cattle from around the world (Ernst et al., 1984; Parker et al., 1984; Hasbullah et al., 1990; Svensson et al., 1993). Soil samples collected from various locations on the heifer’s lot also had a diverse community of Eimeria spp. oocysts. Oocysts of all 12 Eimeria spp., identified in fecal samples collected over the course of the whole study, also were seen in similar proportions in these soil samples. This result, together with the fact that the majority (84%) of oocysts recovered from the soil were sporulated, suggests that oocysts in the environment sporulate and remain viable in proportions similar to that passed in the feces, at least in the warmer months of the year. Most published reports dealing with the epidemiology of coccidia infections in beef cattle focus on cow/calf pairs or freshly weaned calves (Fitzgerald, 1962; Parker et al., 1984; Ernst et al., 1987; Parker and

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Jones, 1987). Little information is available on levels of oocyst shedding 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 suggest that weanling heifers maintain a low level infection with a diverse community of Eimeria spp. Although the pathogenic species (E. bovis and E. zuernii) predominated, no clinical disease was observed throughout the study. Soil samples from the heifer’s pasture indicate that there is a diverse community of infective oocysts present in the animal’s environment. The persistent low level infections and continued reexposure in clinically normal animals suggest that by 9 mo of age, host and parasite have reached a relatively stable equilibrium. We thank the staff of the Virginia Tech Beef Center for coordinating animal husbandry. This research was part of a regional initiative, Pasture-Based Beef Systems for Appalachia, funded in part by USDA– ARS. LITERATURE CITED COXX, D. D., AND A. C. TODD. 1962. Survey of gastrointestinal parasitism in Wisconsin dairy cattle. Journal of the American Veterinary Medical Association 141: 706–709. DIAZ DE RAMIREZ, A., A. HERNANDEZ, A. GARCIA, AND L. N. RAMIREZIGLESIA. 2001. Excretion of oocysts of Eimeria spp. during the first three months of life in calves from diary farms in western Venezuela. Revista Cientifica, Facultad de Ciencia Veterinarias, Universidad del Zulia 11: 207–212. ERNST, J. V., H. CIORDIA, AND J. A. STUEDEMANN. 1984. Coccidia in cows and calves on pasture in north Georgia (U.S.A.). Veterinary Parasitology 15: 213–221. ———, T. B. STEWART, AND D. R. WHITLOCK. 1987. Quantitative determination of coccidian oocysts in beef calves from the coastal plain are of Georgia (U.S.A.). Veterinary Parasitology 23: 1–10. FITZGERALD, P. R. 1962. Coccidia in Hereford calves on summer and winter ranges in feedlots in Utah. Journal of Parasitology 48: 347– 351. HASBULLAH, Y. AKIBA, H. TAKANO, AND K. OGIMOTO. 1990. Seasonal distribution of bovine coccidia in beef cattle herd in the university farm. Japanese Journal of Veterinary Science 52: 1175–1179. LEVINE, N. D., AND V. IVENS. 1986. The coccidian parasites (Protozoa, Apicomplexa) of Artiodactyla. Illinois Biological Monographs No. 55, University of Illinois Press, Urbana, Illinois, 66 p. PARKER, R. J., K. BOOTHBY, I. POLKINGHORNE, AND R. G. HOLROYD. 1984. Coccidiosis associated with postweaning diarrhoea in beef calves in a dry tropical region. Australian Veterinary Journal 61: 181–183. ———, AND G. W. JONES. 1987. The development of Eimerian infections during the first eight months of life in unweaned beef calves in a dry tropical region of Australia. Veterinary Parasitology 25: 1–7. SVENSSON, C., P. HOOSHMAND-RAD, B. PEHRSON, M. TORNQUIST, AND A. UGGLA. 1993. Excretion of Eimeria oocysts in calves during their first three weeks after turn-out to pasture. Acta Veterinaria Scandinavia 34: 175–182. WHITLOCK, H. V. 1948. Some modifications of the McMaster helminth egg-counting technique apparatus. Journal of the Council for Scientific and Industrial Research 21: 177–180.