Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38°C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The original parasite material, derived from an infected Aotus trivirgatus monkey, was diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-day intervals. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours but were no longer highly synchronous. They have remained infective to Aotus.

In vivo and in vitro encystment of the cercariae of Echinochasmus liliputanus and biological activity of the metacercariae were studied. In vivo encystment of cercariae occurred in the gills of goldfish, the second intermediate host. However, the cercariae also encysted in vitro in Locke solution (0.6× to 1.2× strength), 0.7–1.2% NaCl, artificial gastric juice, and human gastric juice. Locke or NaCl solutions were shown to be appropriate for in vitro encystment to occur within 24 hr; however, full-strength Locke solution was shown to be optimal. The 1-day-old metacercariae formed in vivo and treated in 0.1% sodium deoxycholate excystation medium at 37 C for 1 hr showed 88.5% excystation. The metacercariae formed in vitro, however, showed 88.6% and 85.0% excystation for normal and abnormal ones, respectively. Abnormal cysts at room temperature usually die within 10 days. About 70% of the normal cysts, both in vivo and in vitro, can still excyst after being stored in Locke 0.5× solution at 4 C for 3 mo. Cysts formed in vivo and in vitro were equally infective. The encystment of the cercariae in vitro could be inhibited when the cercariae were treated with 1 μmol silver nitrate. Because silver nitrate binds to the papillae, especially to the ciliated papillae, on the cercaria surface, it is suggested that papillary chemoreceptors may be involved in encystment of the cercariae. The finding of E. liliputanus cercariae encysting in vitro, especially in human gastric juice, might be helpful in elucidating mechanisms of the definitive hosts that are directly infected by the cercariae.

The present paper describes the ultrastructure of spermiogenesis and the spermatozoon of Macracanthorhynchus hirudinaceus, an acanthocephalan parasite of the wild boar Sus scrofa. At the beginning of spermatogenesis, spermatocytes exhibit synaptonemal complexes and 2 centrioles. In the spermatid, only 1 centriole remains, generating a flagellum with a 9 2 pattern. Another ultrastructural feature observed during the spermiogenesis of M. hirudinaceus is the condensation of the chromatin, forming a “honeycomb” structure in the old spermatid and a homogeneous, electron-dense structure in the spermatozoon. The mature spermatozoon of M. hirudinaceus presents a reversed anatomy, as has been described previously in other species of the Acanthocephala. The spermatozoon is divided into 2 parts: an axoneme, and a nucleocytoplasmic derivative. The spermatozoon flagellum exhibits a 9 2 or 9 0 pattern. The process of spermiogenesis and the ultrastructural organization of the spermatozoon of M. hirudinaceus are compared with available data regarding other acanthocephalan species.

Neospora caninum, a recently recognized protozoan parasite of animals, is considered to be a major cause of bovine abortion worldwide. Although its life cycle is not completely known, recent studies suggest that the sexual stage occurs in dogs. The prevalence of sexual reproduction in N. caninum, however, is unknown. We investigated the ability of 3 N. caninum isolates (NC-1, NC-SweB1, and NC-Liverpool) to propagate asexually for approximately 250 parasite generations in a cell line in which they had not been cultured previously. The malthusian parameter of fitness was estimated for each isolate from 10 independent replicates of tachyzoites at the beginning as well as at the end of the experimental period. Derived and ancestral values for mean fitness were compared both within and among NC-1, NC-SweB1, and NC-Liverpool isolates. Results showed a significant increase in mean fitness for the 3 N. caninum isolates at the end of the experimental period. These findings indicate that N. caninum can adapt to new environmental conditions without the help of sexual recombination, supporting the idea that this parasite has, at least potentially, the capacity for maintaining clonal propagation in nature.

Host specificity has 2 independent facets: the extent to which different host species are used by a parasite, and the phylogenetic distances among these hosts. Although the number of host species exploited by a parasite commonly is used as a measure of host specificity, it fails to capture ecological and phylogenetic differences among hosts. Here, a new index of host specificity, S_TD*, is developed and illustrated. This index measures the average taxonomic distinctness among the host species used by a parasite, weighted for the parasite's prevalence in the different hosts. For a given number of host species, the index approaches its minimum value when a parasite achieves high prevalence in a few closely related host species, and the index approaches its highest value when a parasite reaches its highest prevalence values in distantly related host species. Simple hypothetical examples are used to demonstrate the index's computation and some of its properties. The new index is influenced independently both by the taxonomic (or phylogenetic) affinities of a set of host species and by the distribution of prevalence values among these hosts. A single value cannot truly capture all the nuances of a phenomenon as complex as host specificity; nevertheless, the proposed index incorporates the features of specificity that are most relevant to parasitologists and will be a useful tool for comparative studies.

Accurate estimates of population-level parameters of parasites, such as prevalence and mean intensity, require large sample sizes. The processing of such samples becomes an overwhelming task when parasites are abundant, as with trematode metacercariae in fishes. In the present study, a subsampling method reduced processing time while maintaining an accurate estimation of metacercariae prevalence and intensity across 3 trematode species and 2 fish species. By double sampling, we generated regression models to predict total intensity from a combination of subsamples. The key to this approach lies in choosing the best strategy from a large number of potential subsampling routines. We selected the most efficient routine by weighing the costs and benefits of each. This approach, however, could not provide an estimate of parasite abundance when no parasites occurred in the initial subsample. To estimate prevalence accurately, our subsampling algorithm prescribed an additional sampling sequence using a new, optimal regression model. In addition, we optimized the technique to measure three parasite species infecting a single host simultaneously. This efficient subsampling procedure decreased the overall processing time per host by up to 91% while obtaining accurate (R² > 0.8) estimates for both prevalence and intensity.

Empruthotrema stenophallus n. sp. (Monogenea: Monocotylidae) is described from specimens from the nasal tissue of the blue-spotted maskray Dasyatis kuhlii (Müller and Henle, 1841) collected in shallow waters off Pulau Banggi and Pulau Mabul, Sabah, Borneo, Malaysia. This is the first monogenean species to be described from an elasmobranch collected from Sabah. E. stenophallus can be distinguished from the other 6 members of the genus by the morphology of the sclerotized male copulatory organ, which is narrow, short, and distally tapered. E. dasyatidis Whittington and Kearn, 1992, previously documented from the nasal tissue of several of elasmobranch species from Australia, is recorded from 8 host species distributed around Malaysian Borneo. These represent new host and locality records for this monocotylid. The difficulties in identifying species of Empruthotrema and the apparent lack of host specificity by some members of the genus are discussed.

In 2000, we initiated an investigation on the tick fauna of Rondônia State, where we collected many specimens of Amblyomma scalpturatum Neumann, 1906 and Amblyomma incisum Neumann, 1906. In addition, we also collected a third group of ticks that were morphologically closely related to those 2 species, but sufficiently different to be considered a distinct species; members of this group were subsequently identified as Amblyomma latepunctatum Tonelli-Rondelli, 1939, through comparison with the type specimens of this taxon. Herein, we redescribe both sexes of A. scalpturatum and A. incisum, the female of A. latepunctatum, and provide the first description of the male of this latter species. Molecular analysis of the second internal transcribed spacer (ITS2) from rDNA of specimens of the 3 species supports morphological results. Examination of both A. scalpturatum and A. incisum deposited in different tick collections revealed that A. latepunctatum appeared relatively frequently in the vials believed to contain specimens of A. incisum or A. scalpturatum. Before this study, A. latepunctatum was considered a synonym of A. scalpturatum. Herein, we provide morphological and molecular evidence to validate the species A. latepunctatum. The South American tapir (Tapirus terrestris) seems to be the primary host for the adult stage of A. latepunctatum, A. scalpturatum, and A. incisum.

Mother sporocysts of Schistosoma mansoni transport exogenously supplied serotonin (5-hydroxytrypamine; 5-HT), and respond to it with increases in motility. In the present study, we investigated the importance of 5-HT transporter activity in the manifestation of these 5-HT-induced motility changes, and further examined the role of 5-HT in the development of daughter sporocysts in vitro. Serotonin-induced motility of in vitro-derived sporocysts is not inhibited by antidepressant compounds, e.g., fluoxetine, that block 5-HT transport, suggesting that the receptors responsible for motility responses to 5-HT are surface exposed. Using a sporocyst in vitro culture system, we show that depletion of larval stores of 5-HT reduces production of daughter sporocysts, the second intramolluscan larval stage. Moreover, we demonstrate a strong correlation between endogenous 5-HT levels and basal mother sporocyst muscle activity. Overall, these data suggest that larval stages of S. mansoni can detect exogenous 5-HT via surface-exposed receptors, and they are consistent with the hypothesis that endogenous stores of 5-HT are important for the proper regulation of muscular contractions in mother sporocysts, and for the successful emergence of daughter sporocysts.

The present study demonstrates that axenic cultures of Leishmania (Viannia) lainsoni produce larger cell masses in NNN-LIT medium, as well as higher amounts of total proteins in cell extracts, than Leishmania (Leishmania) amazonensis. Antigenicity of L.(V.)lainsoni whole promastigotes is similar to that of L. (L.) amazonensis, as demonstrated by an indirect immunofluorescence diagnostic test using sera from human patients and dogs infected with visceral leishmaniasis. Infectivity of the L.(V.)lainsoni strain used in the present work was demonstrated by the detection by transmission-electron microscopy of tissue amastigotes in skin lesion samples from an experimentally infected hamster. Incubation of lesion fragments in NNN-LIT medium allowed us to obtain promastigote forms, which could be cultivated successfully in vitro. Isoenzyme analysis of such promastigotes confirmed the parasite strain as L. (V.) lainsoni, as compared to other Leishmania reference strains. Our data indicate that L. (V.) lainsoni is a useful alternative source for antigen production as well for use in assays that depend on large cell volumes of Leishmania spp. parasites.

The prevalence of Toxoplasma gondii in free-range chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 102 free-range chickens (Gallus domesticus) from Grenada was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 53 (52%) chickens with titers of 1:5 in 6, 1:10 in 4, 1:20 in 4, 1:40 in 4, 1:80 in 15, 1:160 in 9, 1: 320 in 5, 1:640 in 4, and 1:1,280 or greater in 2. Hearts, pectoral muscles, and brains of 43 seropositive chickens with MAT titers of 1:20 or greater were bioassayed individually in mice. Tissues of each of 10 chickens with titers of 1:5 and 1:10 were pooled and bioassayed in mice. Tissues from the remaining 49 seronegative chickens were pooled and fed to 4 T. gondii-free cats. Feces of cats were examined for oocysts; they did not shed oocysts. T. gondii was isolated from 35 of 43 chickens with MAT titers of 1:20 or greater; from the hearts, brains, and pectoral muscles of 2, hearts and brains of 20, from the hearts alone of 11, and brains alone of 2. T. gondii was isolated from 1 of 10 chickens with titers of 1:5 or 1:10. All 36 T. gondii isolates were avirulent for mice. Genotyping of these 36 isolates using polymorphisms at the SAG2 locus indicated that 29 were Type III, 5 were Type I, 1 was Type II, and 1 had both Type I and Type III. Genetically, the isolates from Grenada were different from those from the United States; Type II was the predominant type from the United States. Phenotypically, all isolates from Grenada were avirulent for mice, whereas those from Brazil were mouse-virulent. This is the first report of isolation of T. gondii from chickens from Grenada, West Indies.

Toxoplasma gondii represents the most prominent infectious parasitic organism found in humans. While normally asymptomatic in healthy individuals, toxoplasmosis can cause abortion in patients during pregnancy, or can be fatal in immunosupressed individuals such as persons suffering from acquired immunodeficiency syndrom (AIDS). Toxoplasma gondii infection in humans is routinely assesssed by serological means. Here, we show that detection of anti-T. gondii IgG is also possible using a non-invasive methodology employing saliva. Sera and saliva of 201 healthy volunteers were investigated for the presence of anti-T. gondii-IgG antibodies by immunoblotting. The sera of 59 (29.4%) individuals showed IgG antibodies against T. gondii by ELISA, Vidas, and immunoblotting; 58 (98.3%) of these were also positive for anti-T. gondii IgG in the saliva immunoblot, with diagnostic relevant bands of Mr of 32–35 kDa and 40–45 kDa. The saliva immunoblot test exhibits a specificity of 100% and a sensitivity of 98.5%. Thus, saliva could be used as an alternative, non-invasive means for the detection of specific anti-T. gondii IgG in humans.

Neosporosis is an important cause of pregnancy loss in cattle worldwide. The objective of the present study was to identify Neospora caninum antigens as vaccine candidates using antigen-specific, short-term CD4 T cells established from N. caninum–immunized and –challenged cows. Whole N. caninum tachyzoite lysate was separated into 6 fractions by DEAE anion-exchange chromatography using high-pressure liquid chromatography (HPLC). The CD4 T-cell proliferation assay results indicated that antigenic activity was associated with proteins from HPLC fractions 4–6, with fraction 5 exhibiting the highest antigenic activity. Also, SDS-PAGE analysis revealed a 16-kDa protein in fractions 4–6 that was recognized by anti–N. caninum antibodies. This 16-kDa protein was absent in other fractions, and it may be a target of a T-cell response in cattle. Further identification of immunogenic proteins of N. caninum may facilitate development of subunit vaccines against neosporosis.

From August 1997 to July 2003, we collected 2,103 fecal samples from free-ranging individuals of the 3 colobus monkey species of Uganda—the endangered red colobus (Piliocolobus tephrosceles), the eastern black-and-white colobus (Colobus guereza), and the Angolan black-and-white colobus (C. angolensis)—to identify and determine the prevalence of gastrointestinal parasites. Helminth eggs, larvae, and protozoan cysts were isolated by sodium nitrate flotation and fecal sedimentation. Coprocultures facilitated identification of helminths. Seven nematodes (Strongyloides fulleborni, S. stercoralis, Oesophagostomum sp., an unidentified strongyle, Trichuris sp., Ascaris sp., and Colobenterobius sp.), 1 cestode (Bertiella sp.), 1 trematode (Dicrocoeliidae), and 3 protozoans (Entamoeba coli, E. histolytica, and Giardia lamblia) were detected. Seasonal patterns of infection were not apparent for any parasite species infecting colobus monkeys. Prevalence of S. fulleborni was higher in adult male compared to adult female red colobus, but prevalence did not differ for any other shared parasite species between age and sex classes.

Molecular identification of dorsal-spined larvae (DSL) from fecal samples indicates that the protostrongylid parasite Parelaphostrongylus odocoilei occupies a broader geographic range in western North America than has been previously reported. We analyzed 2,124 fecal samples at 29 locations from thinhorn sheep (Ovis dalli dalli and O. d. stonei), bighorn sheep (Ovis canadensis canadensis and O. c. californiana), mountain goats (Oreamnos americanus), woodland caribou (Rangifer tarandus caribou), mule deer (Odocoileus hemionus hemionus), and black-tailed deer (O. h. columbianus). The DSL were recovered from populations of thinhorn sheep south, but not north, of the Arctic Circle, and they were not recovered from any of the bighorn sheep populations that we examined. In total, DSL were recovered from 20 locations in the United States and Canada (Alaska, Yukon Territory, Northwest Territories, British Columbia, Alberta, and California). The DSL were identified as P. odocoilei by comparing sequences of the second internal transcribed spacer (ITS2) region of ribosomal RNA among 9 protostrongylid species validated by adult comparative morphology. The ITS2 sequences were markedly different between Parelaphostrongylus and other protostrongylid genera. Smaller fixed differences served as diagnostic markers for the 3 species of Parelaphostrongylus. The ITS2 sequences (n = 60) of P. odocoilei were strongly conserved across its broad geographic range from California to Alaska. Polymorphism at 5 nucleotide positions was consistent with multiple copies of the ITS2 within individual specimens of P. odocoilei. This work combines extensive fecal surveys, comparative morphology, and molecular diagnostic techniques to describe comprehensively the host associations and geographic distribution of a parasitic helminth.

The nucleotide sequence of a full-length cDNA encoding phosphofructokinase (PFK) enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2,653 bases comprises a single open reading frame of 2,452 bases and a noncoding region of 201 bases after the stop codon. The mature protein contains 812 amino acids and has a molecular mass of 90,900 Da. The amino acid sequences of several peptides derived from the purified protein show excellent correspondence with the translated nucleotide sequence. Comparison of the amino acid sequence of the protein with those of 3 other worms as well as those of human, rabbit, and bacterial enzymes reveals highly conserved regions interrupted with stretches of lesser sequence similarity. Analyses of the subunit primary structure reveal, as in other eukaryotic PFKs, that the amino-terminal half is homologous to the carboxy-terminal half, supporting the hypothesis that the PFK gene evolved by duplication of the prokaryotic gene and that the allosteric sites arose by mutations at the catalytic site. The location of the phosphorylation site is unique and different compared with other PFKs and plays a key role in regulation of the enzyme activity. Structural motifs such as the putative substrate and effector binding domains and also the key amino acids involved therein are clearly identified by alignment of all the PFK protein sequences.

Parapharyngodon kenyaensis n. sp. and Thelandros samburuensis n. sp. (Nematoda: Pharyngodonidae) from the large intestine of the agamid lizard (Agama caudospina) are described and illustrated. Parapharyngodon kenyaensis n. sp. is the 41st species assigned to the genus, and it differs from other species in that genus by possessing 3 pairs of caudal papillae, cloacal lip adornment, and spicules of 112–120 μm in length. Thelandros samburuensis n. sp. is the 31st species assigned to the genus, and it differs from other species in that genus by possessing swollen posterior annulations, 6 caudal papillae, a smooth anterior cloacal lip, and spicules of 43–52 μm in length. In addition to the 2 new nematode species, Abbreviata ortleppi (Nematoda: Physalopteridae) and Strongyluris ornata (Nematoda: Heterakidae) were found.

Oswaldocruzia costaricensis n. sp. (Strongylida: Molineidae) from the intestines and Rhabdias savagei n. sp. (Rhabditida: Rhabdiasidae) from the lungs of Rana cf. forreri (Anura: Ranidae) are described and illustrated. Oswaldocruzia costaricensis represents the 77th species assigned to the genus and differs from the other Neotropical species in the genus by possessing a Type II bursa and long cervical alae. Rhabdias savagei represents the 47th species assigned to the genus and differs from other Neotropical species in the genus by possession of 4 lips and a postequatorial vulva. Rana cf. forreri was also found to harbor the trematodes, Haematoloechus parcivitellarius and Megalodiscus temperatus, the nematodes, Aplectana incerta, Aplectana itzocanensis, Cosmocerca podicipinus, Foleyellides striatus, Subulascaris falcaustriformis, and a larva of the nematode Brevimulticaecum sp. Cosmocerca panamaensis is considered to be a synonym of Cosmocerca podicipinus.

Procamallanus (Spirocamallanus) halitrophus Fusco and Overstreet, 1978 from the intestines of flounders (Syacium papillosum and Citharichthys macrops) from the coast of the State of Rio de Janeiro, Brazil, is redescribed. This study, which is based on results obtained by light and scanning electron microscopy, clarifies many morphological aspects of this species. Some taxonomic features, such as the cephalic structures, the topography of the cuticle, the vulva, the localization of caudal papillae on males, and the excretory pore, are observed for the first time. C. macrops represents a new host record, and Rio de Janeiro is a new geographical record for this species.

A new species of digenetic trematode and 2 species of ectoparasites from Zalophus wollebaeki Silvertsen, 1953 (Carnivora: Otariidae) in the Galapagos Islands, Ecuador, are reported. These include an eye fluke of Philophthalmus Looss, 1899 (Echinostomata: Philophthalmidae) as well as, to our knowledge, the first report of Antarctophthirus microchir (Trouessart and Neumann, 1888) Enderlein, 1906 (Arthropoda: Anoplura) and Orthohalarachne diminuata (Doetschman, 1944) Newell, 1947 (Arthropoda: Acarina) from this host and location. Philophthalmus zalophi n. sp. differs from the 4 other marine species of Philophthalmus (P. andersoni Dronen and Penner, 1975; P. burrili Howell and Bearup, 1967; P. hegeneri Penner and Fried, 1963; and P. larsoni Penner and Trimble, 1970) by its mammalian host, large body size, lack of tegumental spines, posterior length of seminal vesicle, placement of genital pore, size ratio of oral sucker to acetabulum, shape and size of testes, and size ratio of ovary to testis.

In January 2003, fecal samples from 13 live pygmy rabbits, Brachylagus idahoensis (Merriam, 1891), were collected at the Oregon Zoo in Portland, Oregon, and sent to the University of New Mexico (UNM), Albuquerque, New Mexico, to be examined for coccidia. In July 2004, 14 more fecal samples were collected and sent to UNM, 6 from some of the same rabbits and 8 from 16 other rabbits (4 were pooled samples from siblings). In addition, tissue sections from 3 dead rabbits (2 from the Oregon Zoo, 1 from Washington State University) also were examined. Two of 4 (50%) pooled fecal samples and 8 of 17 (47%) 1-rabbit samples were positive for a single species of Eimeria, which we describe here as a new species. Sporulated oocysts were subspheroidal, 25.6 × 23.8 (22–28 × 21–27) μm, with a length:width (L:W) ratio of 1.1 (1.0–1.2). A micropyle (∼2 μm wide) and 0–1 polar granules were present, but an oocyst residuum was absent. Sporocysts were ellipsoidal, 13.4 × 8.1 (11– 16.5 × 7.5–9) μm, with a L:W ratio of 1.7 (1.3–2.2), and they had a Stieda body and sporocyst residuum. Tissue sections showed a heavy infection of the villous epithelial cells of the proximal and mid-small intestine with coccidial endogenous stages, but no stages were found in liver hepatocytes. Meronts with approximately 46 (26–70) merozoites per infected cell appeared to be fully developed and were subspheroidal, 14.8 × 13.9 (13–18 × 10.5–16.5) μm. Developing macro- and microgamonts were indistinguishable from each other and were spheroidal to subspheroidal, 10.4 × 9.5 (9–11 × 7.5–10.5) μm. Mature macrogamonts were spheroidal to subspheroidal, 14.2 × 13.7 (12–17 × 11–16) μm, and mature microgamonts were smaller and subspheroidal, 11.9 × 10.8 (10.5–13 × 9–12) μm. This eimerian seems to be extremely pathogenic to young pygmy rabbits, and given the precarious nature of this unique genetic population, it appears to be an emerging pathogen that deserves immediate further study.

A new species of Cryptosporidium, C. bovis, is described. Oocysts of C. bovis, previously identified as Cryptosporidium genotype Bovine B (GenBank AY120911), are morphologically indistinguishable from those of C. parvum. They are excreted fully sporulated and contain 4 sporozoites, but lack sporocysts. Oocysts measure 4.76–5.35 μm (mean = 4.89 μm) × 4.17–4.76 μm (mean = 4.63 μm), with a length-to-width ratio of 1.06 (n = 50). Oocysts were not infectious for neonatal BALB/ c mice, but were infectious for 2 calves that were previously infected with C. parvum. Oocysts were not infectious for 2 experimentally exposed lambs less than 1 wk of age and were not detected in 42 lambs 2–3 mo of age, but were detected in a 2-wk-old lamb. In an earlier study, 79 of 840 calves on 14 dairy farms in 7 states were found infected with the new species. Most calves were 2–7 mo of age and none exhibited signs of diarrhea. This new species has been found in 10 of 162 calves aged 9 to 11 mo on a beef farm in Maryland. Fragments of the 18S rDNA, HSP-70, and actin genes were amplified by PCR, and purified PCR products were sequenced. Multilocus analysis of the 3 unlinked loci demonstrated the new species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further evidence of species status. Based on these biological and molecular data, we consider this highly prevalent Cryptosporidium that infects primarily postweaned calves to be a new species and propose the name Cryptosporidium bovis n. sp. for this parasite.

Here we describe the first species of sanguinicolid blood fluke (Trematoda: Digenea) from a polynemid fish. Chaulioleptos haywardi n. gen., n. sp. is described from Filimanus heptadactyla Cuvier, 1829 (Perciformes: Polynemidae), the sevenfinger threadfin from Sandgate, Moreton Bay (southeast Queensland, Australia). Chaulioleptos haywardi differs from existing sanguinicolid genera in the combined possession of the following 7 characters: 2 testes, an entirely postovarian uterus, a uterine chamber, separate genital pores, an H-shaped intestine with abbreviated anterior caeca, tegumental spines in incomplete ventromarginal transverse rows that are continuous along the length of the body, and vitelline follicles that are tightly compacted and subsequently appear to form a solid branching mass occupying the area anterior to intestinal bifurcation and extending posteriorly to the level of the posterior margin of the anterior testis. Chaulioleptos haywardi is most closely related to Paracardicola Martin, 1960 and Adelomyllos Nolan and Cribb, 2004.

Prevalence and disease caused by isosporoid coccidia in passerine birds are well recognized, but confusion about the life cycles of the parasites has led to taxonomic inconsistencies. In this study, we characterized segments of the chromosomal small and large-subunit ribosomal RNA (rRNA) genes of coccidial parasites from 23 species of passerine birds, as well as heat shock protein 70, apicoplast rRNA, and chromosomal 5.8s rRNA genes from a subgroup of these animals, and we correlated genetic data with morphologic findings for different parasite developmental stages, host phylogeny, and overall taxonomic relations within the phylum Apicomplexa. Our findings indicate that isosporoid coccidia of passerine birds are monophyletic but exhibit substantial diversity, with most avian species having one or several unique parasite lineages that underwent synchronous speciation with their hosts, interrupted by sporadic episodes of lateral transmission across species and families. Molecular analyses support a homoxenous life cycle, with sexual forms occurring chiefly in the intestines and asexual merozoites present systemically. Rarely, extraintestinal sexual stages can occur. The passerine coccidia are genetically most closely related to species of Eimeria rather than Isospora. We suggest that these parasites, whether identified from blood merozoite stages or fecal oocysts, be provisionally grouped as a homogeneous clade of individual species in a single taxon and formally named when reliable criteria allowing reclassification of related genera in the suborder Eimeriina are clarified.

A new species of Urotrema inhabits Norops oxylophus and Norops cupreus from the Area de Conservación Guanacaste, Costa Rica. The new species is most similar to Urotrema scabridum but differs by having a relatively longer esophagus, at least 10% of the total body length versus 4–8% in U. scabridum; an oral sucker width : ventral sucker width ratio less than 1 : 1 (averaging 1 : 0.76) versus 1 : 1 or greater; an oral sucker width : pharyngeal width ratio less than 1 : 0.5 (averaging 1 : 0.42) versus greater than 1 : 0.5; and by having many transversely oriented posttesticular uterine loops versus a few vertically oriented posttesticular uterine loops.

In the present study, we used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to compare polypeptides of trypanosomes isolated by hemoculture of squirrel monkeys displaying Trypanosoma saimirii blood trypomastigotes, with other trypanosomes that infect primates to evaluate the validity of T. saimirii. The polypeptide profiles of trypanosomes isolated directly from squirrel monkeys or after their passage in mice were identical to those of 3 standard strains of T. rangeli, but they were distinct from those of T. cruzi, T. conorhini, and T. minasense. These results strengthen previous morphological and biological findings by Rodhain on trypanosomes of the squirrel monkey and lead to the conclusion that T. saimirii is indeed a junior synonym of T. rangeli.

NcMIC1 is a 460 amino acid Neospora caninum microneme protein implicated in host cell adhesion and invasion processes. In this study, we assessed the potential protectivity of NcMIC1-based vaccination against experimental N. caninum infection in mice, employing both recombinant antigen vaccines and DNA vaccines. Recombinant NcMIC1 (recNcMIC1) was expressed in Escherichia coli as gluthatione-S-transferase-fusion protein. The corresponding NcMIC1 cDNA was cloned into the pcDNA3.1 expression plasmid (pcDNA-MIC1), and expression was checked in transfected Vero cells. Mice (10 animals/group) were vaccinated either with recNcMIC1 antigen suspended in Ribi-adjuvant (3 intraperitoneal injections), pcDNA-NcMIC1 (3 intramuscular injections), or pcDNA-NcMIC1 (twice intramuscularly), followed by 1 intraperitoneal recNcMIC1 antigen boost. Control groups included corresponding treatments with adjuvant, pcDNA3.1 without insert, and PBS (= infection control). All vaccinated and control groups were then challenged intraperitoneally with 2 × 10⁶ N. caninum tachyzoites. Animals were inspected daily for a period of 3 wk postinfection (PI). At day 21, all animals were killed and assessed for infection. Before day 21 PI, clinical signs such as walking disorders, rounded back, apathy, and paralysis occurred in infection controls (50% of the mice), pcDNA and adjuvant controls (20% each), and the combined pcDNA-NcMIC1/recNcMIC1-treated group (30%). No clinical symptoms were observed in the recNcMIC1 and pcDNA-NcMIC1 vaccinated groups. All mice were positive for cerebral N. caninum infection as assessed by PCR of brain tissue. However, quantitative real-time PCR revealed that the infection intensity was significantly reduced in the group vaccinated with recNcMIC1 antigen. Immunohistochemistry confirmed these findings. In contrast, the infection intensity was highest in the group vaccinated with the pcDNA-NcMIC1/recNcMIC1 combination, indicating that the sequential application of the DNA vaccine and recombinant antigen had a deleterious effect. Serological analysis showed that only recNcMIC1-immunized animals generated detectable antibody levels recognizing native NcMIC1. Thus, of all protocols applied here, only recNcMIC1 vaccination appears to be suited to reduce cerebral infection in mice challenged with N. caninum tachyzoites.

Avian coccidiosis is an intestinal disease caused by protozoa of the genus Eimeria. To investigate the potential of recombinant protein vaccines to control coccidiosis, we cloned 2 Eimeria sp. genes (EtMIC2 and 3-1E), expressed and purified their encoded proteins, and determined the efficacy of in ovo immunization to protect against Eimeria infections. Immunogen-specific serum antibody titers, parasite fecal shedding, and body weight gains were measured as parameters of disease. When administered alone, the recombinant EtMIC2 gene product induced significantly higher antibody responses, lower oocyst fecal shedding, and increased weight gains compared with nonvaccinated controls following infection with E. tenella. Combined embryo immunization with the EtMIC2 protein plus chicken cytokine or chemokine genes demonstrated that all 3 parameters of vaccination were improved compared with those of EtMIC2 alone. In particular, covaccination with EtMIC2 plus interleukin (IL)-8, IL-16, transforming growth factor-β4, or lymphotactin significantly decreased oocyst shedding and improved weight gains beyond those achieved by EtMIC2 alone. Finally, individual vaccination with either EtMIC2 or 3-1E stimulated protection against infection by the heterologous parasite E. acervulina. Taken together, these results indicate that in ovo vaccination with the EtMIC2 protein plus cytokine/chemokine genes may be an effective method to control coccidiosis.

Hyperimmune anti-Cryptosporidium parvum ovine colostral whey (HOCW) was tested to determine whether it conferred passive immunity to newborn NMRI mice. Three HOCWs (groups IV–VI), 2 nonimmune colostral wheys (groups II and III), and PBS (group I) were administered once (experiment A) and 3 times (experiment B) daily from −1 to 15 days postinfection (PI). Mice in groups I–VI were inoculated with 5 × 10⁵ oocysts (day 0 PI), and group VII mice acted as controls. The percentage and intensity of infection were measured at 6, 9, 12, and 16 days PI. In experiment A, HOCW did not reduce significantly the percentage and intensity of infection except for mice in group VI treated with HOCW with the highest titers of anti-C. parvum antibodies. In contrast, no infection was detected in between 18.7 and 62.5% of the mice in groups IV–VI in experiment B. Furthermore, in these groups, the intensity of the infection decreased significantly, ranging from 83.5 to 97.4%. Thus, HOCW did not completely avoid infection, but a high level of protection was observed, being proportional to the titer of specific antibodies and the amount of whey administered orally. Finally, group VII showed no presence of oocysts.

Effects of repeated treatment with pyrantel pamoate on Enterobius vermicularis infection in chimpanzees were assessed by observing worms discharged in the feces after administration of anthelmintic treatment. Three of 9 chimpanzees reared in a zoological garden in Japan were subjected to fecal worm count and morphometric observation, and all were given oral pyrantel pamoate 6 times at 10-day intervals simultaneously. Following the first and second treatments, more than 30,000 pinworms were discharged from 1 chimpanzee. The number of discharged worms abruptly decreased after the third treatment, and only a few worms were recovered after the fifth treatment, indicating that repeated treatment at short intervals was very effective. Complete eradication was not achieved, however, presumably because of reinfection. The female proportion among discharged worms tended to increase as the treatment was repeated.

We describe a reliable and relatively inexpensive method for detecting and differentiating between the commonly studied avian blood parasite genera Haemoproteus, Plasmodium, and Leucocytozoon. The assay takes advantage of a Haemoproteus-specific restriction site identified by sequencing full mitochondrial genomes from two Haemoproteus and three Plasmodium lineages and an adjacent, genus-specific restriction site identified in Leucocytozoon spp. The assay was sensitive to simulated parasitemias of approximately 8 × 10⁻⁶ per erythrocyte and was 100% accurate in differentiating between parasite genera isolated from a broad geographical and taxonomic sampling of infected hosts.

In many amphibian species, an apparent increase has occurred in the prevalence of limb deformities caused by parasitic trematodes. We are interested in the role of environmental stressors in increasing these infections in amphibians. One mechanism by which environmental stressors could act to increase disease prevalence is to increase circulating levels of glucocorticosteroid hormones, which are released in response to stressors and can be immunosuppressive. In the present study, we treated gray treefroZg tadpoles (Hyla versicolor) with exogenous corticosterone, which is the main glucocorticosteroid “stress” hormone in amphibians. We then exposed treated tadpoles to Alaria sp. cercariae and scored the number of mesocercariae that successfully infected the tadpoles. In addition, we assayed one function of the immune response by counting the number of circulating eosinophilic granulocytes, which are thought to be important in immune responses to macroparasites. Tadpoles treated with exogenous corticosterone developed higher parasite loads than control tadpoles did, and they had lower numbers of circulating eosinophilic granulocytes. These results provide evidence of glucocorticosteroid-mediated immunosuppression in tadpoles that may help to explain apparent increases in the numbers of trematode-induced deformities in amphibian populations during recent decades.

The extracellular products (ECPs) of the oyster parasite Perkinsus marinus have been posed to contain virulence factors, including serine proteases. Supplementation of P. marinus cultures with oyster homogenates enhances infectivity and changes the ECP composition. Therefore, subtractive immunization was used to attempt creation of antibodies to proteins unique to ECPs produced following parasite exposure to oyster homogenates. While control mice remained competent to respond to an unrelated antigen, no serum titers against novel ECP epitopes were detected in experimental mice. Attempts to create discriminatory hybridomas resulted in no clones with anti-ECP specificity. These findings suggest that, because no unique epitopes can be found within ECPs generated following exposure of P. marinus to host homogenates, the changes to ECPs are greatly constrained.

The change in gene expression induced by desiccation in the semiarid, entomopathogenic nematode Steinernema feltiae IS-6, includes induction of transcription of a nucleosome assembly protein, NAP1 homolog, and of casein kinase 2 (CK2) genes. Therefore, one of the events during the dehydration response of S. feltiae IS-6 may be transcriptional activation by S. feltiae IS-6 NAP1 homolog (Sf-Nap1), which is regulated by S. feltiae IS-6 CK2 (Sf-CK2). This regulation necessitates physical interaction between the Sf-Nap1 and Sf-CK2 proteins. In the present study we used yeast 2-hybrid analysis to demonstrate physical interaction between the 2 proteins, thus confirming the involvement of a protein interaction-based step in the desiccation response mechanism of S. feltiae IS-6.

Raccoons (Procyon lotor) are intermediate hosts for Toxoplasma gondii, and clinical toxoplasmosis in raccoons has been reported. A 2-yr serological survey was conducted to determine the prevalence of antibodies to T. gondii in raccoons collected from Fairfax County, Virginia, a suburban/urban area outside Washington, D.C. Serum samples from 256 raccoons were examined for T. gondii antibodies at a 1:50 dilution using the modified direct agglutination test. Results indicated that 216 (84.4%) of the raccoons had been exposed to T. gondii. Our results indicate that raccoons in this area of Virginia are frequently exposed to T. gondii. Domestic cats were common in the study area and may have served as a source of oocysts for raccoons and the food items of raccoons.

Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment with oocysts. T. gondii was identified in a Hawaiian monk seal (Monachus schauinslandi) that had visceral and cerebral lesions. Tachyzoites were found in the lymph nodes, spleen, diaphragm, heart, adrenal glands, and brain. A few tissue cysts were found in sections of the cerebrum. The diagnosis was confirmed serologically, by immunohistochemical staining with T. gondii-specific polyclonal rabbit serum, and by the detection of T. gondii DNA. The genotype was determined to be type III by restriction fragment length polymorphisms of the SAG2 gene. This is the first report of T. gondii infection in a Hawaiian monk seal.

Batillaria minima is a common snail in the coastal estuaries of Puerto Rico. This snail is host to a variety of trematodes, the most common being Cercaria caribbea XXXI, a microphallid species that uses crabs as second intermediate hosts. The prevalence of infection was higher (7.1%) near mangroves than on mudflats away from mangroves (1.4%). Similarly, there was a significant positive association between the proportion of a site covered with mangroves and the prevalence of the microphallid. The association between mangroves and higher trematode prevalence is most likely because birds use mangroves as perch sites and this results in local transmission to snails.

High pressure processing (HPP) has been shown to be an effective non-thermal method of eliminating non-spore forming bacteria from a variety of food products. The shelf-life of the products is extended and the sensory features of the food are not or only minimally effected by HPP. The present study examined the effects of HPP using a commercial scale unit on the viability of Toxoplasma gondii oocysts. Oocysts were exposed from 100 to 550 MPa for 1 min in the HPP unit and then HPP treated oocysts were orally fed to groups of mice. Oocysts treated with 550 MPa or less did not develop structural alterations when viewed with light microscopy. Oocysts treated with 550 MPa, 480 MPa, 400 Mpa, or 340 MPa were rendered noninfectious for mice. Mice fed oocysts treated with no or 100 to 270 MPa became infected and most developed acute toxoplasmosis and were killed or died 7 to 10 days after infection. These results suggest that HPP technology may be useful in the removal of T. gondii oocysts from food products.

This study examined the effects of burn injury on murine immune response against Toxoplasma gondii infection. Male C57BL/6 mice were divided into 3 groups: T. gondii infection (group T), burn injury (group B), and burn injury followed by T. gondii infection (group BT). The survival of group BT was significantly lower than those of group B and group T. Parasite abundance in the tissues was determined by quantitative competitive-polymerase chain reaction. Group BT exhibited significantly higher numbers of T. gondii than group T. Antibody production against T.g.HSP30 in group BT was significantly lower than that in group T, whereas no significant difference was observed in SAG1-specific antibody production. Delayed-type hypersensitivity (DTH) specific for 2,4-dinitrofluorobenzene (DNFB) of both group B and group BT was significantly lower than that of group T. One week after infection, serum interferon-gamma (IFN-γ) and interleukin (IL)-10 levels in group BT were significantly lower, whereas serum IL-6 levels were significantly higher than in group T. Serum TNF-α levels in both group T and group BT were elevated at 1 wk after infection, although there was no significant difference between them. Serum IFN-γ, IL-10, and TNF-α levels in group B were not elevated during the experimental term. In conclusion, the impaired antigen-specific antibody production and DTH response, together with the modulated patterns of cytokine responses, seemed to be strongly involved in the development of burn-induced immunosuppression and the consequent increased susceptibility to T. gondii infection in mice.

The present study was conducted to determine the impact of a caged-trout farm on the helminth parasites of the wild fish puyen chico (Galaxias maculatus) in Lake Moreno, southern Argentina. Samples of water, fish (G. maculatus), and snails (Heleobia hatcheri) were taken at 4 sampling stations in January 2001. Wild fish were parasitized by 8 helminth species, all of which are endemic in the region; therefore, the farm did not introduce any helminth parasite to G. maculatus. Fish captured near the farm were not infected by the digenean Steganoderma szidati, whereas the abundance of the digenean Acanthostomoides apophalliformis in these fish was significantly lower than that in fish captured away from the farm. This lower abundance may be explained by the absence in this area of the snail H. hatcheri, the parasite's first intermediate host, because of the effect of sediments and ammonium produced by the farm. To our knowledge, this is the first study in the Americas linking fish-farm pollution to helminth parasites in wild fish.

This study assessed the effects of a commensal, Chaetogaster limnaei limnaei, and a parasitic trematode, Schistosoma mansoni, on infection patterns and life-history responses in the aquatic snail Biomphalaria glabrata. Prevalence of infection was significantly higher in snails that were devoid of C. limnaei limnaei relative to those that were colonized by the commensal, indicating that the oligochaete may protect the host from trematode infection. This finding appeared to be the direct result of the commensal as opposed to indirect stimulation of the immune system, as hemocyte numbers did not differ between C. limnaei limnaei–colonized and noncolonized snails. Snail growth and reproduction were affected by the presence of C. limnaei limnaei and exposure to S. mansoni. Two-way ANOVA revealed a significant effect of both C. limnaei limnaei presence and trematode exposure on B. glabrata growth over the 5-wk study with C. limnaei limnaei–colonized and parasite-infected snails demonstrating the greatest growth. Snails exposed, but uninfected, by S. mansoni demonstrated the lowest growth regardless of commensal colonization. Chaetogaster limnaei limnaei colonization had no effect on egg production, but S. mansoni–infected snails produced significantly more eggs than individuals from other treatment groups. Survival remained over 85% in all treatment groups. The ecological implications of these results are discussed.

This study reports the molecular characterization of Cryptosporidium spp. isolates identified from intestinal contents of ringed seals (Phoca hispida) from Nunavik (Quebec, Canada). Cryptosporidium spp. fragments of 18S rRNA, HSP-70, and actin loci were amplified by PCR from seal intestinal contents. PCR-positive specimens were sequenced and compared with other Cryptosporidium species and genotypes reported previously. Sequence analysis showed the presence of C. muris and 2 novel genotypes in ringed seals.

A colony of Japanese macaques (Macaca fuscata fuscata) kept by a safari-style zoo in Japan experienced 9 sporadic cases of fatal neurological diseases, such as epilepsy and posterior paralysis, during the 12 yr from 1989 to 2001. This macaque colony consisted of approximately 30 animals, on average, during this period, and the macaques shared their living space with 11 American black bears (Ursus americanus) harboring zoonotic roundworms (Baylisascaris transfuga). Close to this enclosure, a cote for 2–3 raccoons (Procyon lotor) was placed, and raw sewage from this cote ran into a shallow drain in the area for macaques and bears. However, fecal examinations in recent years did not detect the infection of raccoons with zoonotic roundworms (Baylisascaris procyonis). Postmortem histological examination of the latest 2 ill macaques detected multifocal malacia in the brain; 2 ascarid larvae of 60 μm maximum width were encapsulated in the cerebrum and lungs of 1 of the animals. To determine the causative ascarid species of the fatal larva migrans, we analyzed 2 additional encapsulated Baylisascaris larvae collected from formalin-fixed lungs by morphological and molecular approaches. This sporadic outbreak is the second record of Baylisascaris larva migrans in animals in Japan.

Ex vivo lung-stage larvae of Schistosoma mansoni and S. haematobium do not bind specific antibodies in the indirect membrane immunofluorescence test (IF), probably as a result of confinement of the surface membrane antigens in immobile, lipid-rich sites. Treatment with the membrane-impermeable, cholesterol-extracting drug methyl-β-cyclodextrin (MBCD) and staining with filipin III (filipin), a fluorescent polyene antibiotic widely used for the detection and quantitation of cholesterol in biomembranes, allowed us to examine the role of cholesterol in surface membrane antigen sequestration of S. mansoni and S. haematobium ex vivo lung-stage larvae. Treatment of S. mansoni larvae with MBCD elicited appreciable cholesterol depletion as judged by filipin–cholesterol fluorescence diminution, which was accompanied by a considerable increase in specific antibody binding in IF, thus suggesting that cholesterol plays a predominant role in sequestration of the surface membrane antigens of S. mansoni lung-stage schistosomula. Despite that, MBCD induced an almost complete depletion of cholesterol from the outer membrane of S. haematobium larvae; no increase in specific antibody binding in IF was evident, implying that cholesterol is not responsible for masking surface membrane antigens of S. haematobium lung-stage larvae.

Molecular and morphological data permit a rational subdivision of the paraphyletic Isospora into 2 apparently monophyletic groups of parasites, i.e., Isospora and Cystoisospora. Atoxoplasma was determined to be a junior objective synonym for Isospora. Tetrasporozoic, diplosporocystic oocysts possessing Stieda bodies in their sporocysts belong to Isospora (Eimeriidae) and have been described principally from the feces of birds. Tetrasporozoic, diplosporocystic oocysts without Stieda bodies in their sporocysts belong to Cystoisospora (Sarcocystidae).