In order to monitor the early phases of the development of Ascaris suum from domestic pigs, third-stage larvae, retrieved from the liver and the lungs, were studied by analyzing worm growth and length increase of individual transverse annuli in the cuticle. Material for study using light and scanning electron microscopy was obtained from experimental infections. The results show that the third-stage larva (not the second-stage) after emergence from the egg grows continuously, without an ecdysis in the liver. During growth, each annulus is split into a complex of 2 subannuli, each of which attains a bimodal appearance and is a prominent feature during a late phase of the third-stage larva. The results suggest that the first 2 molts occur inside the egg, a synapomorphic feature of the Ascaridoidea. The third-stage larvae of ascaridoids, with some functional similarities of the dauer-larva stage of Caenorhabditis sp., facilitate transmission of these parasitic worms to the digestive tract of the vertebrate final host (utilizing the tracheal route in A. suum), where the third and the fourth molts take place.

The endogenous development of Eimeria minasensis was studied in 9 coccidia-free goat kids inoculated with 10⁵ sporulated oocysts/kg body weight. Kids were killed 4, 7 (2 animals), 10, 13, 16, 18, 19, and 22 days after inoculation (DAI). In tissue sections of the intestines stained with hematoxylin and eosin and examined by light microscopy, 2 generations of meronts, gamonts, gametes, and oocysts were found. The first generation of meronts developed in cells deep in the lamina propria of the jejunum and ileum. Mature giant meronts (299.4 × 243.8 µm) found 16 DAI were visible to the naked eye and contained a large number of crescent-shaped merozoites. The second generation of meronts developed in the epithelial cells of crypts of the ileum and above the host cell nuclei. Mature meronts (11.5 × 10.1 µm) with 18–28 comma-shaped merozoites were first seen 16 DAI. Gametogenesis took place in epithelial cells of the crypts and villi of the terminal part of the ileum, cecum, and colon. Macrogametes (27.8 × 17.6 µm), mature microgamonts (21.3 × 17.0 µm), microgametes, and oocysts (30.5 × 19.4 µm) were found 19 DAI. Sexual stages were below the host cell nucleus.

Both sexes of Sigmactenus timorensis n. sp. are described from Rattus tanezumi and Rattus exulans collected in West Timor, Indonesia. The true host presumably is a native murine rat such as the extant, endemic Rattus timorensis or 1 of several extinct, endemic Timorese rats. Analyses of new collections of Sigmactenus cavifrons and Sigmactenus toxopeusi from New Guinea demonstrate that these fleas show considerable morphological variation. We propose that they represent a single species with the name S. toxopeusi having priority. New collections of Sigmactenus werneri from the Philippines expand the known hosts and geographical distribution of this flea.

The ability of the African tortoise tick, Amblyomma marmoreum, to acquire and transmit Cowdria ruminantium infection was investigated experimentally with transmission trials and with a C. ruminantium-specific polymerase chain reaction (PCR) detection assay. Laboratory-reared A. marmoreum larvae and nymphs were fed on small ruminants with clinical heartwater. After molting, the resultant nymphs were fed on Cowdria ruminantium-naive sheep (n = 3), and the adults were ground and inoculated intravenously into sheep (n = 5). Fatal heartwater developed in the 5 recipient animals, demonstrating larvae–nymph transmission and nymph–adult acquisition of infection. Cowdria ruminantium infection was also detected in adult A. marmoreum by PCR analysis, although at lower frequency (10%) than in Amblyomma hebraeum ticks (43%), the major vector of C. ruminantium in southern Africa, which had been fed simultaneously on the infected animals (P < 0.0001). Amblyomma marmoreum, therefore, can be an effective vector of C. ruminantium. The potential role of this species in heartwater epidemiology and in the spread of the disease to new areas is highlighted by these results and by the fact that immature stages of this tick feed readily on domestic and wild animals susceptible to C. ruminantium.

Genetic and morphological variability of whipworms Trichuris Roederer, 1761 (Nematoda: Trichuridae), parasites of small rodents in southwestern Europe, was studied. Isozyme patterns of natural populations of nematodes parasitizing rodent species of the Muridae (Apodemus sylvaticus, Apodemus flavicollis, Mus musculus) and Arvicolidae (Clethrionomys glareolus, Microtus agrestis, Microtus arvalis) were analyzed at 6 putative loci. Two diagnostic loci were found in T. muris from Muridae and from Arvicolidae. Thus, the existence of 2 species of Trichuris restricted to different host families was indicated. They included Trichuris muris Schrank, 1788, originally described as being from mice, and Trichuris arvicolae n. sp., parasitizing the above species of Arvicolidae. The morphological variability of both species was compared. Although ranges of all morphological characters of the new species overlapped with those of T. muris, stepwise discriminant analysis yielded a 100% accurate classification of females when using vagina length and egg size. Males of T. muris and T. arvicolae cannot be separated entirely. A set of 6 variables yielded 95.7% discrimination; the most discriminating variables were spicule size and body width.

Intraerythrocytic development of the adeleorin apicomplexans Hepatozoon clamatae and Hepatozoon catesbianae were investigated in the bullfrog, Rana catesbeiana, the green frog, Rana clamitans melanota, and the Northern leopard frog, Rana pipiens. Merozoites emerging from hepatic meronts penetrated erythrocytes and underwent 1–3 rounds of binary fission to produce 2–8 merozoites. Following their release from infected erythrocytes, individual merozoites entered new cells and transformed into gamonts. Although this is the first report of intraerythrocytic development for a fully described species of Hepatozoon, a phylogenetic reanalysis of 11 species of Hepatozoon, 6 species representative of the 5 other hemogregarine taxa, 2 species of dactylosomatids, and 2 species of piroplasms, indicates that asexual reproduction of parasites within blood cells of vertebrates has arisen at least 3 times in the apicomplexan lineage that includes adeleorins and piroplasms. This method of asexual development, which is also observed in species of hemospororin genera such as Plasmodium, is discussed in the context of the evolution of apicomplexan life cycles. In addition to supporting the paraphyly of the genus Hepatozoon determined in an earlier study, this phylogenetic analysis featured a monophyletic group, consisting of the sister taxa Hemolivia and Karyolysus, that was the sister group to a clade consisting of the more derived hemogregarines, the dactylosomatids, and the piroplasms.

Five recombinant Toxoplasma gondii antigens, designated B427, C51, C55, V22, and MBP30 were assessed for their potential use in an enzyme-linked immunoassay (EIA) for detection of T. gondii infection in swine. The antigens were evaluated with sera from young pigs that had been fed 1–10,000 T. gondii oocysts of the VEG or GT-1 strains. Results were compared with an EIA using a native T. gondii antigen extract. All 5 recombinant antigens, as well as native antigen, detected antibody responses as soon as 3 wk after infection in pigs inoculated with 1 or 10 oocysts of the VEG strain. This antibody response persisted, at varying levels, for 14 wk when the experiment was terminated. All antigens also detected antibody responses in pigs 4 wk after inoculation with 10,000 oocysts of the GT-1 strain. The antibody response recognized by native antigen remained high through 51 wk after inoculation. However, there was considerable animal-to-animal variation in responses to the individual recombinant antigens. Only antigens C51 and MBP30 consistently detected a positive antibody response over the entire 51-wk course of the experiment. These results suggest that these antigens might be useful for the serological detection of T. gondii infection in pigs.

Superparasitism is a common condition in which a single host is injected with more than 1 parasitoid egg, but only 1 parasite survives to adulthood, and the remaining animals are eliminated. Here, for the first time, we show that supernumerary Ganaspis xanthopoda, endoparasitoids that invade Drosophila melanogaster, are physiologically suppressed during embryonic development. Whereas the suppressed supernumerary embryos can develop to the first larval instar, their subsequent growth is blocked because they become physically trapped within a novel multicellular envelope that is formed during late embryogenesis. Supernumerary embryos can produce this envelope when cultured in vitro even if they are separated from dominant embryos. Our results suggest that physiological suppression programs supernumerary individuals for developmental arrest, starvation, and necrotic or apoptotic death.

The nematode, Mehdinema alii, occurs in the alimentary canal of the decorated cricket Gryllodes sigillatus. Adult nematodes occur primarily in the hindgut of mature male crickets, whereas juvenile nematodes are found in the genital chambers of mature male and female crickets. Here, we present experimental evidence for the venereal transmission of M. alii in G. sigillatus. Infectivity experiments were conducted to test for transmission via oral–fecal contamination, same-sex contact, and copulation. The infective dauers of the nematode are transferred from male to female crickets during copulation. Adult female crickets harboring infective dauers subsequently transfer the nematode to their next mates. Thus, M. alii is transmitted sexually during copulation.

Cercaria cerastodermae I, a digenean parasite of Cerastoderma edule, was recorded for the first time in the Atlantic Ocean off the Iberian peninsula. Sporocysts were present in the hemolymph of the digestive gland, gonad, gills, and foot of the mollusc. Most of the cercariae present within sporocysts were encysted as metacercariae. The corresponding adult stages were obtained after experimental infection of several Diplodus sargus artificially reared in fish farms and that had previously been protected against natural infections. Numerous adult specimens of Monorchis parvus were collected in Diplodus annularis along the French Mediterranean coast. Comparison of wild and experimental adults allowed the adult stage of Cercaria cerastodermae I to be identified as M. parvus. Another monorchid, Monorchis monorchis, a parasite of Spondyliosoma cantharus, was found in the same Mediterranean area and compared with M. parvus. Additionally, ITS1 nuclear ribosomal DNA sequences of C. cerastodermae I and of the adults collected in naturally infected D. annularis and S. cantharus were obtained. Sequence data indicate that C. cerastodermae I corresponds to the adult of M. parvus found in D. annularis and is clearly distinct from M. monorchis found in S. cantharus.

We report the detection and identification of Cryptosporidium and Giardia from 1 of 3 species of pinnipeds. Fecal samples were collected from Pacific harbor seal (Phoca vitulina richardsi), northern elephant seal (Mirounga angustirostris), and California sea lion (Zalophus californianus) in the northern California coastal area. By means of fluorescently labeled monoclonal antibodies, Cryptosporidium oocysts were detected in 3 samples from California sea lions, 1 of which also contained Giardia cysts. Oocysts of Cryptosporidium and cysts of Giardia were morphologically indistinguishable from oocysts of C. parvum and cysts of G. duodenalis from other animal origins. Oocysts and cysts were then purified using immunomagnetic separation techniques and identified by polymerase chain reaction (PCR), from which species-specific products were obtained. Sequence analysis revealed that the 452-bp and 358-bp PCR products of Cryptosporidium isolated from California sea lion had identities of 98% with sequences of their template fragments of C. parvum obtained from infected calves. Based on morphological, immunological, and genetic characterization, the isolates were identified as C. parvum and G. duodenalis, respectively. The findings suggested that California sea lions could serve as reservoirs in the environmental transmission of Cryptosporidium and Giardia.

The transmission of Cryptosporidium parvum between dams and their respective calves was studied. For this purpose, fecal specimens taken from the rectum of preparturient, parturient, and postparturient dams were analyzed for C. parvum oocysts. Fecal specimens were taken from the newborn calf 4 hr after birth. Because the environment can be a source of contamination to the animals, specimens taken from inside and outside the barn were analyzed. The sucrose concentration method together with the Zielh–Nielsen acid-fast staining method were employed to increase the chances of oocyst detection. We are reporting that at parturition, the dams shed a higher number of oocysts by comparison to the preparturient and postparturient periods. Neonates acquire the infection at birth mainly because of the high number of oocysts shed by the dams at parturition. The management practice of moving calves 4 hr after birth away from the dams and the barn reduces the number of clinical cases because they are no longer in contact with an environment that is highly contaminated. We hypothesize that the increase in the number of oocysts sheds by dams at parturition might be due to a depression of the T helper 1-type of immune response during that period.

The taxonomic status of the extraintestinal piscine coccidium Calyptospora funduli is based in part on its requirement of an intermediate host (the daggerblade grass shrimp Palaemonetes pugio). In the present study, grass shrimp fed livers of Gulf killifish (Fundulus grandis) infected with sporulated oocysts of C. funduli exhibited numerous sporozoites suspended in the intestinal contents when fresh squash preparations were examined by light microscopy. Using this method, sporozoites were not seen in intestinal epithelial cells of the grass shrimp or in any other cell types. Ultrastructural examination, however, revealed sporozoites in the cytoplasm of the gut basal cells. Cross-sections of 1–13 sporozoites were seen within a single cell, and those sporozoites each appeared to be situated in individual membrane-bound vesicles, rather than in a single parasitophorous vacuole. These ultrastructural observations indicate that in the grass shrimp intermediate host, sporozoites that develop into an infective stage probably undergo that development in gut mucosal basal cells. Prior studies revealed that these sporozoites modified their structure over 4–5 days and that before that time, they were not infective to the fish host. Following ingestion of an infected shrimp by a killifish, the infective sporozoites apparently reach the liver of their killifish definitive hosts through the bloodstream. Sporozoites were seen in blood smears from the longnose killifish, Fundulus similis, 4 hr after fish were fed experimentally infected grass shrimp. Additionally, coccidian trophozoites and early meronts were seen in hepatocytes from several longnose killifish at 48, 72, and 96 hr postinfection. This study, in conjunction with previous findings, clearly confirms that a true intermediate host is required in the life cycle of C. funduli, that a developmental period of about 5 days in grass shrimp is necessary for sporozoites to become infective to killifishes, and that sporozoites do occur intracellularly in gut basal cells of the grass shrimp.

Blood parasites and nest cavity arthropods associated with the red-cockaded woodpecker (Picoides borealis) were surveyed and the impact of blood-feeding arthropods on woodpecker fitness traits was assessed. Five woodpeckers (8%) were infected with unidentified microfilariae, and 1 woodpecker (2%) was infected with 2 species of haemoproteid (Haemoproteus velans and Haemoproteus borgesi). This is the first record of haemoproteids in this species and the first observation of H. borgesi in North America. We collected representatives of at least 6 families of mites and 12 families of primarily commensal insects from woodpecker cavities. Only a few specimens of blood-feeding insects were recovered. The mite Androlaelaps casalis was the most common hematophagous arthropod (prevalence = 76%, mean density = 51 ± 7 mites/cavity). The number of A. casalis mites increased with cavity age but there was no association between the number of mites and the number of woodpecker eggs laid or the number of hatchlings or fledglings. In conclusion, the prevalence of blood parasites in the red-cockaded woodpecker is low, woodpecker cavities are not heavily infested with blood-feeding insects, and there is no evidence that A. casalis mites affect woodpecker fitness.

The prevalence of malaria parasites was studied in the lizard Anolis gundlachi over a 9-yr period at a site in the wet evergreen forest of eastern Puerto Rico. Three forms of the parasite infected the lizards; these were Plasmodium floridense, Plasmodium azurophilum in erythrocytes, and P. azurophilum in white blood cells. Overall prevalence of infection for 8 samples during the study period was significantly higher for males than females (32% of 3,296 males and 22% of 1,439 females). During the study, the site experienced substantial climatic and physical disturbance including rising temperature, droughts, and hurricanes that severely damaged the forest. Parasite prevalence in the first sample, 8 mo after the massive hurricane Hugo, was slightly, though significantly, lower than for subsequent samples. However, overall prevalence was stable during the 9-yr period. The results show malaria prevalence is more constant at the site than found for 2 studies in temperate forests, and that the Puerto Rico system may be an example of the stable, endemic malaria described by standard models for human malaria epidemiology.

A 36-kDa immunosuppressant protein (Da-p36) was isolated from salivary glands of feeding female ixodid ticks Dermacentor andersoni, using its affinity for UltraLink™ Biosupport Medium (Pierce, Rockford, Illinois)/protein complexes. Using a nested set of forward degenerate oligonucleotide primers corresponding to Da-p36 N-terminal amino acids, a cDNA encoding the immunosuppressant protein was isolated by 3′ rapid amplification of cDNA ends. The resulting 772-base pair cDNA encodes a novel protein with predicted molecular weight of 24.9 kDa. Sequence analysis revealed the presence of 5 potential glycosylation sites and 1 myristylation site. Immunoblot analyses showed native Da-p36 is present in salivary glands and saliva from both male and female D. andersoni but not in salivary glands or saliva from Amblyomma americanum or Ixodes scapularis. Reverse transcription polymerase chain reaction and immunoblot analyses showed that Da-p36 expression is temporally regulated in salivary glands with maximum mRNA levels preceding maximum Da-p36 accumulation that occurred at day 6 of feeding. The levels of Da-p36 mRNA and protein were greatly reduced in salivary glands from near-replete females removed from sheep after 8 days of feeding. These data are consistent with a role of Da-p36 in immunosuppression during feeding.

Toxoplasma gondii was isolated from brain or heart tissue from 15 southern sea otters (Enhydra lutris nereis) in cell cultures. These strains were used to infect mice that developed antibodies to T. gondii as detected in the modified direct agglutination test and had T. gondii tissue cysts in their brains at necropsy. Mouse brains containing tissue cysts from 4 of the strains were fed to 4 cats. Two of the cats excreted T. gondii oocysts in their feces that were infectious for mice. Molecular analyses of 13 strains indicated that they were all type II strains, but that they were genetically distinct from one another.

Reactivation of chronic toxoplasmosis resulting in Toxoplasma encephalitis (TE) is a common event in acquired immune deficiency syndrome (AIDS) patients. Conversion from Toxoplasma gondii bradyzoites to tachyzoites is a prerequisite for reactivation. Until recently, the study of stage conversion in human tissue was not possible due to the lack of antibodies that recognize stage-specific epitopes after long-term formaldehyde fixation. Using the combination of a polyclonal anti-T. gondii antibody, the cyst-stage-specific monoclonal antibody CC2, and a tachyzoite-specific polyclonal antibody (anti-SAG1, recombinant), we tried to demonstrate parasite differentiation in the brain tissue of 10 AIDS patients with clinically suspected TE. Double labeling of the stage-specific antibodies enabled us to demonstrate interconversion between tachyzoites and bradyzoites for the first time in human tissue. The study confirmed that the transformation process is nonsynchronous and that the manifestation of TE depends on the degree and site of tissue destruction caused by invading tachyzoites. The original source of tachyzoites could never be located, but a few samples suggested that tachyzoites may invade by dissemination across the blood–brain barrier. Cyst rupture as the first event in the process of reactivation was not seen. We conclude that the initial site(s) of reactivation will be destroyed by tissue-destructive tachyzoites long before clinical symptoms occur.

From several gnathostome species the complete internal transcribed spacer ITS-2 ribosomal DNA (rDNA) repeat sequence and a fragment of the 5.8S rDNA were obtained by direct polymerase chain reaction cycle-sequencing and silver-staining methods. The size of the complete ITS-1 sequence in agarose gel electrophoresis was also obtained. The ITS-2 enabled the differentiation of Gnathostoma spinigerum from Thailand and Gnathostoma binucleatum from Mexico and Ecuador and confirmed the validity of the latter. Gnathostoma turgidum, Gnathostoma sp. I (=Gnathostoma procyonis sensu Almeyda-Artigas et al., 1994), and Gnathostoma sp. II (=G. turgidum sensu Foster, 1939 pro parte), all from Mexico, proved to be independent species, but Gnathostoma sp. III, also from Mexico, could not be differentiated from G. turgidum. In Mexico and Ecuador, gnathostomes involved in human infection and that had been classified as G. spinigerum belong to G. binucleatum. The 5.8S rDNA sequences of the 6 Gnathostoma species studied were identical. The results of the ITS-1 agreed with those results of ITS-2.

Filarioid nematodes of the genus Litomosoides occur in the abdominal and (or) thoracic cavities of marsupials, rodents, and bats of the Nearctic and Neotropical regions. In this study, the phylogenetic relationships among these nematodes were estimated with a parsimony analysis of morphological characters derived from species descriptions. This nonweighted analysis produced 20 shortest trees. The monophyly of the genus was not supported in that Litomosoides thomomydis and Litomosoides westi failed to group with the other members of the genus. When these 2 taxa (parasites of pocket gophers) were excluded, monophyly of Litomosoides was supported by 2 synapomorphies (structure of the walls and general shape of the stoma); however, ancestor–descendant relationships among the species in the genus were not well resolved. A posteriori reweighting of the characters produced a single tree, different from all 20 most parsimonious trees. Alternative host–parasite evolutionary models were tested against these results supporting the process of host switching as being most important in forming the patterns of mammal–nematode associations that have been detected in this group of nematodes.

Hedruris hanleyae n. sp. (Nematoda: Hedruridae) from the stomach of Hemidactylus garnotii collected in 1989 on Atiu, Cook Islands is described and illustrated. Hedruris hanleyae n. sp. represents the 21st species to be assigned to the genus and is distinguished from other oriental species by the distribution pattern of caudal papillae of the male: 10 pairs posterior subventral papillae; 2 pairs precloacal and 8 pairs postcloacal.

Stylocephalus occidentalis n. sp. (Apicomplexa: Eugregarinida) is described from Trimytis pruinosa (Coleoptera: Tenebrionidae) collected from Keith County in the Sandhills of western Nebraska. Measurements are means in micrometers. Developing trophozoites solitary; epimerite a complex of terminal epimerite and intercalating diamerite; epimerite shallowly ovoid to transversely elliptoid, with transverse basal constriction at junction with diamerite, length 0.5–1 times width, approximately 3–4 times that of diamerite; width approximately equal to that of diamerite; diamerite roughly cylindrical to spindle-shaped, without significant anterior taper, little or no evidence of longitudinal folds, length approximately twice width. Association late, frontal, isogamontic. Gamont protomerite depressed ovoid to very broadly ovoid, length 27.3, width 35.1, anterior distance to widest point 15.4. Protomerite–deutomerite septum clearly marked and constricted, width 34.6. Deutomerite often with distinct marginal crenulation, narrowly obovoid to very narrowly obovoid, length 356.5, maximum width 57.6, anterior distance to widest point 26.3, equatorial width 35.1, ±12.5, 29. Total length 381.5. Nucleus ellipsoid, length 32.5, width 18.8; with 0 or 2 polysomal endosomes. Gametocysts roughly spherical; diameter 205.0; wall desiccating to become paper-like, slightly papillated, dehiscing by simple rupture, releasing oocysts in coiled chains, epispore packet absent, gametocyst residuum present. Oocysts dark brown to black, axially asymmetric, broadly deltoid, gibbous in lateral aspect, slightly keeled in dorsal aspect; length 9.8, height 7.9; with slight terminal protuberances and 2 central, spherical residua.

Nucleotide sequences were determined for the rRNA internal transcribed spacers 1 and 2 (ITS1 and 2) and the 5′ terminus of the large subunit rRNA in selected Gyrodactylus species. Examination of primary sequence variation and secondary structure models in ITS2 and variable region V4 of the small subunit rRNA revealed that structure was largely conserved despite significant variation in sequence. ITS1 sequences were highly variable, and models of structure were unreliable but, despite this, show some resemblance to structures predicted in Digenea. ITS2 models demonstrated binding of the 3′ end of 5.8S rRNA to the 5′ end of the large subunit rRNA and enabled the termini of these genes to be defined with greater confidence than previously. The structure model shown here may prove useful in future phylogenetic analyses.

Attempts were made to develop an animal model for Cyclospora cayetanensis to identify a practical laboratory host for studying human cyclosporiasis. Oocysts collected from stool of infected humans in the United States, Haiti, Guatemala, Peru, and Nepal were held in potassium dichromate solution to allow development of sporozoites. The following animal types were inoculated: 9 strains of mice, including adult and neonatal immunocompetent and immune-deficient inbred and outbred strains, rats, sandrats, chickens, ducks, rabbits, jirds, hamsters, ferrets, pigs, dogs, owl monkeys, rhesus monkeys, and cynomolgus monkeys. Most animals were inoculated by gavage, although some of the primates were fed oocysts on food items. The animals were examined for signs of infection, particularly diarrhea, and stool samples were examined for 4–6 wk after inoculation. None of the animals developed patent infections or signs of infection. We conclude that none of the animals tested is susceptible to infection with C. cayetanensis.

The monotypic Kamegainema n. gen. is proposed for Filaria cingula, a subcutaneous parasite of cryptobranchids. Examination of female specimens recently collected from the Japanese giant salamander, Andrias japonicus, revealed that it belongs to Dracunculidae. Kamegainema is closest to Protenema Petter and Planelles, 1986, the only other dracunculid genus known from Amphibia, but is readily distinguished by having prominent cuticular bosses. Kamegainema cingulum is redescribed.

Nuclear ribosomal sequence data from the internal transcribed spacers (ITS-1 and ITS-2), 5.8S subunit, and regions of the 18S and 28S genes were used to investigate sequence diversity among geographic samples of Nematodirus battus, and to infer phylogenetic relationships among Nematodirus species. Phylogenetic analysis of these data yielded strong support for relationships among species, depicting Nematodirus helvetianus and Nematodirus spathiger as sister-taxa and a clade of these 2 species and Nematodirus filicollis. This tree is consistent with caprine bovids as ancestral hosts, with a subsequent host shift to Bovinae in N. helvetianus. Eleven of 14 N. battus sequences were unique, with 19 variable sites among sequences representing 5 geographic samples. The lowest number of variable nucleotide sites was observed in samples representing apparently recent introductions to the United States and Canada, which is consistent with a population bottleneck concomitant with translocation. Comparison of directly sequenced polymerase chain reaction products and clones revealed evidence for intraindividual variation at some of the sequence sites, and this pattern of variation and that within geographic samples indicates incomplete rDNA repeat homogenization within species. This pattern of variation is not conducive for inferring phylogenetic relationships among sequences representing N. battus or addressing the putative history of introduction.

Eugregarines of the suborder Septatorina are apicomplexan parasites that are found mainly in arthropods. Some exceptions are species in the Metameridae that contains the only 5 septate gregarines recorded from annelids. The type genus is Metamera Duke, 1910 with 2 species, Metamera schubergi Duke, 1910, in European Glossiphonia complanata, and Metamera reynoldsi Jones, 1943, from North American G. complanata. Over the summers of 1995–1998, in Keith County, Nebraska, septate gregarines were found in the glossiphoniid leech Helobdella triserialis. The gregarines were determined to be a new species of Metamera, herein named Metamera sillasenorum. Measurements of size and body proportions of over 600 gregarines and 50 oocysts showed differences from measurements of M. schubergi and M. reynoldsi, and secondary septa in the deutomerite were rarely observed. Field observations indicated that M. sillasenorum is probably host specific. In the laboratory, leeches also exhibited strong feeding preferences; e.g., H. triserialis and G. complanata consumed only snails, whereas Helobdella stagnalis consumed only oligochaetes. Infection experiments demonstrated that freshwater snails ingest the oocysts and are required as mechanical vectors. Oocysts were passed unaltered through the snails' intestines. Glossiphonia complanata did not become infected regardless of heavy exposure to oocysts, although only 5 G. complanata were used in the experiments. The results show that host specificity of M. sillasenorum is most likely due to a combination of host-feeding habits and host–parasite compatibility.

The effect of the dinitroaniline herbicides oryzalin and trifluralin on the growth of Entamoeba histolytica was examined. Oryzalin inhibited the growth of E. histolytica strain HM-1:IMSS. Trifluralin was less effective than oryzalin for this parasite. Entamoeba histolytica was more resistant to these dinitroanilines than other parasitic protozoa examined so far, including Leishmania spp., Trypanosoma brucei, Plasmodium falciparum, Toxoplasma gondii, and Cryptosporidium parvum. Colchicine, a potent microtubule inhibitor of animal cells, was much less effective for E. histolytica, even at very high concentrations. A reptilian parasite, Entamoeba invadens strain IP-1, examined for comparison, was more resistant to these dinitroanilines than E. histolytica. Accumulation of E. histolytica trophozoites in mitosis was observed after culture in 100 μM oryzalin. The inhibitory effect of oryzalin on the growth of E. histolytica trophozoites was abrogated by removal of the drug after exposure to 100 μM for 2 days. In parallel to the recovery of growth after removal of the drug, the percentage of trophozoites in mitosis was reduced to a normal level. The results indicate that treatment of trophozoites with oryzalin arrests mitosis and that its effect is reversible. Therefore, oryzalin is a useful tool for studies relating to the cell cycle of this parasite.

Serum samples of 31 amebic liver abscess (ALA) patients, 8 amebic hepatitis (AH) patients, and 60 controls were tested for anti-amebic IgG by enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody tests (IFAT). Sera of 29 (93.6%) ALA and 6 (75%) AH patients and 2 (3.3%) control subjects were positive by IFAT. Anti-amebic antibody titer above the cutoff point (= 0.168; x̄ 2 SD of control sera) was observed in sera of 27 (87%) ALA, 4 (50%) AH, and 1 (1.7%) control by ELISA. All the 8 pus samples were positive for anti-amebic antibodies by IFAT and ELISA. Sensitivity of ELISA was 87% for ALA, with a positive predictive value of 0.96, and 50% for AH cases, with a positive predictive value of 0.80. The sensitivity of IFAT was 93.6% for ALA, with a positive predictive value of 0.94, and 75% for AH, with a positive predictive value of 0.75. When pus samples were tested, the sensitivity was 100% for both tests. The specificity was 98.3% for ELISA and 96.7% for IFAT. Although not significant, IFAT was found more sensitive than ELISA (P > 0.05).

Permeability of the cercarial tail in Proterometra macrostoma was examined in vitro with 1 mM ³H-glucose, which tails absorb by diffusion alone. Naturally emerged cercariae (bodies withdrawn into tails) were permeable, but they rapidly (3 min) equilibrated with glucose in the bathing medium and maintained steady state for 4 hr. Metabolism of absorbed glucose was not detectable until after 90 min, and radioactivity in bodies dissected from tails after 4 hr was negligible. On the basis of cercarial water content (90% of total weight) and absorbed isotope at steady state, the calculated volume of the equilibrating compartment was 4% of an intact cercaria. This value correlated well with that of the tegument (3–5%), which was 1–2 µm thick as seen by transmission electron microscopy. A continuous, electron-dense basal membrane/lamina separated the tegument from subtegument. We conclude that the glycocalyx and external plasma membrane are freely permeable, whereas the basal membrane is the barrier that effectively isolated the subtegument from exogenous glucose. The basal membrane also may be the primary structure that protects the subtegument and cercarial body from effects of osmotic stress.

Skin lesions caused by the ectoparasite Dermophthirius penneri Benz, 1987 (Monogenea: Microbothriidae) on 2 wild-caught blacktip sharks (Carcharhinus limbatus) from the northern Gulf of Mexico were studied using light and scanning electron microscopy. Grossly, lesions appeared as multifocal, well-demarcated, ovoid or irregularly shaped, light gray patches of skin. Scanning electron microscopy of lesions revealed gaps between placoid scales apparently created by detachment and loss of placoid scales, rotated and tilted placoid scales with blunt distal tips and shallow ridges, and a frayed epithelium that covered some placoid scales and filled some spaces between placoid scales. Light microscopy of lesions revealed epithelial hyperplasia accompanied by dermal infiltrates of moderate numbers of loosely arranged lymphocytes interposed between collagen bundles in the superficial layers of the stratum compactum. This report provides the first details of microbothriid skin lesions on wild sharks. Our results indicate that D. penneri caused chronic skin lesions not associated with bacterial infection or severe, debilitating, skin disease in the studied sharks.

Serum samples from 697 Rocky Mountain bighorn sheep (Ovis canadensis) from North America were examined for antibodies to Toxoplasma gondii by the modified agglutination test incorporating mercaptoethanol and formalin-fixed tachyzoites. Antibodies to T. gondii were found in 25 of 697 (3.6%) sheep in titers of 1:25 (8 sheep), 1:50 (4 sheep), 1:100 (7 sheep), 1:200 (1 sheep), 1:400 (1 sheep), 1:800 (1 sheep), and 1:1,600 (3 sheep). This is the first record of T. gondii exposure in bighorn sheep.

Serum samples from 973 ostriches (Struthio camelus) in Canada were examined for antibodies to Toxoplasma gondii by the modified agglutination test incorporating mercaptoethanol and formalin-fixed whole tachyzoites. Twenty-eight (2.9%) of the 973 birds were found to be seropositive for antibodies to T. gondii at titers of 1:25 in 15 birds, 1:50 in 12 birds, and 1:500 in 1 bird. This is the first record of T. gondii exposure in ostriches, and it supports the hypothesis that all avian species are susceptible to Toxoplasma infection. Nevertheless, the results of this study suggest that the risk of acquiring toxoplasmosis from ostriches as a food source is low.

Sarcocystis speeri Dubey and Lindsay, 1999 from the South American opossum Didelphis albiventris was successfully transmitted to the North American opossum Didelphis virginiana. Sporocysts from a naturally infected D. albiventris from Argentina were fed to 2 γ-interferon knockout (KO) mice. The mice were killed 64 and 71 days after sporocyst feeding (DAF). Muscles containing sarcocysts from the KO mouse killed 71 DAF were fed to a captive D. virginiana; this opossum shed sporocysts 11 days after ingesting sarcocysts. Sporocysts from D. virginiana were fed to 9 KO mice and 4 budgerigars (Melopsittacus undulatus). Schizonts, sarcocysts, or both of S. speeri were found in tissues of all 7 KO mice killed 29–85 DAF; 2 mice died 39 and 48 DAF were not necropsied. Sarcocystis stages were not found in tissues of the 4 budgerigars fed S. speeri sporocysts and killed 35 DAF. These results indicate that S. speeri is distinct from Sarcocystis falcatula and Sarcocystis neurona, and that S. speeri is present in both D. albiventris and D. virginiana.

Sera from 173 turkeys, 108 chickens, and 48 ducks from Giza, Egypt, were tested for the presence of anti-Toxoplasma gondii antibodies by means of the modified agglutination test using mercaptoethanol and formalin-fixed tachyzoites. The prevalence of anti-T. gondii antibodies (>1:25) among turkeys, chickens, and ducks was 59.5%, 47.2%, and 50%, respectively.

When eggs of the trichostrongylid nematode Haemonchus contortus were exposed to thiabendazole, the concentration required to prevent hatching in 90% of the eggs (MIC₉₀) was found to be 0.1 μg/ml (using 1% dimethylsulfoxide [DMSO] as solvent). In contrast, eggs of the free-living rhabditid nematode Caenorhabditis elegans hatched at normal rates at a concentration 200 times higher, i.e., 20 μg/ml, and showed only a partial inhibitory effect at a concentration 1,200 times higher, i.e., 120 μg/ml (in 3% DMSO). Because solubility limitations precluded the testing of higher concentrations of thiabendazole, a more soluble derivative, 5-([1-methylethoxy]carbonylamino)-2-(4-thiazloyl)-1H-benzimidazolyliminoacetic acid N,N-diethylethanamine salt, was tested against C. elegans eggs. The MIC₉₀ was found to be 400 μg/ml, and although the derivative was not tested against H. contortus eggs, this finding further suggests that C. elegans eggs have an exceptionally low degree of benzimidazole sensitivity.

Therapy based on the protective passive immunity of hyperimmune bovine colostrum (HBC) (raised against Cryptosporidium parvum in cows) was applied to 4 Savanna monitors (Varanus exanthematicus) with gastric Cryptosporidium sp. infections. All lizards were moderately emaciated, and their fecal and gastric lavage samples contained moderate numbers of Cryptosporidium sp. oocysts. The first 3 of 7 gastric HBC treatments at 1-wk interval each decreased the numbers of oocysts in the fecal and gastric samples to undetectable levels. Neither feces nor lavages of the HBC-treated lizards contained Cryptosporidium sp. oocysts after the HBC therapy, whereas such samples of a single control lizard remained positive for oocysts. Two of the HBC-treated lizards died spontaneously due to metastasized carcinoma and septicemia of unknown etiology, respectively, and 2 lizards treated and killed during the experiment were histologically negative for developmental stages of Cryptosporidium sp. The control lizard died spontaneously of septicemia of unknown etiology and contained developmental stages of Cryptosporidium sp. in the gastric region. The HBC therapy was efficacious in V. exanthematicus and is recommended for lizards with gastric cryptosporidiosis.

To investigate the role of fleshfly maggots as a paratenic host for Trichinella spiralis larvae, maggots of Sarcophaga argyrostoma (Muscidae, Sarcophagidae) kept at different temperatures (26, 22, 20, 16, 12, 8, and 4 C) were allowed to feed on T. spiralis-infected mouse meat. Trichinella larvae found in maggots kept at 8–26 C were able to cause infection when inoculated in mice. Infective larvae survived in maggots up to 5 days postinfection at 8 C and for shorter periods of time at higher temperatures. The survival time in maggots was negatively related to the temperature of maggot breeding. The results suggest that the role of S. argyrostoma in the dissemination of Trichinella larvae in nature is limited in comparison to the role played by mammals with scavenger and cannibalistic behavior.

High-performance thin-layer chromatography (HPTLC) analysis was done on lutein and β-carotene in the digestive gland–gonad complex (DGG) and whole body of uninfected Cerithidia californica snails and those infected with the larval trematodes Mesostephanus appendiculatis or Euhaplorichis californiensis. HPTLC of the DGG extract on C-18 reversed-phase plates developed in petroleum ether–acetonitrile–methanol (1:2:2) mobile phase showed 2 identifiable pigment zones; the least polar zone had a retention factor (R_f) of 0.07, identical to a β-carotene standard, and the more polar zone had an R_f of 0.41, identical to a lutein standard. Densitometric scanning of the pigment zones in sample versus standard chromatograms showed that the weight percent of lutein in the uninfected DGGs (3.4 × 10⁻³%) was significantly greater (P < 0.05) than that of DGGs infected with either M. appendiculatis (0.35 × 10⁻³%) or E. californiensis (0.82 × 10⁻³%). Changes in β-carotene in the infected DGGs were insignificant compared to the uninfected controls. However, the β-carotene content of whole snails was significantly reduced (P < 0.05) by infection with either trematode.

This is the first study that establishes a standardized western blotting method for the detection of heat shock protein (HSP) 70 from Trichinella spiralis using (selected) monoclonal antibodies (mAbs). Enhancement of HSP transfer onto the supportive membrane and increased retention of protein by the membrane are prominent features of the procedure. The reactivity of T. spiralis HSP70 on western blots was substantially increased by the use of a 10% acrylamide gel, the optimization of conditions during electrotransfer, and transfer onto Immobilon membranes. These data indicate that mAbs actually capable of detecting the agent of interest may be discarded because of nonoptimal testing conditions. We suggest that this method will aid in understanding the role and function of T. spiralis HSP70 in host–parasite (inter)relationship(s).

Eighty stocked lake trout Salvelinus namaycush (Salmonidae), collected from 2 locations in Lake Huron in May 1995, were examined for parasites. The parasite fauna of this top predator in Lake Huron was characterized by only 6 helminth species. Echinorhynchus salmonis infected all lake trout with a mean intensity of 163.9. The intensity of this acanthocephalan species significantly increased with host length and weight. Eubothrium salvelini infected 78 lake trout with a maximum number of 81 scoleces counted. Diplostomum sp., Cyathocephalus truncatus, Capillaria salvelini, and Neoechinorhynchus sp. infrequently infected lake trout. The low parasite species richness in these lake trout is believed to be due to their large size at stocking and to the loss of historical enzootic host–parasite relationships that followed the absence of this fish species in Lake Huron for 26 yr.

The aggregation of parasites among individual hosts is one of the best documented features of parasite populations; we still do not know, however, why certain parasite species are more highly aggregated than other, related species. Here we search for a general explanation of interspecific variation in aggregation levels, based on the relationship between parasite body size and fecundity, transmission success, and intensity-dependent population regulation. We test the prediction that larger-bodied parasite species are more weakly aggregated than smaller-bodied related species, in a comparative analysis across parasitic nematode species. Across species, the variance-to-mean abundance ratio correlated negatively and significantly with nematode body sizes, as predicted. All other tests, however, including the more robust analyses controlling for phylogenetic influences, failed to support this result. This is mainly because the variance in infection levels is almost completely explained by mean parasite abundance. For this reason, it may prove difficult to identify a general biological explanation for interspecific variability in aggregation levels among parasites.

The monospecific Lamanema historically has been assigned to the Nematodirinae within the Molineidae. Inconsistencies in morphological characters, within a phylogenetic context for Nematodirinae, led to a re-evaluation of the putative relationships and taxonomic placement of Lamanema. Among 7 putative synapomorphies for Nematodirinae, Lamanema possesses only 1, large eggs. Large eggs, sporadically present in phylogenetically disparate taxa of trichostrongyles, are equivocal with respect to placement of Lamanema; it is considered that possession of this single homoplasious character alone is insufficient justification to retain the genus in Nematodirinae. Affinities with the Trichostrongylidae (Cooperiinae or Haemonchinae) have also been proposed; however, Lamanema possess neither of 2 synapomorphies that diagnose monophyly of the family. Lamanema is retained in the Molineidae and transferred to the Molineinae as it possesses all characters of the family as currently defined. The origin of Lamanema represents a secondary colonization of ruminants by molineids and provides no context for elucidating the history of the Nematodirinae and Nematodirus.

Because resistance to parasites usually has a cost for host species, it is theoretically expected that, in case of multi-infection, host immune responses should vary according to the levels of parasite pathogenicity. The crustacean gammarid Gammarus aequicauda is the second intermediate host of 4 trematode species. Three of these parasites always encyst in the abdomen of gammarids and have no particular effect on the host. However, 1 of these species is sometimes able to encyst in the cerebroid ganglia of the gammarid and strongly alter its behavior in a way that increases its predation risk by aquatic birds, the definitive hosts. In accordance with the hypothesis that the level of parasite pathogenicity influences the likehood and the degree of host reaction, cases of melanization in our gammarid collection almost exclusively concern the cerebral metacercariae. Our results also indicate that this melanization is able to cancel the behavioral alterations induced by the parasite, suggesting that the cause of the manipulation is not the physical presence of metacercariae in the brain.

Parasites of the fathead minnow, Pimephales promelas, were examined in fish collected from Elk Creek (40.88534°N, 96.83366°W) and West Oak Creek (40.90821°N, 96.81432°W), Lancaster County, Nebraska. These 2 streams are part of the Salt Valley watershed and flow together approximately 2 km downstream from the collection sites to form Oak Creek. This study examined the extent to which the 2 tributaries constitute a continuous habitat with respect to fish hosts. The parasite community included Trichodina sp., Myxobolus sp., Dactylogyrus simplex, D. bychowskyi, and D. pectenatus (all on gills); Gyrodactylus hoffmani (gill and body surface); Posthodiplostomum sp. (neascus, body cavity); and Uvulifer ambloplitis (encysted in skin). Among 46 fish from Elk Creek and 56 fish from West Oak Creek taken on 5 dates during April–July 1998, U. ambloplitis was found in Elk Creek fish at prevalences of 44–100% but in only 2 West Oak fish on 1 date. Prevalence and mean abundance of D. simplex also differed between the 2 sites. On the basis of these observations, fish populations in the 2 streams were considered to be distinct, with little or no fish movement between the tributaries.