Transcripts encoding discrete, cathepsin L-like cysteine proteases, known as SmCL1 and SmCL2, have been reported from the adult stages of the human blood fluke Schistosoma mansoni. However, the physiological roles of these 2 enzymes and their natural substrates remain uncertain and controversial. To determine their localization in adult S. mansoni by immunocytochemical procedures, and thereby to gain insight into their likely functions, polymerase chain reaction-based cDNAs encoding mature SmCL1 and SmCL2 were ligated into Escherichia coli. The bacterially expressed recombinant proteins (bSmSL1, bSmCL2) were used to generate monospecific rabbit antisera. For light microscopy, paraffin-embedded sections were visualized with the fluorophore Cy3. For transmission electron microscopy (TEM), LR White-embedded tissue was visualized with 15 nm gold. Under light microscopy, fluorescence was visible on the luminal surface of the gastrodermis in both sexes for both proteins. For bSmCL1 and bSmCL2, TEM revealed gold particles primarily associated with amorphous deposits within superficial digestive vacuoles on the gastrodermal surface of males and females. Some bSmCL1 reaction product was observed in vesicles within the gastrodermis, and very sparse gastrodermal activity was observed with bSmCL2. By contrast, neither enzyme was immunolocalized in the reproductive organs, vitelline glands, nor gynecorphoric canal. The gut-associated immunolocalization of SmCL1 and SmCL2 indicates that both these endopeptidases participate in hemoglobin proteolysis.

Brugia malayi L3 molt to the L4 stage in serum-free cultures supplemented with arachidonic, linoleic, or linolenic acids and the basidiomycetous yeast Rhodotorula minuta. These fatty acids are capable of entering the eicosanoid pathway of arachidonate metabolism, the pathway responsible for generating a number of biologically active mediators, including prostaglandins, leukotrienes, and lipoxins. To determine whether this pathway was required for L3 development, we added dual inhibitors of cyclooxygenase and lipoxygenase to in vitro cultures containing B. malayi L3. These compounds significantly inhibited L3 molting. To evaluate whether 1 or both of these pathways of arachidonate metabolism were involved in molting, we tested drugs inhibiting either cyclooxygenase or lipoxygenase. Lipoxygenase inhibitors blocked L3 molting, whereas cyclooxygenase inhibitors did not. To assess whether enzymes operating downstream of lipoxygenase were also involved in L3 molting, we added inhibitors of enzymes involved in leukotriene synthesis and found they were also capable of preventing development. We tested the same inhibitor panel on Dirofilaria immitis L3. A single lipoxygenase inhibitor and inhibitors of 2 different enzymes operating downstream of lipoxygenase disrupted D. immitis development. These results demonstrate that a lipoxygenase pathway product is required for molting of the infective stage larvae of filarial parasites.

The spatial distribution of 6 parasite species (Myxobolus sp., Dactylogyrus sp., Sterliadochona ephemeridarum, Plagioporus sinitsini, Allopodocotyle chiliticorum, Allocreadium lucyae) was studied in 5 species of fishes (Oncorhynchus mykiss, Clinostomus funduloides, Notropis chiliticus, Rhinichthys atratulus, Semotilus atromaculatus) in Basin Creek, an Appalachian stream in North Carolina. Nonmetric multidimensional scaling and vector fitting were used to determine if the proximity of sampling sites was related to community similarity. Position along Basin Creek was significantly related to parasite community structure. Breaks in parasite community composition were imposed by waterfalls at upstream areas of Basin Creek that restricted distributions of C. funduloides, N. chiliticus, and S. atromaculatus and at the downstream limit of the study area by a break in the distribution of S. ephemeridarum coincident with the existence of a dam but were independent of suitable piscine host distributions. These discontinuities in parasite community composition imply that the relationship between proximity of sites and community similarity is predictive because distance between sites is related to the probability that fish at different sampling sites recruit parasites from different species pools. This relationship is not the same for all component communities.

Dispersal of avian ectoparasites can occur through either vertical transmission from adult birds to their offspring in the nest or through horizontal transmission between adult birds or through phoresy. In this study, we investigated the importance of the 2 main transmission modes in the colonial European bee-eater and examined whether individual differences in ectoparasite intensity exist in relation to age, sex, and morphological features of the birds. The intensity of 3 chewing lice species was investigated. Almost all adult bee-eaters (98.3%, n = 176) were infested with 1 of the 3 ectoparasite species, whereas only 10.8% (n = 167) of all chicks were infested. Meropoecus meropis was the most frequent ectoparasite species on adult bee-eaters (prevalence 94.3%), whereas Meromenopon meropis was the most common species on chicks (prevalence 9.6%). Our results suggest that chewing lice are mainly horizontally transmitted among adult bee-eaters and mainly among pair members, whereas vertical transmission between parents and nestlings is less frequent. These conclusions were supported by a relation in ectoparasite intensity of pair members and a parasite removal experiment. Ectoparasite intensity was in general low in nestlings and did not correlate with ectoparasite intensity of their parents. Host age, sex, weight, and other morphological features did not explain variation in chewing lice infestation.

Hoplopleura auliscomydis n. sp. is described based on specimens collected from Auliscomys micropus (Waterhouse 1837), from Collón Curá, Neuquén Province, Argentina. Descriptions are provided for the holotype male, female, first nymphal instar, external architecture of eggs (by scanning electron microscopy), and sites of oviposition, providing differences from its close relative Hoplopleura neotomydisCastro, González, and Cicchino, 1995. The geographical ranges of these 2 species are restricted to the Neuquén Province in Argentina. A key to the 9 species of the Hoplopleura travassosi species-group recorded in Argentina is also given.

Metacercariae of the trematode Ornithodiplostomum ptychocheilus cause a conspicuous enlargement of the cranium of juvenile fathead minnows (Pimephales promelas). Minnows sampled from 2 naturally infected ponds in northern Alberta, Canada, had 12% higher and 7% wider craniums compared to fish from an adjacent, uninfected pond. We tested the prediction that cranial distortion was caused by encystment of metacercariae on the brains of slow-growing minnows in a factorial experiment. Juvenile fish were either exposed once to 120 cercariae or 3 times to 40 cercariae; they were then fed either a low- or high-quantity diet for 8 wk. Results showed that after controlling for host size, cranial heights were affected by infection regime and host diet but not by the infection × diet interaction. Cranial distortion was most prominent in minnows exposed once to cercariae, showing that the rapid, simultaneous growth of metacercariae interfered with the normal development of the cranium. Thus, the expression of the parasite-induced phenotype was context dependent, the result of factors associated with the dynamics of cercariae transmission and host growth rate.

Previous investigations of cercarial sensory systems have focused on chaetotaxy and ultrastructure of sensory receptors and have revealed chaetotaxic patterns within families, genera, and species as well as different types of sensory receptors. However, chaetotaxic and ultrastructural observations have rarely been combined. We investigated the ultrastructure of cercarial sensory receptors in conjunction with the chaetotaxy and neuromorphology in 2 allocreadiid species belonging to the genera Crepidostomum and Bunodera. Cercariae were treated with acetylthiocholine iodide and silver nitrate, and for scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Similar cholinergic nerve networks were revealed. Chaetotaxy was consistent with that of other allocreadiids. Seven and 6 types of receptors were distinguished with SEM in Crepidostomum sp. and Bunodera sp., respectively. Types differed in number of cilia (1 or 2), cilium length (short, moderately long, or long), presence or absence of a tegumentary collar and a domelike base, and tegumentary collar length (low, moderately low, or high). TEM of some types revealed unsheathed cilia, basal body, and thickened nerve collars. Some receptor types were site specific. Thus, long uniciliated receptors were concentrated on the dorsal surface. Other types, such as short uniciliated receptors, were widespread throughout most regions. Ultrastructure and site-specificity observations suggest that most receptors are mechanoreceptors.

Antibodies against Eimeria stiedai sporozoites and merozoites were detected in the sera of rabbits immunized with bile obtained from infected rabbits on the 15th day post-infection. The trails made by gliding sporozoites were also detected by the sera. After penetration into the host cell, an antibody-binding region was observed on the parasitophorous vacuole membranes of the parasites. Rabbits administered a combination of the bile and cholera toxin shed fewer oocysts in the feces after infection than control rabbits. The immunized rabbits developed a high level of IgA antibody against soluble antigens in the bile. By immunoblotting, antigens with molecular masses of 32, 37, and 49 kDa were detected in the bile obtained from infected rabbits on the 15th day postinfection. Absorption treatment with sporozoites reduced or abolished the antibody reactivity to the 32-kDa antigen of merozoites and the bile antigens. However, antibody reactivity to the 37- and 49-kDa antigens still remained. These results indicate that soluble antigens are present in the bile of rabbits in the acute phase of infection, and these may be produced and released by merozoites during the host cell invasion process.

The fate of Schistosoma mansoni (Trematoda) sporocysts in its molluscan host Biomphalaria glabrata (Gastropoda) is determined by circulating phagocytes (hemocytes). When the parasite invades a resistant snail, it is attacked and destroyed by hemocytes, whereas in a susceptible host it remains unaffected. We used 3 inbred strains of B. glabrata: 13-16-R1 and 10-R2, which are resistant to the PR-1 strain of S. mansoni, and M-line Oregon (MO), which is susceptible to PR-1. In an in vitro killing assay using plasma-free hemocytes from these strains, the rate of parasite killing corresponded closely to the rate by which S. mansoni sporocysts are killed in vivo. Hemocytes from resistant snails killed more than 80% of S. mansoni sporocysts within 48 hr, whereas sporocyst mortality in the presence of hemocytes from susceptible snails was <10%. Using this in vitro assay, we assessed the involvement of reactive oxygen species (ROS) produced by resistant hemocytes, during killing of S. mansoni sporocysts. Inhibition of NADPH oxidase significantly reduced sporocyst killing by 13-16-R1 hemocytes, indicating that ROS play an important role in normal killing. Reduction of hydrogen peroxide (H₂O₂) by including catalase in the killing assay increased parasite viability. Reduction of superoxide (O₂⁻), however, by addition of superoxide dismutase or scavenging of hydroxyl radicals (·OH) and hypochlorous acid (HOCl) by addition of hypotaurine did not alter the rate of sporocyst killing by resistant hemocytes. We conclude that H₂O₂ is the ROS mainly responsible for killing.

Between 1989 and 1998, 3,504 rodents of the genera Dipodomys and Perognathus were collected from 4 permanent collecting sites on the University of New Mexico's Long Term Ecological Research station, located on the Sevilleta National Wildlife Refuge (SNWR), Socorro County, New Mexico. All animals were killed and examined for endoparasites (acanthocephalans, cestodes, coccidia, and nematodes). The present report focuses on 3 endoparasite groups, cestodes, coccidia, and nematodes. Specific analyses address how prevalence changes were related to abiotic factors such as habitat, season, or precipitation, and how prevalence of each parasite species in each host species differed in relation to host age, host sex, host reproductive status, host body mass, host density, parasite–parasite interactions, and host specificity. A logistic regression was used to determine which host characters and which abiotic factors are correlated with a parasite infection. Significant variables for at least half of the parasites include season, site, and winter precipitation. However, no parasite prevalences were correlated, and significant variables were not identical between parasites, indicating that each parasite species varied independently and that no generalizations can be drawn. The parasite prevalences in these rodents on the SNWR vary in independent and complex ways.

Thirty-six wild boars were inoculated with Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis (USSR), T. pseudospiralis (USA), T. pseudospiralis (AUST), Trichinella murrelli, Trichinella T6, and Trichinella nelsoni. The wild boars were killed at 5 and 10 wk postinoculation (PI), and the number of muscle larvae per g (lpg) of tissue was determined for 18 muscles or muscle groups. Five weeks PI, all Trichinella genotypes had established as muscle larvae, but their infectivity varied widely: T. spiralis established in high numbers (mean = 296 lpg), T. britovi, T. nelsoni, and 1 of the T. pseudospiralis genotypes (AUST) in moderate numbers (mean = 53–74 lpg), whereas the remaining genotypes were poorly infective (mean = 2–16 lpg). Because of considerable weight gain of the wild boars, an estimated total larval burden (live weight × lpg) was calculated for each animal. The total larval burden did not change significantly over time for T. spiralis, T. murrelli, T. britovi, T. nelsoni, and T. pseudospiralis (USA and USSR), whereas a significant reduction could be demonstrated for T. nativa, Trichinella T6, and T. pseudospiralis (AUST). Diaphragm and tongue were predilection sites in wild boars, independent of Trichinella genotype and infection level. At low infection levels, a greater percentage of larvae were found in diaphragm and tongue at 10 wk than 5 wk PI. Antibody responses increased rapidly between weeks 3 and 5 PI. For T. spiralis and T. nelsoni, the high antibody level persisted throughout the experimental period, but for T. nativa, T. britovi, T. murrelli, or Trichinella T6, the levels declined. For T. pseudospiralis, the antibody response increased more gradually between weeks 3 to 10 PI. Infection with all genotypes of Trichinella were detected using any of 7 excretory–secretory antigens, which points to the potential use of 1 common antigen for epidemiological studies on Trichinella in wild boars. In conclusion, T. spiralis is highly infective to wild boars, T. britovi, T. nelsoni, T. pseudospiralis (USA), and T. pseudospiralis (USSR) are moderately infective, and T. nativa, T. murrelli, T. pseudospiralis (AUST), and Trichinella T6 are poorly adapted to this host species.

The aquatic oligochaete Tubifex tubifex is an obligate host of Myxobolus cerebralis, the causative agent of salmonid whirling disease. Tubifex tubifex can become infected by ingesting myxospores of M. cerebralis that have been released into sediments upon death and decomposition of infected salmonids. Infected worms release triactinomyxons into the water column that then infect salmonids. How the dose of myxospores ingested by T. tubifex influences parasite proliferation and the worm host are not well understood. Using replicated laboratory experiments, we examined how differing doses of myxospores (50, 500, 1,000 per worm) influenced triactinomyxon production and biomass, abundance, and individual weight of 2 geographically distinct populations of T. tubifex. Worm populations produced differing numbers of triactinomyxons, but, within a population, the production did not differ among myxospore doses. At the lowest myxospore dose, 1 worm population produced 45 times more triactinomyxons than myxospores received, whereas the other produced only 6 times more triactinomyxons than myxospores. Moreover, total T. tubifex biomass, abundance, and individual weight were lower among worms receiving myxospores than in myxospore-free controls. Thus, T. tubifex populations differ in ability to support the replication of M. cerebralis, and infection has measurable consequences on fitness of the worm host. These results suggest that variability in whirling disease severity observed in wild salmonid populations may partially be attributed to differences in T. tubifex populations.

Heads of 109,597 mosquitoes collected during 1996 and 1997 from Gainesville, Florida (1996, n = 39,131; 1997, n = 34,209), Bartow, Florida (1996, n = 12,000; 1997, n = 12,000), and Baton Rouge, Louisiana (1996, n = 12,257) were tested by a polymerase chain reaction and Southern hybridization-based test for the presence of third-stage larvae of the canine heartworm Dirofilaria immitis. Mosquito heads were pooled (1–200 heads) by month, locality, and species for testing. The test used was species specific for D. immitis and was capable of detecting DNA from a single larva in a pool of 200 mosquito heads. Specificity for the third larval stage was achieved by probing only mosquito heads. One or more D. immitis-infected mosquito heads were detected in each month of the year from Bartow in both 1996 and 1997. No infected mosquito heads were detected from Gainesville or Baton Rouge in December, January, February, or March. These results are in general agreement with previous sentinel dog and model prediction studies that showed heartworm transmission in the warm temperate Gulf coast region of the United States to be seasonal rather than continuous as previously believed.

Divergence of biological performance of a laboratory-reared population of Moniliformis moniliformis (Acanthocephala) was investigated after 31 yr, or approximately 60 generations, of genetic isolation. An isolate of the laboratory-reared population and isolates of 2 wild populations were used to begin 3 independent life cycles that were maintained for 1 generation for interbreeding and life history trait comparison. Both wild population isolates represented populations with open gene flow. One wild population isolate represented a present-day sample of descendants of the parent population of the laboratory isolate. All 3 populations hybridized, and egg production occurred in all mixed-sex pairs of different populations. The 3 populations did not differ significantly in prepatent period, mean daily egg production, or establishment within the definitive host Rattus norvegicus. The 3 populations varied in patent period, but the laboratory-isolated worms differed from the 2 wild population isolates no more than they did from each other. A positive correlation between mean daily egg production and duration of patent period resulted in different cumulative egg productions. A 31-yr period of isolation did not produce greater divergence in a laboratory population of M. moniliformis than occurs between wild populations with respect to the biological parameters measured.

Seventy-four specimens of Falcaustra lowei n. sp. were recovered from the intestines of 9 of 42 (21%) Tarahumara frogs, Rana tarahumarae, from Sonora, Mexico. F. lowei is the 14th Nearctic species to be described and belongs to that group of species possessing a pseudosucker, namely F. catesbeianae, F. chabaudi, F. chelydrae, F. mexicana, and F. wardi. The new species can be readily differentiated from these by the arrangement of caudal papillae and length of spicules. Priority description of F. affinis is established and F. concinnae is removed from synonymy with F. affinis. In addition to F. lowei, 3 species of Digenea, Glypthelmins quieta, Haematoloechus breviplexus, Langeronia macrocirra; 1 species of Eucestoda, Ophiotaenia magna; 7 species of Nematoda, F. inglisi, Foleyellides striatus, Oswaldocruzia pipiens, Rhabdias ranae, Subulascaris falcaustriformis, Physaloptera sp. (larvae); and 1 species of Acanthocephala, an unidentified oligacanthorhynchid cystacanth, were found.

Neospora hughesi was isolated in cell cultures inoculated with homogenate of spinal cord from a horse in Oregon. Tachyzoites of this Oregon isolate of N. hughesi were maintained continuously by cell culture passage and tachyzoites were infective to immunosuppressed mice. Gamma interferon gene knockout (KO) mice injected with tachyzoites developed fatal myocarditis and numerous tachyzoites were seen in lesions. Gerbils (Meriones unguiculatus) inoculated with tachyzoites developed antibodies (≥1:500) as indicated by the Neospora caninum agglutination test but did not develop clinical signs, and Neospora organisms were not demonstrable in their tissues. Tissue cysts were not found in gerbils, nude mice, KO mice, immunosuppressed outbred Swiss Webster mice, or BALB/c mice injected with the Oregon isolate of N. hughesi. Ultrastructurally, tachyzoites of the Oregon isolate from the myocardium of infected KO mice and from cell culture were similar to N. caninum tachyzoites. Western blot analysis using NcSAG1 and NcSRS2 polyclonal and monoclonal antibodies and characterization of the internal transcribed spacer 1 sequences from the equine isolates and different isolates of N. caninum from dogs and cattle indicated that the Oregon isolate of N. hughesi is distinct from N. caninum isolates from cattle and dogs.

Examination of the spiral intestines of 29 whiptail stingrays, Dasyatis brevis, from 6 sites in the Gulf of California, México in 1993 and 1996 resulted in the discovery of 4 new species of Acanthobothrium: Acanthobothrium bullardi, Acanthobothrium dasi, Acanthobothrium rajivi, and Acanthobothrium soberoni. This brings the total number of species of Acanthobothrium known from the eastern Pacific Ocean to 34. With 22 additional species reported from elasmobranchs from the western Atlantic Ocean, and over 100 species globally, the number of interspecific comparisons required to justify the designation of a new species is rather unwieldy. To facilitate these and future comparisons, and in the absence of a phylogenetic hypothesis for this genus, the 56 species of Acanthobothrium from these 2 geographic regions were categorized for 4 characters: total length (≤ or >15 mm), number of segments (≤ or >50 segments), number of testes (≤ or >80), and symmetry of poral and aporal ovarian lobes. These 56 taxa and their categories are presented in tabular form. Based on these characters, A. bullardi, A. dasi, and A. rajivi are category 2 species (they are relatively small, possess few segments, relatively few testes, and exhibit asymmetrical ovaries); A. soberoni is a category 6 species (it is a relatively longer worm with a larger number of segments, but with fewer testes and an asymmetrical ovary). All 4 species differ from 1 another and from species in other geographic regions in further subtleties of these 4 characters as well as hook size and relative length of hook prongs, cirrus sac size, genital pore position, number of columns of testes anterior to the cirrus sac, and number of postvaginal testes. Five specimens that appear to represent a new species of Acanthobothroides were also collected. This species is figured and some details of the morphology are described, but the material was considered to be insufficient to allow for formal description of this species at this time. This is the first record of either of these genera of onchobothriid tapeworms from the Gulf of California.

An epidemiologic study designed to identify the small mammal reservoir for the zoonotic WA1-type babesial parasite resulted in the discovery of a small, intraerythrocytic piroplasm in smeared blood from dusky-footed woodrats (Neotoma fuscipes) in northern California. The woodrat parasites were isolated and compared to other piroplasm parasites based on their morphology, antigenicity, and genetic characteristics. These studies indicated that the woodrat parasites were not the WA1-type babesial agent but were of the genus Theileria. We accordingly named it Theileria youngi. The prevalence in the woodrat population was high (61%). Infection was unrelated to gender or age of the woodrats. Potential vectors for this tick-transmitted parasite included 3 species of ticks recovered from the woodrats, Dermacentor occidentalis, Ixodes woodi, and Ixodes pacificus. Mostly larval or nymphal stages were recovered, suggesting transstadial transmission is possible. This is the first piroplasm fully characterized from a dusky-footed woodrat.

Recent progress in understanding the etiology of proliferative kidney disease (PKD) includes the identification of freshwater bryozoans as the natural hosts of the myxozoan parasite that causes the disease in salmonid fish and formal description of the parasite as Tetracapsula bryosalmonae. This paper presents data on patterns of occurrence of T. bryosalmonae and sequence variation among isolates. T. bryosalmonae infects bryozoans that range from primitive to more derived genera within the Phylactolaemata and that differ in growth form and habits. Infected bryozoans have been collected in diverse habitats including cold, clear streams and warm, eutrophic lakes. Temporal surveys reveal intra- and interannual variation in infection levels, and spatial variation in incidence of infection is implicit by the apparent absence of T. bryosalmonae from many bryozoan populations. The significance of minor variation in partial sequences of 18S rDNA requires further investigation. The information presented here provides the first significant insights into the ecology of T. bryosalmonae.

A phylogenetic analysis was performed on 13 species of digenetic trematodes in the Macroderoididae, including 10 species of Alloglossidium, 2 species of Alloglossoides, and Hirudicolotrema richardsoni. The evolution of the unusual life-cycle patterns in the group was assessed in light of the proposed phylogeny. The results support previous hypotheses that taxa with a 3-host life cycle involving catfish as definitive hosts are basal to taxa with a 2-host life cycle involving invertebrates such as crustaceans and leeches as definitive hosts. Our results also strongly suggest that species maturing in leeches evolved from an ancestor that matured in crustaceans. Our phylogeny places Alloglossoides and Hirudicolotrema within Alloglossidium, showing Alloglossidium to be paraphyletic. To achieve a natural classification, Alloglossoides and Hirudicolotrema are synonymized with Alloglossidium, and a revised generic diagnosis for Alloglossidium is given.

The monotypic Philureter n. gen. (Ancyrocephalinae; Dactylogyridae) is proposed to accommodate Philureter trigoniopsis n. sp. with the following features: presence of a cuplike ventral haptor armed with 14 hooks and 2 anchor/bar complexes; dorsal pair of anchors poorly defined and variable in shape, 1 frequently absent; tandem, intercecal gonads, testis bilaterally lobulated. Philureter trigoniopsis n. sp. is described from the ureters and urinary bladder of Galaxias maculatus (Jenyns, 1842) (Osmeriformes) in Patagonian Andean lakes, Argentina.

Neospora caninum oocysts, passed in the feces of a definitive host (dog), were isolated, and genomic DNA was extracted. A polymerase cahin reaction (PCR) targeting the N. caninum-specific Nc 5 genomic sequence was performed using the isolated DNA. A synthesized competitor molecule containing part of the Nc 5 sequence was included in the assay as a check against false-negative PCR results and to quantify N. caninum oocyst DNA in fecal samples. A standard curve of the ratio of fluorescence intensity of PCR-amplified competitor to that of oocyst DNA was constructed to compare oocyst equivalents from fecal samples containing unknown numbers of N. caninum oocysts and to assess the sensitivity of the assay. The specificity of the assay was determined using the Nc 5–specific primers in PCR assays against other parasites likely to be found in canine feces. Genomic DNA sequences from the canine coccidians Hammondia heydorni, Cryptosporidium parvum, Sarcocystis cruzi, S. tenella, and Isospora ohioensis and the canine helminth parasites Strongyloides stercoralis, Toxocara canis, Dipylidium caninum, and Ancylostoma caninum were not amplified. In addition, genomic DNA sequences from oocysts of coccidian parasites that might contaminate dog feces, such as Hammondia hammondi, Toxoplasma gondii, or Eimeria tenella, were not amplified in the PCR assay. The assay should be useful in epidemiological surveys of both domestic and wild canine hosts and in investigations of oocyst biology in experimental infections.

The effect of jasplakinolide, an actin-polymerizing and filament-stabilizing drug, on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens was examined. Jasplakinolide inhibited the growth of E. histolytica strain HM-1:IMSS and E. invadens strain IP-1 in a concentration-dependent manner, the latter being more resistant to the drug. The inhibitory effect of jasplakinolide on the growth of E. histolytica trophozoites was reversed by removal of the drug after exposure to 1 µM for 1 day. Encystation of E. invadens as induced in vitro was also inhibited by jasplakinolide. Trophozoites exposed to jasplakinolide in encystation medium for 1 day did not encyst after removal of the drug, whereas those exposed to the drug in growth medium for 7 days did encyst without the drug. The process of cyst maturation was unaffected by jasplakinolide. Large round structures were formed in trophozoites of both amoebae grown with jasplakinolide; these were identified as F-actin aggregates by staining with fluorescent phalloidin. Accumulation in trophozoites of both amoebae of actin aggregates was observed after culture in jasplakinolide. Also, E. invadens cysts formed from trophozoites treated with jasplakinolide contained the actin aggregate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis revealed that the jasplakinolide treatment led to an increase in the proportion of F-actin associated with formation of the aggregate. The results suggest that aggregates are formed from the cortical flow of F-actin filaments, and that these filaments would normally be depolymerized but are artificially stabilized by jasplakinolide binding.

Although single-color flow cytometry has been shown to be more sensitive than fluorescence microscopy for the quantification of Cryptosporidium parvum oocysts, this method has not been optimized. Monoclonal antibody OW50, specific to the cell wall of oocysts, was conjugated to superparamagnetic particles, to fluorescein isothiocyanate, and to r-phycoerythrin. The oocysts were then double stained with the fluorochrome-labeled OW50 and were placed in tubes with known numbers of highly fluorescent polystyrene beads, allowing quantification of the oocysts without dependence on acquired sample volume by flow cytometry. Data from 2-color flow cytometry using logical gating of the oocysts and beads showed a linear relationship between dilutions of a purified oocyst suspension and the mean numbers of oocysts detected (r² = 1.00). An average of 15 purified oocysts/ml were counted in a dilution with a theoretical concentration of 12 oocysts/ml. Known numbers of purified oocysts were seeded into normal mouse fecal specimens, captured by OW50-labeled immunomagnetic particles, eluted with 5% potassium dichromate at low pH, and double stained with fluorochrome-labeled OW50. By flow cytometry, the mean recovery was 43.1% (±8.3%), and as few as 133 oocysts were detected. The captured and eluted oocysts were infective in neonatal BALB/c mice. This 2-color flow cytometry method, used in conjunction with the capture and elution of oocysts by and from immunomagnetic particles, provides a powerful tool for not only the quantification and purification of C. parvum oocysts from different sources but also for the characterization of oocysts in vitro and in vivo.

A clinical trial to compare the effectiveness of 4- and 6-mo repeated treatment with albendazole 600 mg (Zentel®, SmithKline Beecham) or mebendazole 600 mg (Vermox®, Janssen) on geohelminth infections was carried out on children in 6 primary schools; the study included 1,186 children, ages 4 to 19 yr. Kato–Katz examination was performed on stool samples before and after treatment. Overall, albendazole produced better cure rates and egg reduction rates for geohelminths. The cure rates for albendazole were 92.4% for hookworm infection, 83.5% for Ascaris lumbricoides, and 67.8% for Trichuris trichiura. Mebendazole given either 2 or 3 times in a year had cure rates of 50 and 55.0% (respectively) for hookworm, 79.6 and 97.5% for A. lumbricoides, and 60.6 and 68.3% for T. trichiura infection. The geometric mean intensity of hookworm eggs per gram (epg) of stool decreased by 96.7% after albendazole treatment compared with 66.3 and 85.1%, respectively, for 2 or 3 doses of mebendazole (P < 0.05) over the same period. Reductions in epg for A. lumbricoides and T. trichiura were comparable for both drugs. Our results indicate that treatment with albendazole at a 6-mo interval was more effective than mebendazole regimens and may be the best choice for use in the control of the 3 geohelminths.

Nodulisporic acid A (NSA) is a novel natural product from a new structural class that was shown previously to have insecticidal activity against blowfly larvae. To determine if there was useful systemic efficacy against fleas (Ctenocephalides felis), NSA was evaluated in an artificial membrane flea feeding device and in dogs. In the artificial membrane flea feeding device, adult C. felis were allowed to feed on bovine blood containing various concentrations of NSA through a Parafilm membrane. NSA killed the fleas with a 50% lethal concentration of 0.68 µg/ml and was approximately 10-fold more potent than the systemic insecticide ivermectin. In the initial probe dog test, a single beagle was challenged with 100 C. felis before oral dosing with 15 mg/kg of NSA. Flea counts conducted at 72 hr postdosing showed an 88% reduction relative to control. Rechallenge of the same dog at 5 days postdosing showed 50% reduction of fleas at day 7, demonstrating some residual flea activity. In a confirmatory study, 8 dogs were challenged with 100 fleas just before oral dosing with 15 mg/kg of NSA (4 dogs) or vehicle (4 dogs). There was 99% reduction of fleas at 48 hr postdosing in the NSA-treated dogs relative to control. Additional challenges with 100 fleas were performed on these 8 dogs at 48-hr intervals to determine the duration of efficacy, and there was 97, 51, and 0% reduction of fleas relative to control on days 4, 6, and 8, respectively. No adverse effects were observed in the dogs in these studies. These data show that NSA has potent oral activity in the dog for the control of fleas, while lacking overt mammalian toxicity.

Fifty-one common mergansers were captured on Douglas Lake (Cheboygan County, Michigan) and their avian schistosome loads were determined by fecal examination. Each bird was given a single dose of 0, 40, or 200 mg/kg of body weight of praziquantel and released. All birds were recaptured within 10 days of drug administration to determine posttreatment schistosome loads. Only the highest dose of praziquantel was found to significantly reduce avian schistosome loads. The potential use of praziquantel in swimmer's itch control programs is discussed.

Fasciola hepatica adult flukes have a native protein complex denoted nFh12 and consisting of fatty acid binding proteins that comprise at least 8 isoforms. It is a potent immunogen because in several animal hosts it induces an early antibody response to F. hepatica infection. It is also a potent cross-protective immunogen because it induces a protective immune response in mice to challenge infection with Schistosoma mansoni cercariae. The gene encoding this protein has been cloned and sequenced. It produces a polypeptide of 132 amino acids with a predicted molecular mass of 14.7 kDa and is denoted rFh15. It also has a significant homology to a 14-kDa S. mansoni fatty acid binding protein (Sm14). In the present study, nFh12 was delipidated with charcoal treatment and then studied by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Additionally, a lipid analysis of nFh12 was undertaken using gas chromatography–mass spectrometry to demonstrate that the nFh12 protein complex is, in fact, a complex of fatty acid binding proteins. Five long-chain saturated and unsaturated fatty acids were detected. The most abundant were palmitic acid (38%), stearic acid (24%), and oleic acid (13%). These fatty acid molecules do not have covalent bonds attached to the protein molecule. Because both nFh12 and Sm14 protect mice against challenge infection with F. hepatica and S. mansoni, it is possible that they have common protective epitopes in which fatty acids could be involved. Further studies are in progress to determine the chemical nature of these potential common epitopes.

A polymerase chain reaction (PCR) screen was used to examine the diversity of myosins in 7 Apicomplexan parasites: Toxoplasma gondii, Plasmodium falciparum, Neospora caninum, Eimeria tenella, Sarcocystis muris, Babesia bovis, and Cryptosporidium parvum. Using degenerate PCR primers compatible with the majority of known myosin classes, putative myosin sequences were obtained from all of these species. All of the sequences obtained showed greatest similarity to previously identified apicomplexan myosins, suggesting that the diversity of myosins in these parasites is limited. Myosin classes that are known to be widespread across the phylogenetic spectrum, e.g., the myosins I, II, and V, were not seen in the Apicomplexa. Thus, like the plants, the Apicomplexa may have evolved their own unique cohort of myosins that are responsible for the myosin-driven cellular functions observed in these parasites.

This report describes a simple filtration technique to isolate the parasite Mikrocytos mackini from oyster tissue. The technique is based on successive filtration through filter papers and polycarbonate membrane filters of decreasing mesh using a low vacuum (<8 cm Hg). This technique allows for the recovery of about 1 × 10⁸ parasites (microcells) from about 2 g of heavily infected oyster tissue. About 99% of the particulate material in the final preparation consisted of intact M. mackini.

Neospora caninum tissue cysts were found in the brains of surgically delivered twin fetuses at 119 days of gestation. In the brains of both fetuses, there was an inflammatory reaction involving perivascular cuffings of mononuclear cells, glial nodules. The dam of these fetuses died because of metritis. Histopathological examination of the ewe revealed N. caninum tissue cysts and focal gliosis with mononuclear cell cuffings. A N. caninum-specific DNA fragment was detected in a brain homogenate of the ewe by the polymerase chain reaction method. This is the first report of N. caninum infection in twin ovine fetuses and in an adult sheep.

A small Babesia gibsoni-like parasite was identified and isolated as the cause of clinical babesiosis in a dog from Oklahoma. Because this was potentially the first documented case of B. gibsoni infection in Oklahoma, further characterization was warranted, and the 18S nuclear small subunit ribosomal RNA gene was sequenced. Sequence comparison with other piroplasms from dogs showed significant nucleotide sequence differences between this isolate and both B. canis and B. gibsoni. These findings demonstrate that in domestic dogs in North America there are at least 2 “small” B. gibsoni-like organisms with distinct nucleotide sequences and that the geographic distribution of the “small” canine Babesia species may be wider than previously recognized.

Prevalence of antibodies to Toxoplasma gondii was determined in 147 barren-ground caribou (Rangifer tarandus groenlandicus) from 5 herds in the Northwest Territories and Nunavut, northern Canada, by the modified agglutination test (MAT). In the mainland herds (Bluenose, Bathurst, and Beverly), antibodies were found in 43 (37%) of 117 caribou, and MAT titers were 1:25 in 10, 1:50 in 24, and 1:500 in 9. In the island herds, only 1 (4.3%) of 23 animals sampled from the North Baffin Island herd was positive (titer = 1:25) and no antibodies were detected in 7 caribou from the Dolphin and Union herd. The high prevalence of antibodies to T. gondii in the mainland caribou herds indicates that caribou meat may contain viable T. gondii.

Microparasite virulence (the potential to cause harm in the host) is thought to be regulated by a direct trade-off with pathogen transmission potential, but it is unclear whether similar trade-offs occur in macroparasites (helminths). In this analysis, the transmission potentials of 5 nematode species (order Strongylida), known to differ in their virulence, were estimated using an index based on egg production and larval survivability. Virulence estimates were based on the minimum number of worms that cause host death. In nematode species where mature adults cause pathology (trichonematidic development), there is a direct relationship between virulence and transmission, suggesting that high virulence is related to parasite fitness in these worms. However, in nematodes where the juvenile stages produce pathology during migration and development (strongylidic development), virulence is not correlated with transmission. These data suggest that trade-offs between transmission and virulence in nematode parasites are not analogous for all species and may depend on the developmental strategy and mechanism of pathogenicity of the parasites.

Outbred goats were fed 10⁵ Toxoplasma gondii oocysts and were monitored twice weekly for 8 wk for rectal temperature, clinical signs, parasitemia, and antibody levels by indirect fluorescence antibody test (IFAT), latex agglutination test (LAT), and indirect enzyme-linked immunosorbent assay (ELISA). After 8 wk, all goats were killed, and samples of heart, skeletal muscle, brain, lymph nodes, kidneys, and liver were bioassayed in mice. Anorexia, fever, and lethargy were observed from day 3 to day 7 postinfection (PI). Parasitemia was detected by bioassay in 50% of infected goats from day 7 to day 14 PI. Viable T. gondii organisms were isolated from all infected goats. Antibodies to T. gondii were detected in some animals on day 10 PI by IFAT and LAT and on day 14 PI by ELISA. The infected goats were seropositive on day 17 PI.

To determine the influence of interferon (IFN)-γ on the organ infectivity and on the genetic susceptibility of susceptible (C57BL/6) and resistant (BALB/c) strains after peroral infection with cysts of Toxoplasma gondii, IFN-γ knockout (KO) mice in C57BL/6 and BALB/c backgrounds were utilized. The kinetics of the changes in T. gondii abundance were evaluated with a quantitative competitive polymerase chain reaction assay in various organs at different times after peroral infection. In IFN-γ KO mice, a T. gondii-specific gene, SAG1, was detected in all organs examined, and the protozoan proliferated much more actively than in wild-type mice. The abundance of T. gondii was much higher in mesenteric lymph nodes and the heart than in other organs. In contrast, in the nervous system organs and kidneys, only a weakly detectable reaction was observed. Toxoplasma gondii grew at a more rapid rate in the organs of IFN-γ KO C57BL/6 mice than in the organs of IFN-γ KO BALB/c mice during the course of infection. Destruction of the IFN-γ gene showed remarkable effects on the infectivity in both susceptible and resistant mice.

Several observations have suggested that the anthelmintic ivermectin can affect nematodes by non-oral entry into the nematode body. To investigate this possibility further, we refrigerated Caenorhabditis elegans at 5 C to prevent its locomotion and to block the pharyngeal pumping that is so prominent a feature of its feeding. Worms were exposed to ivermectin (1–25 µg/ml) at that temperature for 1 hr, after which the medium was replaced by unmedicated medium at room temperature. After 1 hr at room temperature the worms were examined and counted to determine the degree to which irreversible immobilization had occurred. The drug was significantly less effective at 5 C than at room temperature. This reduction in potency could be attributed to a general cold-induced decline in the rate of the biochemical processes involved in drug action. Alternatively, the reduction could be attributed to the cold-induced blockade of pharyngeal pumping, which would suggest that the efficacy of ivermectin is partially the result of oral intake of drug. The fact that antinematodal efficacy was not entirely abrogated and reached a significant level despite blockade of pharyngeal pumping supports the former interpretation and is in accord with earlier indications that ivermectin can enter by non-oral routes. This conclusion is further supported by the observation that ivermectin is active against the nonfeeding third-stage larva of Haemonchus contortus.

To determine whether avian species are susceptible to infection with Gymnophalloides seoi (a human-infecting intestinal trematode), we exposed 7 species of birds with metacercariae obtained from oysters. The birds were necropsied at days 2, 4, and 6 postinfection (PI). The highest worm recovery at day 6 PI was obtained from the Kentish plover (Charadrius alexandrinus; x̄ = 56.0%), followed by the Mongolian plover (C. mongolus; 49.3%), and the grey plover (Pluvialis squatarola; 32.3%). In contrast, no mature worms were recovered from the great knot (Calidris tenuirostris), dunlin (C. alpina), black-tailed gull (Larus crassirostris), and mallard (Anas platyrhynchos). Among the plovers, the worms attained the greatest size at day 6 PI (254.1 × 190.4 µm) in the Kentish plover, with a significantly higher number of eggs in the uterus. The 3 species of plovers are highly susceptible to experimental G. seoi infection, suggesting that they could play a role as definitive hosts for these worms in nature.

The role of iron was evaluated with respect to the virulence of Trichomonas vaginalis in mice. Iron-supplemented and iron-depleted Diamond's trypticase–yeast extract–maltose (TYM) media were prepared by adding 360 µM of ferrous sulfate and 100 µM of 2,2′-dipyridyl. Trophozoites cultivated from normal TYM and iron-supplemented TYM media produced subcutaneous abscesses; however, trichomonads grown in an iron-deficient TYM medium failed to produce any pathology. In addition to the increased virulence of trophozoites in mice, iron affects the level of adherence and the cytotoxicity of trichomonads to HeLa cells, which are significantly reduced in trophozoites grown in iron-deficient medium. In conclusion, it is suggested that under iron-depleted conditions such as that induced by 2,2′-dipyridyl the virulence of T. vaginalis is reduced.

Adoptively transferred resistance to Schistosoma mansoni in the snail intermediate host Biomphalaria glabrata was measured as a function of miracidial challenge dose. Schistosome-susceptible snails implanted with the amebocyte-producing organ (APO) from resistant donors showed 29 and 39% prevalences of infection after challenge with 5 and 10 miracidia, respectively, but 68–83% prevalences when exposed to 25–200 miracidia. Prevalences in control (untampered) susceptible snails ranged from 97 to 100% at the different miracidial doses. Higher infection prevalences at elevated doses suggest that a range of transferred resistance occurs and possibly that low levels of APO-derived plasma factors or hemocytes in some recipients can be overwhelmed by larger numbers of parasites.

A female dioctophymatid nematode larva, presumably belonging to the genus Dioctophyme, was found in a dermal granuloma accompanied by creeping eruption in the left inner thigh of a 26-yr-old Chinese woman who had stayed in Japan for 4 yr. Morphology of the sectioned worm is described in detail. This is the fourth case of dermal infection with dioctophymatid larva in humans.