Our previous investigation demonstrated the expression in Eimeria tenella sporulated oocysts of an aminopeptidase (AP) with strong homology to AP N. To further understand the role of proteases during development, we investigated the molecular and biochemical properties of E. tenella AP. Greater than 95% AP activity was present in a soluble extract during sporulation of oocysts with highest activity in fully sporulated oocysts. The AP activity was inhibited by the AP inhibitors bestatin and 1,6-phenanthroline, but not by serine protease inhibitors. The AP had specificity for synthetic endopeptidase substrates that contain arginine, alanine, or glycine at the N terminus. Partial purification of the enzyme yielded a major protein band with an M_r of about 106 kDa and an isoelectric point (Ip) of 5.1. Reverse transcription-polymerase chain reaction indicated that the gene for AP is expressed during sporulation, but expression is absent or greatly reduced in the sporozoites and merozoites. On the basis of the deduced gene structure, the predicted M_r is 110 kDa with a pI of 5.59. Database search indicates that the E. tenella AP shares significant homology with the AP from Apicomplexan taxa: Toxoplasma gondii, Cryptosporidium parvum, and Cryptosporidium hominis. Together, these results confirm the presence of a cytosolic AP related to AP N, which is expressed and active during sporulation of E. tenella oocysts.

Babesia gibsoni multiplies well in canine red blood cells (RBCs) containing high concentrations of potassium (HK), reduced glutathione, and free amino acids as a result of an inherited high Na,K-ATPase activity, i.e., HK RBCs. To determine the role of Na,K-ATPase in the multiplication of B. gibsoni, the effect of ouabain on the proliferation of the parasites in HK RBCs was investigated. To determine the direct effect of ouabain on the parasites, the proliferation of the parasites in normal canine RBCs containing low potassium (LK) and high sodium concentrations, i.e., LK RBCs, which completely lack Na,K-ATPase activity, was observed. Ouabain at 0.1 mM significantly suppressed the multiplication of B. gibsoni in HK RBCs in vitro, whereas it had no effect on the parasites in LK RBCs. The results suggest that the multiplication of B. gibsoni in HK RBCs depends mainly on the presence of Na,K-ATPase in the cells. Therefore, the effects of ouabain on the intracellular cation and free amino acid composition of the HK RBCs were examined. In HK RBCs incubated with ouabain, a marked decrease in the concentration of potassium and an increase in sodium were observed, together with a decrease in the number of parasitized cells. These results suggest that the intracellular cation composition maintained by Na,K-ATPase might be advantageous to the parasites. Moreover, the concentrations of some free amino acids, i.e., asparagine, aspartate, glutamate, glutamine, glycine, and histidine, were markedly decreased in HK RBCs incubated with ouabain. Decreased concentrations of the free amino acids induced by inhibition of Na,K-ATPase seemed to affect the multiplication of B. gibsoni in HK RBCs. Based on these results, it is clear that the high Na,K-ATPase activity in HK RBCs contributes to the proliferation of B. gibsoni by maintaining high potassium and low sodium concentrations, as well as high concentrations of some free amino acids in the cells.

Parasitism is not well documented for birds found in tropical habitats. Long-distance migratory birds may face additional risks to an already hazardous journey when infected. This study explores the ecology of an ectoparasite infestation in Chiapas, Mexico. During a mist-netting project in 2 different coffee management systems, chigger mites (Acarina: Trombiculidae), ectoparasitic during the larval stage, were found on both resident and migratory birds. Using a rapid assessment protocol, it was observed that 17 of 26 species of long-distance migrants and 33 of 71 resident species had at least 1 infested individual. Infestation prevalences were unexpectedly high on some long-distance migrants, as high as 0.73 for Swainson's thrush (Catharus ustulatus), a value on par with heavily infested resident species. Prevalence was highest during winter sampling: 0.18 overall, 0.16 of migrants, and 0.23 of residents. Prevalence was 0.14 for resident birds during the summer breeding season. Mean abundance and mean intensity of infestation are reported for 97 species captured and inspected during the course of this study. In this region, chigger mite larvae are relatively common on birds and their abundance varies seasonally. High prevalence for some migratory birds suggests that more research and monitoring of ectoparasites are needed, especially in light of emerging diseases.

In collections of ectoparasites from 368 small ground finches, Geospiza fuliginosa, in populations from the islands of Isabela, Santa Cruz, San Cristóbal, and Santa Fé, in the Galápagos Archipelago, Ecuador, we found 8 species of mites. Four mite species were common on all islands sampled, i.e., Mesalgoides geospizae Mironov and Pérez (Psoroptoididae), Xolalges palmai Mironov and Pérez (Xolalgidae), and 2 new species, Trouessartia geospiza n. sp. (Trouessartiidae) and Proctophyllodes darwini n. sp. (Proctophyllodidae). Four other species were represented by single collections from G. fuliginosa, i.e., Pterodectes atyeoi n. sp. (Proctophyllodidae), Strelkoviacarus sp. (Analgidae), Dermoglyphus sp. (Dermoglyphidae), and Dermanyssus sp. (Dermanyssidae). Authorship of new species names is attributed to the 3 authors who prepared the descriptions (B.M.O.C., J.F., D.L.). Trouessartia geospiza and P. atyeoi were also found on previously collected specimens of other Geospiza species in museum collections. For the 4 common species, we found no differences in prevalence among the 4 island populations, but infection prevalence differed among the 4 species. The mean infection prevalence was high for T. geospizae (89%), moderate for M. geospizae (58%) and X. palmai (44%), and low for P. darwini (26%) in all populations. The feather mite fauna of G. fuliginosa was similar to that of other Geospiza species, and generally related to communities found on other emberizid finches.

The chimpanzee pinworm, Enterobius (Enterobius) anthropopitheci (Gedoelst, 1916) (Nematoda: Oxyuridae), is redescribed based on light and scanning electron microscopy of both sexes collected from the feces of chimpanzees, Pan troglodytes, of an introduced population on Rubondo Island, Tanzania. Enterobius (E.) anthropopitheci is characterized by having a small body (males 1.13–1.83 mm long, females 3.33–4.73 mm long), a rather straight spicule with a ventral membranous formation in males, double-crested lateral alae in females, small eggs (53–58 by 24–28 μm), and a smooth eggshell with 3 longitudinal thickenings. Morphological comparison is made between the present and previous descriptions.

We demonstrate polymorphism in the structure of the tail among first-stage larvae of Parelaphostrongylus odocoilei (Protostrongylidae). Two distinct larvae, both with a characteristic dorsal spine, include (1) a morphotype with a kinked conical tail marked by 3 distinct transverse folds or joints and a symmetrical terminal tail spike and (2) a morphotype with a digitate terminal region lacking folds or joints and with an asymmetrical, subterminal tail spike. These divergent larval forms had been postulated as perhaps representing distinct species of elaphostrongyline nematodes. Application of a multilocus approach using ITS-2 sequences from the nuclear genome and COX-II sequences from the mitochondrial genome confirmed the identity of these larvae as P. odocoilei. Additionally, based on scanning electron microscopy (low-temperature field emission), the cephalic region of these larvae consisted of a cuticular triradiate stoma surrounded by 6 single circumoral papillae of the inner circle, 10 papillae of the outer circle (4 paired and 2 single), and 2 lateral amphids. Our's is the first demonstration of structural polymorphism among larval conspecifics in the Metastrongyloidea and Strongylida. The basis for this polymorphism remains undetermined, but such phenomena, if discovered to be more widespread, may contribute to continued confusion in discriminating among first-stage larvae for species, genera, and subfamilies within Protostrongylidae.

Calreticulin (CRT ) is a unique eukaryotic gene. The CRT gene product, calreticulin, was first identified as a calcium binding protein in 1974, but further investigations have indicated that CRT protein performs many functions in cells, including involvement in evading the host's immune system by parasites. Many studies of CRT have been published since the molecule was first discovered; however, the CRT gene exon-intron structure is only known for a limited number of ectoparasite species. In this study, we compared tick CRT genomic sequences to the corresponding cDNA from 28 species and found that 2 exons and 1 intron are present in the tick CRT gene. The intron position is conserved in 28 hard ticks, but intron size and nucleotide sequences vary. Three tick introns possess duplicated fragments and are twice as long as other introns. All tick CRT introns obey the GT-AG rule in the splice-site junctions and are phase 1 introns. By comparing tick CRT introns to those of fruit fly, mouse, and human, we conclude that tick CRT introns belong to the intron-late type. The number and size of CRT introns have increased through the evolution of eukaryotes.

The prevalence of Toxoplasma gondii, in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 46 free-range chickens (Gallus domesticus) from Venezuela was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 16 (32%) chickens with titers of 1:5 in 1, 1:10 in 2, 1:40 in 2, 1:80 in 2, 1:160 in 2, 1:320 in 3, 1: 640 in 2, and 1:1,280 or higher in 2. Hearts, pectoral muscles, and brains of 13 chickens with MAT titers of 1:40 or more were bioassayed individually in mice. Tissues of each of 3 chickens with titers of 1:5 or 1:10 were pooled and bioassayed in mice. Tissues from the remaining 30 seronegative chickens were pooled and fed to 1 T. gondii-free cat. Feces of the cat were examined for oocysts; it did not shed oocysts. Toxoplasma gondii was isolated from 12 of 13 chickens with MAT titers of 1:40 or more. Toxoplasma gondii was isolated from pooled tissues of 1 of 2 chickens with titers of 1:10. Eight of these 13 isolates were virulent for mice. Genotyping of 13 of these isolates using the SAG2 locus indicated that 10 were type III, and 3 were type II. Phenotypically and genetically these isolates were different from T. gondii isolates from North America and Brazil. This is the first report of isolation of T. gondii from chickens from Venezuela.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 61 free-range chickens (Gallus domesticus) from provinces of Santiago del Estero and Entre Rios, Argentina was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and were found in 25 chickens; titers were 1:5 in 6 chickens, 1:10 in 1 chicken, 1:20 in 2 chickens, 1:40 in 1 chicken, 1:80 in 2 chickens, 1:60 in 4 chickens, 1:120 in 2 chickens, 1:640 in 3 chickens, and 1: 1,280 or higher in 4 chickens. Hearts, pectoral muscles, and brains of 22 seropositive (MAT 1:10 or higher) chickens were bioassayed individually in mice. Tissue from 39 chickens with titers of 1:5 or less were pooled and fed to 3 T. gondii-free cats. Feces of cats were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 17 of 22 chickens with MAT titers of 1:10 or higher. Genotyping of these 17 isolates using polymorphisms at the SAG2 locus indicated that 4 were Type I, 3 were Type II, and 10 were Type III. Toxoplasma gondii isolates (2 Type I and 1 Type III) from 3 chickens were virulent for mice and 1 Type I was not mouse virulent. Prevalence of T. gondii antibodies in chickens varied among regions, being 3 times greater in the humid Pampeana region (61.2%) than in the semiarid plain of Santiago del Estero (20%).

Analysis of the Schistosoma mansoni peptidome for immunomodulatory molecules by solvent extraction and reverse-phase HPLC revealed a 27-amino-acid residue peptide from an extract of cercariae. Using matrix-assisted, laser desorption– ionization, time-of-flight mass spectrometry, the peptide yielded a protonated molecular ion [M H] of m/z 2789. The unequivocal sequence was deduced by automated Edman degradation as: DLWNSIKDMAAAAGRAALNAVTGMVNQ. The peptide exhibited an 80.76% identity with dermaseptin 3.1 from the leaf frog Agalychnis annae, and was therefore named Schistosoma mansoni dermaseptin-like peptide (SmDLP). Immunocytochemical staining using a primary antidermaseptin B2 antibody located SmDLP in acetabular glands of cercariae, in and around schistosomula, and in adult worms and their eggs. Dot-blotting confirmed its presence in extracts (cercariae and worms) and excretion/secretion (E/S) products (transforming cercariae and eggs). This was corroborated by use of a MALDI-ToF spectra database of E/S products from cercariae. Functional characterization of the peptide indicated that SmDLP had typical amphipathic antimicrobial peptide properties, i.e., the ability to lyse human erythrocytes causing a decrease in the levels of nitric oxide produced by monocytic cells. This last function strongly suggests that SmDLP plays a vital role in the parasite's immunoevasion strategy. The possibility that schistosomes acquired this gene from amphibians has been discussed by constructing a phylogenetic tree.

We report 2 polymerase chain reaction (PCR)-based methods for distinguishing morphologically similar gregarine species based on amplification of variable regions of the internal transcribed spacer region of ribosomal DNA. The gregarines we investigated were Ascogregarina barretti (Vavra), A. culicis (Ross), and A. taiwanensis (Lien and Levine), parasites of the mosquitoes Ochlerotatus triseriatus (Say), Aedes aegypti (Linnaeus), and Ae. albopictus (Skuse), respectively. These 3 important vector mosquitoes often utilize the same container habitats, where larval development and infection by the parasite occurs, leaving ample opportunity for cross-species gregarine infection. Because previous studies have shown that the parasites A. culicis and A. taiwanensis variably affect fitness in both normal and abnormal mosquito hosts, distinguishing parasite infection and species is important. The task is complicated by the fact that these 2 parasite species are virtually identical in morphology, whereas A. barretti is morphologically distinct. Of the 2 PCR-based assays reported here, the first provides a rapid, sensitive, and straightforward means of general ascogregarine detection based on a single PCR amplification. The second method provides a means of differentiation between A. culicis and A. taiwanensis based on a species-specific PCR assay. Together, these assays allow whole mosquitoes to be tested for the presence of Ascogregarina species as well as identification of both A. culicis and A. taiwanensis singly or in dual infections.

Urbanization has deleterious effects on water quality and biota in stream systems. This project used caged bluegill (Lepomis macrochirus) to assess metazoan fish parasite communities in 2 urbanizing streams of the upper San Antonio River Basin, Bexar County, Texas. Field studies on Leon and Salado creeks were conducted during late summer in 1999 and 2000. Juvenile bluegill, obtained from a local aquaculturist, were held in cages for 10–22 days at middle and lower watershed sites to expose them to in-stream conditions and to allow parasite communities to establish. After removal from cages, fish were examined for metazoan parasites. In 2000, wild Lepomis spp. also were collected at study sites for parasite assessment. In both years, physical and chemical water properties were monitored at each site. Of the 120 fish examined for parasites, 96.7% were infected by at least 1 organism from among the 11 parasitic taxa observed. For caged fish, both diversity and equitability of parasite communities tended to be lower at the more eutrophic downstream sites; accordingly, parasite diversity and equitability were inversely correlated with nitrate concentrations. Ectoparasites were more prevalent in caged fish and endoparasites were more abundant in wild fish. An Ergasilus sp. copepod and a Posthodiplostomum sp. trematode dominated the ecto- and endoparasite faunas, respectively. This study suggests that assessment of watershed health can benefit from comparative cage studies of parasite community development involving sentinel fish species.

Philureter trigoniopsis parasitizes the ureters and urinary bladder of Galaxias maculatus in Patagonian Andean lakes. To investigate factors associated with variation in the prevalence and intensity of this monogenean, fish were sampled periodically over 2 yr in Lake Gutiérrez. Prevalence and mean intensity are higher in smaller fishes than in larger ones. A seasonal pattern was observed, with peak recruitment and peak mean intensity occurring in early spring (September), followed by lows in late summer (January–February). Galaxias maculatus length classes are spatially segregated due to seasonal migrations, so the annual infection cycle is characterized by higher prevalence and intensity from late winter to early summer in the smaller fish from the deep zone of the lake.

In trematodes, vitelline precursor proteins are required for eggshell formation. A cDNA clone of Clonorchis sinensis (CsVpB1) was selected from an EST pool, encoding a polypeptide of 245 amino acids. The CsVpB1 polypeptide demonstrated homology with vitelline precursor proteins from trematodes with high sequential identities. In a phylogenic tree, CsVpB1 clustered with trematode VpB proteins. The CsVpB1 polypeptide was found to be rich in tyrosine residues, including putative pre-dihydroxyphenyl alanine (DOPA) residues, involved in cross-linking of the precursor proteins. Mouse immune sera were raised against a recombinant CsVpB1 protein. In adult C. sinensis, CsVpB1 protein was exclusively localized in vitelline follicles. Based on these results, the CsVpB1 cDNA is believed to encode a VpB of C. sinensis.

A new haploporid trematode, Culuwiya cichlidorum n. sp., is described from the intestine of the black-belt cichlid Vieja maculicauda (=Cichlasoma maculicauda (Regan)) from the Atlantic coast of Nicaragua. It differs from congeneric species from freshwater and estuarine mugilid, cichlid, and gobiid fishes, previously placed to Saccocoelioides Szidat, 1954, and Carassotrema Park, 1938, in possessing the intestinal ceca reaching up to the posterior half of the testis and a bipartite, swollen external seminal vesicle. The new species also is characterized by a small, pyriform body, a well-developed prepharynx, a large pharynx, extensive vitelline follicles, confluent posterior to the testis that is situated near the posterior extremity, a uterus limited to the pretesticular and postacetabular region, and few, but relatively large, eggs (67–81 × 36–47 μm, i.e., about 12–14% of the body length) and miracidia without eye-spots. Culuwiya cichlidorum is also reported from Tomocichla tuba (Meek) in Nicaragua and species of Cichlasoma, Herichthys, Oreochromis, Parachromis, Petenia, Theraps, and Vieja from Mexico.

One species of digenean, Zeylanurotrema sphenomorphi n. sp., and 2 nematode species, Meteterakis crombiei n. sp. and Physalopteroides milnensis n. sp., from the intestine of Sphenomorphus jobiensis, are described and illustrated. Zeylanurotrema sphenomorphi n. sp. represents the second species, Meteterakis crombiei n. sp. the 18th species, and Physalopteroides milnensis n. sp. the 19th species assigned to each respective genus. In addition, 1 species of cestode, Oochoristica javaensis; 7 nematode species, Abbreviata oligopapillata, Aplectana macintoshii, Aplectana zweifeli, Cosmocerca zugi, Maxvachonia adamsoni, Oswaldocruzia bakeri, Parapharyngodon maplestonei; 1 species of acanthocephalan, Acanthocephalus bufonis; and 1 species of pentastome, Kiricephalus sp., were found. With the exception of Abbreviata oligopapillata, Sphenomorphus jobiensis represents a new host record for each species of endoparasite.

Allopharynx daileyi n. sp. (Digenea: Plagiorchiidae) from the small intestine of the tropidurid lizard Uranoscodon superciliosus from Amazonian Brazil is described and illustrated. Of the 11 currently recognized species of Allopharynx, we consider Allopharynx megorchis Simha, 1961 a synonym of Allopharynx mehrai (Gogate, 1935) Price 1938 and Allopharynx puertoricensis Acholonu, 1976 a synonym of Allopharynx riopedrensis Garcia-Diaz; thus, A. daileyi becomes the 10th species assigned to the genus. The trematode species Mesocoelium monas and 2 species of Nematoda, Africana chabaudi and Cosmocerca vrcibradici, were also present.

Fecal samples from 20 Caucasian tree squirrels, Sciurus anomalus (Gmelin, 1778) Güldenstaedt, 1785, were collected in Turkey during February and March, 2004, and all 20 were infected with a single species of coccidia, Eimeria serbica. Sporulated oocysts are ellipsoidal, 27.5 × 17.5 (21–34 × 15–20) with a length:width ratio (L/W) of 1.6 (1.4–1.8); they lack a micropyle and oocyst residuum, but 0–2 polar granules may be present. Sporocysts are ellipsoidal, 11.9 × 6.9 (10.5–14 × 6– 7.5) with a L/W of 1.7 (1.5–2.1); a Stieda body and sporocyst residuum are present.

The Palaeacanthocephala is traditionally represented by 2 orders, Echinorhynchida and Polymorphida, with 10 and 3 families, respectively. To test the monophyly of the class, these 2 orders, and certain families, phylogenies were inferred using nuclear small-subunit (SSU) and large-subunit (LSU) ribosomal DNA sequences obtained for 29 species representing 10 families, 2 other classes of acanthocephalans, and 3 rotifer outgroups. Phylogenetic relationships were inferred by analyzing combined SSU and LSU sequences using maximum parsimony (MP) and maximum likelihood (ML) methods. Parsimony and ML trees inferred from combined analysis of these rDNA data strongly supported monophyly of Palaeacanthocephala and provided good resolution among species. Neither Polymorphida nor Echinorhynchida was monophyletic. Gorgorhynchoides bullocki (Echinorhynchida) was nested within the 6 species representing Polymorphida, and this clade was nested within species representing Echinorhynchida. Three of 4 palaeacanthocephalan families that could be evaluated were not monophyletic, and this finding was strongly supported. These results indicate that the family level classification of palaeacanthocephalans, which is mainly based on combinations of shared characters (not shared derived characters), needs to be reevaluated with respect to comprehensively sampled phylogenetic hypotheses.

Caballerolecythus ibunami n. gen., n. sp. is described from the intestine of 2 species of rodents (Liomys irroratus [Gray, 1868] and Peromyscus difficilis [Allen, 1891]) from Piñonal, El Carmen Tequexquitla, Tlaxcala state, Mexico. These specimens represent a new genus and a new species of Dicrocoeliinae by possession of an extremely short ceca (ending at equatorial level of ovary); vitelline follicles that begin posterior to these structures, without overlapping; and a body that is long and slender (length/width ratio 1:17–21). To the best of our knowledge, this combination of characters has not been reported in other genera of this subfamily (Athesmia Looss, 1899; Unilaterilecithum Oshmarin in Skrjabin and Evranova, 1952; and Pseudathesmia Travassos, 1942).

Individual specimens of Anisakis, Pseudoterranova, and Contracaecum collected from marine mammals inhabiting northern Pacific waters were used for comparative diagnostic and molecular phylogenetic analyses. Forty-eight new sequences were obtained for this study of 14 Anisakis taxa, 8 Pseudoterranova taxa, 4 Contracaecum taxa, and 4 outgroup species. Partial 28S (LSU) and complete internal transcribed spacer (ITS-1, 5.8S, ITS-2) ribosomal DNA was amplified by the polymerase chain reaction and sequenced. Sequences of ITS indicated that Pseudoterranova specimens from Zalophus californianus (California sea lion), Mirounga angustirostris (northern elephant seal), Phoca vitulina (harbor seal), Enhydra lutris (sea otter), and Eumetopias jubatus (Steller's sea lion) exactly matched P. decipiens s. str., extending the host and geographic range of this species. Anisakis from northern Pacific marine mammals were most closely related to members of the A. simplex species complex. Comparison of Anisakis ITS sequences diagnosed the presence of A. simplex C in 2 M. angustirostris hosts, which is a new host record. Anisakis specimens from Phocoena phocoena (harbor porpoise), Lissodelphis borealis (Pacific rightwhale porpoise), and E. jubatus included 3 ITS sequences that did not match any known species. Contracaecum adults obtained from Z. californianus were most closely related to C. ogmorhini s.l. and C. rudolphii, but ITS sequences of these Contracaecum specimens did not match C. ogmorhini s. str. or C. margolisi. These novel Anisakis and Contracaecum ITS sequences may represent previously uncharacterized species. Phylogenetic analysis of LSU sequences revealed strong support for the monophyly of Anisakinae, Contracaecum plus Phocascaris, Pseudoterranova, and Anisakis. Phylogenetic trees inferred from ITS sequences yielded robustly supported relationships for Pseudoterranova and Anisakis species that are primarily consistent with previously published phenograms based on multilocus electrophoretic data.

One-hundred and fifteen anisakid larvae from 3 different fish hosts, Aphanopus carbo, Scomber japonicus, and Trachurus picturatus, caught in Madeiran waters, were identified by PCR-RFLP. Three distinct species were identified in A. carbo, namely Anisakis simplex sensu stricto, Anisakis pegreffii, and Anisakis ziphidarum; 5 in S. japonicus, i.e., A. simplex s.s., A. pegreffii, Anisakis physeteris, Anisakis typica, and A. ziphidarum; and 3 in T. picturatus, i.e., A. simplex s.s., A. pegreffii, and A. typica. Anisakis simplex s.s. was the most frequent species in both A. carbo and S. japonicus (54% and 23.5%, respectively). Anisakis pegreffii and A. physeteris occurred with a frequency of 20.6% in S. japonicus, whereas in T. picturatus the most frequent species was A. typica (41.9%), followed by A. simplex s.s. (32.3%). Furthermore, A. carbo and S. japonicus were infected by an apparently undescribed taxon, provisionally named Anisakis sp. A. Based on estimations of the genetic distance, this new taxon seems to be more similar to A. ziphidarum (0.0335) than to other species of the genus.

A hypothesis-based framework was used to test if 3 genetic strains of Mesocestoides (clades A, B, and C) are distinct evolutionary lineages, thereby supporting their delimitation as species. For comparative purposes, 3 established cestode species, Taenia pisiformis, Taenia serialis, and Taenia crassiceps were assessed using the same methods. Sequence data from mitochondrial rDNA (12S) and the second internal transcribed spacer of nuclear rDNA (ITS-2) revealed derived (autapomorphic) characters for lineages representing clade A (n = 6 autapomorphies), clade B (n = 4), and clade C (n = 9) as well as T. pisiformis (n = 15) and T. serialis (n = 12). Furthermore, multivariate analysis of morphological data revealed significant differences among the 3 genetic strains of Mesocestoides and between T. pisiformis and T. serialis. The level of phenotypic variation within evolutionary lineages of Mesocestoides and Taenia spp. tapeworms was similar. Results from this study support recognizing Mesocestoides clades A, B, and C as separate species, and provide evidence that clade B and Mesocestoides vogae are conspecific.

Ligophorus mugilinus was originally described on the flathead mullet Mugil cephalus from the Gulf of Mexico. The species was later recorded in the Mediterranean Sea on the flathead mullet and in the Caribbean Sea on the white mullet Mugil curema. After examination of the type material and additional specimens of Ligophorus from the northwest Atlantic coast, the Caribbean Sea, and the Mediterranean and Black Sea, we observed morphological differences concerning haptoral and male copulatory structures between L. mugilinus from the northwest Atlantic and the forms from the Mediterranean and Black Seas. Accordingly, the latter specimens are described as a new species, Ligophorus mediterraneus n. sp. A redescription of L. mugilinus, including new morphometric data, is also given. The morphometric analysis of the Caribbean specimens suggested that their ascription to L. mugilinus is uncertain, but additional material is needed to clarify their specific identity. The results of this study, together with previous zoogeographical evidence, indicate that flathead mullets from different seas harbor different species complexes of Ligophorus. Given the coastal habits of the host, it is suggested that open oceans could act as geographical barriers favoring speciation within disjointed flathead mullet populations.

One hundred and seventy snakes were collected in Guatemala and examined for coccidia. Of these, 8 individuals representing 6 host species were positive for Caryospora spp., 6 of which are described as new species. Sporulated oocysts of Caryospora bothriechis n. sp. from Bothriechis aurifer are spheroidal to subspheroidal, 12.7 × 12.5 (12–14 × 12–13) μm, with a length/width (L/W) ratio of 1.0; they lack a micropyle (M) or oocyst residuum (OR), but 1 large polar granule (PG) is usually present. Sporocysts are ovoidal, 9.0–7.5 (8–10 × 7–8) μm, and have a L/W ratio of 1.2, and a Stieda body (SB) and sporocyst residuum (SR). Oocysts of Caryospora coniophanis n. sp. from Coniophanes imperialis are spheroidal to subspheroidal, 18.8 × 18.1 (17–20.5 × 16–20) μm, with a L/W ratio of 1.0; they lack a M and OR, but 1 large PG is usually present. Sporocysts are ovoidal, 13.2 × 9.4 (12–15 × 8–10) μm with a L/W ratio of 1.4, and a SB, substieda body (SSB), and SR. Oocysts of Caryospora conophae n. sp. from Conophis lineatus are spheroid to subspheroidal, 20.4 × 19.5 (17–26 × 17–25) μm, with a L/W ratio of 1.0; they lack a M and OR, but 1 large PG is usually present. Sporocysts are ovoidal, 13.1 × 9.8 (11–15 × 8–11) μm with a L/W ratio of 1.3 and a SB, SSB, and SR. Oocysts of Caryospora guatemalensis n. sp. from Lampropeltis triangulum are spheroidal to subspheroidal, 23.9 × 23.2 (20–27 × 20–26) μm, with a L/W ratio of 1.0; they lack a M and OR, but 1 large PG is usually present. Sporocysts are ovoidal, 14.4 × 10.6 (13–18 × 9–13) μm, with a L/W ratio of 1.4 and a SB, SSB, and SR. Oocysts of Caryospora mayorum n. sp. from Conophis lineatus are spheroidal to subspheroidal, 25.6 × 24.4 (24–27 × 24–25) μm, with a L/W ratio of 1.0; they lack a M and OR, but 1 large PG is usually present. Sporocysts are ovoidal, 16.3 × 11.9 (16–18 × 11–13) μm, with a L/W ratio of 1.4 and a SB, SSB, and SR. Oocysts of Caryospora zacapensis n. sp. from Masticophis mentovarius are spheroidal to subspheroidal, 22.5 × 21.8 (19–25 × 18–25) μm, with a L/W ratio of 1.0; they lack a M and OR, but 1 large PG is usually present. Sporocysts are ovoidal, 14.6 × 11.4 (11–16 × 10–13) μm, with a L/W ratio of 1.3 and a SB, SSB, and SR.

The following Acanthocephala, all Echinorhynchidae, were found on examination of 229 amphibians and 191 reptiles collected in the Eastern Arc Range, Tanzania: Acanthocephalus bufonis from the frogs Arthroleptis stenodactylus and Arthroleptides martiensseni, the toad Nectophnyroides viviparous, and the chameleon Rhampholeon brevicaudatus; Pseudoacanthocephalus betsileo from the frogs Probreviceps macrodactylus and Ptychadero anchietae; and Pseudoacanthocephalus rhampholeontos n. sp. from the chameleons Rhampholeon sp. and Rhampholeon uluguruensis. Pseudoacanthocephalus rhampholeontos differs from the only other acanthocephalan occurring in chameleons, Pseudoacanthocephalus bigueti, in being a larger worm with fewer, smaller hooks in the proboscis armature. The status of the Acanthocephalus and Pseudoacanthocephalus is still problematic and requires further attention and study.

A new species of Petasiger inhabits Pelecanus occidentalis, from the Area de Conservación Guanacaste, Costa Rica. The new species most closely resembles Petasiger novemdecim Lutz 1929 and Petasiger caribbensis Nassi, 1980 by having 19 circumoral spines and vitelline follicles confluent between the ventral sucker and gonads. The new species differs from both of these species by having a cirrus sac that is anteroposteriorly elongate and that reaches posteriorly to the midventral sucker, an ovary and Mehlis' gland that overlap the anterior testis dorsally, a uterus that lies dorsal to the ventral sucker, and a dextromedial genital pore. In P. novemdecim and P. caribbensis the cirrus sac is round and does not extend posteriorly to the anterior margin of the ventral sucker, both the ovary and Mehlis' gland are anterior to the anterior testis, the uterus runs lateral to and not dorsal to the ventral sucker, and the genital pore opens medially and sinistromedially, respectively.

Congenital Trypanosoma cruzi infection is a highly pathogenic and underreported condition. Early recognition is essential for effective treatment. Umbilical chord blood from newborns (n = 302) to infected mothers was analyzed with microhematocrit, hemoculture, and PCR methods. Each subject was then followed serologically. In calibrated suspensions of T. cruzi in blood, the sensitivity of PCR was 27-fold higher than hemoculture. However, this advantage was not reflected during routine testing of samples from maternities, partly because of the uneven distribution of few parasites in small samples. Levels of detection of congenital infection were 2.9% (8/272) for microhematocrit, 6.3% (18/287) for hemoculture, 6.4% (15/235) for PCR, and 8.9% (27/302) for cumulated results. Evaluation against the standard of delayed serology indicates that the regular application of PCR, hemoculture, and microhematocrit to blood samples allows the rapid detection of about 90% of the congenitally infected newborns, in samples that can be obtained before the mother and child leave the maternity ward.

We have demonstrated that fluoroquinolones, a class of antibacterial agents that act through inhibition of type II DNA topoisomerases, exert selective action against intracellular amastigotes of Leishmania (Viannia) panamensis at concentrations that are achievable in vivo. Drug cytotoxicity assays employing the luciferase reporter gene revealed that intracellular amastigotes were 6.6- to 25.9-fold more sensitive than human macrophages (P < 0.05) to second-generation fluoroquinolones in vitro. The most selective agents (enoxacin and ciprofloxacin) exhibited 2 orders of magnitude greater potency against parasites (50% effective dose [ED₅₀] = 54.9–83.4 μM) than host cells (ED₅₀ = 1,425–1,740 μM). Linear regression analysis of ED₅₀ data confirmed a complete lack of correlation (r = 0.001) between the relative drug sensitivities of parasites and host cells. A potential relationship between the structures of fluoroquinolones and their relative leishmanicidal activities was observed. The key substituents of the basic pyridone β-carboxylic acid nucleus accounting for enhanced antiparasite potency and selectivity appear to be a nitrogen at position 8 of the bicyclic nucleus (enoxacin), a cyclopropyl substituent at the R₁ site (ciprofloxacin), and linkage of the R₁ and X₈ groups by a CH₃CHO bridge to form a tricyclic compound (ofloxacin). These findings support the potential of fluoroquinolones and derivatives as novel antileishmanials and encourage their clinical evaluation.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 100 free-range chickens (Gallus domesticus) from Sri Lanka was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 39 chickens with titers of 1:5 in 8, 1:10 in 8, 1:20 in 4, 1:40 in 5, 1:80 in 5, 1:160 in 5, 1:320 in 2, 1:640 or more in 2. Hearts and brains of 36 chickens with MAT titers of 1:5 or more were bioassayed in mice. Tissues of 3 chickens with doubtful titers of 1:5 were pooled and fed to a cat; the cat shed T. gondii oocysts in its feces. Tissues from 61 chickens with titers of less than 1:5 were pooled and fed to 2 T. gondii-free cats; the cats did not shed oocysts. Toxoplasma gondii was isolated from 11 of 36 seropositive chickens by bioassay in mice. All 12 T. gondii isolates were avirulent for mice. Genotyping of 12 isolates using the SAG2 locus indicated that 6 were type III, and 6 were type II. This is the first report of genetic characterization of T. gondii from any host in Sri Lanka.

Bile is a major growth factor for the proliferation of Giardia spp. trophozoites in the small intestine and, at high concentrations, stimulates encystment of trophozoites. This report demonstrates that surgical cholestasis to interrupt the flow of bile from liver to intestine or the use of bile-binding resins in the diet can both dramatically decrease the fecal excretion of Giardia muris cysts. Cholestasis produced a 3 log reduction in excretion of G. muris cysts within 24 hr of surgery and a 4 log reduction after 3 days. Sham controls showed no difference in cyst excretion from presurgical control values. Two isocaloric diets were studied: a control diet (N) of Purina mouse chow containing 5% celufil and an experimental diet (CR) containing 5% cholestyramine, a resin that binds bile. Compared with the N diet, the CR diet was associated with reductions in cyst excretion of 3 logs within 1 day. Despite lowered excretion of G. muris cysts in mice fed the cholestyramine diet, the trophozoite recovery from the duodenum was similar with both diets. Cyclic feeding of the CR diet and the N diet at 3-day intervals produced significant oscillations (changes of 3–4 logs) in fecal cyst shedding. The significant reductions in fecal excretion of cysts observed with agents that bind bile suggests that diets capable of binding bile might be a therapeutic means to minimize the fecal excretion of cysts and thereby may help to reduce the risk of spreading giardiasis through fecal–oral contamination.

Rates of community similarity decay were examined among parasite communities of the bluegill sunfish from isolated populations located throughout the midwestern and southeastern United States. Rates of decay were compared for assemblages composed of all species, autogenic species only, and allogenic species only. Rates of similarity decay were calculated by regressing a matrix of intercommunity distance against a matrix of intercommunity Sorenson similarity for each group of species. Significance was determined with the use of a Mantel test using 1,000 permutations. Significant rates of decay were found for all groups; however, allogenic similarity decayed according to an exponential function, whereas autogenic similarity decayed according to a linear function. Overall, the results suggest that autogenic– allogenic status is an important factor determining the rate at which similarity decays, but illustrate the need for more insight regarding the factors that affect parasite dispersal ability.

High-pressure processing (HPP) has been shown to be an effective means of eliminating bacteria and destructive enzymes from a variety of food products. HPP extends the shelf life of products while maintaining the sensory features of food and beverages. In this study, we examined the effects of HPP on the infectivity of Encephalitozoon cuniculi spores in vitro. Spores were exposed to between 140 and 550 MPa for 1 min in a commercial HPP unit. Following treatment, the spores were loaded onto cell culture flasks or were kept for examination by transmission electron microscopy. No effect was observed on the infectivity of spores treated with 140 MPa. Spores treated with between 200 and 275 MPa showed reduction in infectivity. Following treatment of 345 MPa or more, spores were unable to infect host cells. No morphologic changes were observed in pressure-treated spores with transmission electron microscopy.

Groups of mice were infected with tachyzoites of the RH strain of Toxoplasma gondii, treated with the opioid analgesic buprenorphine, sodium sulfadiazine, a combination of buprenorphine and sodium sulfadiazine, or nothing in the drinking water, on days −1 to 12 postinfection. Mice in the T. gondii-infected buprenorphine-treated group did not live significantly longer (P > 0.05) than mice given T. gondii and not treated with buprenorphine. Clinical observations of mice indicated that buprenorphine treatment reduced distress and pain in mice with acute toxoplasmosis. Mice treated with sodium sulfadiazine alone or sodium sulfadiazine combined with buprenorphine survived the 28-day study. Mice treated with buprenorphine and not infected with T. gondii also survived the 28 days. This study demonstrates that buprenorphine does not adversely interfere with acute T. gondii infection and indicates that buprenorphine can be given to mice to alleviate pain and distress associated with a T. gondii infection, and not adversely influence the results of toxoplasmosis studies. Analgesic (buprenorphine) treatment should now be the standard of care for mice in acute toxoplasmosis studies.

Toxoplasma gondii is an apicomplexan parasite of mammals and birds. Herbivores acquire postnatal infection by ingesting oocysts from contaminated food or water. Toxoplasma gondii infection is common in white-tailed deer, Odocoileus virginianus, but little is known about the prevalence of infection in mule deer, O. hemionus. We examined sera from 89 mule deer from Nebraska for agglutinating antibodies to T. gondii using the modified direct agglutination test (MAT) with formalin-fixed tachyzoites as antigen. Thirty-one (35%) of the samples were positive at dilutions of ≥1:25. Samples were examined from 29 fetuses from these mule deer and none were positive in the MAT. Sera from 14 white-tailed deer from Nebraska were also examined and 6 (43%) were positive for T. gondii. Samples were examined from 5 fetuses from these white-tailed deer and none was positive in the MAT. Our results in both deer species from Nebraska are similar to studies conducted in white-tailed deer from other regions of the United States. Our findings indicate that mule deer are frequently infected with T. gondii and that mule-deer meat may be a source of human infection.

The breast cancer susceptibility protein BRCA2 is implicated in the DNA double-strand break (DSB) repair pathway through its association with Rad51. It is almost a ubiquitous eukaryotic protein; homologs of the BRCA2 gene (BRH2) have been identified in many mammals, as well as nonmammals. As a part of our quest to understand the DNA damage, repair, and recombination process in the parasitic protozoan, Leishmania sp., we have cloned and characterized a BRCA2 homolog from Leishmania sp. (LBRH2). LBRH2 is coded by a single-copy gene (ORF = 3,498 bp) located at the 700-kb chromosome 16. The transcripts in both the promastigotes and the amastigotes are ∼3.9 kilonucleotides (knt) in size, corresponding to a protein with a calculated molecular mass of 128 kDa. The primary transcript of the gene is alternatively trans-spliced to produce 3 distinct mRNAs with altered folded structures at their 5′ ends. This study will contribute toward the understanding of a potential RAD51-mediated DNA recombination/repair pathway in Leishmania sp.

Recent reports of toxoplasmosis in marine mammals raise concern that cold-blooded marine animals are a potential source of Toxoplasma gondii infection. To examine the transmissibility of T. gondii to fish, we observed the development of T. gondii tachyzoites inoculated into oviduct epithelial cells of goldfish (Carassius auratus) microscopically in vitro. Further, the survival period of tachyzoites inoculated into goldfish muscle was bioassayed in mice and through PCR analysis. In cell cultures at 37 C, both RH and Beverley strains of T. gondii tachyzoites had penetrated into cells at 6 hr post inoculation, and were multiplying. In cell cultures at 33 C, many tachyzoites of both strains attached to the host cells, but no intracellular tachyzoites were observed at 24 hr post inoculation. In the T. gondii inoculated goldfish kept at 33 C, tachyzoite DNA was detected in the inoculated region on day 3, but not on day 7. When inoculated goldfish were kept at 37 C, live tachyzoites were seen at the inoculation site on day 3, but not on day 7. These results suggest that T. gondii does not persist in fish.

Equine protozoal myeloencephalitis (EPM), caused by Sarcocystis neurona infection in the central nervous system (CNS), affects up to 1% of all horses during their lifetimes. Neither the protective immune response nor the immunopathology associated with the disease is well understood. To begin to clarify the pathogenesis of the disease, immunohistochemical staining for B and T lymphocytes was performed on spinal cord sections obtained from 17 horses, all of which were all positive for S. neurona based on immunohistochemical staining. Fifteen of the 17 horses included in the study were killed due to neurologic dysfunction; 2 of the 17 horses were killed because of fractures. All 17 horses had histologic changes consistent with S. neurona infection. A significantly greater number of T cells were seen in sections from S. neurona-infected versus control horses. Because this was a small descriptive study, we were not able to determine the mechanisms of enhanced T-cell recruitment in the sections from the S. neurona-infected horses.

Commercial crab populations off the Kamchatka coasts are infested to a considerable degree by the rhizocephalan parasite Briarosaccus callosus: of 769 Lithodes aequispina males examined, 43 (5.7%) were parasitized. Infestations result in the feminization of the crabs, a significant decrease in the cheliped length, and a significant decrease in the carapace length and width. We suggest that commercial selection of healthy males, and the returning of unsuitable crabs, including infested ones, back into the sea, results in an increase of the proportion of infested crabs in the population, their elimination from reproduction, and, eventually, the gradual degradation of a whole population. To minimize as far as possible the negative effects of commercial crab harvesting, all infested crab specimens caught must be destroyed, either aboard or elsewhere, instead of throwing them back into the sea.

The coding sequence for hexokinase enzyme was cloned from Leishmania major. The sequence was found to encode an enzyme with a molecular mass of 51.74 kDa. Amino acid sequence showed maximum homology with known trypanosome and plant hexokinases. It has a calculated isoelectric point of 8.46 and contains an N-terminal peroxisome-targeting signal, the characteristics frequently associated with glycosomal proteins. The sequence indicated the presence of conserved amino acid residues and motifs that are present in plant and mammalian hexokinases; these are apparently involved in the binding of different substrates. The L. major genome was found to have 2 copies of hexokinase coding sequences in tandem with an intergenic spacer of 2.58 kb. Both the genes in the hexokinase locus were transcribed as individual transcripts in a monocistronic form, having the same size as seen by Northern blot analysis. The hexokinase gene was transcribed in large amounts in the promastigote stage, whereas there is only weak expression in the amastigote stage as determined by RT-PCR analysis. Sequencing of hexokinase loci from different Leishmania species (e.g., L. donovani, L. infantum, L. tropica, and L. mexicana) revealed that the hexokinase locus is highly conserved at the DNA and protein levels among species of Leishmania compared with trypanosomes.

Plasmodium-specific polymerase chain reaction (PCR) primers allowed detection of infections with very low-level parasitemia for 3 species of malaria parasites infecting Anolis lizards at 2 Caribbean sites, Puerto Rico and Saba, Netherlands Antilles. A verification study, using a single-tube nested PCR to eliminate contamination, showed that infections as low as 1 parasite per millions of erythrocytes could be detected by amplifying a 673 bp fragment of the cytochrome b gene. Very low-level parasitemia infections, subpatent under the microscope, were common in A. sabanus on Saba sites, with no significant seasonal difference (31% of infections appearing uninfected by microscopic examination in summer were found infected by PCR, 38% in winter). At the Puerto Rico site, the subpatent infections were also common in A. gundlachi, but were more prevalent in winter (53%) than in summer (17%). A similar high frequency of subpatent infections is known from studies on human and bird malaria, but a previous PCR-based study on a temperate lizard malaria system found few such low-level infections. Differences in the prevalence of subpatent infections by site and season suggest transmission biology may select for distinct life history strategies by the parasite.