We produced the Taenia solium triosephosphate isomerase (TPI) in Escherichia coli and compared its biochemical and immunological properties with those of the commercial TPI from Sus scrofa. Taenia solium TPI is a homodimer composed of two 27-kDa monomers, with a specific activity of 5,683 U/mg and a K_m value of 0.758, and S. scrofa TPI is also dimeric with similar monomeric molecular weight, specific activity of 4,227 U/mg, and a K_m value of 0.51. The catalytic parameters for the isomerization of glyceraldehyde 3-phosphate, affinity between TPI monomers, and kinetic thermal denaturation and inactivation were similar for both enzymes. Anti–T. solium TPI antibodies cross-react weakly with Schistosoma mansoni TPI but do not cross-react with S. scrofa, human, or protozoan TPIs. These antibodies inhibited T. solium TPI activity but did not affect S. scrofa enzymatic activity. Immunizations with 1 μg of the T. solium TPI reduced 52% of cysticerci in a mouse–Taenia crassiceps model 1 mo after challenge. Our findings show that T. solium and S. scrofa TPIs possess similar biochemical and enzymatic properties but do not share immunological properties because anti–T. solium TPI antibodies did not recognize S. scrofa TPI. Inhibition of enzyme activity by anti-TPI antibodies suggests that they can be used as inhibitors of the enzyme.

Circulation of 24 macroparasite species among 12 species of fish was evaluated within samples of hosts collected from 9 lakes on an isolated plateau in northern Alberta, Canada. Twenty-seven parasite taxa (24 species plus the larval stages of Triaenophorus crassus, T. stizostedionis, and Raphidascaris acus) had the potential to be circulated among hosts. Sixteen parasite taxa were recovered from a single host species within a lake. Of the 11 remaining nonspecialist taxa, 4 were larval stages that matured in fish or birds and 7 were adults. Eight of the 11 cases of circulation among hosts involved lake whitefish, and this host was involved in the transmission of 5 species to piscivorous fishes. Despite evidence for the circulation of 7 taxa among the 4 species of sympatric Salmonidae, 60–99% of all worms were recovered from just 1 species of host. These results indicated that approximately 60% of the parasite taxa that infected fish in these lakes were absolute host specialists. The remaining 40% of parasite taxa had restricted host ranges, with most examples of parasite circulation limited to the 2 species of sympatric coregonid.

Over the past decade, eastern Europe has experienced a resurgence of trichinellosis. A recent outbreak in Serbia, Yugoslavia, from December 2001 to January 2002, involving 309 people, revealed many of the causes for this reemergence. Epidemiological investigations indicate that the immediate cause of the recent outbreak was the consumption of smoked sausages produced by a small slaughterhouse or meat processor. However, failure of in-house meat inspection procedures and quality assurance as well as oversight by official veterinary control were also responsible. Further analysis of this breakdown in the food safety net revealed additional general factors that have yielded a seriously deficient veterinary control system, and these are factors that are relevant to the problems experienced throughout eastern Europe and other regions. The recent civil war that led to the breakup of the former Federation of Yugoslavia resulted in severe economic and demographic changes, including high inflation and external economic sanctions. This led to (1) the loss of large numbers of experienced veterinary control officers and their replacement with inexperienced personnel, (2) a change in the swine industry with reduction in the number of large establishments with in-house inspection and replacement with more than 1,000 small abattoirs, too small to afford full-time in-house inspection, and (3) an increase in smallholder pig farming with reduced government oversight to ensure high standards in pig-rearing practices (infection risk management). The consequences of these events have been a 300% increase in Serbian pig infection and a concomittant large increase in human outbreaks. Before 1990, swine trichinellosis in Serbia was confined to 4 small districts, but today about one third of the Republic is considered endemic for trichinellosis. The reemergence of trichinellosis in Serbia illustrates the ability of this zoonosis to “leak” through a poorly maintained food safety barrier and the vulnerability of effective veterinary control to national and international events.

Empirical studies of helminth parasites reveal that the distribution of parasite burdens in their host populations is highly aggregated. This aggregation is fundamental to the ecology and epidemiology of helminth parasites. Results from a stochastic model predict that aggregation of helminth parasites is inversely related to the intensity of host immunity. Aggregation also decreases with antigenic heterogeneity and increases with heterogeneity in transmissibility among parasite strains. It is also found that the degree of aggregation is greater when immunity affects parasite fecundity than when immunity acts on host susceptibility. Potential relevance of this result for assessing the influence of vaccines that target either host susceptibility or parasite fecundity on the level of aggregation and consequent effects on drug resistance and disease prevalence are discussed.

The degree of parasitism by the parasitic copepod Sphyrion laevigatum on kingclip Genypterus blacodes in the Falkland Islands was investigated. In a sample of 719 kingclip, ranging in size from 35 to 145 cm total length, the prevalence of infection was 49%. The number of parasites per fish ranged from 0 to 24, with only 2% of the sample having more than 5 parasites. There was a significant increase in mean abundance of parasites with increasing fish age and length (P < 0.01 and P < 0.05, respectively). In addition, there was a significant difference in the prevalence in 1 of 3 geographical areas for the largest size class.

Studies of avian host–parasite interactions rarely include consequences of relationships among hosts for either the host or parasite species. In this study, we examine the ectoparasitic burden of adult and nestling European bee-eaters (Merops apiaster) and rock sparrows (Petronia petronia) in a mixed colony. We found that (1) each bird species had its own species of lice; (2) hematophagous mites parasitized both adults and nestlings of both bird species; (3) Carnus hemapterus, a common parasite of nestling bee-eaters, also infested rock sparrow nestlings, a species not previously described as a host for this dipteran; and (4) whereas C. hemapterus did not show high host specificity within the colony, the emergence of adult flies was synchronized with the start of hatching in bee-eater nests. We suggest that coexistence of these 2 bird species results in parasite exchange, bee-eaters obtaining mites from sparrows and sparrows becoming infested by C. hemapterus. Differences in the detrimental effects of parasite transfer for each host species may result in a process of apparent competition mediated by shared parasites. Interspecific parasite exchange is an important aspect of host–parasite relationships in mixed colonies, which requires further attention.

Laboratory-reared cystacanths of Leptorhynchoides thecatus (Acanthocephala: Rhadinorhynchidae) were used to study the effect of cystacanth size on adult success and the factors that influence cystacanth size within the intermediate host. To assess how host size and intensity of infection influence cystacanth size, infected amphipods (Hyalella azteca) were measured, and sex, length, and width of cystacanths were determined. After a subset of cystacanths was measured, small- and large-size classes of cystacanths were designated. To determine how cystacanth size relates to adult size, green sunfish (Lepomis cyanellus) were fed 10 large or small cystacanths. Fish were dissected 6 wk after infection, and worms were removed. After adult worms were permanently mounted on slides, their length and width were measured. Intensity of infection and amphipod size significantly influenced cystacanth size in that large amphipods harbored larger cystacanths than did small amphipods and heavy infections produced smaller cystacanths than did light infections. Adult worms from the small and large cystacanth–size classes showed no significant difference in size; however, large cystacanths had a significantly higher establishment and survival than did small cystacanths: 40% of large worms and 14% of small worms were recovered. The results of this study indicate that host size and host sharing influence cystacanth size and that cystacanth size is an important factor in determining adult success.

Malaria infection in the mosquito vector can be modulated by the vertebrate host, mosquito factors, and interactions between different parasite populations. Modulation of parasite development can be assessed through the study of gene expression. The present report describes a specific, sensitive, and nonradioactive method that permits assessment of parasite load and quantification of circumsporozoite protein gene expression during the sporogonic stages of Plasmodium yoelii yoelii and P. y. nigeriensis. A decrease in parasite load was observed when comparing DNA of oocysts on day 7 postinfection with that of oocysts and sporozoites on day 19. On day 7, parasites (oocysts) showed a marked increase of circumsporozoite protein expression when compared with that (sporozoites and oocysts) on day 19. The method developed in this work can be a valuable tool to understand parasite interaction mechanisms that are involved in mosquito malaria infections.

Expressed sequence tag (EST) sequencing and analysis is a primary research tool to identify and characterize the Schistosoma mansoni transcriptome. As part of our gene discovery effort, a total of 5,793 ESTs have been generated from clones selected randomly from complementary DNA (cDNA) libraries constructed from male and female adult worms. Assembly analysis of all the 16,813 public S. mansoni ESTs has identified 1,920 distinct tentative consensus sequences (TCs) and 5,571 nonoverlapping ESTs (singletons). Of these, 376 TCs (20%) and 1,449 singletons (26%) are unique to the SUNY/TIGR sequencing effort. Tentative consensus sequences and singletons were distributed into various categories of biological roles associated with cell structure, metabolism, protein fate, signal transduction, transcription, protein synthesis, transporters, and cell growth. The TCs and singletons represent transcripts that can be used as a resource for functional annotation of genomic sequence data, comparative sequence analysis, and cDNA clone selection for microarray projects. The utility of EST analysis is demonstrated by identifying new protease genes, which may be involved in hemoglobin degradation.

To investigate the role of intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) in controlling Cryptosporidium parvum infection, changes in their phenotypes and functional properties were studied after induction of primary and challenge infections in immunocompetent mice. As shown by oocyst-shedding patterns, the challenge-infected group recovered more rapidly from infection than did the primary-infected group. In LPL, proportions of activated CD4 , CD25 , IgG1 , IgA , and CD4 /IFN-γ cells increased significantly in the primary-infected group compared with controls. In the challenge-infected group, proportions of these cells decreased. The antigen-specific IgA level was elevated significantly among LPL of both primary- and challenge-infected groups. Among IEL, proportions of activated CD8 , T cell receptor (TCR) γδ , and CD8 /TCR γδ cells increased significantly in the challenge-infected group compared with controls and the primary-infected group; their cytotoxicity also was enhanced. However, the proportion of IEL expressing Th1 cytokines was lower than that among LPL in both infected groups. The results suggest that LPL play a more important role in protection against a primary infection with C. parvum, through the production of IFN-γ and IgA, whereas IEL are more involved in protection against a challenge infection, through enhanced cytotoxicity.

In order to develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of Cryptosporidium parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green fluorescent protein were integrated into the gG gene of BHV-1. Despite a low frequency of homologous recombination, cloning of the recombinants was easy because of the specific fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis. Rabbits immunized with the recombinant produced IgG against the p23 protein. It was also demonstrated that the sera of immunized rabbits reduced infection of C. parvum sporozoites in HCT-8 cells. The serum of an immunized rabbit reduced infection compared with the normal rabbit serum control. These results indicate that the recombinant BHV-1 induces neutralizing antibodies in rabbits.

Intracellular endosymbionts, Wolbachia spp., have been reported in many different orders of insects and in nematodes but not previously in fleas. This is the first conclusive report of Wolbachia spp. within members of the Siphonaptera. Using nested polymerase chain reaction (PCR) targeting of the 16S ribosomal RNA gene, we screened for Wolbachia spp. in fleas collected from 3 counties in Georgia and 1 in New York. The prevalence of Wolbachia spp. detected varied among the 6 different species screened: 21% in the cat flea Ctenocephalides felis (n = 604), 7% in the dog flea C. canis (n = 28), 25% in Polygenus gwyni (n = 8), 80% in Orchopeas howardi (n = 15), 94% in Pulex simulans (n = 255), and 24% in the sticktight flea Echidnophaga gallinacea (n = 101). Wolbachia spp. infection in fleas was confirmed by sequencing positive PCR products, comparing sequenced 16S ribosomal DNA (rDNA) with Wolbachia spp. sequences in GenBank using BLAST search, and subjecting sequence data to phylogenetic analysis. For further confirmation, 16S rDNA–positive samples were reamplified using the wsp gene.

Under laboratory conditions, 2 modes of transmission of Eustrongylides ignotus (Nematoda: Dioctophymatoidea) to fish were identified. Eastern mosquitofish (Gambusia holbrooki) became infected after ingestion of either eggs of E. ignotus containing first-stage larvae or aquatic oligochaetes (Limnodrilus hoffmeisteri) containing third-stage larvae of E. ignotus. After removal from the uterus of gravid E. ignotus females and incubation for 17–28 days, depending on temperature, it was found that parasite eggs contained first-stage larvae that were infective to fish and oligochaetes. Larvae developed to the third stage in oligochaetes and were infective to fish 35–77 days postinfection (PI) and when fed to fish, developed to the fourth stage between 127 and 184 days PI. Eggs containing first-stage larvae fed directly to fish developed to the fourth stage between 84 and 105 days PI. The amount of time for development from the undifferentiated egg to the fourth-stage larva was 78–156 days shorter when fish ingested eggs containing first-stage larvae than when fish ingested oligochaetes containing third-stage larvae. Three species of large piscivorous fish, including black crappie (Pomoxis nigromaculatus), largemouth bass (Micropterus salmoides), and warmouth (Lepomis gulosus), were fed mosquitofish containing fourth-stage larvae. At necropsy, live E. ignotus larvae were recovered from all 3 species. Several fish had multiple infections after ingesting >1 larva, indicating that bioaccumulation of the parasite in the food chain may occur.

During a long-term survey of the parasites of mammals from all over Bolivia, 44 individuals of Oxymycterus inca and 6 of Oxymycterus paramensis were collected and examined for parasites from the foothills and Eastern Cordillera (Los Yungas) of the Andes of Bolivia. Three species of nematode were found including, from the cecum, a previously unknown genus and species of pinworm, Caroloxyuris boliviensis n. gen, n. sp., aspidoderids representing Nematomystes rodentophilus, and from the stomach, Protospirura numidica criceticola. These helminths occurred in prevalences of 25, 9, and 14%, respectively, in O. paramensis. Caroloxyuris boliviensis resembles species included in the genus Syphacia in the structure of the cephalic mask and copulatory organs, and the extension of lateral alae. However, males of this species possess only 2 mamelons on the ventral surface of the body. Caroloxyuris can be recognized as being distinct from both Helminthoxys and Rauschtineria in the ornamentation of the mamelons and the shape of cephalic mask. A redescription of N. rodentophilus is provided, and Nematomystes scapteromi is transferred from Ansiruptodera to Nematomystes.

One hundred and seventeen ponies were surveyed for the seasonal prevalence of strongyloid parasites, particularly cyathostomes, and for host- and age-related differences in these infections. For 56 ponies, all stages of the cyathostome life cycle, both mucosal and luminal, were enumerated. Total numbers of cyathostomes and percentage of developing larvae (DL) encysted in the mucosa remained constant in all 4 seasons of the year, whereas a significant increase in the percentage of adults in the cyathostome population occurred in fall. In yearling ponies, encysted early third-stage larvae constituted a significantly lower percentage of the cyathostome population, and DL and adults formed a significantly higher percentage, compared with those stages in older ponies, 2–5 yr of age. More species of cyathostomes were present in yearling ponies than in older ponies. Significant differences occurred in fecal egg counts at different seasons of the year, even though adult cyathostome and large strongyle numbers remained constant. Twenty-four species of cyathostomes were found year-round, and 2 rare species were found in only 2 or 3 seasons of the year. Prevalences for these 24 species were not significantly different during any season, although 5 species had significant differences in intensity levels of infection in certain seasons of the year. Three species of large strongyle (strongylinae) adults had significant seasonal variations in intensities. These were Strongylus edentatus and S. vulgaris, which occurred in significantly higher numbers in summer and fall, and Triodontophorus brevicauda, which was more numerous in spring and summer. Fourth- and fifth-stage larvae of S. vulgaris recovered from the mesenteric vasculature were significantly more numerous in winter and spring than in other seasons.

We analyzed the content of the abomasum (n = 79) and small intestine (n = 83) of Spanish ibex from Sierra Nevada Natural Park, southern Spain. Fifteen species of trichostrongylid nematodes were identified, 4 of which were found for the first time in this host, i.e., Nematodirus fillicollis, N. oiratianus, Ostertagia lyrata, and O. ostertagi. Teladorsagia circumcincta and Marshallagia marshalli were the most abundant abomasal species, whereas N. abnormalis, N. davtiani, and N. oiratianus were dominant in the small intestine. Counts of both abomasal and intestinal nematodes were generally low (year-round-median = 292 and 94 worms, respectively), and significantly lower numbers of M. marshalli, N. davtiani, and N. oiratianus were found in summer. No sex-related differences in helminth abundance were found, but young ibex harbored significantly more N. davtiani and N. oiratianus than adults. The presence of scabies was not related to increased nematode counts.

Dirofilaria immitis is a filarial nematode parasite that is currently widely enzootic in dog and coyote (Canis latrans) populations of California. Weak historical evidence suggests that the initial focus of D. immitis in California occurred 3 decades ago in the Sierra Nevada foothills (SNF) and spread to other parts of California thereafter. However, this hypothesis is difficult to evaluate because of the lack of epidemiological studies on heartworm in California before 1970. We investigated this hypothesis by comparing D. immitis prevalence in coyotes between initial (1975–1985) and current (2000–2002) surveys in the SNF and 2 coastal regions. In the SNF, prevalence of heartworm was not significantly different between initial (35%, n = 169) and current (42%, n = 60) surveys (P = 0.17), suggesting the existence of a stable enzootic focus in the initial survey period. In contrast, current prevalence was 4 times higher than initial prevalence in the northern Coast Range foothills (44 vs. 10%; n = 119, 107; P < 0.001) and in the south San Francisco Bay foothills (32 vs. 8%; n = 31, 59; P = 0.005), suggesting that initial surveys were made during the early stages of colonization. Dirofilaria immitis prevalence, intensity, and abundance was similar in a coastal location in Mendocino County between 1994–1996 and 1999–2002, suggesting some degree of stability in this enzootic focus. Collectively, these findings support the hypothesis that D. immitis established itself initially in California coyotes living in the SNF and subsequently expanded its range of enzootic foci in central California.

Seasonal prevalence and abundance of the helminths of bluegill sunfish in a Louisiana oligohaline bayou were measured by a survey and a field “live-box” experiment. The survey took place from the spring of 1997 to the summer of 1998 and examined fish that were <7 cm. Three trematode species, Phagicola nana (Heterophyidae), Ascocotyle tenuicollis (Heterophyidae), and Posthodiplostomum minimum (Diplostomatidae), and 2 nematode species, Camallanus oxycephalus (Camallanidae) and Spinitectus carolini (Cystidicolidae), were examined. Camallanus oxycephalus was the only helminth that showed a distinct seasonal pattern. Abundance and prevalence peaked in summer, which was likely driven by concerted reproductive cycles of females. The survey data indicated that the other helminths fluctuated over time but did not seem to follow a distinct seasonal pattern. The fluctuations could be attributed to the variable nature of the habitat or to the stochastic events that influence transmission dynamics, particularly isolated events such as tropical storms, which cause dramatic changes in salinity. The live-box experiment successfully measured recruitment of A. tenuicollis and suggested a seasonal component of the distribution of this species, which was possibly caused by temperature-dependent emergence of cercariae from the snail intermediate host.

The U6 and U5 snRNA (small nuclear ribonucleic acid) genes were identified in Taenia solium with the aim of characterizing their sequence and genomic structures. They are contained within a shared 1,009-nt tandem genomic repeat and present at approximately 3 copies per haploid genome. The U6 snRNA gene shares 92 and 95% sequence similarity with the U6 homologs from humans and Schistosoma mansoni, respectively. The U5 snRNA gene of T. solium is 70% similar to the human U5 sequence in the 5′ stem and loop 1 domains. The U6 and U5 snRNA genes are on complementary genomic strands and separated by 458 nt at their “heads” and 306 nt at their “tails.” The nucleotides upstream of the U6 gene lack a recognizable TATA box and proximal sequence elements (PSEs), and the putative gene promoter for U5 snRNA does not resemble vertebrate examples. There are short blocks of similarity between the sequences upstream of the U5 and U6 snRNA genes, and these may be sites of shared transcription factor binding at the respective RNA polymerase II and III promoters. It is possible that this unusual allied U5/U6 snRNA genomic repeat may help mediate coordinated regulation of expression of the 2 snRNAs.

We describe the microsporidian Amazonspora hassar n. gen., n. sp. from the gill xenomas of the teleost Hassar orestis (Doradidae) collected in the estuarine region of the Amazon River. The parasite appeared as a small whitish xenoma located in the gill filaments near the blood vessels. Each xenoma consisted of a single hypertrophic host cell (HHC) in the cytoplasm of which the microsporidian developed and proliferated. The xenoma wall was composed of up to approximately 22 juxtaposed crossed layers of collagen fibers. The plasmalemma of the HHC presented numerous anastomosed, microvilli-like structures projecting outward through the 1–3 first internal layers of the collagen fibrils. The parasite was in direct contact with host cell cytoplasm in all stages of the cycle (merogony and sporogony). Sporogony appears to divide by plasmotomy, giving rise to 4 uninucleate sporoblasts, which develop into uninucleate spores. The ellipsoidal spores measured 2.69 ± 0.45 × 1.78 ± 0.18 μm, and the wall measured ∼75 nm. The anchoring disk of the polar filament was subterminal, being shifted laterally from the anterior pole. The polar filament was arranged into 7–8 coils in a single layer in the posterior half of the spore, surrounding the posterior vacuole. The polaroplast surrounded the uncoiled portion of the polar filament, and it was exclusively lamellar. The spores and different life-cycle stages were intermingled within the cytoplasm of the HHC, surrounding the central hypertrophic deeply branched nucleus. The ultrastructural morphology of this microsporidian parasite suggests the erection of a new genus and species.

Icosiella turgeocauda n. sp. from the intestinal mesenteries of Rana cancrivora collected at Luzon, Republic of the Philippines, is described and illustrated. Icosiella turgeocauda n. sp. represents the ninth species to be assigned to the genus and is easily differentiated from all the previously described species by the position of the vulva and the presence of bilateral umbos on the caudal end of the male. Seuratascaris numidica also was found. The Philippines represents a new location record for S. numidica.

Phyllodistomum funduli n. sp. is described from the urinary bladder and ureters of the plains topminnow, Fundulus sciadicus, from Cedar Creek in Keith County, Nebraska (41°11.18′N, 101°21.77′W). Phyllodistomum funduli differs from most other Phyllodistomum species reported in possessing an oral sucker that is larger than the acetabulum. Currently, 7 other species of Phyllodistomum have been reported to possess this trait. A comparison of P. funduli with these 7 other species indicates that P. funduli is a distinct species, lacking notches, posterior body folds, caudal projections, or cephalic glands found in some congeners. There is no published report on parasites other than monogeneans inhabiting the plains topminnow, and there is no other report of members from the Gorgoderidae inhabiting any members of the Fundulidae.

A new species of Spauligodon collected from Cnemidophorus mexicanus and C. deppii is described in this study. The species is placed in Spauligodon because the caudal alae start at the level of the precloacal papillae and embed the adcloacal papillae. The new species is most similar to S. goldbergi, but diagnostic traits of the former are the presence of 2 ridges in lateral alae, flaplike expansions in the caudal end of lateral alae, and a smooth tail for males. This species is the eighth recorded in the neotropics.

Rhabdochona ahuehuellensis n. sp. is described from the Balsas goodeid, Ilyodon whitei. The distinctive characters are cuticular elevations in the middle of the prostom, tricuspid ending of the distal part of the left spicule combined with a scoop shape, and an egg surface with limited, short subpolar filaments. The characters place the present species farther apart from species described from North America, except R. lichtenfelsi. This description brings the total number of species of Rhabdochona in the Mesa Central of Mexico to 4, suggesting that this genus has undergone speciation in response to basin fragmentation within the Mesa Central of Mexico.

Plasmodium pifanoi Scorza and Dagert B., known only from the type host, Ameiva ameiva, is redescribed from Kentropyx calcarata collected in Territorio Amazonas, Venezuela. Schizonts, 6.2 × 4.5 (4–8 × 3–6), produce on average 11.9 (7–16) merozoites. Gametocytes average 12.4 × 6.0 (8–16 × 4–10), with length × width (LW) 72.9 (52–112) and L/W 2.18 (1.1–3.3), and always contain 1–5 prominent vacuoles. Macrogametocytes in active infection are longer than microgametocytes, with greater LW, but gametocytes in chronic infection are not sexually dimorphic in dimension and are slightly smaller. Two additional malarial parasites are described from K. calcarata. Plasmodium lepidoptiformis has small schizonts, 4.6 × 3.2 (3–6 × 2.5–3), that produce 5.1 (4–8) merozoites and commonly resemble a butterfly in appearance. Gametocytes are elongate, 9.0 × 4.3 (7–10 × 3–6), with LW 38.3 (24–51) and L/W 2.2 (1.3–3.3), and sexually dimorphic, with macrogametocytes longer than microgametocytes, with greater LW. Plasmodium minasense calcaratae is characterized by very small, usually fan-shaped, schizonts, 3.4 × 2.6 (2.5–4.5 × 2.0–3.0), that produce 3.9 (3–4) merozoites. Gametocytes are spherical or ovoid, 6.7 × 5.0 (4.5–9.0 × 3.0–7.0), with LW 33.7 (15–54) and L/W 1.4 (1.0–2.3), with no sexual dimorphism in dimensions.

Giardia sp. and Cryptosporidium sp. have emerged as waterborne pathogens of concern in Taiwan. This study examined both parasites in the actual water samples in southern Taiwan. Method 1623 was characterized by a higher recovery rate and lower detection limit compared with the information collection requirement protozoan method. A significant correlation between water turbidity and Cryptosporidium sp. in raw water samples was found in this study.

Cross-reactions between Leishmania braziliensis and Trypanosoma cruzi caused by common antigenic determinants hinder the specific diagnosis of cutaneous and mucocutaneous leishmaniasis (CL and MCL). Therefore, the usefulness of the 70-kDa heat shock protein (Hsp70) from L. braziliensis for differential serodiagnosis was investigated. The single-copy gene encoding Hsp70, consisting of 663 amino acids, was isolated from a genomic DNA library. The antigenicity data show that Hsp70 is an immunodominant antigen highly recognized (84%) by sera of patients with CL and MCL and to a lesser extent by chagasic patients (18.75%). Antigenic mapping of the 5 overlapping fragments into which the protein was split showed that the main antigenic determinants are located in the carboxy-terminal end. The linear antigenic determinants that show cross-reactions with chagasic sera are located in the fragment rLb70(352–518). The carboxy-terminal fragment rLb70(513–663) presents 70% sensitivity and 100% specificity, so it could be a potential candidate for specific serodiagnosis of CL and MCL caused by L. braziliensis.

Small monozoic cysts found in the spleen of dogs infected with Hepatozoon canis are described from naturally and experimentally infected dogs. These forms of H. canis resemble cysts formed by other Hepatozoon species that infect frogs, lizards, and grey squirrels as intermediate hosts. The H. canis cyst stage differs in size and morphology from the large cysts of H. americanum, the second Hepatozoon species known to infect dogs.

We have previously demonstrated that cardiac myocytes isolated from the hearts of adult dogs develop rapid repetitive cytosolic Ca² transients, membrane depolarization, and cell contraction by mobilization of sarcoplasmic reticulum Ca² stores when exposed to a soluble factor from the trypomastigotes of Trypanosoma cruzi. These findings led us to investigate the regulatory mechanisms of cytosolic Ca² in cardiac tissues from dogs chronically infected with T. cruzi. Expression of the plasma membrane calcium pump (PMCA) RNA and protein was determined by Northern and Western blotting, respectively, followed by densitometric analyses. A 642-bp PMCA 1b complementary DNA probe derived from canine epicardial tissue hybridized to 2 major transcripts (7.3 and 5.3 kb) in canine epicardium. Expression of the dominant transcript (7.3 kb) was 77% greater in cardiac tissues obtained from dogs with chronic T. cruzi infection (140 days after inoculation) in comparison with constitutive expression levels in normal dogs. Monoclonal antibody 5F10, known to recognize all isoforms of the PMCA, was used to detect expression of the PMCA protein in epicardial tissue. Expression of a 142-kDa protein was increased by 58% in the cardiac tissues of infected dogs when compared with those from uninfected dogs. To establish a link between the upregulation of PMCA in dogs chronically infected with Chagas disease and the ventricular-based arrhythmias and myocardial failure that occur during this stage of disease both in dogs and humans, further study will be required.

Sarcocystis neurona merozoites were examined for their ability to invade and divide in bovine turbinate (BT) cell cultures after treatment with cysteine (iodoacetamide), aspartic (pepstatin A), metallo- (1,10-phenanthroline and ethylene glycol-bis(aminoethylether)-tetraacetic acid [EGTA]), or serine (4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride [AEBSF], phenylmethane sulphonyl fluoride [PMSF], and tosyl lysyl chloramethyl ketone [TLCK]) protease inhibitors. Significant (P < 0.01) inhibition of serine protease activity by PMSF and TLCK led to a reduction of 86 and 78% in merozoites produced in BT cell cultures, respectively, whereas AEBSF (1 mM) led to a 68% reduction in merozoites produced in BT cell cultures and a reduction of 84 and 92% at higher AEBSF concentrations (2 and 3 mM, respectively). Pepstatin A and iodoacetamide failed to cause any inhibition in merozoite production, whereas 1,10-phenanthroline and EGTA caused slight, but not significant, inhibition at 6 and 17%, respectively. In zymograms, 2 bands of protease activity between 65- and 70-kDa molecular weight were seen. The protease activity was inhibited by AEBSF but not by E-64 (cysteine protease inhibitor), EGTA, iodoacetamide, or pepstatin A. In native zymograms, the protease activity was highest between a pH range of 8 and 10. These data suggest that merozoites of S. neurona have serine protease activity with a relative molecular weight range between 65 and 70 kDa and optimal pH range between 8 and 10, which is essential for host cell entry at least in vitro. The protease activity described here could be a potential target for chemotherapy development.

We examined temporal and geographical distribution of Haemoproteus sp. and Plasmodium sp. parasites in Swedish willow warblers, Phylloscopus trochilus. Parasite lineages were detected with molecular methods in 556 birds from 41 sites distributed at distances up to 1,500 km. Two mitochondrial lineages of Haemoproteus sp. were detected, WW1 in 56 birds and WW2 in 75 birds, that differed by 5.2% sequence divergence. We discuss the reasons behind the observed pattern of variation and identify 3 possible causes: (1) variation in the geographic distribution of the vector species, (2) the degree of parasite sharing with other bird species coexisting with the willow warbler, and (3) timing of transmission. Our results support a fundamental and rarely tested assumption of the now classical Hamilton–Zuk hypothesis of sexual selection, namely, that these parasites vary in both time and space. Such fluctuations of parasites and the selection pressure they supposedly impose on the host population are likely to maintain variation in immune system genes in the host population.

The importance of B cells in host resistance to, and recovery from, Cryptosporidium parvum infection was examined in gene-targeted B cell–deficient (μMT−/−) mice. Neonatal μMT−/− mice infected with C. parvum at 5 days of age completely cleared the infection by day 20 PI. The kinetics of infection and clearance were similar to those seen with age-matched C57BL/6 control mice. Furthermore, B cells were not required to clear existing C. parvum infection in adult mice. Reconstitution of persistently infected Rag-1−/− adult mice with spleen cells from μMT−/− donor mice resulted in significant reduction of infection, as in the results seen with spleen cells from C57BL6 donors. These findings indicate clearly that B cells are not essential for host resistance to, and recovery from, C. parvum infection in mice.

A water buffalo (Bubalus bubalis) was fed 5.0 × 10⁵ Sarcocystis hominis sporocysts from a human volunteer who had ingested S. hominis cysts from naturally infected cattle. A necropsy was performed on the buffalo 119 days after inoculation, and a large number of microscopic sarcocysts (∼5,000/g) were found in skeletal muscles. Ultrastructurally, the sarcocyst wall from buffalo muscles has upright villar protrusions measuring about 5.6 × 0.8 μm with numerous microtubules that run from the base to the apex. Sarcocysts from this buffalo were infective to 2 human volunteers, confirming their identity as S. hominis. Therefore, we believe that buffaloes can act experimentally as the intermediate host for S. hominis.

The prevalence of Toxoplasma gondii in free-range chickens from Campos dos Goytacazes, Rio de Janeiro State, Brazil, was examined to evaluate environmental contamination by oocysts. Antibodies against T. gondii were assayed by the modified agglutination test (MAT) in sera of chickens. Antibodies against the parasite were found in 129 of 198 chickens with MAT titers ≥1:25. Brains and hearts of 86 of the 198 chickens were bioassayed in mice for the presence of T. gondii. Viable parasites were isolated from 61 (70.9%) of the 86 chickens. Importantly, viable T. gondii were recovered even from seronegative chickens (MAT titer ≤1:10). The distribution of parasite-positive chickens by MAT titer was 4 of 17 (titer ≤1:10), 3 of 4 (titer of 1:20), 2 of 6 (titer of 1:40), and 52 of 59 (titer ≥1:80). Thus, the high recovery rate of T. gondii observed in mice is indicative of high levels of environmental contamination of free-range chickens by T. gondii oocysts in this area that is endemic to humans.

The number of Sarcocystis species that infect sea otters (Enhydra lutris) is unknown. Sea otter tissues were recently shown to harbor sarcocysts of S. neurona and of unidentified species of Sarcocystis. Whereas sarcocysts of S. neurona have walls 1–3 μm thick with type 9 villar protrusions, ultrastructure of a distinct thin-walled sarcocyst (0.5–0.7 μm thick) lacking villar protrusions, but instead exhibiting minute type 1 undulations on the sarcocyst wall, is described in this report. Parasites characterized from a sea otter infection were inferred to be related to, but distinct from, other species belonging to Sarcocystis, based on sequencing and phylogenetic analysis of a portion of the beta subunit of the plastid-encoded RNA polymerase gene.

Within the coccidia, morphological features of the oocyst stage at the light microscope level have been used more than any other single characteristic to designate genus and species. The aim of this study was to conduct morphometric analysis on a range of Cryptosporidium spp. isolates and to compare morphological data between several genotypes of C. parvum and a second species C. canis, as well as a variation within a specific genotype (the human genotype), with genetic data at 2 unlinked loci (18S ribonucleic deoxyribonucleic acid and HSP 70) to evaluate the usefulness of morphometric data in delineating species within Cryptosporidium. Results indicate that morphology could not differentiate between oocysts from C. parvum genotypes and oocysts from C. canis, whereas genetic analysis clearly differentiated between the two. The small size of the Cryptosporidium spp. oocyst, combined with the very limited characters for analysis, suggests that more reliance should be placed on genetic differences, combined with biological variation, when delineating species within Cryptosporidium.

Degenerate oligonucleotide primers derived from conserved serine protease inhibitors were used to amplify a 90–base pair (bp) amplicon from an Ancylostoma caninum adult-stage complementary deoxyribonucleic acid (cDNA) library by polymerase chain reaction (PCR). The amplicon was labeled and used as a probe to screen the library, and a 2,300-bp cDNA clone was identified. The 5′ end of the molecule was obtained from adult cDNA by 5′-RACE. The complete sequence named A. caninum Kunitz-type protease inhibitor (Ac-kpi-1) was 2,371 bp and encoded a 759–amino acid open reading frame. The deduced amino acid sequence had a calculated molecular weight of 84,886 Da and contained an amino terminal signal peptide, suggesting that the protein is secreted. Analysis of the predicted protein sequence indicates 12 highly conserved Kunitz-type serine protease inhibitor domains connected by short, conserved spacers. On the basis of sequence analysis, the first 11 domains are predicted to be active serine protease inhibitors based on the P1 amino acid. Domains 5–8 have identical amino acid sequences, and the remaining domains are 38–88% identical. Domain 12 lacks several of the conserved cysteine residues and has an atypical amino acid in the P1 position, suggesting that it is nonfunctional. Reverse transcriptase–PCR indicated that the Ac-kpi-1 messenger ribonucleic acid is present in egg, L₁, L₃, and adult stages but is most abundant in the adult stage. Ac-KPI-1 is most similar in domain architecture to several extracellular matrix proteins involved in cellular remodeling during insect development. In addition, there are 44 nematode proteins containing one or more Kunitz domains in GenBank, including several with multiple domains.

A human case of creeping eruption due to Gnathostoma nipponicum was confirmed indirectly by finding infective advanced third-stage larvae in leftover largemouth bass meat. This is the first report indicating that the largemouth bass (Micropterus salmoides) serves as a source of G. nipponicum infection in humans.

The haemoprotozoan Babesia canis has been recognized in Australia for many years, and a second, smaller species has recently been discovered. Amplification and sequencing of a partial region of the 18S small subunit ribosomal RNA (rRNA) gene enabled detection and characterization of the large and small canine babesiae of Australia for the first time. Isolates from northern Australia were genetically characterized to be 99% homologous to Babesia canis vogeli, confirming previous speculation about the subspecies of B. canis endemic to Australia. The partial 18S rRNA gene sequence amplified from isolates obtained in southeastern Australia was genetically identical to Babesia gibsoni, a species not previously known in Australia. The polymerase chain reaction (PCR) used was shown to be specific to Babesia and had a high sensitivity, detecting DNA at a parasitemia of approximately 0.0000027%. This study also reports the first known detection and characterization of B. canis DNA in Rhipicephalus sanguineus ticks using PCR.

The occurrence of cutaneous trematode Collyriclum faba in wild birds was monitored in the central European Carpathians from 1996 to 2001. A total of 5,414 birds, representing 86 species, was examined. Collyriclum faba was found at 7 sites (5 in Slovakia, 1 in Poland, and 1 in the Czech Republic), and prevalences at the sites varied from 1 to 16%. Ten species of passerine birds were infected: blackcap (Sylvia atricapilla) (16 positive/622 tested, 2% prevalence), black redstart (Phoenicurus ochruros) (2/25, 8%), chaffinch (Fringilla coelebs) (7/113, 6%), common blackbird (Turdus merula) (1/143, 1%), common redstart (Phoenicurus phoenicurus) (1/30, 3%), dipper (Cinclus cinclus) (1/9, 11%), European robin (Erithacus rubecula) (103/838, 12%), goldcrest (Regulus regulus) (1/76, 1%), grey wagtail (Motacilla cinerea) (5/25, 20%), and yellowhammer (Emberiza citrinella) (1/73, 1%). Cutaneous cysts of C. faba were found in the birds from the end of May to mid-September, with the prevalence peaking in July and August. One to 21 cysts per bird were found. In black redstart, chaffinch, common redstart, European robin, and yellowhammer, cysts were most frequently observed on the legs, particularly in the crural region. In blackcap, common blackbird, dipper, and grey wagtail, almost all the cysts were found around the vent and on the abdomen. In goldcrest, the cyst was located above the coccygeal gland. Collyriclum faba is a common parasite in birds during summer in central European Carpathians. Collyriclum faba was observed for the first time in Poland. It appears that C. faba may be fatal for some of the bird hosts.

Experimental crosses between Schistosoma mansoni and S. rodhaini have shown that hybrid offspring are viable, yet, until now, no naturally occurring hybrid has been identified. A collection of freshwater snails from Nyamlebi-Ngoma, Ukerewe Island, Lake Victoria, Tanzania, yielded a mixed infection within a single Biomphalaria sudanica of S. mansoni females and S. mansoni–S. rodhaini hybrid males. The hybrids were identified using deoxyribonucleic acid (DNA) sequences. Mitochondrial DNA 16S and 12S sequences of the hybrids match those of S. mansoni, whereas their nuclear ribosomal DNA ITS1 and ITS2 sequences match those of S. rodhaini. The identification of hybrids in Tanzania highlights the possibility that the genetic identity of either parasite species might be modified by introgression.

We have recently developed recombinant subunit vaccine consisting of second-generation schizont (2GS) membrane protein (rR7) of Leucocytozoon caulleryi. Chickens immunized with rR7 antigen acquired clear resistance to challenge by Leucocytozoon sporozoites. We examined the induction of cellular immune responses in vaccinated chickens. Spleen adherent cells from vaccinated chickens showed significantly higher phagocytic activity against 2GS-coated latex particles than did cells from adjuvant-inoculated or untreated control birds. Anti-R7 chicken IgG significantly increased the phagocytic rate of adherent cells from these 3 groups. These results show that specific cellular immune responses are induced by recombinant R7 subunit vaccine consisting of L. caulleryi 2GS protein, which suppresses the growth of parasites in the host in association with antiparasite antibodies to 2GS antigen.

The extent to which wild ruminant populations are exposed to infective helminth larvae on their natural pastures is relatively undetermined. In the present study, a modified method for sampling of herbage and isolation of trichostrongyle infective third-stage larvae from natural pastures was used successfully in a muskox habitat in low-Arctic Greenland. The method, a revision of the Macro-Baermann method, is particularly aimed at fieldwork under primitive conditions.

The objective of this study was to compare a polymerase chain reaction (PCR) assay and a monoclonal antibody–based immunofluorescence assay (IFA) for detection of Cryptosporidium parvum in cat feces. Eight C. parvum–naive DSH cats were orally inoculated with 1 × 10⁶ oocysts of a C. parvum human isolate. Fecal samples were collected before inoculation, daily for the next 30 days, and twice weekly until day 85. Methylprednisolone acetate was administered at 20 mg/kg i.m. on days 85, 92, and 99. From days 86 to 115, feces were collected daily and then up to twice weekly until day 126. Immunofluorescence assay was performed after collection of the samples, and then the samples were frozen at −70 C until assayed by PCR. Cryptosporidium parvum was detected by PCR in 101 of 353 samples and by IFA in 52 of 353 samples: 27 samples were PCR positive, IFA positive; 74 samples were PCR positive, IFA negative; 25 samples were PCR negative, IFA positive; and 227 samples were PCR negative, IFA negative. The percentage of concordance between IFA and PCR was 72%. Results of this study suggest that this PCR assay is more sensitive than IFA for detection of C. parvum in cat feces.