Many parasites induce specific changes in host behavior that promote the transmission of their infective stages between hosts. Toxoplasmosis in rodents is known to be accompanied by specific behavioral changes (shift in activity level, learning capacity, and novelty discrimination) that can theoretically increase the chance of infected animals being eaten by the definitive host, the cat. However, toxoplasmosis is also accompanied by many pathological symptoms. It is not known whether the behavioral changes are products of manipulation activity of the parasite or only nonspecific by-products of pathological symptoms of toxoplasmosis. Here, we compared the dynamics of development of behavioral and pathological changes in Toxoplasma gondii-infected mice. The results showed that the maximum reduction of mouse activity corresponded with the peak of pathological symptoms, and also that maximum increase of reaction times corresponded with the peak of development of tissue cysts in the brains of infected mice. Behavioral changes were only transient and disappeared before the 12th wk postinoculation. The results suggest that the behavioral changes in infected mice reported by many authors and observed in our experiments could be nonspecific by-products of pathological symptoms of toxoplasmosis rather than specific products of manipulation activity by the parasite.

Some parasites have been shown to manipulate host behavior so that parasite transmission to the next host is enhanced. Infection with Triaenophorus crassus Forel (Cestoda) caused alterations in the activity and microhabitat selection of the first intermediate host Cyclops strenuus Fischer (Copepoda) in the laboratory. Infected copepods made more starts to swim but spent less time swimming than uninfected copepods. These changes were independent of the intensity of infection. In a water column illuminated from above, infected copepods approached the surface, whereas uninfected ones remained close to the bottom. In the dark both infected and uninfected copepods stayed near the bottom. Finally, infection with T. crassus increased the probability of C. strenuus being eaten by the second intermediate host, whitefish (Coregonus lavaretus L. s.l.), in the laboratory. In experimental infections, 10-day-old procercoids had significantly lower infectivity for whitefish than older (12-, 14-, and 21-day-old) procercoids. Behavioral changes were detected in infected copepods containing procercoids 12 days old or older but not in experiments with 10-day-old procercoids. These results may indicate that T. crassus changes the behavior of the copepod host only after it has become infective to the next host, which is consistent with the active manipulation hypothesis.

Schizonts of Sarcocystis speeri Dubey and Lindsay, 1999 were cultured in vitro in bovine monocyte and equine kidney cell cultures inoculated with infected tissues of nude and γ-interferon knockout mice fed sporocysts from opossums, Didelphis albiventris. At least 1 asexual cycle was completed in 3 days. In vitro-grown merozoites were structurally and antigenically distinct from those of Sarcocystis neurona and Sarcocystis falcatula. Culture-derived merozoites of S. speeri were not infective to budgerigars (Melopsittacus undulatus).

The mortality of Boophilus annulatus, Hyalomma excavatum, Rhipicephalus bursa, and Rhipicephalus sanguineus adult ticks was recorded after their exposure in petri dishes to 5 entomopathogenic nematode strains. The strains used were DT and Mexican of Steinernema carpocapsae, Hb HP88 of Heterorhabditis bacteriophora, and IS-3 and IS-5 of Heterorhabditis sp. The most rapid killing rates (LT₅₀ and LT₉₀) were observed for B. annulatus ticks (0.8–5.0 days). Most unfed males died 0.3–2.8 days after the unfed females, whereas even more time (0.8–8.0 days) passed before engorged females died. In most bioassays, the IS-3 and IS-5 strains of Heterorhabditis sp. nematodes appear to be the most pathogenic for ticks and, in most cases, killed ticks several days before the other 3 nematode strains. Unfed adult ticks exposed to nematodes died within less time than it takes for adults to complete their prefeeding period after molting.

Representatives of Leptorhynchoides thecatus (Acanthocephala) inhabit ceca of green sunfish but cannot survive in the anterior intestine. The influence of elevated cecal protein concentrations, pH, and amounts of lumenal materials on the microhabitat specificity of L. thecatus was investigated. An attempt was made to alter the distribution of worms in starved fish, in fish of which cecal pH was reduced, and in fish of which intestinal protein concentration was elevated. Protein concentration and pH showed no effect on worm distribution. Starving hosts had no effect on worm number or distribution but resulted in retardation of worm growth and development, providing a mechanism by which worms may overwinter and by which peak egg production may coincide with abundance of the amphipod intermediate host. None of the factors investigated is solely responsible for the microhabitat specificity of L. thecatus. It is suggested that helminth site specificity is characterized by long histories of adaptation to specific habitats with many physiological adaptations being facilitated synergistically. Maximization of sexual congress may exert an important selective pressure favoring this establishment of microhabitat specificity.

This study presents and analyzes positional data on 18 helminth species forming the intestinal infracommunities of 59 Cichlasoma synspilum from southeastern Mexico. Interspecific interactions were found between 4 pairs of host specialists: Crassicutis cichlasomae (Digenea) with Neoechinorhynchus golvani (Acanthocephala), Spirocamallanus rebecae (Nematoda) with N. golvani, and Raillietnema kritscheri (Nematoda) with N. golvani. The interactions were expressed as negative correlations, positional shifts, and reductions in realized distribution. It is suggested that N. golvani produces unsuitable habitats for C. cichlasomae, S. rebecae, and R. kristscheri. All interactions were found among phylogenetically unrelated helminth specialists of cichlids.

A new genus and species of piscicolid leech in the Platybdellinae inhabits the oral cavity of Dasyatis akajei in the northwestern Pacific Ocean near Tanabe, Japan. The genus Rhopalobdella n. gen. is characterized externally by very small oral and caudal suckers and a smooth body that is widest just posterior to the clitellum. Eyespots and ocelli are lacking. The coelom is spacious with large segmental connecting sinuses between dorsal and ventral sinuses. There are 5 pairs of testisacs, an unusually extensive epididymis, and a very large bursa. Conducting tissue is absent. There are 2 pairs of esophageal diverticula and very well developed nephridia. Rhopalobdella japonica n. gen. n. sp. is characterized by a urosome that tapers strongly to the caudal sucker and by a single gonopore; the common oviduct opens into the posterior portion of the bursa. The coelomic and excretory systems resemble Aestabdella, but in other respects the genera are quite different. This is the first marine leech reported from rays in the northwestern Pacific.

Following the discovery of establishment of the African tortoise tick Amblyomma marmoreum in Florida, the present study was undertaken to determine the extent of introduction of exotic ticks into Florida on imported reptiles. Exotic ticks were identified on 29 (91%) of 32 reptile premises in 18 counties of Florida. The ticks, found on a variety of imported tortoises, snakes, and monitor lizards, belonged to 4 Amblyomma species (A. marmoreum, Amblyomma nuttalli, Amblyomma sabanerae, and Amblyomma sparsum) and 4 Aponomma species (Aponomma exornatum, Aponomma flavomaculatum, Aponomma latum, and Aponomma varanensis). The most commonly encountered ticks were A. latum and A. marmoreum. The identifications of A. marmoreum on 8 premises in 7 counties, and of A. sparsum on 1 premises, are of great concern because both species are vectors of heartwater, a lethal disease of cattle, sheep, goats, and deer.

Sarcocystis sporocysts were found in intestinal scrapings from 24 (54.5%) of 44 opossums (Didelphis virginiana). The number of sporocysts varied from a few (<10,000) to 245 million. Sporocysts from 23 of 24 opossums were fed to captive budgerigars (Melopsittacus undulatas), and the inocula from 21 opossums were infective, indicating the presence of Sarcocystis falcatula. Sporocysts from 24 opossums were fed to gamma-interferon-knockout (KO) or nude mice; inocula from 14 opossums were infective to mice. Sarcocystis neurona was detected in tissues of KO mice by specific staining with anti-S. neurona antibodies, and the parasite was cultured in vitro from the brains of KO mice fed sporocysts from 8 opossums. Sarcocystis speeri was identified by specific staining with anti-S. speeri antibodies in tissues of KO mice fed inocula from 8 opossums; 3 opossums had mixed S. neurona and S. speeri infections. Thus, the prevalences of sporocysts of different species of Sarcocystis in opossums were: S. falcatula 21 of 44 (47.7%), S. neurona 8 of 44 (18.1%), and S. speeri 8 of 44 (18.1%) opossums. Sarcocystis neurona alone was found in 1 opossum, and S. speeri alone was found in 1 opossum. Mixed Sarcocystis infections were present in 21 opossums.

Intermediate filaments (IFs) make up the cytoskeleton of most eukaryotic cells. In vertebrates, a number of IF proteins have been identified, showing distributions unique to tissue or cell type. Information on helminth IFs is limited to some nematode species. To observe immunofluorescent localization of IFs in helminth tissues, we selected a murine hybridoma clone producing IgM antibody to multiple types of mammalian IF proteins and examined cross-reactivity to helminth proteins. The selected monoclonal antibody (HUSM-9) cross-reacted well with IFs from nematode species such as Toxocara canis, Dirofilaria immitis, Anisakis simplex, and Trichinella britovi; strong immunofluorescence on cryostat sections was detected in the hypodermis, cords, body muscle, smooth muscle of the uterus, and other epithelial structures. In platyhelminths, i.e., adult Schistosoma mansoni, larval Taenia taeniaeformis, adult Taenia crassiceps, and Echinococcus multilocularis protoscolex, the reactivity was weaker than in nematodes, and localized in the body wall muscle and subtegumental tissue. Western blotting of 8 M urea extracts of parasites with the antibody detected a pair of clear bands in nematodes but not in S. mansoni or the cestodes. These results might be explained by sparse distribution of IFs in platyhelminths, or low affinity of the used antibody to platyhelminth IF proteins, or both.

The region between the 28S and 18S rRNA genes, including the intergenic spacer (IGS) region and the 5S rRNA gene, from 32 strains of Toxoplasma gondii and the NC1 strain of Neospora caninum was amplified and used for DNA sequencing and/or restriction fragment length polymorphism (RFLP) analysis. The 5S rDNA sequences from 20 strains of T. gondii were identical. The IGS region between the 5S and 18S rRNA genes (nontranscribed spacer 2 or NTS 2) showed 10 nucleotide variations. Six of the 10 variant positions correlated with the murine virulence of the strains. Intraspecific polymorphisms distinguished the virulent strains of zymodemes 5, 6, and 8 from other virulent strains (in zymodeme 1). RFLP methods (IGS-RFLP) were developed and used to characterize the virulent and avirulent patterns among 29 T. gondii strains. Sequence diversity of 19.8% was found between T. gondii and N. caninum when comparing a region of 919 bp at the 3′ end of NTS 2. The sequence variation in ribosomal IGS could therefore be a useful marker for Toxoplasma strain identification and for distinguishing N. caninum from T. gondii.

It is known that Fasciola sp. from Japan and the Republic of Korea consist of diploids (2n = 2x = 20), triploids (2n = 3x = 30), and mixoploids with diploid and triploid cells. Triploids are distributed over Asia and Hawaii. Abnormal spermatogenesis and parthenogenetic reproduction are the main characteristics of Fasciola sp. Here we measured 21 different morphological parameters of diploid and triploid flukes of Fasciola sp. obtained from Japan and the Republic of Korea. Statistical analysis showed that diploid and triploid flukes were morphologically different. No bivalents or trivalents could be detected in diploid and triploid flukes, respectively. Based on our findings, we speculate that parthenogenetic diploids, triploids, and mixoploids (2x/3x) of Fasciola sp. are genetically related to each other.

Negatively charged liposomes, proposed as potential vaccine adjuvants, have been extensively studied in association with various antigens. In the present study, we investigated the adjuvanicity of negatively charged liposomes to enhance the protective immunity of membrane antigens of Leishmania donovani promastigotes (LAg). In comparison to the control mice immunized with phosphate-buffered saline and empty liposomes, immunization with free LAg led to significant levels of protection against infection with virulent promastigotes. Encapsulation of LAg in liposomes also induced effective protection. However, the level of protection by LAg–liposome was not significantly different from that induced by free LAg. Investigation of the immune responses showed, in contrast to free LAg, that immunization with LAg–liposome elicited strong antibody responses. IgG isotype analysis revealed the presence of all 4 isotypes. However, the titer of IgG1 was significantly higher than IgG2a, IgG2b, and IgG3. Following infection, stimulation of IgG and IgG isotypes did not differ in the different immunization groups. Delayed-type hypersensitivity (DTH) analysis after immunization showed significant induction by LAg and LAg–liposomes, in comparison to controls. With infection, again, the level of DTH in all the groups became almost comparable. Stimulation of insufficient cellular response, as reflected by DTH and potentiation of IgG1 over IgG2a, IgG2b, and IgG3 suggest a dominance of Th2 response with this liposome–antigen formulation, resulting in weak protection against visceral leishmaniasis.

We characterized the cytokine response and T-cell requirements of mice infected with the intraerythrocytic parasites Babesia microti and WA1. WA1 infections were fatal, whereas B. microti infections were resolved. We measured production of tumor necrosis factor (TNF)-α, interferon-γ, interleukin (IL)-10, and IL-4 by splenic CD4 , CD8 , and γδ T cells using flow cytometry. WA1 inoculation stimulated TNF-α production, whereas resolving B. microti infections were characterized by increased IL-10 and IL-4. The role of TNF-α in WA1 infections was further investigated by inoculating TNFRp55^−/− mice with a lethal dose of WA1. A survival rate of 90% in the TNFRp55^−/− mice indicated that a disruption in the TNF-α pathway abrogated the pathologic mechanism of WA1. Inoculation of WA1 into CD4^−/− and CD8^−/− mice resulted in survival rates of 60% and 78%, respectively, whereas WA1 infection in γδ^−/− and control mice was fatal. These results suggest that CD8 T cells may contribute to the WA1-associated disease. Babesia-infected CD4^−/− mice experienced a longer duration of parasitemia, indicating that CD4 T cells participate in parasite elimination. These studies demonstrate differences in immune responses during fatal or resolving Babesia infections, and they identify TNF-α as an important mediator of the WA1-associated pathogenesis.

We investigated the emergence of third-stage larvae (L3) of Umingmakstrongylus pallikuukensis from the slugs Deroceras laeve, Deroceras reticulatum, and the snail Catinella sp. in the laboratory and from D. laeve on the tundra. Third-stage larvae emerged from 8 of 8 D. laeve and 8 of 8 D. reticulatum housed at 20 C in darkness and from 9 of 10 D. laeve and 5 of 5 Catinella sp. housed at 21 C with 10–12 hr of light/day. Larvae emerged from D. laeve and D. reticulatum over a wide range of infection intensities (2–179 and 20–65, respectively), and the patterns of emergence were independent of intensity. The majority of the L3 emerged from most of the Deroceras spp. by 58 or 60 days postinfection (PI). Lower rates of emergence were observed from Catinella sp. Larvae emerged from D. laeve on the tundra by 10 wk PI and were recovered from the vegetation in some experimental enclosures the following year. Third-stage larvae survived in tap and distilled water at 0–4 C for 13 mo. Emergence of L3 of U. pallikuukensis from the intermediate host may increase the temporal and spatial availability of L3 and enhance their survival and transmission.

Sixty-two brown anoles, Anolis sagrei, from Oahu, Hawaii were examined for helminths. Anolis sagrei was introduced to Hawaii, presumably from the Caribbean. Two species of trematodes, Mesocoelium monas and Platynosomum fastosum, 3 species of nematodes, Atractis scelopori, Physaloptera squamatae, and Physocephalus sp., 1 acanthocephalan, Acanthocephalus bufonis, and 1 pentastome, Raillietiella frenatus, were found. Atractis scelopori and P. squamatae, previously unknown in Hawaii, are widely distributed in the Caribbean and were most likely transported to Hawaii with the introduced anoles. Mesocoelium monas, P. fastosum, Physocephalus sp., A. bufonis, and R. frenatus have been previously reported from Hawaiian herptiles; A. sagrei most likely acquired infections of these parasites from Hawaiian populations. This study indicates that helminths can be transported with their introduced hosts and become established in the colonized areas and that introduced lizards may quickly acquire species of previously established helminthes.

Seven donkeys (Equus asinus) from North-West and Mpumalanga Provinces in South Africa were examined at necropsy. Quantitative samples were collected from the gastrointestinal tract for recovery of helminth parasites from the stomach, small intestine, cecum, ventral colon, dorsal colon, descending colon, and cranial mesenteric artery. Fifteen genera and 29 species of helminths were identified comprising 27 species of nematodes in the Ascarididae, Atractidae, Habronematidae, Onchocercidae, Oxyuridae, and Strongylidae; 1 species of cestode in the Anoplocephalidae; and 1 species of trematode in the Paramphistomatidae. In addition, 2 species of oestrid fly larvae in the Gastrophiliidae were identified. The most abundant group in number of species was the cyathostomes (small strongyles) and, of these, Cyathostomum montgomeryi, Cylicocyclus sp. (a), and Cylicostephanus minutus were the most numerous. The most prevalent cyathostomes were C. montgomeryi and Cylicocyclus sp. n. (a). Strongylus vulgaris was the most abundant and prevalent large strongyle species. The occurrence of small strongyle species and their prevalences in this study are compared with 3 other studies on donkeys in Africa.

Six female reindeer calves were inoculated intraruminally with various doses of Trichinella muscle larvae. Four calves were inoculated with T. nativa, receiving 15,000 (n = 1), 5,000 (1), and 2,500 (2) larvae each. Two calves were inoculated with 5,000 T. spiralis larvae each. Blood samples were collected twice per week for total white blood cell (WBC) and differential counts and for serology using enzyme-linked immunosorbent assay (ELISA) based on T. spiralis excretory–secretory antigen. On day 56, the calves were slaughtered and muscle samples were examined according to the standard digestion method for Trichinella larvae. Blood samples were also collected twice a week from 4 uninoculated, but otherwise similar, reindeer calves corralled separately. Both the total WBC and eosinophil counts of the inoculated animals were, on average, higher during the experimental period. All the inoculated calves seroconverted, showing an increase in the optical density (OD) in the ELISA starting between day 23 and day 27 postinoculation. Very few muscle larvae (<0.08 larvae/g [lpg]) were to be found from the animals inoculated with T. nativa, but about 4 and 6 lpg were recovered from the masseter muscles of those inoculated with T. spiralis.

The fellodistomid Proctoeces lintoni is a common parasite of the gonads of key-hole limpets Fissurella spp. (Archaeogastropoda). It has also been found in the mantle of Octopus vulgaris and as an intestinal parasite of haemulid and gobiesocid fishes. Fissurella crassa, a host for progenetic P. lintoni, can be found from Huarmey, Perú (10°S) to Chiloé, Chile (42°S). Proctoeces lintoni has been found parasitizing fishes and molluscs from Callao, Perú (12°S) to Valdivia, Chile (39°S). Progenesis is thought to be a latitude-dependent phenomenon, and high progenesis is expected at higher latitude. In the present article, the association between latitude and progenesis was examined over a latitudinal gradient of about 3,000 km. Data suggest that progenesis of P. lintoni infecting F. crassa was not associated with latitude. Low levels of progenesis found in the Peruvian population could be a consequence of parasite-induced mortality rather than of low latitude, as would be predicted by the latitude dependence hypothesis.

A philophthalmid species from Israel using the freshwater snail Melanoides tuberculata as intermediate host was studied. The biological and morphological characteristics of all developmental stages of the life cycle of this philophthalmid were described, and compared to those of Philophthalmus lucipetus Rudolphi, 1819 from Israel, Philophthalmus gralli Mathis and Leger, 1910 from Jordan, Philophthalmus palpebrarum Looss, 1899, Philophthalmus nocturnus Looss, 1907, Cercaria distomatosa Looss 1896 from Egypt, and Philophthalmus lucknowensis Baugh, 1962 from India. The possible identity with 1 of these species is discussed. On the basis of comparative analysis of the data for all parasite stages in the life cycle, geographical distribution, snail hosts, and snail host specificity, we propose to designate the Israeli Melanoides tuberculata-transmitted eye fluke to Philophthalmus distomatosa n. comb. (Looss, 1896), (Digenea: Philophthalmidae).

The eukaryotic translation initiation factor eIF-4A is an ATP-dependent RNA helicase involved in ribosome attachment to the 5′ end of mRNAs. Employing as a probe a Cryptosporidium parvum genomic amplicon encoding a partial polypeptide related to eIF-4A, we screened a C. parvum sporozoite cDNA library to clone the full length of the gene. Two complete cDNAs were characterized, Cp.F6 and Cp.F10, which consisted of 1,900 and 1,418 bp, respectively. The overlapping portions of the sequences shared 100% identity and encoded a polypeptide of 405 amino acids whose identity to known eIF-4A molecules ranged between 77 and 39%. The 2 cDNAs differed in the length of their respective 3′ untranslated regions, of 577 bp in Cp.F6 and 72 bp in Cp.F10, in both of which a putative polyadenylation signal was identified. The structure of the cloned cDNAs, along with genomic Southern blot data indicating that eIF-4A is encoded by a single copy gene, strongly suggested that Cp.F6 and Cp.F10 reflect a differential 3′ end processing of mRNA precursors, not observed so far in C. parvum. Northern blot analysis confirmed that the sporozoites express 2 eIF-4A mRNAs and showed that the lower molecular weight transcript is 10- to 20-fold more abundant. We also investigated the polymorphism of the eIF-4A gene and defined a novel polymerase chain reaction–restriction fragment length polymorphism marker discriminating between C. parvum isolates of genotypes 1 and 2.

To assess muscle breakdown during avian coccidiosis, the level of the nonmetabolizable amino acid 3-methylhistidine (3MH) was determined in muscle, plasma and excreta from chickens infected with Eimeria acervulina. The changes in 3MH levels during infection were assessed at 1–29 days postinoculation (DPI) in animals given 5 × 10⁵ oocysts per bird. The effect of levels of parasitism were evaluated at 8 DPI in birds receiving 5 × 10³, 5 × 10⁴, 5 × 10⁵ or 1 × 10⁶ oocysts each. The 3MH levels of plasma, muscle, and excreta samples were determined by high-pressure liquid chromatography after derivatization with fluorescamine. Weight gains, breast muscle weight, eviscerated weight, plasma carotenoid levels, dry weight of muscle, and gross lesion scores were also determined. Infected birds had significantly elevated plasma and muscle 3MH at 4 and 8 DPI following a single dose of E. acervulina. The increase in 3MH levels had an inverse relationship with the time course of weight gain and plasma carotenoid levels. Plasma and muscle 3MH levels returned to control values by 15 DPI and remained unchanged from control values through the remainder of the experiment (29 DPI). Breast weight was decreased in infected birds, but the ratio of breast weight to eviscerated body weight was unchanged. Excretion of 3MH decreased relative to controls at 4 and 8 DPI and returned to control levels on 15 DPI. The plasma and muscle levels of 3MH were related to severity of infection; however, levels of excreted 3MH were not. The results suggested that muscle breakdown, as assessed by plasma and muscle levels of 3MH, increased during the acute stage of E. acervulina infection. The underlying causes for this muscle breakdown was unclear but could involve a physiological response to anorexia and decreased food intake during the acute phase of infection. Levels of excreted 3MH did not increase during infection and this may be the result of decreased excreta output during infection. Plasma and muscle levels of 3MH were correlated with severity of E. acervulina infections but may not be as sensitive an indicator of infection as plasma carotenoid levels or other physiological parameters.

Metastatic disease is a major concern of dermal leishmaniasis caused by Leishmania of the Viannia subgenus. The golden hamster provides an experimental model of systemic dissemination and cutaneous metastasis of Leishmania Viannia. We have exploited this model to examine the expression of parasite virulence in cloned populations derived from a strain of L. guyanensis previously shown to be highly metastatic in the hamster. Metastatic capacity manifested as dissemination throughout the lymphoid organs; cachexia and secondary cutaneous lesions were found to differ among clones, yielding a spectrum of virulence. The metastatic phenotype of clonal populations was stable over 5 sequential passages in hamsters. In addition, the low or high propensity to disseminate and produce cutaneous metastatic lesions was reproduced. Capacity to disseminate from the inoculation site was conserved following subcloning of metastatic clones that had been passaged in culture for several generations; clinical manifestations, cachexia, and cutaneous metastatic lesions were variably expressed. Dissemination of parasites and cachexia were significantly related (P = 0.004). Overall, cachexia was an earlier manifestation of dissemination than cutaneous metastases (P < 0.001). The reproducible expression of virulence phenotypes by discrete populations of Leishmania in the golden hamster provides an experimental model for clinically relevant expression of virulence in human leishmaniasis.

The mechanisms of epithelial injury in giardiasis remain unknown. The effects of live Giardia lamblia on cellular G-actin, F-actin, α-actinin, and electrical resistance of human intestinal epithelial monolayers were investigated using SCBN and Caco2 cell lines grown on chamber slides or Transwell™ filter membranes. In separate experiments, some monolayers were also exposed to sonicated trophozoites, some to supernatant from live G. lamblia cultures, and some with or without the Ca² channel blocker verapamil. After 2, 24, or 48 hr of coincubation with G. lamblia, monolayers were assessed for cytoskeletal arrangement under fluorescence and confocal laser microscopy, and transepithelial electrical resistance was measured. Exposure to live G. lamblia trophozoites induced localized condensation of F-actin and loss of perijunctional α-actinin while G-actin remained unchanged. Confocal laser microscopy indicated that F-actin rearrangement was not affected by verapamil and was localized within the terminal web area. Coincubation of monolayers with G. lamblia lysates or with spent medium alone similarly rearranged F-actin. Verapamil alone did not alter F-actin. Electrical resistance of SCBN and Caco2 monolayers exposed to G. lamblia was significantly decreased versus controls regardless of whether live or lysed trophozoite samples were used. The results indicate that G. lamblia-induced epithelial injury is associated with F-actin and α-actinin rearrangements in the terminal web area via mechanisms independent of extracellular Ca² . These alterations are associated with reduced transepithelial electrical resistance and are due at least in part to trophozoite products.

During a parasitological survey of aquatic birds in Mar Chiquita coastal lagoon (Buenos Aires province, Argentina), specimens of Contracaecum Raillet & Henry, 1912 sp. were found in the esophagus and stomach of the great egret Egretta alba (Gmelin, 1789) (Aves: Ardeidae). The nematodes were identified as Contracaecum multipapillatum (Drasche, 1882) on the basis of the following features: ratio of ventricular appendage length to esophagus length, ratio of intestinal caecum length to esophagus length, spicule lengths, number and arrangement of postcloacal papillae (3–4 pairs of adanal papillae, 1 double pair, and a subterminal group of 2 subventral and 3 sublateral pairs). A complete description of the adult specimens of C. multipapillatum is given in the present paper, together with a discussion regarding the validity of other species of Contracaecum reported from Argentina. Contracaecum philomultipapillatum Labriola and Suriano, 1996 is reduced to a junior synonym of C. multipapillatum, and Contracaecum longicaecum Schuurmans Sterkhoven, 1952 is considered a species inquirenda. A revision of Contracaecum spheniscus Boero and Led, 1970 based on appropriate material is necessary.

A new species of Sprostoniella is described from the gills of Chaetodipterus zonatus in Chamela Bay, on the west coast of Mexico. The new species differs from the 2 previously described congeners by having smaller testes each of which is not nearly as long as the ovary. It further differs from the type species, Sprostoniella multitestis, by having a poorly developed pair of accessory sclerites and the cecal diverticula are not confluent posteriorly. The new species also differs from Sprostoniella micrancyra, the other species of the genus, by having a shorter peduncle, the glands of Goto are smaller than the testes and ovary, and by having a slightly different haptoral structure, with 1 pair of hamuli rather than 2 pairs, and 1 central haptoral loculus instead of 2 loculi.

Neopolystoma elizabethae n. sp. is described from the conjunctival sac of the western painted turtle Chrysemys picta belli (Gray), from the Upper Peninsula of Michigan. This is the first species found in this location from chelonians in North America. The new species differs from all other species of Neopolystoma in possessing a circle of 8 genital spines that are recurved and possess a crescent-shaped base. Eight additional species of helminths were found in the 5 turtles examined in this study. All are common parasites of North American freshwater turtles. An additional species of Monogenea (Polystomoides pauli) was found in the oral cavity. Four species of Digenea (Eustomos chelydrae, Allassostomoides chelydrae, Spirorchis kirki, and Spirorchis parvus) and 3 species of Nematoda (Spiroxys contorta, Serpinema trispinosus, and Amphibiocapillaria serpentina) were also found. The following are reported from Michigan for the first time: P. pauli, S. kirki, and A. serpentina.

Two different Rhabditida species (Nematoda: Rhabditidae) Parasitorhabditis platidontus n. sp. and Cruznema campestris n. sp. are described and illustrated from the larvae of Cyclocephala signaticollis (Coleoptera: Scarabaeidae) from Buenos Aires province, Argentina. Parasitorhabditis platidontus n. sp. is characterized by having 4 odontoplates in the stoma and the vulva is at 80% of the length of the body. Cruznema campestris n. sp. can be distinguished by the presence of 4 odontoplates and the arrangement of the male genital papillae, 9 pairs, of which 4 are preanal and 5 postanal.

A set of oligonucleotide primers I and II was developed by analyzing the specificity of a cloned kinetoplast DNA (kDNA) fragment of Leishmania donovani and sequencing the fragment. Polymerase chain reaction (PCR) was conducted with the primers to amplify a minicircle kDNA fragment (297 bp) to detect L. donovani in the bone marrow (22 samples), whole blood (16 samples), and serum (17 samples) of 22 patients with visceral leishmaniasis. All of 22 patients were diagnosed by microscopic identification. Control samples of bone marrow, whole blood, and serum were obtained from patients with leukemia and from healthy volunteers. In addition, 12 dogs were infected with L. donovani promastigotes for the PCR test. The total number of patients positive by PCR testing was 95.5% (21/22), with 91.0% (20/22) from the bone marrow, 68.8% (11/16) from the blood, and 29.4% (5/17) from the sera. Similar results were obtained in infected dogs. No amplification products were seen in control samples from humans or dogs. Our results suggest that PCR may be useful in detecting kDNA in the bone marrow and blood of patients with visceral leishmaniasis.

A quantitative competitive polymerase chain reaction (QCPCR) assay was developed for the oyster parasite Perkinsus marinus. PCR primers for the rRNA gene region of P. marinus amplified DNA isolated from P. marinus but not from Perkinsus atlanticus, Crassostrea virginica, or the dinoflagellates Peridinium sp., Gymnodinium sp., or Amphidinium sp. A mutagenic primer was used to create a competitor plasmid molecule identical to the P. marinus target DNA sequence except for a 13-bp deletion. Both P. marinus and competitor DNA amplified with equivalent efficiencies. Each of 25 oysters was processed by 5 P. marinus diagnostic methods—Ray's fluid thioglycollate medium (FTM) tissue assay, FTM hemolymph assay, whole oyster body burden assay, QCPCR of combined gill and mantle (gill/mantle) tissue, and QCPCR of hemolymph. The QCPCR assay enabled detection of 0.01 fg of P. marinus DNA in 1.0 µg of oyster tissue. QCPCR of gill/mantle tissue or hemolymph as well as the body burden assay detected infections in 24 of 25 oysters. Ray's FTM tissue assay detected only 19 infections. The FTM hemolymph assay detected only 22 infections. Regression analysis of QCPCR results and FTM results indicated that the QCPCR assays were effective in quantitating P. marinus infections in oyster tissues.

The genetic variability of 10 Cryptosporidium parvum isolates of human and animal origin was investigated using amplified fragment length polymorphism (AFLP). Analysis of fluorescent dye-labeled amplified products was carried out using an ABI PRISM™ 377 DNA sequencer and ABI PRISM™ GeneScan software. One-hundred and twelve primer combinations were evaluated using a single C. parvum isolate. The patterns generated were highly reproducible. For subsequent study, a subset of 9 primer pairs that yielded 30–90 DNA fragments after the polymerase chain reaction, within the size range of 50–500 bp, was used to screen the 10 C. parvum isolates, including 7 bovine, 1 equine, and 2 of human origin. The animal isolates produced identical fingerprint patterns with every primer combination tested. Of the 2 human isolates tested, 1 of the isolates, passaged in calves, generated the same AFLP DNA banding patterns as the animal isolates, whereas the other isolate, obtained directly from human feces, produced unique patterns. Polymorphism, detected by comparison of the fingerprint patterns of the latter human isolate with the common pattern shared by all other isolates, ranged from 17 to 35% for the 9 primer pairs. The results show that AFLP is a useful method for differentiating C. parvum isolates into 2 distinct genotypes.

Injection of the rat with guinea pig myelin basic protein (MBP) induces an inflammatory demyelination that leads to development of a condition mimicking human multiple sclerosis (MS), including severe depressions in mobility, coordination, and strength in the affected animal. This model was used to observe and compare the anti-inflammatory effects of the intestinal and late migratory phases of infection with Trichinella pseudospiralis on development of MBP-induced, MS-like debilitation in rats. Animal performance was measured in an activity monitor and in a series of physical tests designed to assess animal coordination and strength. Uninfected animals injected with MBP showed declines in mobility, coordination, and strength typical for this model. These changes were similar in rats infected so that the intestinal phase of infection coincided with the peak of MBP-induced debilitation. Rats infected so that the late migratory phase of infection occurred during the period of peak MBP-induced debilitation showed significantly higher performance scores in mobility, coordination and strength compared to the latter 2 groups. These findings demonstrate the potency of the anti-inflammatory effects of elevations in host corticosteroids seen during the migratory phase of infection with T. pseudospiralis.

In this paper, we report a method for isolation of high molecular weight DNA from Leishmania promastigotes. This technique is especially indicated for small-scale purification of DNA suitable for the construction of highly representative genomic libraries. In our protocol, lysis buffer is compatible with RNase treatment, avoiding an additional precipitation step and consequent shearing of DNA. In order to prove the quality of the DNA isolated by this method, a Leishmania braziliensis genomic library was constructed, and an L. braziliensis KMP-11 gene was cloned after screening the library with a heterologous probe.

Archeological deposits from the 19th century company town of Fayette, Michigan were analyzed for evidence of endoparasitic infection in the human population residing in the town between 1867 and 1891. Three privies were associated with upper-income and middle-income neighborhoods; 2 household refuse disposal areas were found in a predominately lower-income immigrant working class neighborhood. Sediment samples from 2 privies associated with dwellings in the middle-income neighborhood were positive for eggs of the human whipworm Trichuris trichiura. The parasite was probably also present among residents of the lower income neighborhood, but the shallow nature of the refuse deposits in that locality precluded preservation of the eggs. Contemporary epidemiologic studies of helminth infections support the belief that T. trichiura may have been a common parasite of 19th century school-age children given the natural inclination of young children to defecate indiscriminately, play freely in the dirt, and eat without washing their hands.

Species of Lecithodesmus (Campulidae) occur almost exclusively in baleen whales throughout a wide geographical distribution. Other campulids occur only in odontocetes and, secondarily, in pinnipeds and the sea otter. Therefore, the ancestor of Lecithodesmus might have either cospeciated with mysticetes during the early divergence of mysticete and odontocete cetaceans or originated later via host switching. We evaluate both possibilities based on a phylogenetic analysis. The ND3 mitochondrial gene sequence of a species of Lecithodesmus was included in a previous partial molecular phylogeny of the Campulidae. Fasciola hepatica and Dicrocoelium dendriticum were used as outgroups. Maximum parsimony, neighbor-joining, and maximum likelihood methods indicated a nonbasal position of Lecithodesmus sp. in the tree, suggesting that the ancestor of Lecithodesmus colonized mysticetes from campulids of odontocetes. This result emphasizes the importance of host-switching processes in the development of the helminth fauna of marine vertebrates, as previously suggested.

Clams (Dosinia exoleta, Ruditapes philippinarum, Venerupis pullastra, Venerupis rhomboideus, Venus verrucosa), mussels (Mytilus galloprovincialis), and oysters (Ostrea edulis) were tested for the presence of Cryptosporidium sp. oocysts using various stain techniques and a commercially available kit containing fluorescein isothiocyanate-conjugated monoclonal antibodies. All molluscs were harvested in northwest Spain (Galicia) except for R. philippinarum, which was from Italy, and 1 of the 6 oyster samples, which was from England. The results showed the presence of Cryptosporidium sp. oocysts in all of the molluscan species destined for human consumption.

The relationship between host size and sex and the abundance, prevalence, species richness, diversity, and total number of parasites in the infracommunities of Sebastes capensis was analyzed. Nine helminth species were obtained from 180 red rockfish captured off Coquimbo Bay, Northern Chile. These included the digeneans Pseudopecoelus sp., Diphtherostomum sp., Derogenes sp., Podocotyloides sp., and Lecithochirium sp., the acanthocephalan Corynosoma sp., and the nematodes Ascarophis sebastodis, Hysterothylacium sp., and Anisakis sp. The abundances of Pseudopecoelus sp. and Anisakis sp. were significantly and positively correlated with the size of S. capensis, while Corynosoma sp. correlated negatively with host size. Only prevalence of Anisakis sp. increased with host size. The number of parasite individuals increased significantly with size of S. capensis. However, species richness and diversity of the infracommunities was not affected by host size. These results suggest that red rockfish >18 cm do not undergo significant ontogenetic changes in their diet. Host sex did not seem to affect abundance and prevalence of infections. The presence of Derogenes sp. and Podocotyloides sp. are new records of digeneans for S. capensis.

The presence of Toxoplasma gondii DNA was detected in sera of acutely infected mice by polymerase chain reaction. Adult mice were inoculated intraperitoneally with 5 × 10³ T. gondii RH strain tachyzoites. Five mice were killed every 3 hr from 3 to 21 hr post infection (PI) and every day from 1 to 7 days PI. Toxoplasma gondii DNA was first detected in 60% of the infected mice 18 hr PI and in 100% of the animals 21 hr PI and from 1 to 7 days PI. No mice survived longer than 7 days.

This report examines the presence of proteolytic activity detected in media collected from in vitro cultures of Giardia intestinalis, and the partial characterization by gelatin-substrate polyacrylamide gel electrophoresis and inhibition studies. Gelatin-substrate polyacrylamide gel electrophoresis revealed 6 bands with proteolytic activity, with estimated molecular weights of 36, 59, 63, 72, 103, and 175 kDa. These bands were not present in the control medium. On the other hand, G. intestinalis trophozoite lysates showed proteolytic bands at 16, 20, 66, 82, 108, and 120 kDa, thus indicating that intracellular proteases could be different from the excretory/secretory (E/S) products. Based on inhibition studies, 2 bands of 59 and 63 kDa were inhibited by iodoacetic acid, indicating the presence of cysteine proteases. Partial inhibition of a band of 36 kDa was found with EDTA, a metal-chelating agent, suggesting the possible presence of metalloproteases. The presence of aspartic and serine proteases were not detected under the assay conditions used. As G. intestinalis E/S may be involved in differentiation mechanisms of the parasite and also be responsible for the mucosal alterations that occur in giardiasis, the characterization of these proteases may facilitate their evaluation as targets in the therapy of the disease.

An analysis of antibody recognition of Trypanosoma cruzi exoantigens by immunoblotting revealed a unique banding pattern that seems to be characteristic of each strain or isolate. Trypomastigote excreted–secreted antigens (TESA) present in supernatants of LLC-MK2 cells infected with 5 strains and 10 isolates of T. cruzi produced 13 different immunoblotting patterns. The same bands were observed when probed with acute-phase Chagas' disease serum or with serum from a rabbit immunized with the repetitive domain of T. cruzi transialidase recombinant protein (anti-shed acute-phase antigens). Three similar patterns were observed with TESA from 3 human isolates that probably belong to the same T. cruzi strain. When clone CL Brener, clone CL-14, and CL parental strain were analyzed, the same bands were observed, although they presented different biological behavior. These results suggest that immunoblotting analysis of TESA may be a useful tool for characterization of T. cruzi strains and isolates.

Flatfish tissue samples exhibiting X-cell pseudotumors were tested with a number of ribosomal DNA (rDNA) general primers in polymerase chain reactions (PCRs). Microsporidian primers resulted in the amplification of an rDNA fragment and molecular phylogenetic analysis indicated that although the organism did not relate closely with any current microsporidian genera, it was most similar to Nucleospora salmonis and branched within the Enterocytozoonidae. Re-examination of the original tissues used for DNA extractions revealed the presence of putative microsporidian spores in PCR-positive samples. These observations reiterate the highly sensitive diagnostic feature of PCR, allowing detection of organisms overlooked by conventional methods and demonstrate the occurrence of rare, coinfecting organisms.

The marine polychaete Nereis (Hediste) diversicolor (Annelida) from shallow water in the Øresund, Denmark, was found to be infected with an actinosporean stage of a myxozoan parasite. The body length of the pyriform actinospore is 12–16 μm and its maximum width is 10–12μm. The spore is triangular in apical view, with the 3 spherical polar capsules distally. The spore is without caudal processes. Eight spores develop in each pansporocyst. Free spores and pansporocysts were found in the musculature and parapodia but not in the intestine. The myxosporean stage in fish is unknown. This is the first record of an actinosporean stage in a marine polychaete, but because marine oligochaetes are rare, compared with polychaetes, the latter are believed to play an important role as invertebrate (alternate) hosts in marine myxozoan life cycles.

The role of oxidative metabolism in resistance to macrocyclic lactones in Haemonchus contortus was examined by measuring activities toward 2 model cytochrome P450 substrates, aldrin and ethoxycoumarin, in a susceptible and a resistant isolate of the parasite. Microsomal preparations from larvae and adults of the 2 isolates showed no differences in levels of NADPH- or cumene hydroperoxide-supported aldrin epoxidase or ethoxycoumarin O-deethylase activities. Intact adult nematodes showed an ability to catalyze the epoxidation of significant amounts of aldrin, although the nature of the enzyme group responsible was unknown. This epoxidase activity was greater in adults of the susceptible isolate. It is apparent that oxidase activities toward the 2 substrates are not enhanced in the resistant isolate, suggesting that the observed resistance to macrocyclic lactones may not involve enhanced oxidative metabolism.

The cystozoites of Sarcocystis muris were infective to other mice after peroral inoculation. They transformed into gamonts and after fertilization underwent sporulation with the production of infectious oocysts/sporocysts in the lamina propria of the small intestine. The present study demonstrated that S. muris possesses both diheteroxenous and dihomoxenous life cycle and can be transmitted by the cannibalism among mice.

Prevalence of antibodies to Toxoplasma gondii was determined in 203 muskoxen (Ovibos moschatus) from 3 geographically distinct areas of northern Canada (near the hamlets of Kugluktuk and Cambridge Bay, Nunavut and Holman, Northwest Territories) by the modified agglutination test (MAT). Antibodies were found in 13 (6.4%) of 203 animals with MAT titers of 1:25 in 2, 1:50 in 7, 1:200 in 2, 1:400 in 1, and 1:800 in 1. The 4 muskoxen with MAT titers ≥1:200 were adult females and were among 10 animals examined from a mainland population near Kugluktuk. The seroprevalence was lower in Victoria Island muskoxen collected near Cambridge Bay (4.6% of 151) and Holman (4.8% of 42). This is the first serologic survey for T. gondii infection in muskoxen.

A time course for the heterogonic development of Strongyloides stercoralis is described and a method for distinguishing the early larval stages of this nematode is proposed. The number of cells in the developing gonad were counted at various time intervals of incubation, along with the percentage of larvae in molt at each interval. The time course of growth of the gonad follows a pattern comparable to that reported for body length in an idealized general nematode. A model for the heterogonic development of S. stercoralis is proposed, which, although similar to other nematode developmental models, is stage specific for S. stercoralis, allowing the otherwise morphologically similar rhabditiform stages (L1, L2) to be distinguished.

The present study was designed to determine the minimum number of Cryptosporidium parvum oocysts capable of producing patent infections in immunosuppressed C57BL/6N adult mice. Sixty-four female mice were divided into 6 groups of 8 mice each, except group 1 that contained 24 mice. Mice in groups 1–3 were immunosuppressed with dexamethasone and inoculated with 1, 5, and 10 oocysts per mouse, respectively. The accuracy of the inoculum size was microscopically confirmed. Mice in groups 4–6 served as controls: they received either only oocyst inoculation (group 4), or immunosuppression (group 5), or no treatments (group 6). Fecal oocyst shedding was monitored daily for each mouse using an indirect immunofluorescent assay. Parasite colonization in the terminal ileum of each mouse was evaluated histologically. Four of 24 mice in group 1 developed patent infections, with a prepatent period of approximately 6 days. All mice in groups 2 and 3 developed patent infections, with prepatent periods ranging from 4 to 7 days. Mice in groups 4–6 remained uninfected. Parasite colonization was observed in the terminal ilea of all mice in groups 1–3 that shed fecal oocysts. The present study experimentally demonstrates that a single viable oocyst can induce patent C. parvum infections in immunosuppressed C57BL/6N adult mice and indicates that this mouse model could be used for the parasite genotype or isolate cloning.

Genetic characterization of a total of 15 Giardia lamblia isolates, 8 from Anhui Province, China (all from purified cysts) and 7 from Seoul, Korea (2 from axenic cultures and 5 from purified cysts), was performed by polymerase chain reaction amplification and sequencing of a 295-bp region near the 5′ end of the small subunit ribosomal DNA (eukaryotic 16S rDNA). Phylogenetic analyses were subsequently conducted using sequence data obtained in this study, as well as sequences published from other Giardia isolates. The maximum parsimony method revealed that G. lamblia isolates from humans in China and Korea are divided into 2 major lineages, assemblages A and B. All 7 Korean isolates were grouped into assemblage A, whereas 4 Chinese isolates were grouped into assemblage A and 4 into assemblage B. Two Giardia microti isolates and 2 dog-derived Giardia isolates also grouped into assemblage B, whereas Giardia ardeae and Giardia muris were unique.

Pulmonate snails (Helisoma anceps) of varying sizes were exposed to 2, 4, 8, or 16 eggs of Halipegus occidualis and observed over several months to determine the duration of the prepatent period. Infection probability was positively correlated with number of eggs ingested. The number of eggs ingested did not, however, have a significant effect on the duration of the prepatent period, presumably because low infectivity of the eggs (15%) dictated that most patent infections arose from a single miracidium. There was a significant, positive correlation between snail length (measured at time of exposure) and duration of prepatent period, suggesting that density-dependent effects are, in part, responsible for the initiation of cercariae production in H. occidualis.

Jirds (Meriones unguiculatus) were infected with 700 infective larvae (L3) of Trichostrongylus colubriformis. The fecal egg counts (epg) were followed regularly until 340 days postinoculation, and worm numbers were determined on necropsy at 315 and 350 days postinoculation. The epg increased to a peak of 2,600 on day 24 postinoculation. Thereafter, the epg showed continuous decrease from day 31 until day 318 postinoculation. The rates of inoculum recovery were 0.29–1.43% on day 315 and 0.142–0.428% on day 350 postinoculation. On day 315 postinoculation, 12 male worms were recovered from 2 of 3 animals necropsied. On day 350 postinoculation, all 6 animals were infected, and 6 female and 4 male worms were recovered. The systemic IgM, IgG, and intestinal mucous IgA responses were significantly higher in infected groups compared with negative control groups necropsied on 315 and 350 days postinoculation (P < 0.001). These results confirm that the T. colubriformis–jird system is a useful model for studies on the various host–parasite relationships of chronic trichostrongylosis.