The pheromone fraction that elutes at Kav 0.64 in gel filtration showed significantly reduced activity for the male's locomotor response when obtained from 10-day-old, or older, female Nippostrongylus brasiliensis. This fraction, obtained from males and females that were admixed prior to extraction, revealed a sex-ratio dependent increase in pheromone activity at 50%:50% male:female according to the male's locomotor response. The female's response to mixed sex pheromone at Kav 0.64 showed no distinct sex-ratio effect. The pheromone that was contained within the female helminths as the Kav 0.64 elutant exhibited little variation in its level, based on collection period studies up to 3 hr. However, exposure of females to male worms altered the amount of pheromone at Kav 0.64 that was recovered from maceration and gel filtration of female helminths, presumably owing to pheromone release.
The effect of storage of Toxoplasma gondii tissue cysts in various NaCl solutions at different temperatures was studied. Tissue cysts from rodent brains were suspended in 0.85%, 2.0%, 3.3%, and 6.0% aqueous NaCl solutions. After storage at 4-20 C for various time intervals, brains were bioassayed in mice for viability of T. gondii. At 4 C, tissue cysts survived for at least 56 days in 0.85% NaCl, for 49 days in 2.0% NaCl, and for 21 days in 3.3% NaCl solutions. At 10 C, tissue cysts survived for at least 21 days in 0.85%, 2.0%, and 3.3% NaCl solutions. At 15 C, tissue cysts survived for at least 21 days in 0.85% NaCl, 14 days in 2.0%, and 3.3% NaCl solutions. At 20 C, tissue cysts survived for 14 days in 0.85% NaCl, 7 days in 2.0% NaCl, and 3 days in 3.3% NaCl solutions. Tissue cysts generally did not survive in 6.0% NaCl solution at any temperature.
Schistosoma mansoni is 1 of the causative agents of schistosomiasis, an endemic disease in 76 countries of the world. The study of its genome, estimated to be 270 Mb, is very important to understanding schistosome biology, the mechanisms of drug resistance, and immune evasion. Repetitive elements constitute more than 40% of the S. mansoni genome and may play a role in the parasite evolution. The retrotransposons Boudicca, a long terminal repeat (LTR), and Perere 03, a non-LTR, are present in a high number in the S. mansoni genome and were localized with the use of fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS). Bacterial artificial chromosomes (BAC) clones containing the retrotransposons Boudicca and Perere 03 were selected by bioinformatic analysis and used as probes in FISH. Using metaphase chromosomes from sporocysts and the FISH and PRINS techniques, we were able to map these retrotransposons. Perere 03 was localized in the euchromatic regions of the short arm of chromosome 2 and Boudicca in the euchromatic regions of the short arm of chromosomes 2 and Z.
The antioxidant used in commercial animal feeds, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin, Santoquin, or EMQ) had an inhibitory effect on larval nematode development. Increasingly fewer larvae were recovered from fecal cultures as the quantity of EMQ fed to infected calves, or mixed with feces from infected calves, was increased from the amount commonly used as a feed preservative (1/4 lb/ton) up to 100 times that amount. For example, the number of Cooperia pectinata larvae recovered from fecal cultures from a calf fed untreated chopped alfalfa hay was 40.4% as compared to 9.5% when 100 x EMQ was mixed with the hay. Similarly, 25 x EMQ resulted in reductions as follows: Trichostrongylus colubriformis from 33.2 to 13.5%, T. axei from 28.3 to 10.8%, and Oesophagostomum radiatum from 45.3 to 4.8%. Comparing commercial pelleted alfalfa hay with and without EMQ showed reductions, varying from 17% for C. punctata to 64% for T. colubriformis. Larval development in feces-vermiculite cultures treated with various concentrations of EMQ showed reductions as follows: T. axei from 27.9 to 0.0% with 500 x, C. pectinata from 22.8 to 0.8% with 30 x, and T. colubriformis from 24.6 to 15.3% with 4 x.
The identification of parasites from ancient cultures expands our list of parasites infective to extant humans. A partially mummified human body from the archeological site of Lapa do Boquete, Minas Gerais State, Brazil, was recently discovered. It was interred between 600 and 1,200 yr ago. Dietary analysis showed that the mummified body was from a society that had a mixed subsistence of agriculture and gathering of wild foods. Coprolites from the body contained numerous helminth eggs. The eggs were identified as those of Echinostoma sp. and hookworm. Hookworm infection in pre-Columbian populations is already established, but this is the first evidence of Echinostoma sp. eggs found in human coprolites. The diagnosis of a true infection, as opposed to false parasitism, is discussed. The possibility of Echinostoma ilocanum infection is discussed, as this is a common species found in humans in the Asiatic region, which could have been introduced in South America in the pre-Columbian period. Alternative possibilities are also considered, including indigenous Brazilian Echinostoma species.
The pharmacokinetic basis for the efficacy of 4-amino-6-trichloroethenyl-1,3-benzenedisulfonamide (L-631,529) against mature Fasciola hepatica was studied in experimentally infected rats. Single oral doses of the sulfonamide from 12.5 to 100 mg/kg were 84-100% effective against 16-20-week-old infections. Efficacy decreased below 12.5 mg/kg as the drug displayed only moderate activity at 6.25 mg/kg. Pharmacokinetic studies using 14C-labeled sulfonamide at 6.25 and 12.5 mg/kg showed that the drug rapidly entered the blood, attained a maximum concentration approximately 4 hr after dosing and then decreased exponentially. Approximately 75% of the circulating drug was in the plasma and the remainder was in the erythrocytes. Drug levels in F. hepatica paralleled those in the blood after a lag in time. Drug concentration in the flukes rose rapidly, peaked 8-12 hr post-administration and decayed exponentially. Gross observations indicated that onset of drug clearance from the flukes appeared to coincide with cessation of feeding by the parasites. The sulfonamide was shown to be a potent inhibitor of rat erythrocyte carbonic anhydrase and virtually complete inhibition occurred 5 to 8 hr after treatment at both 6.25 and 12.5 mg/kg. The inhibition of carbonic anhydrase activity then decreased in parallel with the drug level in the erythrocytes. Calculations indicated that binding of the sulfonamide to carbonic anhydrase in a 1:1 stoichiometry can account for 70-100% of the drug in the erythrocytes. Intravenous administration of 14C-labeled L-631,529 absorbed onto rat red blood cells demonstrated that bound drug was directly incorporated into the parasites. The drug did not disassociate from the red cells into the plasma. The sulfonamide probably reaches the liver flukes via a mechanism which in part involves ingestion of the red cells containing the drug bound to the carbonic anhydrase.
Chagas disease, caused by Trypanosoma cruzi, is a widespread infection in Latin America. Currently, only 2 partially effective and highly toxic drugs, i.e., benznidazole and nifurtimox, are available for the treatment of this disease, and several efforts are underway in the search for better chemotherapeutic agents. Here, we have determined the trypanocidal activity of 2,3-diphenyl-1 ,4-naphthoquinone (DPNQ), a novel quinone derivative. In vitro, DPNQ was highly cytotoxic at a low, micromolar concentration (LD50 = 2.5 microM) against epimastigote, cell-derived trypomastigote, and intracellular amastigote forms of T. cruzi, but not against mammalian cells (LD50 = 130 microM). In vivo studies on the murine model of Chagas disease revealed that DPNQ-treated animals (3 doses of 10 mg/kg/day) showed a significant delay in parasitemia peak and higher (up to 60%) survival rate 70 days post-infection, when compared with the control group (infected, untreated). We also observed a 2-fold decrease in parasitemia between the control group (infected, untreated) and the treated group (infected, treated). No apparent drug toxicity effects were noticed in the control group (uninfected, treated). In addition, we determined that DPNQ is the first competitive inhibitor of T. cruzi lipoamide dehydrogenase (TcLipDH) thus far described. Our results indicate that DPNQ is a promising chemotherapeutic agent against T. cruzi.
A new anthelmintic agent 1,4 Bis(2-diethylaminoethoxy) anthraquinone dihydrochloride (Ro 2-9009) was found to be more active than quinacrine in clearing mice infected with Hymenolepis nana (CD50 per os: 56 mg/kg vs. 156 mg/kg) and more effective than piperazine in removal of S. obvelata from naturally infected mice (CD50 per os: 39 mg/kg vs. 100 mg/kg). Ro 2-9009 was about as effective as thiabendazole and quinacrine and superior to piperazine and trichlorophenol in preventing the migration of Ascaris suum larvae to the lungs of mice but was without effect against Nematospiroides dubius. Ro 2-9009 given for 3 days at 200 mg/kg per os produced complete destrobilization of H. nana in mice and removed the scolices or late-developing cysticerci from approximately 60% of the treated animals. The same dose and treatment schedule completely eliminated adult and immature S. obvelata from 80% of treated mice and significantly reduced the numbers of pinworms in animals not completely cured. In vitro Ro 2-9009 was inactive against N. dubius adults, slightly active against A. suum larvae, and active against adult H. nana. The uptake of Ro 2-9009 could be followed by fluorescence microscopy and all of the helminths tested absorbed the compound with varying degrees of affinity although drug absorption was not directly related to removal of the worms in vivo or to death in vitro.
It is generally accepted that metabolic changes of different intensity take place during the transformations in the life cycle of Trypanosomo cntzi, a human parasite protozoan, and that such changes are especially important during metacyclogenesis (Wood and Schiller, 1975, Experimental Parasitology 38: 202-2071' C6ceres and Fernandes, 1976, Revista Brasileira de Biologia 36: 397-410; Contreras et al., 1985, Molecular and Biochemical Parasitology l4z 83-96). The study of these metabolic changes during the transformation process deserves special attention because the metacyclic forms are involved in the infection mechanism.
A Schistosoma mansoni cercarial cDNA expression library, constructed in lambda gt11, was screened using the IgG fraction of sera taken from rabbits vaccinated with irradiated cercariae. A positive cDNA clone (1,431 base pairs) was selected and characterized. The amino acid sequence predicted from the cDNA sequence identified a polypeptide of 363 amino acids that showed significant homology to different family members of the enzyme fructose-1,6-bisphosphate aldolase (EC 18.104.22.168). The identity was 66% and 65% with human C and A isoenzymes, respectively. Active sites and substrate-binding determinant analysis suggest that the isolated enzyme in terms of function resembles type A aldolase. The recombinant protein expressed in the vector pGEX-2T was found to be active enzymatically. Antibodies raised against the purified recombinant protein recognized a 40-kDa band in extracts from cercariae, schistosomula (5 and 25 days), adult worms, and eggs. Using immunocytochemistry, aldolase localized to the tegumental region of the adult worms.
The Kav 1.0 pheromone from incubation of mouse-adapted females of Nippostrongylus brasiliensis was soluble in organic solvents, as shown by bioassay of males and presence of a peak at Kav 1.0 during gel filtration. Recovery of the biological activity from the females was enhanced when the Kav 1.0 component was purified by organic extraction-gel filtration, C-18 cartridge separation with 50% methanol, C-18 cartridge-gel filtration, and C-18 cartridge-TLC. The activity recovered from organic extraction, TLC, or gel filtration of solutions in which females had been incubated was less than the above combined techniques. Reverse-phase cartridges retained activity of 20,000 female-hr of incubation without loss and enabled storage of the pheromone component for 3 days at 4 C. The HPLC separation of incubate from females revealed an active peak(s) with a 4- to 5-min retention time. This peak(s) was present also in regions containing activity from other purification procedures.
Biological characteristics of Trichinella isolate (wolverine: 55 degrees 00'N, 100 degrees 00'W, 1979) were established in Crl: COBS CFW (SW) mice. Comparison of the wolverine isolate's biological characteristics with another Trichinella isolate (polar bear), both from closely related geographic areas, revealed there were stable and reproducible genetic differences between isolates. Differences were most pronounced for degree of infections causing a 50% mortality of mice, larval production by females in vitro, reproductive capacity indices, and survival of muscle larvae. Species and strain of host altered characteristics of the wolverine isolate such as worm position, sex ratios, % recovery, larval production by females in vitro, and reproductive capacity indices. Genetic differences between isolates of Trichinella and interaction with host genetics raises interesting questions on Trichinella speciation. Differences reported here, are best interpreted for the time being at least, as part of the normal biological variability of the species Trichinella spiralis.
Stephanurus dentatus was grown to advanced parasitic stages in a new static in vitro system, free of bacteria and fungi. Criteria for evaluation were based on anatomical characters of the head, stoma, excretory system, and intestine. Descriptions of these characters and observations on morphogenesis not previously reported from in vivo specimens are given in detail. Development to late fourth stage was obtained in cultures consisting of primary monolayers of swine kidney cells overlaid with the following medium (KW-1S): yeast extract (BBL), 112.5 mg; Bacto-peptone (Difco), 140.65 mg; dextrose (BBL), 140.65 mg; swine serum, 50 ml; NCTC 109, 50 ml; and antibiotics to give a final concentration of 1,000 units penicillin G potassium, 1 mg dihydrostreptomycin, and 10 μg Amphotericin B per ml of medium. When bovine serum was substituted (KW-1) development halted at mid fourth stage. In addition, with both fluid media, the following were observed: only larvae in fourth stage fed on the kidney cell cultures; a definite feeding pattern occurred; larvae fed on rolled cell layers (presumably dead) as well as living cells; digestion and egestion took place, and there was a correlation between feeding behavior and anatomical development of the stoma, excretory system, and intestine. In either medium, without the cell culture, only early fourth stage was attained and yields of this phase of development were reduced to one-fortieth. Media KW-1 and KW-1S evolved from attempts to culture S. dentatus in modifications of Pitts' and Ball's (1953) medium, or in NCTC 109 (McQuilkin et al., 1957) used alone or supplemented with calf serum. The prototype cell-free media, one of which was also supplemented with nonliving tissue, had the following effects on development: (1) the most advanced stage attained was early fourth stage; (2) larvae advanced to late parasitic third stage in media supplemented with either NCTC 109 or serum; (3) development proceeded to early fourth stage when both NCTC 109 and serum were present; (4) a more rapid rate of development to third molt and early fourth stage ensued when NCTC 109 and serum were present at concentrations of 50%, each; and (5) although fourth-stage larvae ingested nonliving tissues when available, development was not enhanced.
The expression of surface lipophosphoglycan (LPG) and the lipophosphoglycan-associated kinetoplastid membrane protein (KMP)-11 was studied in the strain AG83 of Leishmania donovani in axenic culture. The expression of LPG and KMP-11 decreased along with parasite virulence as a function of the time of the subculture.
Abstract : An experimental excystation assay was used to test the potential species isolating effects of excystation signaling among gregarines. Oocysts of a single gregarine species, Blabericola migrator , were tested for activation, excystation, and sporozoite motility by using intestinal extracts from 11 species of cockroaches representing a cohesive phylogeny of 7 genera, 3 subfamilies, and 2 families of Blattodea. Sporozoite activation, excystation, and motility were observed for all excystation assay replications using intestinal fluid from blaberid hosts, but delayed activation or excystation was observed for all assay replications using intestinal fluid from hosts in the family Blattidae. The results illustrate a trend toward a generalized excystation signal among gregarines that is conserved across the host clade at a subfamily or family level but that is unlikely to play a significant role as a species-isolating mechanism among sibling gregarine species.
Parasites are intimately connected to the host in which they live, and some may be affected by the polluted environment of their host. The present study describes the effect of a steroid hormone (11-ketotestosterone) on the sex ratio of the invasive hematophagous nematode Anguillicola crassus Kuwahara, Niimi & Itagaki, 1974, when experimentally injected to European eels, Anguilla anguilla. Our results showed that this steroid induced a significant male-biased ratio in the nematode A. crassus infrapopulations, suggesting that the presence of endocrine disruptors in the environment may lead to skewed sex ratios among parasites.
Entamoeba histolytica is a protozoan parasite that can invade the intestinal mucosa. Infection induces production of secretory immunoglobulin A (SIgA) antibodies that can diminish the adhesion between E. histolytica trophozoites and epithelial cells in vitro and reduce the rate of new infections in children. SIgA antibodies produced by asymptomatic cyst carriers could play a protective role against the damage caused by E. histolytica. To identify membrane antigens capable of inducing SIgA response in E. histolytica cyst carriers, salivary SIgA antibodies were confronted with blotted plasma membrane proteins from amebae. A surface 115-kDa ameba protein was recognized by 62% of the human SIgA antibodies tested. The 115-kDa protein is not a mannose-containing glycoprotein and has no protease activity. Rabbit anti-115-kDa protein antibodies were capable of reducing erythrophagocytosis but were unable to protect culture cells from the cytopathic damage caused by E. histolytica. However, anti-115-kDa protein antibodies induced surface receptor redistribution.
Oogram studies have been carried out on mice, hamsters, and Cebus monkeys experimentally infected with Schistosoma mansoni and treated with R.D. 12,869 (6-chloro-5-β-diethylamino-ethylamino-8-methylquinoline). In mice, treated at the dose levels of 120, 60, and 30 mg/kg/day x 5, per os, oogram changes occurred in all animals and 100% of the schistosomes were shifted to the liver. At the dose level of 15 mg/kg/day x 5, 66.6% of the mice presented oogram changes and 82.1% of the schistosomes were found in the liver. No antischistosomal activity could be detected in hamsters dosed with R.D. 12,869 at the level of 120 mg/kg/day x 5, administered per os and intraperitoneally. Parasitological cure was achieved in Cebus monkeys treated with 120, 60, and 30 mg/kg/day x 5, per os. The encouraging results obtained in laboratory animals strongly suggest the desirability of conducting pharmacological clinical studies with this new antischistosomal agent.
An average of only 2.8% of the infective larvae of a rat strain (RS) of N. brasiliensis developed to maturity in adult Syrian hamsters. Prolonged serial passage of the rat parasite in hamsters resulted in a population, called hamster strain (HS III) which had an average development of 44.9% in hamsters. This level of development was reached by 30 passages and was maintained through 90 additional generations in hamsters. HS III was still highly infective for rats, although perhaps slightly less so than RS for rats. Furthermore, after 39 generations in hamsters followed by 40 passages through rats, the high infectivity of HS III for hamsters was unchanged. Development of both strains was greater in mature male than female hamsters but with the RS the difference was 16.7-fold as compared to only 1.2-fold with HS III. No sex difference was apparent in immature hamsters infected with either strain. Mature hamsters of both sexes were significantly less susceptible than immatures to RS, whereas HS III appeared to develop equally well in hamsters that ranged in age from 2 to 44 weeks. Substantial numbers of larvae were found in the lungs of RS-infected rats and HS-infected hamsters on the 2nd day of infection, but on the 4th, 6th, and 10th days most of the worms recovered were in the gut. By comparison, the vast majority of worms found in RS-infected hamsters, at all times, were third-stage larvae in the lungs. Female worms were significantly more numerous than males in rats that received RS, but in hamsters given RS the reverse was found. However, in hamsters, as well as rats infected with HS III, female worms predominated. It is suggested that the adaptation of N. brasiliensis of rats to the hamster occurs through the process of natural selection.
In a study on the genetics of resistance to schistosomiasis in WEHI 129/J mice, susceptibility to either Schistosoma mansoni or Schistosoma japonicum was shown to be unequivocally dominant in F1 hybrid crosses between genetically resistant WEHI 129/J and susceptible BALB/c mice. The operation of only 1 or 2 genes in the expression of resistance to S. mansoni was suggested by backcross analysis. Thus, approximately 25% of (BALB/c x WEHI 129/J) F1 x WEHI 129/J mice were resistant to S. mansoni infection, whereas resistance was manifest in approximately 50% of WEHI 129/J mice. The data are consistent with resistance being controlled by 1 recessive gene having 50% penetrance. We also report that 129/J mice obtained directly from the Jackson Laboratories (Bar Harbor, Maine) (designated JAX 129/J), differ from locally bred WEHI 129/J in being entirely susceptible to S. mansoni infection. However, both WEHI 129/J and JAX 129/J are relatively resistant to S. japonicum infection.
A portion of mitochondrial 12S rDNA sequences (337-355 base pairs) and 63 morphological characters of 36 hard-tick species belonging to 7 genera were analyzed to determine the phylogenetic relationships among groups and species of Rhipicephalus and between the genera Rhipicephalus and Boophilus. Molecular and morphological data sets were first examined separately. The molecular data were analyzed by maximum parsimony (MP), maximum likelihood, and neighbor-joining distance methods; the morphological data were analyzed by MP After their level of congruence was evaluated by a partition homogeneity test, all characters were combined and analyzed by MP. The branches of the tree obtained by combining the data sets were better resolved than those of the trees inferred from the separate analyses. Boophilus is monophyletic and arose within Rhipicephalus. Boophilus species clustered with species of the Rhipicephalus evertsi group. Most of the clustering within Rhipicephalus was, however, consistent with previous classifications based on morphological data. Morphological characters were traced on the molecular reconstruction in order to identify characters diagnostic for monophyletic clades. Within the Rhipicephalus sanguineus complex, the sequences of specimens morphologically identified as Rhipicephalus turanicus were characterized by a high level of variability, indicating that R. turanicus-like morphology may cover a spectrum of distinct species.
Given the constraints of classical diagnostic methods, i.e., morphological and isoenzymatic studies of proglottids, a polymerase chain reaction test complemented with restriction enzyme analysis has been modified by redesigning one of the primers to reduce nonspecific amplifications experienced when using field samples. The use of these new, highly cestode-specific primers and the restriction enzyme Ddel led to the development of a diagnostic assay that clearly distinguishes between Taenia saginata and T. solium proglottids in field samples. This assay confirms the presence of T. saginata in Ecuador. DNA amplification of some of these taeniids showed different patterns, suggesting the possibility that strain differences exist. These results demonstrate the need for development of useful molecular assays as reliable tools for epidemiological studies on cestodes.
Abstract Molecular genetic variability of 12S rRNA gene on the basis of partial primary sequence, and in silico predicted secondary structures among Dermacentor reticulatus Fabricius ticks was studied in the Chernobyl Nuclear Power Plant Exclusion Zone. A total of 20, 20, and 25 ethanol-preserved specimens previously collected at 3 sites with 0.76, 1.91, and 4.5 mSv/hr ionising radiation background were examined. The primary sequence analysis generated 4 haplotypes defined by 3 polymorphic sites. The most common haplotype 1 was found in all 3 locations, representing 86.2% of all sampled individuals. Haplotype 4 (10.8%) was detected at the 1.91 and 4.50 mSv/hr sites. The unique haplotypes 2 (1.5%) and 3 (1.5%) were detected only at the 1.91 and 4.50 mSv/hr sites, respectively. The haplotype diversity, nucleotide diversity, and pairwise nucleotide differences for 2 tick populations at the 1.90 and 4.50 mSv/hr sites were 0.279, 0.00085, 0.289, and 0.397, 0.00122, 0. 413, respectively. No polymorphism was detected in ticks collected at the 0.76 mSv/hr site. The primary sequences of 12S rRNA were folded into the secondary structures, and the free energy of haplotypes was calculated. The free energy at 37 C (ΔG) of the nonmutant haplotype 1 and the mutant haplotypes 2, 3 and 4 were -45.79, -44.17, -39.56, and -45.79 kcal/mol, respectively. Considering the correlation between structural profile similarity of 12S rRNA and point mutations, haplotypes 1 and 4 have similar secondary structure profiles and have received 0.999219 similarity score in the cluster tree. The unique haplotypes 2 and 3 have differences in the secondary structure in comparison with the haplotypes 1 and 4, and the similarity scores were 0.914747 and 0.169431, respectively. Further studies using more genetic markers are warranted to ascertain the genetic variability and population genetic structure within D. reticulatus tick populations in the Chernobyl Nuclear Power Plant Exclusion Zone, and to resolve their vector capacity.
Rhinonastes pseudocapsaloideum n. sp. (Dactylogyridae, Ancyrocephalinae) is described from the nasal cavity of Prochilodus nigricans Agassiz (Cypriniformes, Prochilodontidae) in Brazil. Rhinonastes n. gen. is proposed for species possessing a dextroventral genital pore, a bilobed testis, a ventral C-shaped ovary lying between the 2 testicular lobes, and a disc-shaped haptor armed with a ventral anchor-bar complex and 14 hooks.
Grossly visible sarcocysts were seen in the skeletal muscles of 1 of 12 13-lined ground squirrels, Spermophilus tridecemlineatus tridecemlineatus, collected in Nebraska. The tissue cyst wall was up to 5.0 microm thick and contained spikelike projections. Transmission electron microscopy of tissue cysts revealed they were similar to Sarcocystis campestris Cawthorn, Wobeser, and Gajadhar, 1983, previously known only from experimental infections in Richardson's ground squirrel Spermophilus richardsonii. Prominent electron-dense bodies were observed lining the microfilaments present in the spikelike projections of the sarcocyst wall. This is the first report of S. campestris in a natural intermediate host and the first report of this parasite outside of Saskatoon, Canada.
Adults of 4 of the 6 species constituting the subgenus Carios and of 3 of the 4 species constituting the subgenus Chiropterargas were studied by scanning electron microscopy. All species parasitize Old World cave-dwelling insectivorous bats (Microchiroptera). The anterior pit setae number 10 in Carios and 10 or 11 in Chiropterargas. In most Carios, the setiform seta is replaced by a second serrate seta. In 2 of the 3 studied Chiropterargas species, 1 of the 2 grooved setae is exceptionally long. Porose setae number 3 in Carios and 3 or 4 in Chiropterargas. The Haller's organ roof in both subgenera is solid, lacking perforations; the aperture is narrowly transverse in Carios, irregularly wide or wide and transverse in Chiropterargas; uniquely, 1 or 2 sensilla protrude from the aperture of Chiropterargas species. The protruding sensilla and long grooved seta of Chiropterargas suggest a probably distinctive sensory-behavior pattern common to these ticks. Other morphological characters are discussed and compared to show relationships between these 2 subgenera and the subgenera Argas and Persicargas and distinctive characters present only in adult and/or larval Carios and Chiropterargas.
Length measurements of Trichinella spiralis were made daily (from 5 to 13 days postinoculation) on larvae recovered from adult females from the small intestine, abdominal cavity fluid, thoracic duct lymph, blood, and the skeletal muscle of male albino rats. Mean length measurements obtained were as follows: newly born larvae (95 µ); larvae from abdominal fluid (111 µ); larvae from lymph (105 µ); larvae from blood (115 µ); and larvae from skeletal muscle on days 5 to 7 postinoculation (106 µ). Analysis of the above data using Student's t test indicated significant differences (P 0.10). Using a 2-factor analysis of variance, effects of the two main migratory pathways (blood vs. abdominal fluid), days postinoculation, and individual rats were significant (P
Cesium-137, becoming a more readily available ionizing gamma radiation source for laboratory use, was shown to effectively attenuate Schistosoma mansoni cercariae for vaccine production. In parallel comparison studies with the murine model, cesium-137 attenuated cercariae consistently afforded better (P greater than 0.05) protection than did the cobalt-60 prepared vaccine. Dose-response data indicated that the optimal total irradiation with cesium-137 was between 45 and 50 Krad.
Currently available candidate vaccines against schistosomiasis elicit only partial protection. In addition, the type of immune response that could lead to the highest level of protection against schistosomes has not yet been described. Thus, efforts should be made in both the identification of novel proteins essential for the parasite cycle and in the modulation of immune responses against these novel candidates through the combined use of immunomodulatory molecules. Several parasites have 14-3-3 proteins, and these proteins are known to play a key role in parasite biology. In the present work, we report the isolation and characterization of a new 14-3-3 gene from Schistosoma bovis and offer new information regarding the genetic structure of the gene. In addition, we have produced the corresponding recombinant protein. Finally, we describe the immune responses elicited by this protein when combined with 4 different immunomodulators in immunized mice.
14C-praziquantel was rapidly taken up by Schistosoma mansoni, Fasciola hepatica, Hymenolepis nana, and isolated strobilocerci of Taenia taeniaeformis. Schistosoma mansoni lost praziquantel rapidly to drug-free medium. Chromatography of extracts prepared after incubation of S. mansoni and H. nana yielded no indication that praziquantel was metabolized. Autoradiography revealed a uniform distribution of praziquantel throughout the tissues of S. mansoni and H. nana. Uptake was considerably slower in the nematode Heterakis spumosa and apparently via the oral route.
Metacercariae, in vivo, absorbed more glucose-U-14C and incorporated more radioactivity into glycogen than did in vitro forms. Metacercariae, in vivo and in vitro, incorporated glucose-U-14C carbon into alanine, glutamic acid, glutamine, proline, and alpha ketoglutaric, citric, fumaric, lactic, pyruvic, and succinic acids. The labeling patterns obtained indicate that the metacercariae of Clinostomum campanulatum possess the components of a TCA cycle.
The incorporation of carbon from D-glucose-U-14C by Cooperia punctata grown in vitro in Ae medium was studied. Results indicate that 2-week-old cultures of C. punctata, consisting mainly of 4th-stage worms, incorporated carbon from glucose of the Ae medium. Of the 14C of glucose origin recovered from the worms, 42% was found in protein, 24% in glycogen, 21% in the nonlipid fraction, 2% in lipids, and 11% in the nucleic acid fraction. Carbon-14 assay of the amino acids revealed high activity in aspartic acid (21%), alanine (28%), and glutamic acid (28%), while lower activity was distributed among 9 other amino acids and 2 unidentified ninhydrin-positive spots. C. punctata stores some carbohydrate as glycogen, and glycogenesis occurs utilizing carbon provided as glucose in Ae medium.
Following a 60 min in vitro incubation of Schistosoma mansoni with D-[14C-U]-glucose 76% of the radiocarbon was incorporated into metabolic end products and excreted back into the medium. In the presence of 5-HT uptake of glucose increased 61%; excreted end products accounted for 87% of the radiocarbon, indicating increased levels of energy utilization. Substantial amounts of radiolabelled carbon from D-[14C-U-]-glucose were incorporated into glycogen, lipids, amino acids and proteins, suggesting the utilization of glucose carbon in the biosynthetic processes of the parasite. Incorporation of glucose carbon was diminished in the presence of 5-HT, indicating the priority of energy generation over biosynthesis to meet the demands of increased muscular activity.
Mature male worms from fasted animals absorbed more glucose-U-C14 and incorporated more radioactivity into glycogen than did female worms. The rate of glycogenolysis was slower in males than in females, based on the rate of disappearance of radioactivity in glycogen, when worms, after short incubations in the isotope, were reincubated in buffer without glucose. Moniliformis incorporated glucose-U-C14 carbon into alanine, aspartic acid, serine, and malic, lactic, succinic, and fumaric acids, but not into citric, aconitic, glutamic acids, or glycine. Other radioactive compounds presumed to be glycolytic intermediates were also found.
Ultrastructural and quantitative studies were conducted to determine the in vitro effects of mannan and cytochalasin B (CB) on the transport of horseradish peroxidase (HRP) and [14C]sucrose by epimastigotes of Trypanosoma cruzi (strain Y). Time-dependent changes in HRP uptake were observed in cells incubated with the actin inhibitor CB. After 60 min incubation in CB, HRP and sucrose uptakes were inhibited by 48 +/- 15.4% and 16.5 +/- 3.96%, respectively. Morphological changed included HRP reaction product on the cell surface and a reduction in the number of HRP-positive reservosomes when compared to controls. After 120 min incubation, no inhibition was measured for either molecule. However, electron microscopy revealed HRP reaction product on the surface of the cells and in the cytosol. Also, perturbation of the plasma membrane was evident, suggesting that CB compromised the integrity of the plasma membrane, allowing HRP and sucrose to diffuse into the cytosol, giving misleading quantitative results. Mannan displayed a concentration-dependent inhibitory effect on HRP uptake but had little effect on sucrose uptake. Electron microscopic analysis revealed no change in the number of reservosomes per cell but reduction in the amount of HRP in reservosomes concomitant with mannan concentration. These results suggest that T. cruzi epimastigotes transport HRP by receptor-mediated and fluid-phase pinocytosis.
We are attempting to design a simpler assay based on synthetic or recombinant antigens to replace the labor-intensive enzyme-linked immunoelectrotransfer blot (EITB-C), which is currently used to diagnose Taenia solium cysticercosis. From the lentil lectin-bound fraction of cyst glycoproteins (the LLGP fraction used in the EITB-C), we previously identified and purified 2 related polypeptides of 14- and 18-kDa that demonstrated diagnostic usefulness. Using degenerate oligonucleotide primers corresponding to amino acid sequences of these polypeptides and a cDNA library prepared from T. solium cysticerci, we amplified cDNA clones that represent the 14- and 18-kDa polypeptides. These clones share sequence homology at the nucleotide and amino acid levels. Synthetic polypeptides that represented the full-length, mature proteins (sTS14 and sTS18) were assessed for serologic potential using an ELISA. sTS14, but not sTS18, demonstrated utility as a diagnostic antigen. sTS14 was recognized by antibodies in a majority of the sera from patients with cysticercosis and none of the sera from persons with other helminth infections or uninfected human sera. Furthermore, polyclonal antibodies to sTS14 reacted with 6 discrete proteins present in the LLGP cyst fraction, suggesting that TS14 is a subunit of other previously described antigens used for diagnosing cysticercosis.
Three experiments are described. In the first, mice were injected twice into the footpads with an antigen prepared from larvae and mixed in equal volume with Freund's complete adjuvant. After an infection with T. spiralis, these mice harbored significantly fewer adult worms than noninjected controls thereby proving the immunizing effectiveness of the two antigen injections. In the last 2 experiments, donor mice, treated as the experimentais in the first experiment, were killed to collect spleen cells for transfer into recipients, which were challenged with T. spiralis 1, 3, 7, 14, or 21 days after transfer of the cells. The counts of adult worms in these recipients and their respective controls were not significantly different in the case of the 1-day and 3-day groups, but the 7, 14, and 21-day recipients harbored significantly fewer worms than their controls. On the basis of these results, it is concluded that the 2 injections of this antigen preparation produced effects that caused a significant expulsion of worms after challenge, and that the spleen cells from mice treated in this way were responsible for a similar effect noted in recipients challenged as early as 7 days after cell transfer.
The purpose of this study was to measure the incorporation of intraperitoneally administered ¹⁴C tryptophan into the free pool fraction of a number of organs in voles (Microtus montanus) chronically infected with Trypanosoma brucei gambiense (Wellcome TS strain). Infected voles develop a low, fluctuating parasitemia and exhibit pronounced lethargy and ataxia; they therefore appear to represent a good experimental model for the study of the human infection. The results obtained suggest that free pool tryptophan may be reduced in the organs of trypanosome infected voles as a result of either decreased uptake from the blood or stimulated incorporation into protein.