Characterization of Otostrongylus circumlitus from Pacific harbor and northern elephant seals.

Otostrongylus circumlitus (Railliet, 1899) from Pacific harbor seals (Phoca vitulina richardsi) and northern elephant seals (Mirounga angustirostris) were examined using morphological and molecular methods to determine whether northern elephant seals along the central California coast are infected by the same species of Otostrongylus as are Pacific harbor seals in the same area. Fixed nematodes were examined and measured using light microscopy. The polymerase chain reaction (PCR) was used to amplify and sequence the second internal transcribed spacer (ITS-2) and D3 expansion (26S) regions of ribosomal DNA of O. circumlitus from Pacific harbor and northern elephant seals. The ITS-2 region was also amplified from Parafilaroides sp. from the Pacific harbor seal, northern elephant seal, and California sea lion (Zalophus californianus) and used for restriction fragment length polymorphism (RFLP) analysis. Morphologically, it was not possible to distinguish O. circumlitus from Pacific harbor and northern elephant seals, and over a consensus length of 443 base pairs (bp) for ITS-2 and 321 bp for D3 the sequences of O. circumlitus from both hosts were identical. With the PCR-RFLP assay, it was possible to distinguish O. circumlitus from Parafilaroides sp. The results suggest that O. circumlitus is the same species in Pacific harbor and northern elephant seals, and molecular methods make it possible to distinguish this nematode from related nematodes.

Venereal worms: sexually transmitted nematodes in the decorated cricket.

The nematode, Mehdinema alii, occurs in the alimentary canal of the decorated cricket Gryllodes sigillatus. Adult nematodes occur primarily in the hindgut of mature male crickets, whereas juvenile nematodes are found in the genital chambers of mature male and female crickets. Here, we present experimental evidence for the venereal transmission of M. alii in G. sigillatus. Infectivity experiments were conducted to test for transmission via oral-fecal contamination, same-sex contact, and copulation. The infective dauers of the nematode are transferred from male to female crickets during copulation. Adult female crickets harboring infective dauers subsequently transfer the nematode to their next mates. Thus, M. alii is transmitted sexually during copulation.

Molecular systematics of Mesocestoides sPP (cestoda: mesocestoididae) from domestic dogs (Canis familiaris) and coyotes (Canis latrans).

The genus Mesocestoides Vaillant, 1863 includes tapeworms of uncertain phylogenetic affinities and with poorly defined life histories. We previously documented 11 cases of peritoneal cestodiasis in dogs (Canis familiaris L.) in western North America caused by metacestodes of Mesocestoides spp. In the current study, DNA sequences were obtained from metacestodes collected from these dogs (n = 10), as well as proglottids from dogs (n = 3) and coyotes (Canis latrans Say, 1823 [n = 2]), and tetrathyridia representing laboratory isolates of M. corti (n = 3), and these data were analyzed phylogenetically. Two nuclear genetic markers, 18S ribosomal DNA and the second internal-transcribed spacer (ITS 2), were sequenced. Phylogenetic analysis of the 18S rDNA data recovered a monophyletic group composed of all samples of Mesocestoides spp., distinct from closely related outgroup taxa (Amurotaenia Akhmerov, 1941 and Tetrabothrius Rudolphi, 1819). Initial analysis of the ITS 2 data resolved 3 clades within Mesocestoides. Two proglottids from dogs formed a basal clade, a second clade was represented by tetrathyridial isolates, and a third clade included all other samples. Interpretation of these data from an apomorphy-based perspective identified 6 evolutionary lineages. We also assessed whether metacestodes from dogs (n = 4) are capable of asexual proliferation in laboratory mice. One tetrathyridial and 2 acephalic isolates from dogs proliferated asexually. Further investigation is warranted to determine which of the lineages represent distinct species and to determine the life history strategies of Mesocestoides spp.

Arginine kinase and phosphoarginine, a functional phosphagen, in the rhabditoid nematode steinernema carpocapsae.

Moderate activity of arginine kinase was found in Steinernema carpocapsae, an entomopathogenic nematode. In the forward reaction, 4.60 and 3.12 micromol ATP/min/mg protein was produced in infectious third-stage juveniles (J3s) and adult nematodes, respectively. For the reverse reaction, 3.20 and 2.27 micromol phosphoarginine/min/mg protein was produced by J3s and adults, respectively. The K(m)s for phosphoarginine and ADP were 0.73 and 0.42 mM, respectively, in the forward reaction, whereas in the reverse reaction, the K(m)s were 0.37 and 2.35 mM for arginine and ATP, respectively, for the enzyme from J3s. The pH optimum for the forward reaction was 7.2 and 7.3 in J3s and adults, respectively. The pH optimum was elevated for the reverse reaction, 7.8 and 7.9-8.5 in J3s and adults, respectively. In the J3s, the in vitro optima for arginine kinase activity was correlated with the in vivo tissue pH in hypoxic (6.9) and aerobic (7.5) J3s estimated by in vivo flow 31P-NMR.

Morphological, molecular and biological characterization of Mehdinema alii (Nematoda: Diplogasterida) from the decorated cricket (Gryllodes sigillatus).

The nematode Mehdinema alii was recovered from the decorated cricket Gryllodes sigillatus (Walker). Morphometric comparisons are presented from 3 populations. The nematode is characterized by dense arrays of spines on the cuticle of the anterior half of the body and a highly elongate, tubular stoma with a dorsal denticle in the glottoid region. Females have a protruding vulva. Young females are amphidelphic, but the anterior ovary disappears in older females bearing multiple developing juveniles. The male is monorchic with asymmetrically placed genital papillae, distally fused spicules, and a highly complex gubernaculum bearing 2 cuticularized thorns that protrude through a separate, postcloacal opening. Adult nematodes are located primarily in the hindgut, whereas juveniles or dauers occur mainly in the genital chamber of both male and female crickets. Male crickets are significantly more likely to be infected than females. This male-biased infection may be linked to the venereal transmission mechanism of the dauers. Although morphologically unusual in many respects, placement of M. alii in Diplogasterida is supported by both the morphology of the anterior digestive tract as well as analysis of its 18S rDNA sequence. These sequence data suggest that M. alii groups most closely with members of the Cylindrocorporidae.

Diagnostic procedures and treatment of eleven dogs with peritoneal infections caused by Mesocestoides spp.

An 8-year-old spayed Schnauzer with a distended abdomen was examined because of straining to urinate and suspected urinary tract infection. Abdominal radiography revealed a ground-glass appearance, and ultrasonography revealed numerous cystic structures in the peritoneal cavity. Examination of an aspirate of abdominal fluid revealed tissues consistent with metacestodes. Tissues were definitively identified as Mesocestoides spp on the basis of polymerase chain reaction amplification of restriction fragment length polymorphisms. The dog required several courses of treatment with fenbendazole to eliminate the infection. This was 1 of 11 dogs infected with Mesocestoides metacestodes. Treatment involving the use of praziquantel and albendazole were ineffective, but fenbendazole successfully cleared Mesocestoides infections in 5 of 6 dogs.

[Effects of temperature on the viability and infectivity of preparasitic larvae of Romanomermis yuanenesis].

Romanomermis yuanenesis (Mermithidae: Nematoda) was found in Henan, China (Song and Peng, 1987), which has a broad host range in Culicinae mosquito and has been used successfully in field test for control of culex tritaeniorhynchus, culex fatigans and Aedes albopictus in Sichuan, Yunnan, Guangxi and Henan Provinces. This study was attempted to determine the viability and infectivity of preparasitic larvae in various temperatures. The cultures containing R. yuanenesis eggs were flooded 2h with distilled water, filtered and blocked with 1% agarose. Put the filter paper into water, then the motile preparasites separated from the unhatched eggs and got through the agarose membrane into water. About 200ml water containing preparasites free from eggs were held at 26 degrees C-28 degrees C, 16 degrees C-18 degrees C and -2 degrees C to 2 degrees C for test. The motility or lack of motility was used as the criterion to distinguish the living and dead nematodes. The rate of infection of mosquitoes and the rate of parasitism of nematodes were used to show the infectivity of the preserved preparasites. The results showed that at -2 degrees C to 2 degrees C, more than 90% of preparasitic larvae of R. yuanenesis survived for 8 days and the rate of mosquito infection was 87.5% to 100%, but at 26 degrees C-28 degrees C and 16 degrees C-18 degrees C the survival times of 90% preparasites were only 24 hours and 48 hours respectively. It indicates the low temperature preservation may prolong the survival time and keep the infectivity of these preparasitic larvae.

Characterization of Key Glycolytic and Oxidative Enzymes in Steinernema carpocapsae.

The enzyme activities of isocitrate dehydrogenase (ICDH, NADP-specific), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), phosphoenolpyruvate carboxykinase (PEPCK), phosphofructokinase (PFK), pyruvate kinase (PK), and fructose-l,6-bisphosphatase (FBPase) were studied in the third-stage juveniles of Steinernema carpocapsae. Reaction requirements, pH optima, substrate and cofactor kinetic constants were similar to those reported previously from other parasitic helminths with the exception of LDH, which was unstable and could not be characterized for specific activity and kinetic constants. The respective pH optima were 7.5 for ICDH, 8.8 for MDH, 6.5 for PEPCK, 7.3 for PFK, 7.2 for PK, and 7.5 for FBPase. The specific activities for ICDH, MDH, PEPCK, PFK, PK, and FBPase at pH 7.5 were 4.8, 1,300, 22, 25, 35, and 6.8 (nmoles substrate min(1) mg protein(1)), respectively. In summary, the infective juveniles of S. carpocapsae display the metabolism typical of a facultative aerobe.

High energy phosphate metabolites observed by NMR in infective larvae of Haemonchus contortus.

Phosphorus resonances consistent with phosphoarginine and ATP were observed in the in vivo 31P-nuclear magnetic resonance (NMR) spectra of infective larvae of Haemonchus contortus. The level of phosphoarginine quickly declined when nematode suspensions were purged with nitrogen and was restored upon return to aerobic conditions. Saturation transfer NMR demonstrated forward and reverse exchange of phosphorus between phosphoarginine and ATP.

Rapid miniprep isolation of mitochondrial DNA from metacestodes, and free-living and parasitic nematodes.

A method, based on one to isolate supercoiled plasmid DNA from bacterial cells, has been developed to purify mitochondrial DNA (mtDNA) from cestode and nematode tissue easily and efficiently. Starting with as little as 100 mg of helminth tissue, sufficient mtDNA for electrophoretic analysis was extracted. This DNA was essentially free of nuclear DNA and readily digested by restriction endonucleases. Approximately 20% of the mtDNA in helminth tissue was recovered, which is a significant improvement over previously available techniques.

Phosphoarginine-adenosine triphosphate exchange detected in vivo in a microscopic nematode parasite by flow 31P FT-NMR spectroscopy.

In vivo flow 31P NMR spectroscopy of a microscopic nematode, Steinernema carpocapsae, is described. Long-term viability was maintained during analysis by continuous circulation of an oxygenated suspension of the parasite through an NMR spectrometer. Saturation transfer and inversion recovery were employed under flowing conditions to investigate the kinetics of phosphoarginine<-->adenosine triphosphate exchange. The kinetic constants for the forward and reverse reactions were 0.37/s and 1.45/s, respectively. This report is the first to demonstrate a functional phosphagen kinase in the metabolism of a parasitic helminth.

[Susceptibility of thirty-one species of mosquitoes to Romanomermis yunanensis].

A new species of Mermithidae was found parasitizing the larvae of Culex tritaeniorhynchus and Culex fatigans in Henan, China and then named Romanomermis yunanensis. Thirty-one species of Mosquitoes involving six genera have been tested for susceptibility to R. yunanensis and Culicinae mosquito have consistently been highly susceptible. At a 1:5 ratio of mosquito larvae to nematode juveniles, the parasitisms of Aedes aegypti, Ae. albopictus, Culex fatigans, Culex tritaeniorhynchus, Anopheles sinensts, Psorophora columbiae and Culesta inornata were 98.2%, 98.5%, 98.6%, 97.1%, 0%, 98.8%, and 99.0% respectively. R. yunanensis presented a phenomenon of retarted development in Anopheles maculatus, which remained at the parasitic stage both in the larvae and pupa of An. maculatus. The parasitism of An. dirus was 89.7%, but most (88.8%) parasitizing mermithids underwent melanization and died. The comparative susceptibility of some North American mosquitoes to R. yunanesis and R. culicivorax at a 1:5 infective ratio showed that both percentage infection and intensity of infection statistically supported the generalization that, under laboratory conditions, R. yunanesis is a more vigorous and aggressive parasite of Culicinae.

A simple, inexpensive electroelution device for the recovery of nucleic acid fragments from agarose gels.

A simple, inexpensive apparatus for the electroelution of nucleic acids is described. It is constructed from disposable supplies commonly found in molecular biology laboratories.

In vivo flow 31P FT-NMR spectroscopy of a nematode parasite.

31P NMR spectroscopy of a parasitic nematode. Steinernema carpocapsae, is described. In vivo spectra were generated from nematode suspensions continuously circulating through the spectrometer. By flowing the organism, saturation effects were avoided and short interpulse delays significantly reduced spectral generation time. To maximize sensitivity, 90 degrees pulses were employed. Changes in energetic status in response to oxygen availability were easily and rapidly monitored in circulating nematodes.

Optimization of Mitochondrial DNA-based Hybridization Assays to Diagnostics in Soil.

Nucleic acid hybridization among root-knot nematode mitochondrial DNAs can be used to identify several Meloidogyne species. Research was initiated to optimize mitochondrial DNA-based molecular diagnostics for the demanding environments likely to be encountered in field isolates. DNA hybridization using reconstituted DNA-soil mixtures revealed a loss of assay sensitivity ranging from 34% to 92% with four agronomic soils tested. This problem was alleviated by the addition of exogenously added DNA. Variation in nematode egg lysis procedures also affected hybridization efficiency, with NaOC1 treatment most effective at disrupting Meloidogyne eggs. These optimized conditions permit detection of mtDNA released from one to five Meloidogyne eggs using standard nucleic acid hybridization procedures.

Romanomermis culicivorax: penetration of larval mosquitoes.

In the presence of second larval instars of three mosquito species the preparasites of Romanomermis culicivorax swam near the water surface in an orthokinetic manner. When the preparasites were ca. 1 mm from the host, they stopped and swam klinotactically toward the host. During this phase, the preparasites secreted a small amount of a putative adhesive material from the anterior region and host contact was completed. The adhesive appeared to aid in attachment of the preparasites to the host and initiation of the search-boring phase. The preparasites glided over the host until a suitable penetration site was found. The penetration phase was initiated by probing with the odontostyle. This was followed by partial paralysis, decreased intestinal peristaltic movement, and temporary cardiac arrest in all host mosquitoes which was probably related to injection of esophageal secretions. SEM observations showed that the abdominal walls were the most frequent site for penetration. As the preparasites entered through the penetration hole, microorganisms adhering to the cuticle of the preparasites were retained by the adhesive which accumulated around the penetration site. Thus, microbial contamination of the host was avoided by a mechanical cleansing mechanism. Penetration was usually completed in less than 10 min.

The IgE and IgG subclass responses of mice to four helminth parasites.

To investigate whether the formation of IgE is linked in vivo to an IgG subclass, mice were infected with four helminth parasites, Nippostrongylus brasiliensis (Nbr), Mesocestoides corti, Taenia crassiceps and Trichinella spiralis, and the changes in the serum levels of the different Ig isotypes as well as the antibody response to M. corti and T. crassiceps antigen extracts were determined by radioimmunoassays. All four parasites induced a concomitant increase of the IgE and IgG1 serum levels and usually a decrease of the IgG2a level. They also induced an increase of the IgM level but had little effect on the IgG2b, IgG3, and IgA serum levels. The specific antibodies to an M. corti antigen extract were mainly of the IgG1 subclass, whereas it was of both IgG1 and IgG2a subclasses to T. crassiceps. Injections of dead M. corti induced an increase of all IgG subclasses and similar levels of IgG1 and IgG2a anti-parasite antibodies. Subcutaneous instead of intraperitoneal infection with T. crassiceps induced higher IgG2a than IgG1 levels and 10-fold lower IgE levels than the natural ip infection; however, despite the greater IgG2a polyclonal response, anti-parasite antibodies were predominantly of the IgG1 subclass. The data demonstrate that natural infection with four different helminth parasites induces a concomitant polyclonal IgG1 and IgE response. These in vivo observations corroborate the recent in vitro findings demonstrating that interleukin-4 induces lipopolysaccharide-activated murine B cells to secrete both IgG1 and IgE, suggesting that the regulation of these two isotypes is linked.

Phosphoglycerides and derivatives in Taenia crassiceps examined by 31P NMR spectroscopy.

Examination of the larval stage of the tapeworm, Taenia crassiceps, by 31P NMR spectroscopy revealed the presence of a major phosphoglyceride component. However, using saturation transfer, no exchange between glycerophosphorylcholine and phosphoglyceride or any other NMR-detectable phosphorus metabolites was detected.