Trichinella zimbabwensis n.sp. (Nematoda), a new non-encapsulated species from crocodiles (Crocodylus niloticus) in Zimbabwe also infecting mammals

Since 1995, Trichinella larvae have been detected in 39.5% of farmed crocodiles (Crocodylus niloticus) in Zimbabwe. Morphological, biological, biochemical and molecular studies carried out on one isolate from a farmed crocodile in 2001 support the conclusion that this parasite belongs to a new species, which has been named Trichinella zimbabwensis n.sp. This species, whose larvae are non-encapsulated in host muscles, infects both reptiles and mammals. The morphology of adults and larvae is similar to that of Trichinella papuae. Adults of T. zimbabwensis cross in both directions with adults of T. papuae (i.e. male of T. zimbabwensis per female of T. papuae and male of T. papuae per female of T. zimbabwensis), producing F1 offspring which produce very few and less viable F2 larvae. Muscle larvae of T. zimbabwensis, like those of T. papuae, do not infect birds. Three allozymes (of a total of 10) are diagnostic between T. zimbabwensis and T. papuae, and five are diagnostic between T. zimbabwensis and Trichinella pseudospiralis, the third non-encapsulated species. The percentage of the pairwise alignment identity between T. zimbabwensis and the other Trichinella species for the cytochrome oxidase subunit I gene, the large subunit ribosomal-DNA (mt-lsrDNA) gene and the expansion segment five, shows that T. zimbabwensis is more similar to the two non-encapsulated species T. papuae (91% for cytochrome oxidase I; 96% for mt-lsrDNA; and 88% for expansion segment five) and T. pseudospiralis (88% for cytochrome oxidase I; 90% for mt-lsrDNA; and 66–73% for expansion segment five) than to any of the encapsulated species (85–86% for cytochrome oxidase I; 88–89% for mt-lsrDNA; and 71–79% for expansion segment five). This is the first non-encapsulated species discovered in Africa. The finding of a new Trichinella species that infects both reptiles and mammals suggests that the origin of Trichinella parasites dates back further than previously believed and can contribute to understanding the phylogeny and the epidemiology of the genus Trichinella.     

Trichinella nativa and Trichinella T9 in the Hokkaido island, Japan

Trichinella sp. muscle larvae were isolated from the thigh muscle of two red foxes (Vulpes vulpes) captured in Sapporo and Otofuke, Hokkaido, Japan, in 2003. Multiplex PCR designed for genotyping the genus Trichinella revealed that the Sapporo isolate showed a specific pattern to T. britovi complex (T. britovi, Trichinella T8 and Trichinella T9) and the Otofuke isolate showed that to T. nativa. Nucleotide sequences of a part of the mitochondrial cytochrome oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) of the Sapporo isolate showed the highest similarity to those of Trichinella T9, a species detected in the mainland of Japan. This study shows that both T. nativa and Trichinella T9 are circulating in wildlife of the Hokkaido island.     

The presence of the potentially toxic genera Ostreopsis and Coolia (Dinophyceae) in the North Aegean Sea, Greece

The examination of macrophyte, water and sediment samples, collected at depths less than 1.5 m from 50 different sites along the North Aegean coasts, has revealed, for the first time in Greek coastal waters, the presence of two Ostreopsis species (O. ovata and O. cf. siamensis) and Coolia monotis in the majority of the sampling sites (94% and 100%, respectively). Other epiphytic dinoflagellates of the genera Prorocentrum and Amphidinium and diatoms were accompanying species in this epiphytic community. Morphometric features, plate formula and thecal ornamentation were used for species identification. O. ovata cells were smaller in dorsoventral (DV) diameter and width (W) (26.18–61.88 μm and 13.09–47.60 μm, respectively) in comparison with O. cf. siamensis (35.70–65.45 μm and 23.80–49.98 μm, respectively). In contrast, the anterioposterior (AP) diameter of O. cf. siamensis was smaller (14.28–26.18 μm) resulting in DV/AP ≈ 3, whereas the above ratio for O. ovata was less than 2 (AP ranging between 14.28–35.70 μm). Moreover, the theca of O. ovata cells was ornamented with scattered pores, which fluctuated in a wider range (0.07–0.32 μm) than those of O. cf. siamensis (0.23–0.29 μm). Coolia monotis cells were almost round with average DV diameter 26.88 μm, AP 25.66 μm and width 26.76 μm. Small and large cells were recorded in both field and culture populations of Ostreopsis spp. and C. monotis, while hyaline cysts were observed for O. ovata. The presence of O. ovata and O. cf. siamensis exhibited a clear seasonal pattern dominating (maximum abundance up to 4.05 × 10⁵ cells gr⁻¹ fwm) the period from midsummer to late autumn in years 2003 and 2004, while C. monotis was found also in winter and spring months.     

Galanin-induced relaxation in gastric smooth muscle cells is mediated by cyclic AMP

Galanin has numerous effects on gastrointestinal motility in different species; however, its cellular basis of action in mediating these effects is unclear. Dispersed gastric smooth muscle cells have been shown to possess high-affinity galanin receptors that increase cAMP and cause relaxation. Recent studies show some smooth muscle relaxants such as VIP cause relaxation by both cAMP-dependent and -independent mechanisms. It is unknown if galanin's cellular basis of relaxation is similar or different from that of VIP. To investigate galanin's relaxant effect and compare it to VIP's effect, dispersed smooth muscle cells from guinea pig stomach were prepared by collagenase digestion. The mean length in resting cells was 110 ± 2 μm and, with carbachol treatment, contracted to 89 ± 2 μm. VIP and galanin alone had no effect on cell length, but each caused a dose-dependent inhibition of carbachol-induced contraction and both had an EC₅₀ of 3–7 nM. Galanin (1 μM) and VIP (1 μM) increased cellular cAMP from 118 ± 10 pmol/10⁶ cells in control to 212 ± 14 and 214 ± 12 pmol/10⁶ cells, respectively. The protein kinase A inhibitor, Rp-cAMPS, at 100 μM, completely inhibited the relaxant effect of an EC₅₀ concentration of galanin (3 nM), but only inhibited that by VIP by 80% (p < 0.05). Adding the nitric oxide inhibitor, -NNA ( ), at 100 μM did not alter the length of resting cells or inhibit carbachol-induced contraction. However, -NNA (100 μM) decreased VIP-induced relaxation by 45%, whereas it had no effect on galanin-induced relaxation. To determine the ability of each peptide to activate nitric oxide, the incorporation of [³H]arginine into [³H]citrulline was determined. Galanin (1 μM) did not cause nitric oxide generation whereas VIP (1 μM) increased nitric oxide generation above the control by 97 ± 14% (p < 0.01). These results demonstrated that with galanin, in contrast to VIP, nitric oxide is not involved in its ability to cause gastric smooth muscle cell relaxation. The relaxant action of galanin can be accounted for completely by its ability to activate protein kinase A and therefore resembles recent results with β-adrenergic agents.     

Settlement of the serpulid polychaete Hydroides elegans (Haswell) on the arborescent bryozoan Bugula neritina (L.): evidence of a chemically mediated relationship

The tube building polychaete Hydroides elegans Haswell was found living attached to colonies of the arborescent bryozoan Bugula neritina (L.) in Port Shelter, Hong Kong. Field data collected during the period of January through May 1996, showed that H. elegans density reached 77.6 individuals of H. elegans per g wet weight of B. neritina. Density of H. elegans on B. neritina at depths from the surface to 0.5 m was lower than that at depths below 1 m. In January–March, when there were no H. elegans settling on PVC plates or found on natural substrata, numbers on B. neritina were ca. 5 per g wet weight. H. elegans settled on B. neritina and grew rapidly as mean diameter of tubes increased from 605 μm in February to 936 μm in March. In laboratory experiments, larvae of H. elegans settled and metamorphosed on branches of B. neritina and on the bottom of dishes containing B. neritina leachate. Compounds extracted from the leachate of B. neritina induced 74% of H. elegans larvae to metamorphose at a concentration of 16 μg/ml seawater, compared to 5% in dishes containing only filtered seawater (controls). Metabolites from the leachate of B. neritina which were bound to amberlite XAD-2, indicating they are lipophilic in nature, induced over 70% metamorphosis in H. elegans larvae at 56 μg/ml seawater. A biofilm from one of four strains of bacterial isolates associated with the surface of B. neritina induced low levels of metamorphosis in H. elegans larvae, while other bacterial isolates were detrimental to the survival of juvenile H. elegans. Field experiments further demonstrated that H. elegans settled preferentially on Phytagel discs embedded with whole extracts of B. neritina over control Phytagel discs. Metabolites from B. neritina deterred feeding on alginate pellets by assemblages of local fishes in field assays. Metabolites originating from B. neritina, bacteria colonizing B. neritina, and the complex structure of B. neritina contributed to the recruitment of H. elegans to B. neritina surfaces. Hydroides elegans may gain a refuge from predation by associating with B. neritina colonies both from its structural and chemical attributes.     

Effects of geometry on transmission and sensing potential of tapered fiber sensors

Geometry of tapered fiber sensors critically affects the response of an evanescent field sensor to cell suspensions. Single-mode fibers (nominally at 1300 nm) were tapered to symmetric or asymmetric tapers with diameters in the range of 3–20 μm, and overall lengths of 1–7 mm. Their transmission characteristics in air, water and in the presence of Escherichia coli (JM101 strain) at concentrations of 100, 1000, 7000 and 7 million cells/mL were measured in the 400–800 nm range and gave rich spectral data that lead to the following conclusions. (1) No change in transmission was observed due to E. coli with tapers that showed no relative change in transmission in water compared to air. (2) Tapers that exhibited a significant difference in transmission in water compared to air gave weak response to the presence of the E. coli. Of these, tapers with low waist diameters (6 μm) showed sensitivity to E. coli at 7000 cells/mL and higher concentration. (3) Tapers that showed modest difference in water transmission compared to air, and those that had small waist diameters gave excellent response to E. coli at 100–7000 cells/mL. In addition, mathematical modeling showed that: (1) at low wavelength (470 nm) and small waist diameter (6 μm), transmission with water in the waist region is higher than in air. (2) Small changes in waist diameter (0.05 μm) can cause larger changes in transmission at 470 nm than at 550 nm at waist diameter of 6 μm. (3) For the same overall geometry, a 5.5 μm diameter taper showed larger refractive index sensitivity compared to a 6.25 μm taper at 470 nm.     

Two new coccidia (Apicomplexa:Eimeriidae) from the green peacock (Pavo muticus) from Saudi Arabia

Two new species of Eimeria were found from faecal samples of ten green peacocks (Pavo muticus) collected at Al-Kharj area, a central region of Saudi Arabia. Sporulated oocysts of Eimeria mutica n.sp. are ellipsoidal 23.1×17.4 (22.4–25.0×16.7–18.9) μm, with a smooth bilayered wall. A micropyle and bilobed polar body are present, but without an oocyst residuum. The sporocyst is an elongated-ovoid 13.7×6.2 (12.0–14.2×5.4–6.7) μm, with a Stieda body and a residuum. Sporulated oocysts of E. kharjensis n.sp. are subspherical 20.3×17.7 (19.0–21.5×16.2–18.7) μm, with a two layered wall and a single polar body. The micropyle is covered by a dome-shaped cap and the sporocyst is an elongate-ovoid 12.7×6.3 (11.9–13.5×5.4–6.8) μm, with a Stieda body. The sporocyst residuum is present as several small granules.     

Trichinella spiralis and Trichinella pseudospiralis induce collagen synthesis by host fibroblasts in vitro and in vivo

Immunoperoxidase staining of muscle infected with Trichinella spiralis for murine collagen types I and IV provided both qualitative and quantitative evidence of extensive synthesis of both types of collagen by fibroblasts in infected muscle compared to that seen uninfected muscle. Moreover, fibroblasts in muscle infected with T. pseudospiralis, a nonencapsulating species, showed significantly less staining for both types of collagen compared to muscle from mice infected with T. spiralis. Analysis of collagen composition of isolated nurse cells using an ELISA specific for either type I or type IV murine collagen suggested that of these 2 types of collagen, only type IV basement membrane collagen is found in Trichinella capsular collagen. Excretory/secretory products of T. spiralis and T. pseudospiralis induced extensive synthesis of exclusively type IV collagen by 3T3 murine fibroblasts in vitro.     

Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis

The effects of flavan-3-ols (the monomer units of condensed tannins (CT)) and their galloyl derivatives on the viability of eggs, the development of first stage (L1) larvae, and the viability of the infective larvae of Trichostrongylus colubriformis were investigated under in vitro conditions. Each of the flavan-3-ol gallates showed some inhibition of egg hatching at 100 μg/ml, and 100% inhibition at 1000 μg/ml, with epigallocatechin gallate being the most effective in the egg hatch (EH) assay. In contrast, none of the flavan-3-ols were able to completely inhibit egg hatching. The flavan-3-ols and galloyl derivatives dose-dependently inhibited the development of infective larvae as assessed by the larval development (LD) assay. A larval migration inhibition (LMI) assay was used to assess the effect of flavan-3-ols and their galloyl derivatives on the motility of the infective third-stage (L3) larvae of T. colubriformis. In general, the flavan-3-ol gallates were more effective than the flavan-3-ols at immobilising the infective larvae as evidenced by their ability to inhibit more (P<0.05–0.01) larvae from passing through the LMI sieves. At 500 μg/ml, epigallocatechin gallate inhibited significantly more (P<0.1) larvae from passing through the sieves than did catechin gallate, epicatechin gallate, or gallocatechin gallate. Comparisons were made between the flavan-3-ols and their galloyl derivatives with the in vitro effects of CT extracts from several forage legumes, which have exhibited effects on parasites in vivo. The forage legumes tested at 200–500 μg/ml reduced the proportion of eggs that hatch, with comparable results to those obtained using the flavan-3-ols. The activities may be influenced by the prodelphinidin: procyanidin (PD:PC) ratios: CT extracts from Lotus pendunculatus and sainfoin have PD:PC ratios of 70:30 and 77:23, respectively, whereas the less active CT extract from Lotus corniculatus has a PD:PC ratio of 27:73. The active CT extracts from forage legumes have epigallocatechin as the dominant flavan-3-ol extender unit, and epigallocatechin is the most active flavan-3-ol in both the EH and LD assays.     

Trichinellosis of animals in Chukotka, USSR: A general review

During an examination, for trichinellosis, of 1489 mammals of 12 different species, in the territory of Chukotka National district (regions of Anadyr', Bering, Iul'tin, Chaun and Chukotka) and in the region of North-Even of Magadan province, Trichinella larvae were detected in 236 (16·4%) of these animals from 6 of the species: in 109 (44·8%) of the blue polar foxes kept in cages, in 12 (15·8%) of the silver-black foxes kept in cages, in 84 (18·2%) of the white polar foxes, in 17 (17·5%) of the common foxes, in 13 (12·1%) of the dogs and in the one European bear examined. Trichinella larvae were not found in 190 marine mammals examined: 178 ringed seals, 7 whales, 4 walruses, and 1 bearded seal, nor in the 8 Siberian squirrels and 159 domestic pigs examined. The high incidence of Trichinella infection amongst blue polar foxes and silver-black foxes on fur-animal farms in Chukotka, where the food ration of these animals is predominately the flesh of marine mammals (walruses and other pinnipeds), suggests that marine mammals may constitute one of the sources of infection to caged animals. In Chukotka, as in other regions of the Extreme North and North-East, the circulation of trichinae in nature may occur between two ecologically different groups of animals: terrestial and marine mammals. A wide distribution of trichinellosis among animals in nature, or kept in cages and among dogs in Chukotka National district and in the North-Even region of Magadan province provides a constant threat of infection to man. Thus the flesh and sub-products of potential hosts of trichinae (including dogs, fur animals, European and polar bears, marine mammals, etc.) should undergo thorough trichinelloscopy examination before being consumed by man. The flesh of sea mammals should be examined for trichinellosis before being used as food for animals; the presence of trichinae in it requires its sterilization.     

The Calcitonin Gene-Related Peptide Activates Both cAMP and NO Pathways to Induce Relaxation of Circular Smooth Muscle Cells of Guinea-Pig Ileum

Rekik, M., M. Delvaux, J. Frexinos, and L. Bueno. Calcitonin gene-related peptide activates both cAMP and NO pathways to induce relaxation of circular smooth muscle cells of guinea-pig ileum. Peptides 18(10) 1517–1522, 1997.—The direct effects and the intracellular pathways of rCGRP were investigated on smooth muscle cells (SMC) isolated by enzymatic digestion from the circular and longitudinal layers of guinea-pig ileum. In circular SMC, rCGRP inhibited CCK8-induced contraction in a concentration-dependent manner (Cmax = 100 μM and EC₅₀ = 0.7 ± 0.4 nM). Preincubation of SMC with 1 μM Rp-cAMPs, a cAMP antagonist, abolished the relaxing effect of rCGRP; moreover, preincubation of SMC with 100 μM L-NAME, an inhibitor of NOS, inhibited the relaxing effect of rCGRP. hCGRP(8-37), a selective antagonist of rCGRP receptors, inhibited the rCGRP-induced relaxation in a concentration dependent manner whereas the vasoactive intestinal polypeptide (VIP) antagonist had no significant effect. In longitudinal SMC, rCGRP-induced relaxation was abolished by Rp-cAMPs, whereas L-NAME had no effect. In conclusion, rCGRP triggers different intracellular pathways to induce relaxation of circular or longitudinal intestinal SMC; cAMP is involved in cells from both layers while nitric oxide (NO) is involved only in relaxation of circular SMC.     

Taxonomic characterization of two marine peritrichous ciliates, Epicarchesium corlissi n. sp. and Pseudovorticella jiangi n. sp. (Ciliophora: Peritrichia), from northern China

Two new marine peritrich ciliates, Epicarchesium corlissi n. sp. and Pseudovorticella jiangi n. sp., were discovered in mariculture waters on the coast of northern China near Qingdao. Their morphology, infraciliature and silverline system were investigated based on both living and silver-impregnated specimens. E. corlissi is characterized as follows: marine Epicarchesium with dichotomously branched stalk; zooids elongate, approximately 60–70×25–35 μm in vivo; peristomial collar double-folded; macronucleus J-shaped; single, small contractile vacuole ventrally positioned; more than 60 striations between peristome and aboral trochal band, 13–18 from aboral trochal band to scopula; abstomal end of row 1 of infundibular polykinety 3 terminating at same level as rows 2 and 3 of infundibular polykinety 3; rows 2 and 3 of infundibular polykinety 3 much longer than row 1 and converging adstomally with infundibular polykinety 1. The new species P. jiangi is diagnosed as follows: marine Pseudovorticella; zooid inverted bell-shaped, approximately 80×60 μm in vivo and with a broad, flat, thin peristomial collar that measures approximately 90 μm across; pellicle with transparent cortical vesicles; macronucleus J-shaped; number of silverlines between peristome and aboral trochal band 20–24, from aboral trochal band to scopula 9–11; abstomal end of row 1 of infundibular polykinety 3 diverges from the other two rows of this polykinety and ends alongside row 3 of infundibular polykinety 2.     

A new floating sensor array to detect electric near fields of beating heart preparations

A new flexible sensor for in vitro experiments was developed to measure the surface potential, Φ, and its gradient, E (electric near field), at given sites of the heart. During depolarisation, E describes a vector loop from which direction and magnitude of local conduction velocity θ can be computed. Four recording silver electrodes (14 μm × 14 μm) separated by 50 μm, conducting leads, and solderable pads were patterned on a 50 μm thick polyimide film. The conductive structures, except the electrodes, were isolated with polyimide, and electrodes were chlorided. Spacer pillars mounted on the tip fulfil two functions: they keep the electrodes 70 μm from the tissue allowing non-contact recording of Φ and prevent lateral slipping. The low mass (9.1 mg) and flexibility (6.33 N/m) of the sensor let it easily follow the movement of the beating heart without notable displacement. We examined the electrodes on criteria like rms-noise of Φ, signal-to-noise ratio of Φ and E, maximum peak-slope recording dΦ/dt, and deviation of local activation time (LAT) from a common signal and obtained values of 24–28 μV, 46 and 41 dB, 497–561 V/s and no differences, respectively. With appropriate data acquisition (sampling rate 100 kHz, 24-bit), we were able to record Φ and to monitor E and θ on-line from beat-to-beat even at heart rates of 600 beats/min. Moreover, this technique can discriminate between uncoupled cardiac activations (as occur in fibrotic tissue) separated by less than 1 mm and 1 ms.     

Paralygodium meckertii sp. nov. (Schizaeaceae) from the Upper Cretaceous (Coniacian) of Vancouver Island, British Columbia, Canada

More than 50 specimens of permineralized fertile pinnules with abaxially borne sporangia have been discovered in calcareous marine nodules from the Upper Cretaceous (Coniacian) Comox Formation from the Eden Main localities on Vancouver Island, British Columbia, Canada. Isolated pinnules 1.6–3.0 mm wide × 1.6–2.8 mm long are lobed and abaxially enrolled to form irregular globose structures. Pyriform sporangia 216–300 μm wide × 360–468 μm long occur in two rows on the abaxial surface of pinnule lobes. Sporangia have an apical annulus of 15–18 cells. Spores are tetrahedral and trilete, 33–42 μm in diameter, with straight to concave interradial sides, laesurae extending nearly to the equator, and a psilate exine. Spores are assignable to the sporae dispersae genus Deltoidospora. Fertile pinnules are compared to fossils of Anemia poolensis and two previously described species of Paralygodium, and show closest similarities to P. vancouverensis from the Eocene of British Columbia. The Cretaceous Eden Main specimens differ in number of pinnule lobes and their morphology and are described as a new taxon: P. meckertii sp. nov. This discovery extends the Cretaceous geographic range of Paralygodium from Japan to North America and adds to our knowledge of the diversity of extinct schizaeaceous ferns.     

Experimentally induced effects of water temperature and immersion time on reproductive output of bivalves in the Wadden Sea

Results of experiments are reported on the effects of water temperature and immersion time in winter on egg size and egg numbers in three intertidally living bivalves in the Dutch Wadden Sea, the Baltic tellin Macoma balthica, the common cockle Cerastoderma edule and the common mussel Mytilus edulis. Macoma (14–17 mm shell length) produced large eggs (diameter of 107 μm) in relatively small numbers (20 000–70 000) in early spring. Later in spring, Cerastoderma (28–33 mm shell length) produced smaller eggs (77 μm, excluding the surrounding jelly layer) in tenfold larger numbers (200 000–700 000). Mytilus (45–55 mm shell length) spawned even smaller eggs (72 μm) in high (but not easily assessed) numbers over a more extended period. In Macoma egg size was not affected by winter temperatures or immersion time. Effects of winter–spring temperatures and immersion time on egg size could be demonstrated in Cerastoderma. Smaller eggs were produced at the higher temperatures. Effects of immersion time were non-consistent: at lower water temperatures larger, but at higher temperatures smaller eggs were produced by animals kept at longer immersion times. In Mytilus, no temperature effects were observed. However, a longer immersion time resulted in larger eggs. In Macoma as well as in Cerastoderma significantly more eggs were produced at the lower temperature. Immersion time effects were most pronounced at the lower temperature, where more eggs were produced at the subtidal level than at the tidal level. At the higher water temperature differences between egg numbers produced at the two tidal levels were small. Just prior to spawning, egg numbers were strongly positively related to body mass at a certain shell length.     

Determination of sodium cromoglycate in human plasma by liquid chromatography–mass spectrometry in the turbo ion spray mode

A highly sensitivity liquid chromatography–tandem mass spectrometry method has been developed for the quantitation of sodium cromoglycate (SCG) in human plasma. The method was validated over a linear range of 0.100–50.0 ng/ml, using ¹³C₄ sodium cromoglycate as the internal standard. Compounds were extracted from 1.0 ml of lithium heparin plasma by methanol elution of C₁₈ solid-phase extraction cartridges. The dried residue was reconstituted with 100 μl of 0.01 N HCl, and 30 μl was injected onto the LC–MS–MS system. Chromatographic separation was achieved on a C8 (3.5 μm) column with an isocratic mobile phase of methanol–water–0.5 M ammonium acetate (35:64.8:0.2, v/v/v). The analytes were detected with a PE Sciex API 3000 mass spectrometer using turbo ion spray with positive ionization. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 469.2 (precursor ion) to m/z 245.1 (product ion) for SCG and m/z 473.2 (precursor ion) to m/z 247.1 (product ion) for ¹³C₄ SCG (I.S.). The average recoveries of SCG and the I.S. from human plasma were 91 and 87%, respectively. The low limit of quantitation was 0.100 ng/ml. Results from a 4-day validation study demonstrated excellent precision (C.V.% values were between 1.9 and 6.5%) and accuracy (−5.4 to −1.2%) across the calibration range of 0.100–50.0 ng/ml.     

Oocysts of Neospora caninum, Hammondia heydorni, Toxoplasma gondii and Hammondia hammondi in faeces collected from dogs in Germany

Faecal samples of 24,089 dogs were examined coproscopically in two veterinary laboratories in Germany between March 2001 and October 2004. In 47 dogs, oocysts of 9–14 μm size were found. Their morphology was similar to those of Hammondia heydorni and Neospora caninum. Samples of 28 of these dogs were further examined by inoculation into gerbils: seven isolates induced a specific antibody response against antigens of N. caninum NC-1 tachyzoites. This response suggests that the isolates contained N. caninum. In addition to H. heydorni (12 times isolated), Toxoplasma gondii occysts (twice) and Hammondia hammondi oocysts (twice) were observed in dog faeces. The latter findings suggest that coprophagia with a subsequent intestinal passage by dogs plays a role in the dissemination of coccidian parasites for which cats are definitive hosts. Five of the seven N. caninum (NC-GER2, NC-GER3, NC-GER4, NC-GER5, NC-GER6) and the two T. gondii isolates (TG-dgGER1, TG-dgGER2) were successfully passaged into cell culture and are now available for detailed characterization. In contrast to oocysts of other parasites, N. caninum oocysts were predominantly found between January and April (Fisher exact; P=0.038). In the sera of dogs shedding N. caninum, no reactions against the immunodominant antigens with apparent molecular weights of 19, 29, 30, 33 and 37 kDa of N. caninum tachyzoites were observed 3–5 weeks after shedding. However, the animals recognized a 152-kDa N. caninum antigen. Compared with those identified as H. heydorni, T. gondii or H. hammondi, N. caninum oocyst isolates were significantly smaller in length with the 75th percentiles ≤10.7 μm when measured in concentrated sucrose solution and smaller length–width ratios with the 75th percentiles ≤1.06. It may thus be possible to develop criteria for a preliminary identification of N. caninum in dog faeces based on the oocyst morphology.     

Ultrastructure of a novel tube-forming, intracellular parasite of dinoflagellates: Parvilucifera prorocentri sp. nov. (Alveolata, Myzozoa)

We have characterized the intracellular development and ultrastructure of a novel parasite that infected the marine benthic dinoflagellate Prorocentrum fukuyoi. The parasite possessed a combination of features described for perkinsids and syndineans, and also possessed novel characters associated with its parasitic life cycle. Reniform zoospores, about 4 μm long, possessed a transverse flagellum, alveoli, a refractile body, a mitochondrion with tubular cristae, a syndinean-like nucleus with condensed chromatin, micronemes, bipartite trichocysts with square profiles (absent in perkinsids) and oblong microbodies. Like Parvilucifera, the zoospores also possessed a shorter posterior flagellum, a heteromorphic pair of central microtubules in the anterior axoneme and a reduced pseudoconoid positioned directly above an orthogonal pair of basal bodies. Early developmental stages consisted of a sporangium about 5–15 μm in diam that contained spherical bodies and amorphous spaces. The undifferentiated sporangium increased to about 20–25 μm in diam before being enveloped by a wall with a convoluted mid-layer. The sporangium differentiated into an unordered mass of zoospores that escaped from the cyst through a pronounced germ tube about 4–5 μm in diam and 10–15 μm long. Weakly developed germ tubes have been described in Perkinsus but are absent altogether in Parvilucifera and syndineans. Comparison of these data with other myzozoans led us to classify the parasite as Parvilucifera prorocentri sp. nov., Myzozoa. Although we were hesitant to erect a new genus name in the absence of molecular sequence data, our ultrastructural data strongly indicated that this parasite is most closely related to perkinsids and syndineans, and represents an intriguing candidate for the cellular identity of a major subclade of Group I alveolates.     

Differential effects of sugar-mimic alkaloids in mulberry latex on sugar metabolism and disaccharidases of Eri and domesticated silkworms: enzymatic adaptation of Bombyx mori to mulberry defense

Mulberry leaves (Morus spp.) exude latex rich in sugar-mimic alkaloids, 1,4-dideoxy-1,4-imino-d-arabinitol (d-AB1) and 1-deoxynojirimycin (DNJ), as a defense against herbivorous insects. Sugar-mimic alkaloids are inhibitors of sugar-metabolizing enzymes, and are toxic to the Eri silkworm, Samia ricini, a generalist herbivore, but not at all to the domesticated silkworm, Bombyx mori, a mulberry specialist. To address the phenomena, we fed both larvae diets containing different sugar sources (sucrose, glucose or none) with or without sugar-mimic alkaloids from mulberry latex. In S. ricini, addition of sugar-mimic alkaloids to the sucrose (the major sugar in mulberry leaves) diet reduced both growth and the absorption ratio of sugar, but it reduced neither in B. mori. The midgut soluble sucrase activity of S. ricini was low and inhibited by very low concentrations of sugar-mimic alkaloids (IC₅₀=0.9–8.2 μM), but that of B. mori was high and not inhibited even by very high concentrations (IC₅₀>1000 μM) of sugar-mimic alkaloids. In S. ricini, the addition of sugar-mimic alkaloids to the glucose diet still had considerable negative effects on growth, although it did not reduce the absorption ratio of glucose. The hemolymph of S. ricini fed sugar-mimic alkaloids contained sugar-mimic alkaloids. The trehalose concentration in the hemolymph increased significantly in S. ricini fed sugar-mimic alkaloids, but not in B. mori. The trehalase activities of S. ricini were lower and inhibited by lower concentrations of sugar-mimic alkaloids than those of B. mori. These results suggest that sugar-mimic alkaloids in mulberry latex exert toxicity to S. ricini larvae first by inhibiting midgut sucrase and digestion of sucrose, and secondly, after being absorbed into hemolymph, by inhibiting trehalase and utilization of trehalose, the major blood sugar. Further, our results reveal that B. mori larvae evolved enzymatic adaptation to mulberry defense by developing sucrase and trehalase that are insensitive to sugar-mimic alkaloids.     

Development and validation of an in vitro Trichostrongylus colubriformis motility assay

Development and validation of an in vitro Trichostrongylus colubriformis motility assay. International Journal for Parasitology 17: 1441–1444. An in vitro Trichostrongylus colubriformis motility assay involving the use of a micromotility meter has been developed and validated. Four commercially available ruminant anthelmintics (albendazole, ivermectin, levamisole hydrochloride, and coumaphos) and an investigational hydrazone compound (p-toluoyl chloride phenylhydrazone) were evaluated in this assay at four concentrations each. At 100 μg ml^-1, all five treatments significantly (P 0.05) reduced the motility of ensheathed L-3 T. colubriformis larvae, thereby indicating anthelmintic activity. At this concentration, coumaphos was significantly less active than any of the other four treatments. At 10 μg ml^-1 albendazole, ivermectin, levamisole hydrochloride and the hydrazone compound were active, but coumaphos was not. At 1 μg ml^-1 albendazole, ivermectin and levamisole hydrochloride remained significantly active, but neither coumaphos nor the hydrazone compound showed significant activity. At all three of the higher concentrations (1,10 and 100 μg ml^-1), levamisole hydrochloride indicated greater activity than any of the other treatments. This difference was statistically significant at the 1 and 10 μg ml^-1 concentrations. None of the five treatments showed significant activity at the lowest concentration (0.1 μg ml^-1). The in vitro T. colubriformis motility assay proved to be sensitive, accurate, rapid, and repeatable. This assay system should be another valuable addition to the tests used to identify potential anthelmintics, monitor helminth resistance to drugs, and define the kinetics and mode of action of drugs.