Development of Dipetalonema gracile in the squirrel monkey (Saimiri sciureus), with notes on its biology

Infective larvae of Dipetalonema gracile, which had developed in Culicoides hollensis, were inoculated into 4 laboratory-born squirrel monkeys, Saimiri sciureus. Weekly blood sampling revealed the mean prepatent period to be 297 days. All 4 monkeys developed patent infections in which peak microfilaremias were reached 13 to 18 wk after patency. Two laparotomies, performed at 27 and 64 wk, were conducted to evaluate pathological involvement and, at that later time, to recover adult parasites. Slight capsular fibrosis was observed on the spleen of 2 of the animals but fibrous adhesions were absent. Microfilaremias in the 4 monkeys ranged from 15 to 250 mf/20 mm3 of blood and the number of adult parasites recovered varied from 7 to 13. However, the level of microfilaremia did not correlate directly to the number of adult parasites recovered.     

Development of Taenia asiatica cysticerci to infective stage and adult stage in Mongolian gerbils

The development of metacestodes and adult worms of Taenia asiatica in Mongolian gerbils (Meriones unguiculatus) were observed. Cysticerci were recovered from gerbils subcutaneously injected with hatched oncospheres. The recovery rate ranged from 0.1 to 3.2%. No cysticerci were recovered from the orally inoculated gerbils. The infectivity of the cysticerci recovered at 48 weeks post-infection was evaluated. Tapeworms were recovered on day 14 post-infection from the small intestine of 5 of 11 gerbils, with a recovery rate of 27% (6 worms recovered/22 worms inoculated). Three and four adult worms were recovered from two human volunteers who ingested five cysticerci after 4 months post-infection. In worms recovered from gerbils, segmentation and genital primordia in the posterior proglottids and hooklets in the residual rostellum were observed. The results indicate that gerbils can serve as an alternative intermediate host and that partial development of the adult worm stage occurs in gerbils.     

Dipetalonema viteae: phosphorylcholine and non-phosphorylcholine antigenic determinants in infective larvae and adult worms

The humoral immune response of Balb/c mice to live infective larvae or adult worm extract of Dipetalonema viteae is composed of two antibody populations either with or without specificity for phosphorylcholine. Absorption of immune serum on phosphorylcholine-Sepharose and separation of the antibody population demonstrated that anti-larvae serum contains a larger ratio of phosphorylcholine versus non-phosphorylcholine antibodies as compared to anti-adult serum. Immunofluorescence on crossections of female worms revealed that antigen expressing phosphorylcholine determinants were mainly found on certain internal structures, like egg, uterine, and intestinal membranes, but not on the cuticle. Immunoblotting using an adult worm extract demonstrated that protein bands reacted with either one or both populations of antibodies. The patterns were heterogeneous and moreover differed between the anti-larvae serum and the anti-adult serum.     

Divergent evolution of arrested development in the dauer stage of Caenorhabditis elegans and the infective stage of Heterodera glycines

Background

The soybean cyst nematode Heterodera glycines is the most important parasite in soybean production worldwide. A comprehensive analysis of large-scale gene expression changes throughout the development of plant-parasitic nematodes has been lacking to date.

Results

We report an extensive genomic analysis of H. glycines, beginning with the generation of 20,100 expressed sequence tags (ESTs). In-depth analysis of these ESTs plus approximately 1,900 previously published sequences predicted 6,860 unique H. glycines genes and allowed a classification by function using InterProScan. Expression profiling of all 6,860 genes throughout the H. glycines life cycle was undertaken using the Affymetrix Soybean Genome Array GeneChip. Our data sets and results represent a comprehensive resource for molecular studies of H. glycines. Demonstrating the power of this resource, we were able to address whether arrested development in the Caenorhabditis elegans dauer larva and the H. glycines infective second-stage juvenile (J2) exhibits shared gene expression profiles. We determined that the gene expression profiles associated with the C. elegans dauer pathway are not uniformly conserved in H. glycines and that the expression profiles of genes for metabolic enzymes of C. elegans dauer larvae and H. glycines infective J2 are dissimilar.

Conclusion

Our results indicate that hallmark gene expression patterns and metabolism features are not shared in the developmentally arrested life stages of C. elegans and H. glycines, suggesting that developmental arrest in these two nematode species has undergone more divergent evolution than previously thought and pointing to the need for detailed genomic analyses of individual parasite species.     

Motility of Marichromatium gracile in response to light, oxygen, and sulfide.

The motility of the purple sulfur bacterium Marichromatium gracile was investigated under different light regimes in a gradient capillary setup with opposing oxygen and sulfide gradients. The gradients were quantified with microsensors, while the behavior of swimming cells was studied by video microscopy in combination with a computerized cell tracking system. M. gracile exhibited photokinesis, photophobic responses, and phobic responses toward oxygen and sulfide. The observed migration patterns could be explained solely by the various phobic responses. In the dark, M. gracile formed an approximately 500-microm-thick band at the oxic-anoxic interface, with a sharp border toward the oxic zone always positioned at approximately 10 microM O(2). Flux calculations yielded a molar conversion ratio S(tot)/O(2) of 2.03:1 (S(tot) = [H(2)S] + [HS(-)] + [S(2-)]) for the sulfide oxidation within the band, indicating that in darkness the bacteria oxidized sulfide incompletely to sulfur stored in intracellular sulfur globules. In the light, M. gracile spread into the anoxic zone while still avoiding regions with >10 microM O(2). The cells also preferred low sulfide concentrations if the oxygen was replaced by nitrogen. A light-dark transition experiment demonstrated a dynamic interaction between the chemical gradients and the cell's metabolism. In darkness and anoxia, M. gracile lost its motility after ca. 1 h. In contrast, at oxygen concentrations of >100 microM with no sulfide present the cells remained viable and motile for ca. 3 days both in light and darkness. Oxygen was respired also in the light, but respiration rates were lower than in the dark. Observed aggregation patterns are interpreted as effective protection strategies against high oxygen concentrations and might represent first stages of biofilm formation.     

Vital staining of glycoprotein secreted by infective third stage larvae of Haemonchus contortus prior to exsheathment.

Staining of infective third stage larvae of Haemonchus contortus prior to exsheathment shows that protein is exuded from the head region and spreads between the cuticles and that a carbohydrate-containing substance is also found between the L2 and L3 cuticles throughout, at exactly the same sites in this nematode. This glycoprotein, which is either secreted from the mouth or the amphids or both is exuded whether or not the larvae have been stimulated to exsheath. Slight staining was also sometimes detected at the secretory-excretory pore and to an even lesser extent at the anus. This glycoprotein is thought to function as a lubricant to prevent abrasion between the cuticles. It accumulates in areas where there is greatest space between the cuticles such as the head, tail and where bending of the nematode body occurs.     

Identification of penile inputs to the rat gracile nucleus

Neurons in the medullary reticular formation (MRF) of the rat receive a vast array of urogenital inputs. Using select acute and chronic spinal cord lesions to identify the location of the ascending neural circuitries providing either direct or indirect inputs to MRF from the penis, our previous studies demonstrated that the dorsal columns and dorsal half of the lateral funiculus convey low- and high-threshold inputs, respectively. In the present study, the gracile nucleus was targeted as one of the likely sources of low-threshold information from the penis to MRF. Both electrophysiological recordings and neuroanatomical tracing [injection of cholera toxin B subunit (CTB) into a dorsal nerve of the penis] were used. After discrimination of a single neuron responding to penile stimulation, testing for somatovisceral convergence was done (mechanical stimulation of the distal colon and the skin over the entire hindquarters). In 12 rats, a limited number of neurons (43 in total) responded to penile stimulation. Many of these neurons also responded to scrotal stimulation (53.5%, dorsal and/or ventral scrotum) and/or prepuce stimulation (46.5%). Histological reconstruction of the electrode tracks showed that the majority of neurons responding to penile stimulation were located ventrally within the medial one-third of the gracile nucleus surrounding obex. This location corresponded to sparse innervation by CTB-immunoreactive primary afferent terminals. These results indicate that neurons in the gracile nucleus are likely part of the pathway that provides low-threshold penile inputs to MRF, a region known to play an important role in mating processes.     

In vitro migration of Dictyocaulus viviparus larvae: migration of the infective stage inagar gel.

The infective stage of the cattle lungworm, Dictyocaulus viviparus, was able to migrate in agar gel when activated by bile. The number of larvae which penetrated the upper surface of a 2 mm thick agar layer was counted and found to be independent from the agar concentration. Larvae which had migrated out of the agar remained on the surface and did not re-enter. The agar migration process was temperature dependent. Influence of time as well as dependency of agar thickness was investigated. The significance of these findings is discussed.     

A note on the correlation between rainfall and the prevalence of Gnathostoma spp. infective stage larvae in swamp eels in Thailand

Gnathostoma spinigerum is the major causative agent of human gnathostomiasis, a parasitic zoonosis with a great variety of clinical manifestations. Generally, humans are infected by consumption of third-stage larvae (L3) of G. spinigerum in infected hosts in the form of partially cooked or uncooked food. Surveys of the contamination of Gnathostoma spp. L3 in swamp eels are useful for prevention and control of diseases and have been continuously performed in Thailand. The author performed a retrospective study on 33 previous cross-sectional surveys with geographical data and the prevalence of Gnathostoma spp. L3 that covered 12 provinces in Thailand. The relation between rainfall (derived from the geographical data) and the prevalence of Gnathostomo spp. L3 in swamp eels (derived from the overall infection rate of Gnathostoma spp. L3) was investigated. The least-square equation plot rainfall (y) versus prevalence (x) is y= 9.68x + 1,035.12 (r = 0.83; p < 0.01). A significant correlation was discerned between rainfall and the prevalence of eel infection but not for the season of the survey. Similar to the previous study, the prevalence of eel infection may depend on rainfall rather than season. However, this study focused on only 33 cross-sectional surveys in Thailand; further similar study in other countries to assess the correlation between rainfall and the prevalence of infection is required to substantiate this conclusion.     

Hormonal therapy for stage D cancer of the prostate.

Adenocarcinoma of the prostate is the most common malignant neoplasm occurring in men. About half of patients present with metastatic disease. The mainstay of the treatment of stage D cancer of the prostate is hormonal therapy. Bilateral simple orchiectomy remains the gold standard with which other therapies must be compared. Luteinizing hormone-releasing hormone analogues and antiandrogens are now most commonly used but are costly. Initiating hormonal therapy immediately on diagnosing metastatic disease appears to have some advantage over delaying therapy until a patient is symptomatic. Total androgen blockade also appears to be beneficial in terms of survival but at high cost.     

The axenic cultivation of insect forms of Trypanosoma (Duttonella) vivax and development to the infective metacyclic stage

A method for axenic cultivation of epimastigote and metacyclic forms of Trypanosoma (Duttonella) vivax at 27 degrees C in vitro is described. Iscove's medium was supplemented with specific concentrations of foetal bovine serum, L-proline, L-glutamine hypoxanthine, adenosine, pyruvate, and 2-mercaptoethanol. Bloodstream form parasites rapidly transformed into epimastigote forms that grew as surface-adherent colonies in plastic culture flasks. Transformation of epimastigotes to metacyclic forms was first observed 9-12 days after initiation of cultures. Percentages of metacyclics varied: East African T. vivax ranged up to 40% and West African T. vivax ranged up to 24%. Subcultures were made at two-week intervals and maintained for several months. Transformation of bloodstream forms to epimastigotes depended on initial attachment to the bottom of culture flasks and the presence of L-proline. The number and maturity of metacyclic forms was influenced by the concentrations of foetal bovine serum, L-proline, L-glutamine, and 2-mercaptoethanol. Trypanosomes from cultures were cryopreserved, revived, and used to re-establish fresh axenic cultures. These results represent a significant advance in cultivation of T. vivax insect forms that should enable studies to be accomplished on metabolism, differentiation, and pharmacology of this parasitic protozoan, free from the influence of extraneous cells.     

Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in the present study. After in vitro maturation (IVM) of cumulus-oocyte complexes for 48 h, 75.4% of them extruded a visible polar body (PB). Most of the oocytes with a first polar body (PB+ group) were at the metaphase-II (M-II) stage (91.4%). Most of the oocytes without a visible polar body (PB− group) appeared to be arrested at the germinal vesicle (GV) (41.6%) and metaphase-I (M-I) (34.0%) stages. After IVF of oocytes (day of IVF = Day 0), there was no difference between PB⁺ and PB⁻ groups in rates of sperm penetration, mono-spermy, however oocyte activation rate after penetration was greater in the PB+ than in the PB− group (P < 0.05). On Day 2, there was no difference between rates of embryos cleaved at the 2–4 cell stages in PB+ and PB− groups (42.1 ± 48.8% and 33.6 ± 2.1%, respectively). On Day 4, the rate of PB+ embryos developing beyond the 4-cell stage was greater than that of PB− embryos (P < 0.05, 31.7 ± 3.9% and 14.1 ± 1.5%, respectively), and PB+ embryos had more cells than the PB− embryos (P < 0.05, 8.3 ± 0.4 and 6.0 ± 0.8 cells, respectively). On Day 6, a greater proportion of PB+ embryos developed to the blastocyst stage than did PB− embryos (P < 0.05, 34.6 ± 2.4% and 20.7 ± 2.8%, respectively). However, when the GV oocytes of the PB− group were not included in recalculations, there was no difference in blastocyst rates between M-I arrested and M-II oocytes (35.3 and 34.6%, respectively). The number of blastomere nuclei in embryos obtained from the PB+ group (52.0 ± 2.5) was greater than that from the PB− group (P < 0.05, 29.1 ± 2.8). The proportion of degenerated parts in the blastocysts, as determined by morphological appearance, was the same in the PB+ and PB− groups. Although the quality of PB+ embryos was enhanced as compared with that of the PB− group, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ (1:1.9 and 1:2.2, respectively). Chromosome analysis revealed that PB+ blastocysts had more diploidy (P < 0.05, 69.7%) than did PB− blastocysts (44.0%), whereas PB− blastocysts had more triploid cells (P < 0.05, 34.0%) than did PB+ oocytes (8.4%). These results indicate that pig oocytes arrested before the M-II stage (M-I oocytes) undergo cytoplasmic maturation during maturation culture and have the same ability to develop to blastocysts after IVF as M-II oocytes, but some of them resulted in degeneration or delayed development with poor embryo quality.     

Ultrastructural changes in the gracile nucleus of the spontaneously diabetic BB rat.

The present study describes the structural changes in the gracile nucleus of the spontaneously diabetic BB rat. At 3-7 days post-diabetes, axons, axon terminals and dendrites showed electron-dense degeneration. Degenerating axons were characterized by swollen mitochondria, vacuolation, accumulation of glycogen granules, tubulovesicular elements, neurofilaments and dense lamellar bodies. Degenerating axon terminals consisted of an electron-dense cytoplasm containing swollen mitochondria, vacuoles and clustering of synaptic vesicles. These axon terminals made synaptic contacts with cell somata, dendrites and other axon terminals. Degenerating dendrites were postsynaptic to normal as well as degenerating axon terminals. At 1-3 months post-diabetes, degenerating electron-dense axons, axon terminals and dendrites were widely scattered in the neuropil. Macrophages containing degenerating electron-dense debris were also present. At 6 months post-diabetes, the freshly degenerating neuronal elements encountered were similar to those observed at 3-7 days. However, there were more degenerating profiles at 6 months post-diabetes compared to the earlier time intervals. Terminally degenerating axons were vacuolated and their axoplasm appeared amorphous. It is concluded that degenerative changes occur in the gracile nucleus of the spontaneously diabetic BB rat.     

Development of the embryonic chick wing bud from stage 24 to stage 32

If a graft is placed in an early chick wing bud, the location of the graft after several days of further development cannot be predicted solely from the rate of proximal-distal outgrowth. The movement of the graft depends on the rate of outgrowth of the wing but also on morphogenetic tissue movements intrinsic to the wing and on accommodation to the growth and morphogenetic movements of the body of the embryo. Numerous experiments have been reported in which tissue grafted into ectopic sites in the wing causes abnormal wing development. These experiments have been discussed in terms of pattern formation or positional information. However, until the movement of wing tissue during normal development is understood, it cannot be known in what way the development of grafts placed in ectopic sites is abnormal. Previous experiments have demonstrated that carbon particles placed in the wing move in the same manner as grafts of wing mesenchyme, but the carbon particles do not affect normal wing development. Carbon particles were placed in the wing, dorsal to the base of the wing, and cranial and caudal to the wing, to plot the expected movement of a graft and to discover how this movement can be predicted from the tissue movements at the base of the wing. It is concluded that three tissue movements are responsible for the movement of a graft. These are outgrowth at a rate determined by the rate of cell division, formation of the shoulder through caudal movement of the tissues cranial to the wing, and ventral movement of prospective flank ventral to somite 19. These three tissue movements and their influence on normal wing development are discussed.