Sharing of antigens among filarial species revealed by antibody dependent cell-mediated reactions

Antisera raised in albino rats against microfilariae ofLitomosoides carinii, Brugia pahangi, Brugia malayi and sera fromBancroftian elephantiasis patients promoted rat neutrophil-mediated adherence and cytotoxicity to the microfilariae. Pre-treatment of the immune sera, with microfilarial antigen at a final concentration of 5 and 25μg per ml blocked cellular adherence and cytotoxicity to the microfilariae indicating the presence of cross-reactive antibodies. The heterologous immune sera were effective in eliminating the circulatingLitomosoides carinii microfilariae inMastomys natalensis. Contribution No. 766 from Hindustan CIBA-GEIGY Research Centre.     

Effect of thiamine deficiency on the filarial infection of albino rats with Litomosoides carinii

Induction of thiamine deficiency in albino rats led to greater susceptibility to infection with the filarial parasite, Litomosoides carinii. The patency of the infection was prolonged and there was a greater worm burden and a higher peak microfilaraemia in the deficient animals. The haemagglutinating antibody response to the infection was significantly reduced. The mitogenic response of the lymphocytes to PHA and Con A decreased progressively in infected, pair-fed and deficient animals in that order, suggesting the immunosuppressive effect of the infection and the synergistic role of thiamine deficiency on this effect. At the onset of latency to the infection, the serum from animals of all groups promoted antibody-dependent adhesion of splenic cells to microfilariae.     

Schistosoma mansoni and Schistosoma haematobium: Differences in development

Growth and maturation of the Puerto Rico strain of Schistosoma mansoni in mice and the Ghana strain of Schistosoma haematobium in hamsters were compared beginning 19 days after infection. In S. mansoni, optimum development was determined, with copulation first observed on Day 25, egg shell protein formation observed on Day 28, and oviposition occurring on Day 30. In infections of S. haematobium, copulation first occurred on Day 29. Egg shell proteins were first formed on Day 45, and egg production occurred on Day 60. Examination of unisexual and bisexual infections showed that maturation of the vitellaria can be more easily assessed by autofluorescence of the protein globules than by the traditional diazonium salt stains. Autofluorescence of living worms with mature vitellaria allows subsequent examination by electron microscopy, and therefore permits evaluation at a subcellular level.     

Plasmodium chabaudi: Adoptive transfer of immunity with different spleen cell populations and development of protective activity in the serum of lethally irradiated recipient mice

Groups of lethally X-irradiated NIH mice were injected with either glass wool-filtered (g.w.) immune spleen cells or nylon wool enriched immune T cells from syngeneic mice immune to Plasmodium chabaudi, or g.w. normal spleen cells. After cell recipients were infected with P. chabaudi the three groups reached similar mean peak parasitaemias on Day 11. In passive transfer tests serum obtained from mice sacrificed at this time gave little protection compared to normal serum. On Day 14 g.w. immune spleen cell recipients had subpatent infections and enriched immune T-cell recipients had a lower mean parasitaemia than g.w. normal spleen cell recipients. Serum obtained on Day 14 from g.w. immune spleen cell recipients gave better protection after passive transfer than sera from enriched immune T-cell or g.w. normal spleen cell recipients. Day 14 serum from enriched immune T-cell recipients, but not from g.w. normal spleen cell recipients, produced some initial protection after passive transfer. These results suggest that the transferred immune spleen cells contributed to the observed humoral immunity in lethally irradiated recipient mice.     

Comparative morphology and ecology of two species of Litomosoides (Nematoda: Filarioidea) of rodents in Florida, with a key to the species of Litomosoides Chandler, 1931

From April 1970 to August 1971, 235 cotton rats (Sigmodon hispidus) and 281 rice rats (Oryzomys palustris) were trapped in salt and fresh water marshes in north-central Florida and examined for natural infections of filarial worms, Litomosoides spp. Cotton rats from both types of marshes were infected (28–44 per cent prevalence), whereas only rice rats from the salt marsh were infected (56 per cent prevalence). Older animals were more commonly infected than younger ones. In cotton rats the worms were located in the pleural cavity, whereas in rice rats the worms were located primarily in the abdominal cavity. Filarial worms from rice rats were transmitted experimentally to laboratory-reared rice rats and cotton rats, but worms from cotton rats were transmitted only to cotton rats. Morphological studies on adult forms and microfilariae indicated that the worms in rice rats were distinct from those in cotton rats and are therefore described as Litomosoides scotti sp. n. The cotton rat filariids were referable to Litomosoides carinii (Travassos, 1919) Vaz, 1934. L. scotti differs from L. carinii in the ratio of the spicules, in the shape of the distal end of the right spicule and in having shorter microfilariae.     

Plasmodium berghei: The spleen in sporozoite-induced immunity to mouse malaria

A/J mice were splenectomized (Splx) or sham-splenectomized (SSplx) prior to administration of a single injection of irradiated sporozoites. Following challenge 7 days after immunization, it was found that none of the splenectomized mice were protected whereas 50% of the sham-splenectomized and intact animals were resistant to challenge. In another series of experiments similar groups, along with mice splenectomized just prior to challenge, were injected with 1.5 × 10⁵ irradiated sporozoites over a 5 week period. This resulted in protection of (1) 60–100% of the animals splenectomized before immunization, and (2) 90–100% protection of the animals splenectomized prior to challenge, as well as the intact and sham-splenectomized mice. Serum levels of antisporozoite antibodies (CSP and SNA) increased during immunization of the intact animals. Only 15–20% of the animals splenectomized prior to immunization presented positive CSP reactions and little if any sporozoite neutralizing activity (SNA) was detected. Serum from intact animals immunized and found resistant to sporozoite challenge was used for passive transfer studies. Immune serum recipients were challenged with small numbers of sporozoites. Only one out of 18 recipients was protected against sporozoite challenge.     

Strongyloides ratti: Dissociation of the rat's protective immunity into systemic and intestinal components

Analysis of the early stages of a challenge infection with Strongyloides ratti has shown that protection is expressed against the developing third-stage larval worms (L₃) and prevents the maturation to adulthood of most larvae. Challenge after an immunizing infection that was restricted to the parenteral L₃ migratory phase showed that some 10–40% of overall protection could be ascribed to systemic antilarval immunity. Some larvae were trapped in the skin at the site of injection whereas others failed to migrate to the head and lung of immune rats. Larvae arriving in the intestine at Days 3, 4, and 5 did not persist beyond Day 7 and 8. Studies using [⁷⁵Se]methionine-labeled L₃ showed a significant increase in fecal label in rats immunized by a complete infection. This loss did not occur to the same extent in rats immunized only with parenteral larvae. Significant rejection of worms transplanted to the intestine also indicated intestinal protection. The possible existence of large numbers of worms in a state of “arrested development” was excluded by their failure to appear after cortisone treatment and the absence of worm accumulation in radiolabeling studies. It is concluded that at least two responses operate against larval S. ratti, one is systemic and the other operates in the intestine against larvae in a manner that resembles the “rapid expulsion” rejection of Trichinella spiralis in immune rats.     

Interorder transfer of mycoplasma-like microorganisms between Drosophila paulistorum and Ephestia kuehniella: Tissues,dosages, and effects

Testis and head extracts of sterile male intersemispecific hybrid Drosophila paulistorum are pathogenic when injected into Ephestia kuehniella larvae. The time and frequency of death of the recipients is a function of the extract concentration. Extracts of Ephestia recipients are pathogenic and will kill E. kuehniella larvae and D. paulistorum adults. Larval Drosophila recipients of these extracts survive through pupation. A majority of these recipients fail to eclose. Those animals which do survive to the adult stage and which are fertile demonstrate the same pattern of sterility among their progeny as is found in D. paulistorum hybrids and recipients of D. paulistorum extracts. The results indicate that the pathogenicity of the extracts is due to the mycoplasma-like microorganisms which are responsible for the male intersemispecific hybrid sterility in D. paulistorum. The mycoplasma-like microorganisms grow rapidly in E. kuehniella hosts, with a division time of approximately 1.8 hr, and they retain their host specificity for the semispecies of D. paulistorum.     

Schistosoma mansoni and Schistosoma haematobium: Emergence of schistosome cercariae from snails with darkness and illumination

Biomphalaria glabrata and Bulinus globosus were infected with Schistosoma mansoni and Schistosoma haematobium, respectively, and the effect of different illumination conditions at 25 C on cercarial output was observed for 4 days. In both species, a dark period of 10–14 hr on Day 2 of the observation period resulted in an emergence pattern on Day 3 similar to the regular pattern recorded for Day 1. Total cercarial output on Day 3 was within 30% of the control (Day 1) output. A dark period of between 0 and 8 hr resulted in suppression of cercarial emergence and in abolishment of the regular hourly emergence pattern on Day 3. A dark period of 16–20 hr resulted in an emergence pattern with two peaks, the first occurred at Hour 1, and the other at Hour 5 of the subsequent light period. Interjection of a 1-hr dark period during the light period of Day 3, following short (2–8 hr) exposure to dark on the preceding day, produced an increase in cercarial shedding of S. mansoni immediately after restitution of the light conditions. On the other hand, in S. haematobium, cercarial output was stimulated during the interposed dark period itself.     

Pneumocystis carinii: Freeze-fracture study of stages of the organism

The morphology and life cycle of Pneumocystis carinii were studied in cortico-steroid-treated rats by ultrathin section and freeze-fracture electron microscopy. The following stages of P. carinii were noted: trophic, precyst, and cyst. The crescent-shaped cysts appeared to be intermediate forms between precyst and cyst. The cell wall of the trophic stage showed membrane structures suggestive of protozoan endocytosis, whereas the surface of the precyst stage was smooth. The cell wall of the cyst lacked the specialized structural differentiation of yeasts and resembled that of Plasmodium spp. We conclude that P. carinii belongs to the Protozoa, and is presumably Rhizopoda.     

Dipetalonema viteae: Response of spleen cells in experimental mouse filariasis to mitogens and antigens

Balb/c mice were infected by transplanting 3, 5, 10, or 20 female adult Dipetalonema viteae under the dorsal skin. The microfilaremias resulting from infections with 3 or 5 adult worms were of lesser magnitude and of shorter duration than those produced in infections with 10 or 20 worms. Spleen cells taken from these mice at various intervals after infection were assayed in vitro for their ability to respond to phytohemagglutinin (PHA) or lipopolysaccharide (LPS). There was no depression in the response to LPS or to PHA in mice given infections of 3 or 5 D. viteae adult worms. In contrast, the response to PHA was significantly depressed in groups receiving 10 or 20 adult female worms 12 days after infection and by Day 25, the depression was severe. Thereafter the PHA responsiveness recovered gradually to reach control values on Day 60. In mice transplanted with 10 or 20 D. viteae adult worms there was no significant depression in the response to LPS at any time during the infection, but the response was increased slightly sporadically during infection. These results indicate that in mice, this infection causes an initial suppression in the function of PHA-sensitive T cells but has little effect on the B cells which respond to LPS. A factor present in serum taken on Day 25 from mice infected with 10 or 20 adult worms inhibited the proliferative response to PHA by spleen cells from normal mice. The recovery of PHA responsiveness in mice given the heavier infections coincided with death of the adult worms, but mitogen reactivity and microfilaremia were unrelated. Antigens from male or female worms induced cell division in spleen cells taken from infected mice after microfilaremia had ceased whether they were implanted with 3 or 10 adult worms.     

A new clade of Mesorhizobium nodulating Alhagi sparsifolia

We isolated 33 nodule bacteria from the legume Alhagi sparsifolia growing in the desert of northwest China. They fell into three groups by restriction analysis of their rrs (small subunit ribosomal RNA) genes, and these, together with dnaK and dnaJ genes, were sequenced from representative isolates to assess their taxonomic position by phylogenetic analysis. The bacteria in each group belonged to different lineages that might represent three different new Mesorhizobium species, two of which form a novel clade very distinct from other species in the genus. Most A. sparsifolia symbionts harboured closely related nodA and nodC genes forming new lineages. The presence of these closely related symbiosis genes in various genomic backgrounds and the incongruence observed between the different gene phylogenies indicate a history of horizontal gene transfer of symbiosis genes between the A. sparsifolia symbionts.     

Impact of RTS,S/AS02A and RTS,S/AS01B on Genotypes of P. falciparum in Adults Participating in a Malaria Vaccine Clinical Trial

Objective

RTS,S, a candidate vaccine for malaria, is a recombinant protein expressed in yeast containing part of the circumsporozoite protein (CSP) sequence of 3D7 strain of Plasmodium falciparum linked to the hepatitis B surface antigen in a hybrid protein. The RTS,S antigen is formulated with GSK Biologicals'' proprietary Adjuvant Systems AS02_A or AS01_B. A recent trial of the RTS,S/AS02_A and RTS,S/AS01_B vaccines evaluated safety, immunogenicity and impact on the development of parasitemia of the two formulations. Parasite isolates from this study were used to determine the molecular impact of RTS,S/AS02_A and RTS,S/AS01_B on the multiplicity of infection (MOI) and the csp allelic characteristics of subsequent parasitemias.

Design

The distribution of csp sequences and the MOI of the infecting strains were examined at baseline and in break-through infections from vaccinated individuals and from those receiving a non-malarial vaccine.

Setting

The study was conducted in Kombewa District, western Kenya.

Participants

Semi-immune adults from the three study arms provided isolates at baseline and during break-through infections.

Outcome

Parasite isolates used for determining MOI and divergence of csp T cell–epitopes were 191 at baseline and 87 from break-through infections.

Results

Grouping recipients of RTS,S/AS01_A and RTS,S/AS02_B together, vaccine recipients identified as parasite-positive by microscopy contained significantly fewer parasite genotypes than recipients of the rabies vaccine comparator (median in pooled RTS,S groups: 3 versus 4 in controls, P = 0.0313). When analyzed separately, parasitaemic individuals in the RTS,S/AS01_B group, but not the RTS,S/AS02_A group, were found to have significantly fewer genotypes than the comparator group. Two individual amino acids found in the vaccine construct (Q339 in Th2R and D371 in Th3R) were observed to differ in incidence between vaccine and comparator groups but in different directions; parasites harboring Q339 were less common among pooled RTS,S/AS vaccine recipients than among recipients of rabies vaccine, whereas parasites with D371 were more common among the RTS,S/AS groups.

Conclusions

It is concluded that both RTS,S/AS vaccines reduce multiplicity of infection. Our results do not support the hypothesis that RTS,S/AS vaccines elicit preferential effects against pfcsp alleles with sequence similarity to the 3D7 pfcsp sequence employed in the vaccine construct.     

Narrow host range of some streptococcal R plasmids

Nine R plasmids originally harbored by Streptococcus faecalis (pIP614, pIP655, pIP685, pIP686, pIP 1075, pIP1017),S. faecium (pIP716, pIP991), and group B Streptococcus (pMV120) wild-type hosts were transferred by conjugation into various recipients in order to study the extent of their intraspecies, interspecies, and intergeneric host range. Recipients were streptococci of groups A, B, C, D (S. faecalis, S. faecium, S. durans, S. bovis), and G, S. sanguis, two S. pneumoniae strains (encapsulated and nonencapsulated), and two strains of different genera, Staphylococcus aureus and Listeria inocua. The plasmids carried different antibiotic resistance markers: tetracycline, high levels of gentamicin and kanamycin or of streptomycin and kanamycin, and chloramphenicol. These R plasmids displayed narrow host ranges. They transferred into S. faecalis recipients and plasmid DNA could be detected in these transconjugants. Occasionally, the R plasmids also transferred into one or more other recipients, but no detectable plasmid DNA could be demonstrated in the new hosts.     

Monitoring preantral follicle survival and growth in bovine ovarian biopsies by repeated use of neutral red and cultured in vitro under low and high oxygen tension

The development and optimization of preantral follicle culture methods are crucial in fertility preservation strategies. As preantral follicle dynamics are usually assessed by various invasive techniques, the need for alternative noninvasive evaluation tools exists. Recently, neutral red (NR) was put forward to visualize preantral follicles in situ within ovarian cortical fragments. However, intense light exposure of NR-stained tissues can lead to cell death because of increased reactive oxygen species production, which is also associated with elevated oxygen tension. Therefore, we hypothesize that after repeated NR staining, follicle viability and dynamics can be altered by changes in oxygen tension. In the present study, we aim (1) to determine whether NR can be used to repeatedly assess follicular growth, activation, and viability and (2) to assess the effect of a low (5% O₂) or high (20% O₂) oxygen tension on the viability, growth, and stage transition of preantral follicles cultured in vitro by means of repeated NR staining. Cortical slices (n = 132; six replicates) from bovine ovaries were incubated for 3 hours at 37 °C in a Leibovitz medium with 50 μg/mL NR. NR-stained follicles were evaluated in situ for follicle diameter and morphology. Next, cortical fragments were individually cultured in McCoy's 5A medium for 6 days at 37 °C, 5% CO₂, and 5% or 20% O₂. On Days 4 and 6, the fragments were restained by adding NR to the McCoy's medium and follicles were reassessed. In both low and high oxygen tension treatment groups, approximately 70% of the initial follicles survived a 6-day in vitro culture, but no significant difference in follicle survival on Day 4 or 6 could be observed compared with Day 0 (P > 0.05). A significant decrease in the number of primordial and increase in primary and secondary follicles was observed within 4 days of culture (P < 0.001). In addition, a significant increase of the mean follicle diameter in NR-stained follicles was observed (P < 0.001), resulting in an average growth of 11.82 ± 0.81 μm (5% O₂) and 11.78 ± 1.06 μm (20% O₂) on Day 4 and 20.94 ± 1.24 μm (5% O₂) and 19.12 ± 1.36 μm (20% O₂) on Day 6 compared with Day 0. No significant differences in follicle growth rate or stage transition could be observed between 5% and 20% O₂ (P > 0.05). In conclusion, after repeated NR staining, we could not find a difference between low and high oxygen tension in terms of follicle viability, stage transition, or growth. Therefore, under our culture conditions follicle dynamics are not determined by the oxygen tension in combination with quality assessment protocols using repeated NR staining.     

Hormonal control of seasonal morph determination in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

Seasonal morphs (spring and sumer forms) of Papilio xuthus L. are determined coincidentally with diapause and non-diapause in pupae by larval exposure to short days and long days respectively. The neuroendocrine principle underlying seasonal-morph determination was studied using surgical operations in P. xuthus.When recipient 0-day old or chilled diapause pupae were joined to donor 0-day old non-diapause pupae, the recipients developed into summer or intermediate morphs. When the same kinds of recipients used above were joined to 0-day old or chilled diapause pupae, there were no significant effects on the adult morph. In contrast, recipient non-diapause pupae all developed into summer morphs, regardless of groups of the type of donors.Furthermore, when the brain of 5th-instar larvae, pharate pupae or pupae, predetermined to be diapause, was transplanted into the abdomen of 0-day old, 30-day old or chilled diapause pupae, the recipients developed into summer or intermediate morphs.The results indicate that the brain of non-diapause pupae secretes a humoral factor producing the summer morph. In non-diapause pupae, the factor may be secreted at about the stage of larval-pupal ecdysis coincidentally with that of prothoracicotropic hormone.     

A new ovine Babesia species transmitted by Hyalomma anatolicum anatolicum

The pathogenicity and morphology of a large Babesia species, Babesia sp. Xinjiang, are described here. The parasite has very low virulence for sheep, and caused no detectable clinical symptoms. Splenectomized sheep infected with the parasite showed mild fever and low parasitemia and would recover gradually. If splenectomized sheep were immuno-suppressed with dexamethasone, the parasitemia could reach 8.5%, and death occurred. A splenectomized calf could not be infected with the Babesia species. Paired parasites were the typical form of the Babesia species in erythrocytes and the average size of a pair of parasites was 2.42 (±0.35) μm × 1.06 (±0.22) μm. Merozoites were found in the gut, salivary gland, haemolymph, ovary and eggs of female Hyalomma anatolicum anatolicum engorged on sheep infected with the parasites. The results of experimental transmission showed that the larval, nymph and adult stages of H. a. anatolicum could transmit the Babesia species to sheep.     

Molecular Imaging of Induced Pluripotent Stem Cell Immunogenicity with In Vivo Development in Ischemic Myocardium

Whether differentiation of induced pluripotent stem cells (iPSCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection, is unclear. Here, we dynamically demonstrated the immunogenicity and rejection of iPSCs in ischemic myocardium using bioluminescent imaging (BLI). Murine iPSCs were transduced with a tri-fusion (TF) reporter gene consisting of firefly luciferase-red fluorescent protein-truncated thymidine kinase (fluc-mrfp-tTK). Ascorbic acid (Vc) were used to induce iPSCs to differentiate into cardiomyocytes (CM). iPSCs and iPS-CMs were intramyocardially injected into immunocompetent or immunosuppressed allogenic murine with myocardial infarction. BLI was performed to track transplanted cells. Immune cell infiltration was evaluated by immunohistochemistry. Syngeneic iPSCs were also injected and evaluated. The results demonstrated that undifferentiated iPSCs survived and proliferated in allogenic immunocompetent recipients early post-transplantation, accompanying with mild immune cell infiltration. With in vivo differentiation, a progressive immune cell infiltration could be detected. While transplantation of allogenic iPSC-CMs were observed an acute rejection from receipts. In immune-suppressed recipients, the proliferation of iPSCs could be maintained and intramyocardial teratomas were formed. Transplantation of syngeneic iPSCs and iPSC-CMs were also observed progressive immune cell infiltration. This study demonstrated that iPSC immunogenicity increases with in vivo differentiation, which will increase their chance for rejection in iPSC-based therapy.     

Hymenolepis diminuta: The origin of protective antigens

Mice were infected orally with 1,6, or 30 cysticercoids of Hymenolepis diminuta. These were allowed to develop for different periods of time before elimination with anthelminthic, thus exposing the hosts to antigens from the prestrobilate, early strobilate, or fully strobilate worms. Other groups of mice were immunized by intraperitoneal (ip) implantation of a live strobilate worm or by ip implantation of live worms from cysticercoids excysted in vitro. Strong protection against challenge with a surgically transplanted strobilate worm was achieved by prior infection with 6 or 30 worms eliminated as early as Day 3 of infection. By this time these worms would not have strobilated. Conversely, a single worm, strobilating extensively over 16 days, stimulated only weak protection. Parenteral implantation of excysted worms protected mice but parenteral implantation of a strobilate worm had no effect. It is suggested that (i) the tapeworm protective antigens are primarily related to the scolex and/or the germinative region; (ii) the number of worms and the duration of antigenic stimulation in an immunizing infection determine the magnitude of a protective secondary response.