Comparison of the effects of irradiation and splenectomy on Babesia rodhaini infection in mice

Comparison of the effects of irradiation and splenectomy on Babesia rodhaini infection in mice. International journal for Parasitology 3: 773–781. Babesia rodhaini infection was compared in irradiated, splenectomized and control mice. Although irradiation reduced the weight of the spleen by as much as 95 per cent, this reduction in size did not result in parasitaemia levels comparable to those seen in splenectomized mice, which were consistently higher. Parasitaemias were similar in irradiated and control mice, but the mean survival time in control mice was longer than that of irradiated or splenectomized mice, which were comparable. Splenectomy generally resulted in higher parasitaemias than those seen in non-splenectomized mice.

Since B. rodhaini has a predeliction for invading reticulocytes, the apparent failure of irradiated mice to develop parasitaemias comparable to those of splenectomized mice, may have been due to the selective destruction of these immature red cells by irradiation.     

Babesia microti: Molecular and antigenic characterizations of a novel 94-kDa protein (BmP94)

A novel gene, BmP94, encoding 94-kDa protein of Babesia microti was identified by immunoscreening of the cDNA expression library. The full-length of BmP94 was expressed in Escherichia coli (rBmP94), which resulted in insoluble form with low yield, and the truncated hydrophilic C-terminus region of the gene was expressed as a soluble protein (rBmP94/CT) with improved productivity. Antiserum raised against rBmP94/CT recognized the 94-kDa native protein in the parasite extract by Western blot analysis. Next, an ELISA using rBmP94/CT was evaluated for diagnostic use, and it demonstrated high sensitivity and specificity when tested with the sera from mice experimentally infected with B. microti and closely related parasites. Moreover, the immunoprotective property of rBmP94/CT as a subunit vaccine was evaluated in BALB/c mice against a B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmP94 could be a promising candidate for diagnostic use for human babesiosis.     

Babesia bigemina: In Vitro and in VivoEffects of Curdlan Sulfate on Growth of Parasites

Igarashi, I., Njonge, F. K., Kaneko, Y., and Nakamura, Y. 1998.Babesia bigemina:In vitroandin vivoeffects of curdlan sulfate on growth of parasites.Experimental Parasitology90, 290–293.     

Comparison of injectable iron complexes in their ability to iron load tissues and to induce oxidative stress

Iron and copper homeostasis have been studied in various tissues after iron-loading with the polynuclear ferric hydroxide carbohydrate complexes, iron dextran, iron polymaltose, iron sucrose and iron gluconate for four weeks. There were significant increases in the iron content of the different rat tissues compared to controls, with the exception of the brain, which showed no change in its iron content following iron loading. However, the level of iron loading in the different tissues varied according to the preparation administered and only iron dextran was able to significantly increase the iron content of both broncho-alveolar macrophages and heart. The hepatic copper content decreased with iron loading, although this did not reach significance. However the copper content did not alter in the iron loaded broncho-alveolar macrophages. Despite such increases in hepatic iron content, there was little evidence of changes in oxidative stress, the activities of cytosolic (apart from iron dextran) or mitochondrial hepatic superoxide dismutase, SOD, were similar to that of the control rats, confirming the fact that the low reduction potential of these compounds prevents the reduction of the ferric moiety. It was not necessary for macrophages to significantly increase their iron content to initiate changes in NO^. release. Iron gluconate and iron sucrose increased NO^. release, while iron polymaltose and iron dextran decreased NO^. release although only the latter iron preparation significantly increased their iron content. It may be that the speciation of iron within the macrophage is an important determinant in changes in NO^. release after ex vivo stimulation. We conclude that tissues loaded with iron by such polynuclear iron complexes have variable loading despite the comparable iron dose. However, there was little evidence for participation of the accumulated iron in free radical reactions although there was some evidence for alteration in immune function of broncho-alveolar macrophages.     

DBA/2J and DBA/2Ha lymphocytes differ in their ability to induce graft-vs-host disease

Graft-vs-host disease (GVHD) is a common occurrence after bone marrow transplantation despite the use of MHC-matched donors and recipients. This indicates that non-MHC loci play an important role in the regulation and development of GVHD. Non-MHC loci have been shown to regulate GVHD in a murine model where acute GVHD results from i.v. injection of C57BL/6J spleen cells into B6D2F1/J [C57BL/6J X DBA/2J)F1) recipients while chronic GVHD results from injection of DBA/2J spleen cells. In contrast to the hyperproduction of Ig and auto-antibodies that is characteristic of the chronic GVHD that occurs after injection of DBA/2J cells, injection of DBA/2Ha cells was found to induce CTL and suppressor cells characteristic of the acute GVHD that results from injection of C57BL/6 cells into B6D2F1/J recipients. Genetic analysis indicated that one autosomal locus is responsible for the different GVHD responses of DBA/2J and DBA/2Ha cells and that the DBA/2Ha allele is dominant. Further studies indicate that the different responses by DBA/2J and DBA/2Ha cells is not due to functional differences between the two sets of cells but by a radiosensitive B6D2F1 recipient immune response which discriminates between the DBA/2J and DBA/2Ha spleen cells.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome c₁ and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c₁ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c₁ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c₁ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Mutants of Agrobacterium tumefaciens with temperature sensitivity in respect to their tumor inducing ability

Summary The isolation of six mutants of Agrobacterium tumefaciens which can induce tumors at low temperatures (22°C) but fail to do so at 28°C is described. At the nonpermissive temperature the following characteristics of the mutants are the same as those of the wild type: growth rates in vitro, growth rates in planta, and sensitivity towards agrocin 84, a marker for the presence of the virulence-plasmid. The tumors induced by the mutants at low temperature grow without addition of hormones at both 22°C and 28°C. The induction of the tumors but not the maintenance of the tumorous phenotypes are affected in the mutants isolated.     

Identification and immunoprotective analysis of an in vivo-induced Edwardsiella tarda antigen

Edwardsiella tarda is a severe aquaculture pathogen that can infect many important fish species cultured worldwide. The aim of this study was to evaluate the vaccine potential of an E. tarda antigen, Eta21, which was identified from a pathogenic E. tarda strain via the method of in vivo-induced antigen technology (IVIAT). Eta21 is 510-amino acid in length and shares 58% sequence identity with a putative peptidase of several bacterial species. eta21 was subcloned into Escherichia coli, and recombinant Eta21 was purified as a histidine-tagged protein. When used as a subunit vaccine, purified recombinant Eta21 was effective against lethal E. tarda challenge in a Japanese flounder model. In order to improve the immunoprotective efficacy of Eta21, the chimera AgaV-Eta21 was constructed, which consists of Eta21 fused in-frame to the secretion domain of AgaV, an extracellular β-agarase. E. coli DH5α harboring plasmid pTAET21, which constitutively expresses agaV-eta21, was able to produce and secret AgaV-Eta21 into the extracellular milieu. Vaccination of Japanese flounder with live DH5α/pTAET21 elicited immunoprotection that is significantly higher in level than that induced by vaccination with purified recombinant Eta21. Vaccination with DH5α/pTAET21 and recombinant Eta21 both induced the production of specific serum antibodies at four to eight weeks post-vaccination. Taken together, these results demonstrate that Eta21, especially that delivered by DH5α/pTAET21, is an effective vaccine candidate against E. tarda infection.     

Evidence for recombination and segregation of virulence to pine in a hybrid cross between Gibberella circinata and G. subglutinans

Two species associated with the Gibberella fujikuroi species complex, G. circinata (the cause of pitch canker in pines) and G. subglutinans (avirulent on pine), were found to have limited interfertility in hybrid crosses. MAT idiomorphs, polymorphisms in the histone H3 gene, vegetative compatibility, and virulence phenotypes were used to verify recombination. The MAT idiomorphs appeared to be assorting independently, but the histone H3 haplotype was disproportionately represented by that of the G. subglutinans parent. Ninety-eight percent (45/46) of the progeny tested were vegetatively incompatible with both parents. All F₁ progeny were avirulent to pine, but a wide range of virulence was restored through a backcross to the virulent parent (G. circinata). Attempts at hybrid crosses using other isolate combinations were rarely successful (1/26). This limited interfertility supports retention of G. circinata and G. subglutinans as separate species, but offers opportunities to characterize the inheritance of virulence to pine.     

Production and characterization of monoclonal antibodies to Acanthamoeba castellanii and their application for detection of pathogenic Acanthamoeba spp

Fourteen monoclonal antibodies (mAbs) were produced against a strain of Acanthamoeba castellanii isolated from a human cornea. The reactivity of the mAbs to reference strains of Acanthamoeba was examined by an indirect fluorescence antibody test (IFA) and Western immunoblot analysis. Nine mAbs reacted specifically with a known pathogenic reference strain of A. castellanii, but not with a non-pathogenic strain or other Acanthamoeba spp. The antigen recognized by these mAbs had a molecular mass of 17 kDa. The remaining five mAbs reacted with A. castellanii and A. polyphaga, members of group II (Pussard and Pons) but not with A. astronyxis (group I) or A. culbertsoni (group III). Western immunoblot analysis revealed that the latter mAbs stained many protein bands ranging from 30 to 150 kDa. None of the 14 mAbs reacted with Naegleria gruberi, N. fowleri, or Entamoeba histolytica. These observations suggest that an antigen common in group II as well as a pathogenic A. castellanii-specific antigen are present. Slot blot reactivity was comparable to the IFA. Under certain circumstances, therefore, slot blot analysis with a panel of mAbs should be helpful in the detection of keratitis-producing strains of Acanthamoeba.     

Connection of Gnomonia intermedia to Discula betulina and its relationship to other taxa in Gnomoniaceae

Discula betulina is a foliar pathogen on birch (Betula) and Gnomonia intermedia is found on overwintered birch leaves. Perithecia of G. intermedia developed in vitro on colonies of D. betulina isolated from birch tissues in late summer, and single ascospores of G. intermedia consistently developed into colonies similar to D. betulina, producing typical D. betulina conidia. Isolates of D. betulina could be grouped into two mating types, which produced fertile perithecia of G. intermedia when mated with each other. Mycelia from single-ascospore and single-conidial isolates were inoculated onto shoots of downy birch, causing lesions and die-back from which D. betulina was consistently isolated. ITS region ribosomal DNA sequence analysis confirmed the results of the morphological and mating studies, and found that the closest known relatives of G. intermedia/D. betulina are Gnomoniella nana and Sirococcus clavigignenti-juglandacearum. The conclusion from these studies is that D. betulina is the anamorph of G. intermedia.     

Multiple in vivo passages enhance the ability of a clinical Helicobacter pylori isolate to colonize the stomach of Mongolian gerbils and to induce gastritis

The Mongolian gerbil is an excellent animal model for Helicobacter pylori-induced gastritis in humans. In this study, initially low colonization rates of the H. pylori strains ATCC 43504, SS1, or HP87 inoculated into gerbils caused difficulties in establishing this model. In order to increase the colonization ability and pathogenicity, the clinical HP87 isolate was selected for adaptation to the gerbil stomach by multiple in vivo passages through gerbils. Development of gastritis was examined histologically at 4-52 weeks after infection. The proportion of gerbils which tested positive for H. pylori by culture at four weeks after inoculation gradually increased from 11.1% of gerbils inoculated with HP87 without prior in vivo passage (P0) to 100% of gerbils inoculated with HP87 with seven in vivo passages (P7). In addition, adaptation of HP87 resulted in more severe histopathological changes. Gerbils infected with adapted HP87 (P7) exhibited severe infiltration by monomorphonuclear and polymorphonuclear leukocytes in the mucosa, submucosa, and subserosa of the gastric antrum, as well as epithelial changes consisting of hyperplasia, erosion, and ulceration. Histopathological changes increased in severity from four to 52 weeks after infection. Adaptation of HP87 during its passages through gerbils could be due to genetic changes in bacterial colonization factors. Identification of these changes might be useful to understand the underlying mechanism of gastric adaptation and pathogenesis of H. pylori.     

Relationship between in vivo synchronicity of Plasmodium falciparum and allelic diversity

Plasmodium falciparum cells tend to grow in synchronicity during their cyclic intraerythrocytic development in vivo. Both host and parasite factors appear to be involved in this synchronization. We examined the link between mixed-allelic-family P. falciparum infection and synchronicity in parasitized red blood cells (PRBC) from symptomatic children.The distribution of rings and trophozoites in each PRBC sample was determined by standard microscopy. P. falciparum was genotyped by using a polymerase chain reaction (PCR) targeting three loci (merozoite surface proteins (MSP) 1 and 2, and 175-kD erythrocyte binding antigen (EBA), allowing us to distinguish parasite clones belonging to a single-allelic family (SAF) and those belonging to a mixed-allelic family (MAF). Parasite development was considered synchronous when peripheral blood contained at least 95% of rings or 95% of trophozoites.Parasite development was synchronous in 22 (21.2%) of the 104 children studied. Twenty (90.9%) of these infections were SAF and two (9.1%) were MAF. Rings and trophozoites predominated in respectively 12 (60%) and 8 (40%) SAF infections. Respectively 17.1% and 82.9% of the 82 asynchronous cases corresponded to SAF and MAF infection. Parasite synchronicity was therefore significantly related to single-allelic-family infection (p < 2 × 10^− 10).Twenty different MSP-1 alleles and thirteen different MSP-2 alleles were identified. Only three isolates from patients with SAF infection comprised a single allele or genotype, the other isolates harboring at least two alleles. The mean number of alleles or clones was respectively 3.0 and 10.0 in SAF and MAF infection. These results reflect the allelic diversity of the MSP loci and show that SAF infection can correspond to multiple parasite clones (or genotypes) but, in general, fewer than in MAF infection (p ≤ 0.0007).These results confirm the extensive polymorphism of P. falciparum vaccine candidates MSP-1 and -2 in southeastern Gabon and demonstrate that parasite synchronicity in vivo is strongly associated with single-allelic-family infection.     

Differences in the virulence of Heterorhabditis bacteriophora and Steinernema scarabaei to three white grub species: The relative contribution of the nematodes and their symbiotic bacteria

Because susceptibility of white grub species to entomopathogenic nematodes differs, we compared the virulence of Photorhabdus temperata and Xenorhabdus koppenhoeferi, the symbiotic bacteria of the nematodes Heterorhabditis bacteriophora and Steinernema scarabaei, respectively, to the three white grub species, Popillia japonica, Rhizotrogus majalis, and Cyclocephala borealis. Both bacteria were pathogenic to all three grub species even at 2 cells/grub. However, the median lethal dose at 48 h post injection and median lethal time at 20 cells/grub showed that P. temperata was more virulent than X. koppenhoeferi to C. borealis. Although H. bacteriophora is less pathogenic than S. scarabaei to R. majalis and P. japonica, their symbiotic bacteria did not differ in virulence against these two grub species, and they also showed similar growth patterns both in vitro and inside R. majalis larvae at 20 °C. We then tested the pathogenicity of oral- and intrahemocoel-introduced H. bacteriophora to R. majalis to determine whether nematodes are able to successfully vector the bacteria into the hemolymph. Hemocoel injected H. bacteriophora was pathogenic to R. majalis indicating successful bacterial release, but orally introduced H. bacteriophora were not. Dissection of grubs confirmed that the orally introduced H. bacteriophora were unable to penetrate into the hemolymph through the gut wall. We conclude that the low susceptibility of R. majalis to H. bacteriophora is not due to the symbiotic bacteria but rather to the nematode’s poor ability to penetrate through the gut wall and the cuticle to vector the bacteria into the hemolymph.     

Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta

The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His₆-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP™ based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni–nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P. angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2 mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2 mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.     

Seasonal and interannual dynamics of polyphenols in Myriophyllum verticillatum and their allelopathic activity on Anabaena variabilis

In 4 successive years, we investigated the seasonal and interannual variability of the total polyphenolic pool and of the individual polyphenolic compounds in Myriophyllum verticillatum, as well as their allelopathic activity in a small eutrophic lake. We tested whether nutrient availability explained interannual and seasonal changes in the production of polyphenols. There were no strong interannual variations in plant tissue carbon, nitrogen and phosphorus concentrations, while total phenolic compounds (TPC) significantly differed between years, especially in apical meristems (range: 38–122 mg g⁻¹  dry weight (DW)). Seasonal patterns, with maxima between May and July, changed between years. Partially confirming the carbon-nutrient balance hypothesis sensu Bryant et al. [Bryant, J.P., Chapin III, F.S., Klein, D.R., 1983. Carbon/nutrient balance of boreal plants in relation to vertebrate herbivory. Oikos 40, 357–368], we found correlations between TPC and the C/N (carbon/nitrogen) ratio in some but not all years, especially in apical meristems. Plant tissue phosphorus content accounted also for the variability in TPC in some years. Crude extracts of apical meristems always inhibited the growth of Anabaena variabilis, used as a target cyanobacterium. Plant TPC concentration and allelopathic activity were significantly correlated in all years except in 2005. Bioassay-directed fractionation of M. verticillatum extracts coupled with LC–MS analyses of the respective fractions revealed several isomers of HHDP-di- and -tri-galloylglucose apparently responsible for the allelopathic effects. The individual active compounds revealed a more distinct seasonal pattern compared to the pool of phenolic compounds in M. verticillatum, with a clear maximum in May, the ecologically most relevant period for inhibitory effects of submerged macrophytes on phytoplankton.     

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

Host preference in Pseudacteon phorid flies: response of P. tricuspis and P. curvatus to black, red and hybrid imported Solenopsis fire ants in multiple choice bioassays

Host preferences in both sexes of Pseudacteon tricuspis Borgmeier (Jaguariuna biotype) and Pseudacteon curvatus Borgmeier (Formosa biotype) and their relative attraction to the imported fire ants (IFA), Solenopsis invicta Buren (red IFA), Solenopsis richteri Forel (black IFA) and S. invicta × S. richteri hybrids (hybrid IFA) were investigated in two separate experiments utilizing multiple choice flight bioassays. The results of both experiments clearly showed that both sexes of the Jaguariuna biotype of P. tricuspis could distinguish among the three IFA species and demonstrated greater preference for hybrid IFA and red IFA. This conclusion is supported by a variety of data collected on the number of fly visits, attack rate, and hovering duration (Experiment 1), and on the number of trapped flies (Experiment 2), which showed that black IFA is the least preferred of the three species. Similar results were recorded for the Formosan biotype of P. curvatus, although the data were not as strongly conclusive. Females of this biotype spent a significantly greater amount of time in hovering mode over red IFA and hybrid IFA compared to black IFA, but the other data were not significant. The red IFA is the natural host of both phorid fly biotypes and our results suggest that both biotypes may have evolved a specialized relationship with red IFA including an ability to discriminate it from related fire ants. These results are discussed in relation to the possible role of fire ant chemicals in mediating host preferences in phorid flies, contributions of male phorid flies to fire ant biocontrol, and the practical implications of the key findings.     

Correlation between screening procedures to select root endophytes for biological control of Fusarium verticillioides in Zea mays L

Biological control is an alternative to pesticides for protection against crop diseases. A key to progress in the field of biological control to protect maize against Fusarium verticillioides is to select in vitro the best agent to be applied in the field. This research was undertaken to evaluate the correlation between different screening methods and to suggest an adequate procedure that could be used to select bacterial agents with potential biocontrol against F. verticillioides in the maize rhizosphere. The results show an extensive Pearson correlation coefficient analysis between different screening procedures carried out for Arthrobacter spp., Azotobacter spp., Pseudomonas spp., and Bacillus spp. paired with 13 F. verticillioides strains isolated from maize endorhizosphere. The following screening methodologies were correlated: niche overlap index (NOI), indices of dominance, growth rate, lag phase, antibiosis, and fumonisin production. It was observed that NOI and antibiosis methodologies did not show any correlation. They were used to choose the best bacteria to apply under greenhouse conditions. Azotobacter armeniacus RC2 showed a NOI > 0.9 and inhibited all F. verticillioides strains assayed. Seed maize bacterization with A. armeniacus RC2 at 10⁶ and 10⁷ inoculum level resulted in significantly lower values of F. verticillioides counts compared to the control without bacteria at the root levels assayed. The analysis of variance indicated that there were significant interactions between two fungal repression assays. Antibiosis assays significantly correlated with greenhouse conditions (p < 0.01) at two inoculum levels tested. Therefore, the selection system adopted was adequate to choose the best bacterial biocontrol agent. The application of this methodology shows a way to outline the best biocontrol candidate.