Medicarpin confers powdery mildew resistance in Medicago truncatula and activates the salicylic acid signalling pathway

Powdery mildew (PM) caused by the obligate biotrophic fungal pathogen Erysiphe pisi is an economically important disease of legumes. Legumes are rich in isoflavonoids, a class of secondary metabolites whose role in PM resistance is ambiguous. Here we show that the pterocarpan medicarpin accumulates at fungal infection sites, as analysed by fluorescein‐tagged medicarpin, and provides penetration and post‐penetration resistance against E. pisi in Medicago truncatula in part through the activation of the salicylic acid (SA) signalling pathway. Comparative gene expression and metabolite analyses revealed an early induction of isoflavonoid biosynthesis and accumulation of the defence phytohormones SA and jasmonic acid (JA) in the highly resistant M. truncatula genotype A17 but not in moderately susceptible R108 in response to PM infection. Pretreatment of R108 leaves with medicarpin increased SA levels, SA‐associated gene expression, and accumulation of hydrogen peroxide at PM infection sites, and reduced fungal penetration and colony formation. Strong parallels in the levels of medicarpin and SA, but not JA, were observed on medicarpin/SA treatment pre‐ or post‐PM infection. Collectively, our results suggest that medicarpin and SA may act in concert to restrict E. pisi growth, providing new insights into the metabolic and signalling pathways required for PM resistance in legumes.     

Aphid induction of phytohormones in <Emphasis Type="Italic">Medicago truncatula</Emphasis> is dependent upon time post-infestation,aphid density and the genotypes of both plant and insect

This study examined the induction of the defence-related hormones jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA) and the phytoalexin medicarpin in Medicago truncatula when challenged by the pea aphid Acyrthosiphon pisum. There was some induction of hormones in the compatible interaction between A. pisum clone N116 and M. truncatula cultivar DZA315, whereas JA, SA and medicarpin exhibited more significant increases in foliage concentration during the incompatible interaction between A. pisum clone PS01 and M. truncatula cultivar Jemalong A17. Foliar concentration of JA, SA and medicarpin exhibited a positive relationship with aphid density after 3-day feeding, whereas ABA was not affected by the presence of aphids. When aphids were restricted to a single leaf using plastic tubes, JA, SA and medicarpin displayed strong local induction, whereas there were no significant systemic increases in uninfested leaves. Medicarpin and SA appeared to increase with duration of aphid feeding, whereas JA showed a more transient increase in concentration 24 h after challenge commenced. Results suggest that increases in JA, SA and medicarpin are associated with M. truncatula resistance to particular clones of A. pisum. The variation in concentration of the defence-related compounds recorded with regard to aphid density, duration of challenge, genotypes of plant and aphids, and between locally challenged and distant leaves reinforces the need for consideration of these experimental factors when generalizing about the plant defence processes that occur during aphid–plant interactions.     

Flavonoids from Glycyrrhiza pallidiflora hairy root cultures

Three flavonoids named licoagrosides D, E and F together with four known flavonoids, medicarpin 3-O-glucoside, calycosin 7-O-glucoside, formononetin 7-O-(6"-malonylglucoside) and 2'-hydroxyformononetin 7-O-glucoside were isolated from Glycyrrhiza pallidiflora hairy root cultures. Their structures were determined on the basis of spectroscopic evidence. Licoagrosides E and F are the first examples of a 6a-hydroxypterocarpan glycoside and an alpha-O-glycosidic alpha-hydroxydihydrochalcone, respectively.     

New features on the survival of human-infective Trypanosoma rangeli in a murine model: Parasite accumulation is observed in lymphoid organs

Trypanosoma rangeli is a non-pathogenic protozoan parasite that infects mammals, including humans, in Chagas disease-endemic areas of South and Central America. The parasite is transmitted to a mammalian host when an infected triatomine injects metacyclic trypomastigotes into the host′s skin during a bloodmeal. Infected mammals behave as parasite reservoirs for several months and despite intensive research, some major aspects of T. rangeli-vertebrate interactions are still poorly understood. In particular, many questions still remain unanswered, e.g. parasite survival and development inside vertebrates, as no parasite multiplication sites have yet been identified. The present study used an insect bite transmission strategy to investigate whether the vector inoculation spot in the skin behave as a parasite-replication site. Histological data from the skin identified extracellular parasites in the dermis and hypodermis of infected mice in the first 24 hours post-infection, as well as the presence of inflammatory infiltrates in a period of up to 7 days. However, qPCR analyses demonstrated that T. rangeli is eliminated from the skin after 7 days of infection despite being still consistently found on circulating blood and secondary lymphoid tissues for up to 30 days post-infection. Interestingly, significant numbers of parasites were found in the spleen and mesenteric lymph nodes of infected mice during different periods of infection and steady basal numbers of flagellates are maintained in the host′s bloodstream, which might behave as a transmission source to insect vectors. The presence of parasites in the spleen was confirmed by fluorescent photomicrography of free and cell-associated T. rangeli forms. Altogether our results suggest that this organ could possibly behave as a T. rangeli maintenance hotspot in vertebrates.     

Medicarpin and maackiain 3-O-glucoside-6′-O-malonate conjugates are constitutive compounds in chickpea (Cicer arietinum L.) cell cultures

Summary The pterocarpan phytoalexin conjugates medicarpin 3-O-glucoside-6-O-malonate and maackiain 3-O-glucoside-6-O-malonate were isolated from cell suspension cultures of chickpea (Cicer arietinum L.) cultivar ILC 3279 and structurally elucidated. Both pterocarpan conjugates are constitutive metabolites of the chickpea cell cultures. Upon application of an elicitor from yeast to the cell cultures a substantial increase in the level of the phytoalexin aglycones medicarpin and maackiain was observed although a delayed but significantly higher rise of the conjugates also occurred. The significance of the pterocarpan conjugates for phytoalexin production is discussed.Abbreviations MeGM medicarpin 3-O-glucoside-6-O-malonate - MaGM maackiain 3-O-glucoside-6-O-malonate - MeG medicarpin 3-O-glucoside - MaG maackiain 3-O-glucoside - FGM formononetin 7-O-glucoside-6-O-malonate - BGM biochanin A 7-O-glucoside-6-O-malonate - IFR NADPH: 2-hydroxyisoflavone oxidoreductase - PTS pterocarpan synthase - IGT UDP-glucose: isoflavone 7-O-glucosyltransferase - IMT malonyl-coA: isoflavone 7-O-glucoside-6 -O-malonyltransferase - RT retention time - sh shoulder - d duplette - m multiplette - s singulette     

Accumulation of phytoalexins and isoflavone glucosides in a resistant and a susceptible cultivar of Cicer arietinum during infection with Ascochyta rabiei

After infection with spores of a virulent strain of Ascochyta rabiei the chickpea (Cicer arietinum) cultivars ILC 1929 (susceptible) and ILC 3279 (resistant) were compared with regard to pterocarpan phytoalexin and isoflavone accumulation. Quantitative HPLC analyses of total extracts of aerial parts were used to measure the induced formation of the phytoalexins medicarpin and maackiain and the accumulation of the constitutive isoflavones biochanin A and formononetin together with their, 7-0-glucosides and their 7-0-glucoside-6″-0-malonates. The two cultivars showed no significant difference in the level of isoflavones and isoflavone conjugates. On the other hand, the resistant cultivar ILC 3279 rapidly accumulated large amounts of both, phytoalexins (20–26 nmole g^?1 fr.w.) whereas cultivar ILC 1929 only produced very small amounts (5 nmole g^?1 fr.w.) of medicarpin. The data are discussed with regard to isoflavonoid metabolism and the significance of induced and constitutive levels of phytoalexins and isoflavones in resistance of chickpea towards A. rabiei.     

Metabolomics reveals novel pathways and differential mechanistic and elicitor-specific responses in phenylpropanoid and isoflavonoid biosynthesis in Medicago truncatula cell cultures

High-performance liquid chromatography coupled to ultraviolet photodiode array detection and ion-trap mass spectrometry was used to analyze the intra- and extracellular secondary product metabolome of Medicago truncatula cell suspension cultures responding to yeast elicitor (YE) or methyl jasmonate (MeJA). Data analysis revealed three phases of intracellular response to YE: a transient response in mainly (iso)flavonoid metabolites such as formononetin and biochanin-A that peaked at 12 to 18 h following elicitation and then declined; a sustained response through 48 h for compounds such as medicarpin and daidzin; and a lesser delayed and protracted response starting at 24 h postelicitation, e.g. genistein diglucoside. In contrast, most compounds excreted to the culture medium reached maximum levels at 6 to 12 h postelicitation and returned to basal levels by 24 h. The response to MeJA differed significantly from that to YE. Although both resulted in accumulation of the phytoalexin medicarpin, coordinated increases in isoflavonoid precursors were only observed for YE and not MeJA-treated cells. However, MeJA treatment resulted in a correlated decline in isoflavone glucosides, and did not induce the secretion of metabolites into the culture medium. Three novel methylated isoflavones, 7-hydroxy-6,4'-dimethoxyisoflavone (afrormosin), 6-hydroxy-7,4'-dimethoxyisoflavone (alfalone), and 5,7-dihydroxy-4',6-dimethoxy isoflavone (irisolidone), were induced by YE, and labeling studies indicated that the first two were derived from formononetin. Our results highlight the metabolic flexibility within the isoflavonoid pathway, suggest new pathways for complex isoflavonoid metabolism, and indicate differential mechanisms for medicarpin biosynthesis depending on the nature of elicitation.     

Repetitive somatic embryogenesis in Medicago truncatula ssp. Narbonensis and M. truncatula Gaertn cv. Jemalong

Medicago truncatula ssp Narbonensis and four genotypes of M. truncatula Gaertn cv. Jemalong were tested for their somatic embryogenesis potential using a two-step protocol. In the first step, embryogenic callus was induced in folioles isolated from shoots grown in vitro and cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid and zeatin. In the second step, somatic embryos were allowed to develop from the induced callus in MS growth-regulator-free medium. Individual somatic embryos were then isolated and transferred again to growth regulator free medium where they formed secondary somatic embryos in repetitive cycles. Conversion of somatic embryos into plantlets was achieved by isolating late-torpedo-phase somatic embryos with distinct cotyledons and reculturing them onto MS growth regulator free medium. The system of repetitive somatic embryogenesis in M. truncatula described here represents a permanent source of embryogenic material that can be used for the genetic modification of this species. Received: 7 August 1997 / Revision received: 22 December 1997 / Accepted: 20 January 1998     

Invasive Non-Typhoidal Salmonella Typhimurium ST313 Are Not Host-Restricted and Have an Invasive Phenotype in Experimentally Infected Chickens

Salmonella enterica serovar Typhimurium Sequence Type (ST) 313 is a major cause of invasive non-Typhoidal salmonellosis in sub-Saharan Africa. No animal reservoir has been identified, and it has been suggested that ST313 is adapted to humans and transmission may occur via person-to-person spread. Here, we show that ST313 cause severe invasive infection in chickens as well as humans. Oral infection of chickens with ST313 isolates {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 and Q456 resulted in rapid infection of spleen and liver with all birds infected at these sites by 3 days post-infection. In contrast, the well-defined ST19 S. Typhimurium isolates F98 and 4/74 were slower to cause invasive disease. Both ST19 and ST313 caused hepatosplenomegaly, and this was most pronounced in the ST313-infected animals. At 3 and 7 days post-infection, colonization of the gastrointestinal tract was lower in birds infected with the ST313 isolates compared with ST19. Histological examination and expression of CXCL chemokines in the ileum showed that both {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 (ST313) and 4/74 (ST19) strains caused increased CXCL expression at 3 days post-infection, and this was significantly higher in the ileum of {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 vs 4/74 infected birds. At 7 days post-infection, reduced chemokine expression occurred in the ileum of the {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 but not 4/74-infected birds. Histological analysis showed that {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 infection resulted in rapid inflammation and pathology including villous flattening and fusion at 3 days post-infection, and subsequent resolution by 7 days. In contrast, 4/74 induced less inflammation and pathology at 3 days post-infection. The data presented demonstrate that ST313 is capable of causing invasive disease in a non-human host. The rapid invasive nature of infection in the chicken, coupled with lower gastrointestinal colonization, supports the hypothesis that ST313 is a distinct pathovariant of S. Typhimurium that has evolved to become a systemic pathogen that can cause disease in several hosts.     

Feasibility of Histological Scoring and Colony Count for Evaluating Infective Severity in Mouse Vaginal Candidiasis

Qualitative measurement of the infective level is relatively difficult in experimental vaginal candidiasis. Female BALB/c mice aged 8 to 10 weeks were randomly divided into E1, E2 and E0 groups, which received subcutaneous injection of 0.05 mg, 0.1 mg of estradiol benzoate or 0.1 ml soybean oil 3 days before vaginal inoculation, respectively, and hormone treatment continued every other day thereafter. Each group was further divided into infected and noninfected subgroups. The infected mice were inoculated intravaginally with 10 µl (5 × 10⁴ conidia) of Candida albicans suspension, while the noninfected mice were inoculated with 10 µl phosphate-buffered saline. Direct microscopic examination, colony count and vaginal histopathology including infection degree and inflammation extent were performed at 3, 7 and 14 days post inoculation. Estrogen treatment increased the vaginal fungal burden and extent of infection and inflammation compared with the control group, and 0.3 mg/week estrogen generally induced more severe infection and inflammation than 0.15 mg/week estrogen did. Colony count peaked on day 3 and decreased remarkably after 7 days. Infection score increased gradually during the first 7 days and decreased on day 14, while inflammation extent exacerbated progressively over the course of 14 days. This study demonstrates that the modified histological scoring system might be more feasible than colony count for evaluation of infectivity and dynamic change in experimental vaginal candidiasis.     

Consumption of a nectar alkaloid reduces pathogen load in bumble bees

Diet has a significant effect on pathogen infections in animals and the consumption of secondary metabolites can either enhance or mitigate infection intensity. Secondary metabolites, which are commonly associated with herbivore defense, are also frequently found in floral nectar. One hypothesized function of this so-called toxic nectar is that it has antimicrobial properties, which may benefit insect pollinators by reducing the intensity of pathogen infections. We tested whether gelsemine, a nectar alkaloid of the bee-pollinated plant Gelsemium sempervirens, could reduce pathogen loads in bumble bees infected with the gut protozoan Crithidia bombi. In our first laboratory experiment, artificially infected bees consumed a daily diet of gelsemine post-infection to simulate continuous ingestion of alkaloid-rich nectar. In the second experiment, bees were inoculated with C. bombi cells that were pre-exposed to gelsemine, simulating the direct effects of nectar alkaloids on pathogen cells that are transmitted at flowers. Gelsemine significantly reduced the fecal intensity of C. bombi 7 days after infection when it was consumed continuously by infected bees, whereas direct exposure of the pathogen to gelsemine showed a non-significant trend toward reduced infection. Lighter pathogen loads may relieve bees from the behavioral impairments associated with the infection, thereby improving their foraging efficiency. If the collection of nectar secondary metabolites by pollinators is done as a means of self-medication, pollinators may selectively maintain secondary metabolites in the nectar of plants in natural populations.     

Expression profile of immune-related genes in Lates calcarifer infected by Cryptocaryon irritans

Cryptocaryon irritans causes Cyptocaryonosis or white spot disease in a wide range of marine fish including Lates calcarifer (Asian seabass). However, the immune response of this fish to the parasite is still poorly understood. In this study, quantitative polymerase chain reaction (qPCR) was performed to assess the expression profile of immune-related genes in L. calcarifer infected by C. irritans. A total of 21 immune-related genes encoding various functions in the fish immune system were utilized for the qPCR analysis. The experiment was initiated with the infection of juvenile fish by exposure to theronts from 200 C. irritans cysts, and non-infected juvenile fish were used as controls. Spleen, liver, gills and kidney tissues were harvested at three days post-infection from control and infected fish. In addition, organs were also harvested on day-10 post-infection from fish that had been allowed to recover from day-4 up to day-10 post-infection. L. calcarifer exhibited pathological changes on day-3 post-infection with the characteristic presence of white spots on the entire fish body, excessive mucus production and formation of a flap over the fish eye. High quality total RNA was extracted from all tissues and qPCR was performed. The qPCR analysis on the cohort of 21 immune-related genes of the various organs harvested on day-3 post-infection demonstrated that most genes were induced significantly (p < 0.05) in all tissues, particularly liver (11/21 genes) and kidney (11/21). The expression profile demonstrated that induction of the MHC Class IIα gene was the highest compared to the other genes followed by serum amyloid A, CC chemokine and hepcidin-2 precursor genes. In fish that were allowed to recover from the C. irritans infection (10 days post-infection), expression of the immune-related genes was down-regulated to levels similar to the control fish. These results provide insights into the interaction between C. irritans and L. calcarifer and suggest that the innate immune system plays an important role in early defence against parasite infection allowing the fish to eventually recover from the infection.     

Oxidative stress in mice infected with Angiostrongylus cantonensis coincides with enhanced glutathione-dependent enzymes activity

This study aimed to estimate reactive oxygen species (ROS) production, antioxidants activity, and biomarkers level of oxidative damage to protein and DNA in the cerebrospinal fluid (CSF) of C57BL/6 mice infected with Angiostrongylus cantonensis. The mean ROS concentration in the CSF of infected mice increased gradually, and the increase in ROS in CSF became statistical significance at days 12-30 post-infection compared to that before infection (P < 0.001), and then ROS returned to normal level at day 45 after infection. In parallel with the increase in ROS in the CSF, infected mice showed similar of changes in reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST) as that in ROS in the CSF. GSH, GR, GPx, and GST in the CSF of infected mice were all significantly higher than they were before infection during days 12-30 post-infection. However, protein carbonyl content and 8-hydroxy-2′-deoxyguanosine, biomarkers of oxidative damage to protein and DNA, respectively, were also significantly higher in the CSF of infected mice during this period. These results suggest that oxidative stress occur in the cells of central nervous system of mice infected with A. cantonensis during days 12-30 after infection due to ROS overproduction in CSF despite the increase in antioxidants during this period.     

Stress responses in alfalfa (Medicago sativa L.) III. Induction of medicarpin and cytochrome P450 enzyme activities in elicitor-treated cell suspension cultures and protoplasts

Summary Cell suspension cultures of alfalfa (Medicago sativa L.) accumulated phenolic secondary metabolites in a pattern similar to that seen in alfalfa roots. Upon treatment with a crude elicitor preparation from the bean pathogen Colletotrichum lindemuthianum, the pterocarpan phytoalexin medicarpin accumulated in cells and culture medium. The extractable activities of six enzymes involved in medicarpin biosynthesis (including three cytochrome P450 activities) were induced by treatment with elicitor, and their induction kinetics correlated with the rate of medicarpin accumulation. However, protoplasts prepared from these cultures accumulated neither medicarpin nor other secondary products after treatment with elicitor. The cytochrome P450 activities were induced during the preparation of the protoplasts, but could be further induced by treatment with fungal elicitor. The results are discussed in relation to the use of alfalfa protoplasts as a system for functional analysis of cloned defense genes.Abbreviations AUFS absorption unit full scale - CHI chalcone isomerase (EC 5.5.1.6) - CHS chalcone synthase (EC 2.3.1.74) - C40H cinnamic acid 4-hydroxylase (EC 1.14.13.11) - CLE elicitor from Colletotrichum lindemuthianum - IFOH isoflavone 2-hydroxylase - IFS isoflavone synthase - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Flavonoids in roots of white clover: interaction of arbuscular mycorrhizal fungi and a pathogenic fungus

The effects of two arbuscular mycorrhizal fungi (AMF) (Glomus mosseae and G. claroideum) and a pathogenic fungus (Pythium ultimum) on the production of eight flavonoids in roots of two white clover (Trifolium repens L.) cultivars were evaluated. Quantification of AM and pathogenic fungi in the roots showed that the AM symbiosis significantly reduced P. ultimum biomass and in some cases prevented infection. The flavonoid productions in clover roots varied depending on the presence of beneficial and/or pathogenic fungi, fungal isolate or plant cultivar. Only plants colonized with G. claroideum showed detectable concentrations of either coumestrol or kaempferol (cultivar-dependant). In addition, inoculation with G. claroideum resulted in significantly higher concentrations of coumestrol in cv. Sonja and medicarpin in cv. Milo. A low production of coumestrol and kaempferol in mycorrhizal plants may be G. mosseae-specific. Only the concentrations of formononetin and daidzein increased in clover roots in response to infection with P. ultimum. These flavonoids are supposedly stress metabolites, synthesized or produced from glycosides in response to pathogen infection. However, the presence of one or both AMF significantly lowered the formononetin and daidzein concentrations, and overruled the inductive effect of P. ultimum. Therefore the antagonistic action of AM against the pathogen must take place through another mechanism.     

Leaf rust induced volatile organic compounds signalling in willow during the infection

Plants are known to emit volatile organic compounds (VOC) in response to various biotic or abiotic stresses. Although the VOC emission in the case of insect attacks is well described, there is only little known about the impact of pathogens on plant emission. In the present study, we used a willow-leaf rust system to describe the effects of a biotrophic fungal infection on the VOC emission pattern of willow leaves. We detected that isoprene emissions from rust-infected leaves decreased threefold compared to control. The total monoterpene emissions did not change although a stress-signalling compound (Z)-β-ocimene showed an increase in infected plants on several days. The infection also increased the emission of sesquiterpenes and lipoxygenase products (LOX) by factors of 175-fold and 10-fold, respectively. The volatile emission signals showed two clear peaks during the experiment. At 6, 7 and 12 days post-infection (dpi), the relative volatile emission signal increased to about sixfold compared to uninfected plants. These time points are directly connected to rust infection since at 6 dpi the first rust pustules appeared on the leaves and at 12 dpi necrosis had developed around several pustules. We present correlations between LOX and sesquiterpene emission signals, which suggest at least two different steps in eliciting the volatile emission.     

Photosynthesis in localised regions of oat leaves infected with crown rust (Puccinia coronata): quantitative imaging of chlorophyll fluorescence

Localised changes in photosynthesis in oat leaves infected with the biotrophic rust fungus Puccinia coronata Corda were examined at different stages of disease development by quantitative imaging of chlorophyll fluorescence. Following inoculation of oat leaves with crown rust the rate of whole-leaf gas exchange declined. However, crown rust formed discrete areas of infection which expanded as the disease progressed and these localised regions of infection gave rise to heterogeneous changes in photosynthesis. To quantify these changes, images of chlorophyll fluorescence were taken 5, 8 and 11 d after inoculation and used to calculate images representing two parameters; Φ_II, a measure of PSII photochemical efficiency and ΔFm/Fm′, a measure of non-photochemical energy dissipation (qN). Five days after inoculation, disease symptoms appeared as yellow flecks which were correlated with the extent of the fungal mycelium within the leaf. At this stage, Δ_II was slightly reduced in the infected regions but, in uninfected regions of the leaf, values of Φ_II were similar to those of healthy leaves. In contrast, qN (ΔFm/Fm′) was greatly reduced throughout the infected leaf in comparison to healthy leaves. We suggest that the low value of qN in an infected leaf reflects a high demand for ATP within these leaves. At sporulation, 8 d after inoculation, Φ_II was reduced throughout the infected leaf although the reduction was most marked in areas invaded by fungal mycelium. In the infected leaf the pattern of non-photochemical quenching was complex; qN was low within invaded regions, perhaps reflecting high metabolic activity, but was now much higher in uninfected regions of the infected leaf, in comparison to healthy leaves. Eleven days after inoculation “green islands” formed in regions of the leaf associated with the fungal mycelium. At this stage, photosynthesis was severely inhibited over the entire leaf; however, heterogeneity was still apparent. In the region not invaded by the fungal mycelium, Φ_II and qN were very low and these regions of the leaf were highly fluorescent, indicating that the photosynthetic apparatus was severely damaged. In the greenisland tissue, Φ_II was low but detectable, indicating that some photosynthetic processes were still occurring. Moreover, qN was high and fluorescence low, indicating that the cells in this region were not dead and were capable of significant quenching of chlorophyll fluorescence.     

Eimeria pragensis infection alters the gut microenvironment to favor extrinsic shiga toxin-producing Escherichia coli O157:H7 colonization in mice

We examined the effects of Eimeria pragensis infection on intestinal peristalsis, goblet cell proliferation and intestinal flora in C57BL/6 mice. Intestinal peristalsis was evaluated by radiography using barium at 7 days post-infection (p.i.). The intestinal peristalsis of E. pragensis-infected mice was significantly suppressed compared with uninfected control mice. Twenty-three mice were divided into 5 groups of 4 or 5 mice each; 2 groups of mice were infected with E. pragensis and the others were kept uninfected. At 7 days p.i., E. pragensis-infected and -uninfected mice were sacrificed to examine goblet cell numbers in the intestines, and significant decreases were observed only in the infected mice. Shiga toxin-producing Escherichia coli (STEC) O157:H7 was inoculated orally in mice both infected and uninfected with E. pragensis at 7 days p.i., with the remaining mice used as uninoculated controls. When mice were sacrificed at 2 days after STEC inoculation, STEC was only detected in the intestines of E. pragensis-infected mice. Colonization of STEC was also confirmed by immunohistochemistry on the surface of epithelial cells in concurrently infected/inoculated mice. Also, an overgrowth of residential E. coli was observed only in E. pragensis-infected mice. These results suggest that E. pragensis induces the suppression of intestinal peristalsis and modifies the intestinal environment to facilitate artificially introduced STEC colonization and multiplication, in addition to residential E. coli overgrowth.     

Protective cellular responses to Burkholderia mallei infection

Burkholderia mallei is a Gram-negative bacillus causing the disease glanders in humans. During intraperitoneal infection, BALB/c mice develop a chronic disease characterised by abscess formation where mice normally die up to 70 days post-infection. Although cytokine responses have been investigated, cellular immune responses to B. mallei infection have not previously been characterised. Therefore, the influx and activation status of splenic neutrophils, macrophages and T cells was examined during infection. Gr-1⁺ neutrophils and F4/80⁺ macrophages infiltrated the spleen 5 h post-infection and an increase in activated macrophages, neutrophils and T cells occurred by 24 h post-infection. Mice depleted of Gr-1⁺ cells were acutely susceptible to B. mallei infection, succumbing to the infection 5 days post-infection. Mice depleted of both CD4 and CD8 T cells did not succumb to the infection until 14 days post-infection. Infected μMT (B cell) and CD28 knockout mice did not differ from wildtype mice whereas iNOS-2 knockout mice began to succumb to the infection 30 days post-infection. The data presented suggests that Gr-1⁺ cells, activated early in B. mallei infection, are essential for controlling the early, innate response to B. mallei infection and T cells or nitric oxide are important during the later stages of infection.