Biodiversity of Clostridium botulinum type E associated with a large outbreak of botulism in wildlife from Lake Erie and Lake Ontario

The genetic relatedness of Clostridium botulinum type E isolates associated with an outbreak of wildlife botulism was studied using random amplification of polymorphic DNA (RAPD). Specimens were collected from November 2000 to December 2008 during a large outbreak of botulism affecting birds and fish living in and around Lake Erie and Lake Ontario. In our present study, a total of 355 wildlife samples were tested for the presence of botulinum toxin and/or organisms. Type E botulinum toxin was detected in 110 samples from birds, 12 samples from fish, and 2 samples from mammals. Sediment samples from Lake Erie were also examined for the presence of C. botulinum. Fifteen of 17 sediment samples were positive for the presence of C. botulinum type E. Eighty-one C. botulinum isolates were obtained from plants, animals, and sediments; of these isolates, 44 C. botulinum isolates produced type E toxin, as determined by mouse bioassay, while the remaining 37 isolates were not toxic for mice. All toxin-producing isolates were typed by RAPD; that analysis showed 12 different RAPD types and multiple subtypes. Our study thus demonstrates that multiple genetically distinct strains of C. botulinum were involved in the present outbreak of wildlife botulism. We found that C. botulinum type E is present in the sediments of Lake Erie and that a large range of bird and fish species is affected.     

Identification of a sporozoite-specific antigen from Toxoplasma gondii

Reduction of risk for human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. tissue cyst consumption) in horizontally transmitted toxoplasmosis. Existing serological assays can determine previous exposure to the parasite, but not the route of infection. We have used difference gel electrophoresis, in combination with tandem mass spectroscopy and Western blot, to identify a sporozoite-specific protein (T. gondii embryogenesis-related protein [TgERP]), which elicited antibody and differentiated oocyst- versus tissue cyst-induced infection in pigs and mice. The recombinant protein was selected from a cDNA library constructed from T. gondii sporozoites; this protein was used in Western blots and probed with sera from T. gondii -infected humans. Serum antibody to TgERP was detected in humans within 6-8 mo of initial oocyst-acquired infection. Of 163 individuals in the acute stage of infection (anti- T. gondii IgM detected in sera, or < 30 in the IgG avidity test), 103 (63.2%) had detectable antibodies that reacted with TgERP. Of 176 individuals with unknown infection route and in the chronic stage of infection (no anti- T. gondii IgM detected in sera, or > 30 in the IgG avidity test), antibody to TgERP was detected in 31 (17.6%). None of the 132 uninfected individuals tested had detectable antibody to TgERP. These data suggest that TgERP may be useful in detecting exposure to sporozoites in early T. gondii infection and implicates oocysts as the agent of infection.     

Genetic control of the target structures recognized in hybrid resistance

Hybrid resistance of lethally irradiated (C57BL/6 × DBA/2)F₁ and (C57BL/10 × C3H)F₁ hybrid mice to the engraftment of parental C57BL/6 or C57BL/10 bone marrow cells is controlled by the H-2-linked Hh-1 locus. This resistance can be specifically blocked or inhibited by the injection of irradiated spleen cells from lethally irradiated, marrow reconstituted donor mice of certain strains. By testing the ability of regenerating spleen cells from various donor strains to block the resistance, we studied the genetic requirements for the expression of putative cell-surface structures recognized in hybrid resistance to H-2^b marrow cells. Strains of mice bearing informative intra-H-2 or H-2/ Qa-Tla recombinant haplotypes provided evidence that the Hh-1 locus is located telomeric to the H-2S region complement loci and centromeric to the H-2D region class I locus in the H-2 ^b chromosome. Two mutations that affect the class I H-2D ^b gene have no effect on Hh-1 ^b gene expression. The H-2D region of the H-2 ^S haplotype contains an allele of the Hh-1 locus indistinguishable from that of the H-2D ^b region, as judged by the phenotypes of relevant strains and F₁ hybrids. Collectively these data indicate that the Hh-1 locus is distinct from the class I H-2D (L) locus in the H-2 ^b or H-2 ^s genome, and favor the view that the expression or recognition of the relevant determinants is not associated with class I gene products.Abbreviations used in this paper BM(C) bone marrow (cells) - CML cell-mediated lympholysis - CTL cytotoxic T lymphocytes - FBS fetal bovine serum - HBSS Hanks' balanced salt solution - SC spleen cells from irradiated, bone marrow-reconstituted mice Address correspondence to: Dr. I. Najamura, Department of Pathology, School of Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA     

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

Site of OH radical attack on dihydrouracil and some of its methyl derivatives

The site of attack of OH radicals on dihydrouracil and five of its methylated derivatives was determined by pulse radiolysis using N,N,N',N'-tetramethylphenylenediamine (TMPD) to detect oxidizing radicals and tetranitromethane (TNM) as well as K3Fe(CN)6 to detect reducing radicals. In the case of dihydrouracil OH radicals abstract preferentially an H atom at C(6) to give the 6-yl radical (greater than or equal to 90 per cent) which at pH approximately 6.5 reduces TNM and K3Fe(CN)6 at almost diffusion-controlled rates. Only a small fraction of OH radicals abstract the H atom at C(5) (less than or equal to 10 per cent). The resulting 5-yl radical oxidizes TMPD to TMPD+ at pH 7-8. With the methylated derivatives of dihydrouracil, OH radicals react less selectively, especially in the case of N(1)-methyl derivatives. This methyl group is activated to a similar degree as the methylene group at C(6). In 1-Medihydrouracil the yield of N(1)-CH2 radicals is about 29 per cent, which has been deduced from the yield of formaldehyde formed after oxidation of this radical by TNM at pH approximately 6.5 and the subsequent hydrolysis. Radicals at the other methyl substituents are generated to a lesser extent (less than or equal to 10 per cent) and are relatively unreactive towards oxidizing agents such as TNM and K3Fe(CN)6 as well as towards the reducing agent TMPD. Although methyl substitution opens new routes for OH attack the preferred site of H abstraction remains C(6) (greater than 60 per cent).     

On advection in phytoplankton models

Scale analysis is used to deduce a new non-dimensional number, $\text{S =}\text{C}\text{[(A}{}_{\mathrm{H}}\text{τ)}\text{1}\text{2}\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{]}$ which defines the importance of advection of phytoplankton by organized fluid motion in spatial models of marine food chains. When S ? 1, advection greatly affects biological productivity; when S ? I, advection may be neglected. When S is order one, advection and biological productivity play competing roles in determining the spatial configuration of the plankton biomass.     

Potential biomarkers of an exaggerated response to endotoxemia

Serial plasma protein analysis was used to study the acute plasma proteome response to endotoxemia (presence of toxic bacterial products called endotoxins in the blood stream). Plasma samples from healthy volunteers before and multiple time points up to 24 h following administration of low-dose endotoxin were evaluated. Plasma protein profiles were obtained by rapid extraction of whole plasma followed by analysis with matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The profiles were unique to each individual and stable over the time of the experiment. Administration of low-dose endotoxin caused profound change in six of 18 individuals. At 8 h many proteins showed quantitative oxidation, in addition to the appearance of new components and disappearance of common baseline components. An exceptionally intense new component at 4154 mass units was identified as the activation peptide of C1 esterase inhibitor. While recovery of baseline protein structure was nearly complete by 24 h, serum amyloid A, an acute-phase reactant, was still increasing and minor profile changes persisted. Clinical features did not distinguish these extreme responders from others, suggesting that plasma proteome changes offered unique insights into and potential biomarkers of subclinical events following endotoxin exposure.     

Comparative Large-Scale Analysis of Interactions between Several Crop Species and the Effector Repertoires from Multiple Pathovars of Pseudomonas and Ralstonia

Bacterial plant pathogens manipulate their hosts by injection of numerous effector proteins into host cells via type III secretion systems. Recognition of these effectors by the host plant leads to the induction of a defense reaction that often culminates in a hypersensitive response manifested as cell death. Genes encoding effector proteins can be exchanged between different strains of bacteria via horizontal transfer, and often individual strains are capable of infecting multiple hosts. Host plant species express diverse repertoires of resistance proteins that mediate direct or indirect recognition of bacterial effectors. As a result, plants and their bacterial pathogens should be considered as two extensive coevolving groups rather than as individual host species coevolving with single pathovars. To dissect the complexity of this coevolution, we cloned 171 effector-encoding genes from several pathovars of Pseudomonas and Ralstonia. We used Agrobacterium tumefaciens-mediated transient assays to test the ability of each effector to induce a necrotic phenotype on 59 plant genotypes belonging to four plant families, including numerous diverse accessions of lettuce (Lactuca sativa) and tomato (Solanum lycopersicum). Known defense-inducing effectors (avirulence factors) and their homologs commonly induced extensive necrosis in many different plant species. Nonhost species reacted to multiple effector proteins from an individual pathovar more frequently and more intensely than host species. Both homologous and sequence-unrelated effectors could elicit necrosis in a similar spectrum of plants, suggesting common effector targets or targeting of the same pathways in the plant cell.