Preparation of Ehrlichia ruminantium challenge material for quantifiable and reproducible challenge in mice and sheep

The causative agent of heartwater, Ehrlichia ruminantium, is a tick-transmitted pathogen that infects bovine endothelial cells. Due to the obligate intracellular nature of this organism obtaining pure material in sufficient quantities for challenge studies is difficult. A murine model is frequently used to study potential vaccine candidates but giving reproducible challenges in this model for heartwater has always been problematic. We have therefore performed a series of experiments to optimize the parameters governing the reproducibility of challenge material. Two cryoprotectants were compared for the preparation of challenge material, buffered lactose peptone (BLP) and sucrose-potassium-glutamate (SPG). In addition two sources of virulent E. ruminantium were used, infected bovine endothelial cultures and infected mouse spleen homogenates. We also examined practical parameters affecting the reproducibility of challenge experiments: the time it takes to deliver the challenge material, the length of time a mouse remains immune to E. ruminantium challenge, and the effect of a given challenge dose. Finally, we performed a pilot study to determine whether mice could be used to titrate challenge material to be used for experiments in sheep. We found that: (a) E. ruminantium-infected mouse spleen homogenate provides more reproducible challenges than tissue culture material; (b) SPG is a better cryoprotectant than BLP; (c) challenge material should be used within 20min of thawing; (d) it is not essential to use syngeneic material for murine challenge experiments; (e) Balb/c mice are more sensitive to E. ruminantium challenge than C57BL/6J mice; (f) mice immunized by infection and treatment for use as positive immune controls should be challenged within 3 months of immunization; and (g) mice should be challenged with a dose not exceeding 10 LD(50)s.     

Redesigning sugarcane for resistance to Australian canegrubs: a potential IPM component

Current management options to minimize the impact of canegrubs (larvae of melolonthine scarabs) on Australian sugarcane are heavily dependent on the use of synthetic insecticides. In developing a successful integrated pest management (IPM) programme for canegrubs, control options must be broadened. Plant resistance offers the potential for an easily deliverable and environmentally acceptable management option. Previous anecdotal reports suggest that Australian cultivars vary in their resistance to canegrubs. Many sugarcane clones (Saccharum spp. complex hybrids) have been screened and our studies have shown that there is variability in resistance through the reduction of the amount of tops, roots and stubble and in their effects on canegrub development and survival. Proteinase inhibitors, lectins and avidin have been identified as having activity against Australian canegrubs and are being introduced into the sugarcane genome. Toxins from Bacillus thuringiensis have shown little potential and are not being used further. We present a rationale for using resistant plants as a control option and for the incorporation of plant resistance into the breeding programme. Projected research will screen more sugarcane clones for resistance, study the nature of inheritance, identify chemical mechanisms involved in antibiotic effects, screen further antimetabolites for possible incorporation into the genome, prove the potential resistance of genetically engineered plants and incorporate plant resistance into an IPM programme for canegrubs.     

Predicted and observed droplet deposition on tsetse flies (Glossina morsitans) following aerosol application from aircraft

A non-toxic formulation containing fluorescent tracer was dispersed as an aerosol over savannah woodland in stable meteorological conditions, with only light wind, shortly after sunrise. Droplet deposition on wild caught tsetse flies (Glossina morsitans) was measured by means of a fluorescent tracer technique and compared with the deposit levels predicted from rotary sampler measurements using droplet collection efficiency data for samplers and flies derived from a laboratory wind tunnel study. Good correlation was found between the predicted and observed dose. An additional comparison, based only on rotary sampler data, between a typical spray (measured v.m.d. 27 μm) and a spray with smaller drop size (measured v.m.d. 21 μm) suggested that the latter might be significantly more effective as a means of control.     

Infertility in a Dog due to Proximal Cytoplasmic Droplets in the Ejaculate: Investigation of the Significance for Sperm Functionality In Vitro

A 4-year-old Basque Shepherd male dog was presented for breeding soundness evaluation after the dog failed to impregnate the three bitches he had mated. Clinical examination showed no anomaly of the reproductive system. Semen evaluation showed normal sperm count (640 x 10(6)), 80% had progressively motile spermatozoa, and 96% had morphologically abnormal sperm of which 84% had proximal cytoplasmic droplet and 12% had proximal droplet plus other anomaly. A zona pellucida-binding assay, using canine oocytes derived from frozen-thawed ovaries, was performed in order to investigate the zona-binding ability of dog spermatozoa with proximal cytoplasmic droplets. For the zona pellucida-binding assay, ovaries were thawed and minced in phosphate-buffered saline + 0.4% bovine serum albumin, the oocytes recovered were divided into two groups of 35-40 oocytes to be, respectively, used with the infertile dog and with a control fertile dog. Spermatozoa were capacitated in Canine Capacitating Medium (CCM) at 38.5 degrees C and 5% CO(2) in air for 2 h before oocyte insemination. Groups of five to six oocytes placed in 45 microl droplets of CCM were incubated for 1 h. Afterwards, 5 microl of CCM containing 25,000 spermatozoa were added to each droplet and co-incubated for 2 h before fixation and evaluation of the complexes. After oocyte insemination, sperm motility and viability were evaluated: the sample from the infertile dog had 85% sperm motility with fast and linear progressive movement, and sperm viability of 92%. The sample from the control dog showed 40% sperm motility with fast and highly curvilinear and erratic movement, high degree of sperm agglutination and sperm viability of 32%. For the infertile dog the mean number of bound spermatozoa/oocyte was 0.33 whereas for the control dog it was 1.80. It was concluded that dog sperm with proximal cytoplasmic droplets seem to lack normal capacitating ability in vitro, and consequently, they may have reduced capacity to bind to the zona pellucida of canine oocytes.     

Extensive genetic recombination occurs in the field between different genotypes of Ehrlichia ruminantium

The intracellular bacterium Ehrlichia ruminantium is the causative agent of heartwater throughout sub-Saharan Africa, Madagascar, and some islands of the Caribbean. The disease is tick-borne and causes substantial livestock losses, threatening food security and productivity in both the commercial and small-scale farming sectors in endemic areas. Immunization by infection and treatment is currently practised in South Africa, and it is known that a variety of immunotypes of the organism occur in the field, and that cross-protection between them varies widely from total to minimal. Future vaccines may therefore need to incorporate components from different genotypes so it is essential to have information on the extent of genetic variation among isolates. To obtain this information we amplified and sequenced a panel of eight core function genes from 12 different cultured stocks originally isolated in different areas of Africa and the Caribbean. Phylogenetic trees inferred from the sequences yielded different branching orders for different genes, and the reason for this inconsistency appears to be that extensive recombination takes place between different genotypes in the field. It is possible that recombination occurs during the period when the organisms are extracellular within the tick, immediately after feeding and before intracellular infection is established, although detection of more than one genotype in DNA from single ticks is encountered infrequently. The results of the analysis show that the phylogenetic variation is greatest among the isolates of southern African origin, suggesting that this is the region where the parasite first evolved. It also appears likely that the Gardel genotype, isolated in the Caribbean, originally came from west central Africa, not from west Africa as had long been assumed.     

History of atmospheric lead deposition since 12,370 (14)C yr BP from a peat bog, jura mountains, switzerland

A continuous record of atmospheric lead since 12,370 carbon-14 years before the present (14C yr BP) is preserved in a Swiss peat bog. Enhanced fluxes caused by climate changes reached their maxima 10, 590 (14)C yr BP (Younger Dryas) and 8230 (14)C yr BP. Soil erosion caused by forest clearing and agricultural tillage increased lead deposition after 5320 (14)C yr BP. Increasing lead/scandium and decreasing lead-206/lead-207 beginning 3000 (14)C yr BP indicate the beginning of lead pollution from mining and smelting, and anthropogenic sources have dominated lead emissions ever since. The greatest lead flux (15.7 milligrams per square meter per year in A.D. 1979) was 1570 times the natural, background value (0.01 milligram per square meter per year from 8030 to 5320 (14)C yr BP).     

Recalibrated Mariner 10 Color Mosaics: Implications for Mercurian Volcanism

Recalibration of Mariner 10 color image data allows the identification of distinct color units on the mercurian surface. We analyze these data in terms of opaque mineral abundance, iron content, and soil maturity and find color units consistent with the presence of volcanic deposits on Mercury's surface. Additionally, materials associated with some impact craters have been excavated from a layer interpreted to be deficient in opaque minerals within the crust, possibly analogous to the lunar anorthosite crust. These observations suggest that Mercury has undergone complex differentiation like the other terrestrial planets and the Earth's moon.     

Transition-State Spectroscopy of Cyclooctatetraene

The 351-nanometer photoelectron spectrum of the planar cyclooctatetraene radical anion (COT·-) shows transitions to two electronic states of cyclooctatetraene (COT). These states correspond to the D4h 1A1g state, which is the transition state for COT ring inversion, and the D8h 3A2u state. The electron binding energy of the 1A1g transition state is 1.099 ± 0.010 electron volts, which is lower by 12.1 ± 0.3 kilocalories per mole than that of the 3A2u state. The photoelectron spectrum shows that the singlet lies well below the triplet in D8h COT and confirms ab initio predictions that the molecule violates Hund's rule. Vibrational structure is observed for both features and is readily assigned by use of a simple potential energy surface.