Canine visceral leishmaniasis: comparison of in vitro leishmanicidal activity of marbofloxacin, meglumine antimoniate and sodium stibogluconate

The control of canine leishmaniasis largely depends on the success of treatment. Drugs currently available to treat this disease are toxic and partially effective. The curative effect of marbofloxacin, a third-generation fluoroquinolone developed for veterinarian individual treatment, was evaluated in vitro in the presence of Leishmania infantum promastigotes and dog-monocyte-derived macrophages; meglumine antimoniate and sodium stibogluconate were used as comparative treatments. We observed that the killing of Leishmania promastigotes and intracellular amastigotes by marbofloxacin was dose-dependent. We demonstrated that successful treatment of canine infected macrophages for 48 h was possible with 500 microg/ml of marbofloxacin. Leishmanicidal activity acted through a TNF-alpha and nitric oxide pathway and correlated with the generation of nitric oxide (NO(2)) production by monocytes derived macrophages from infected (23+/-5 microM) or healthy (21+/-6 microM) dogs, in comparison with NO(2) concentration in infected/non-treated macrophages (< 3 microM, P<0.01). This significant induced parasiticidal effect correlated with extensive elimination of amastigotes by macrophages derived from infected (11+/-5) and healthy dogs (6+/-2), when compared to infected/non-treated macrophages (530+/-105 and 472+/-86 amastigotes, respectively, P< 0.01). Marbofloxacin was shown to be non-toxic at 500 microg/ml in vitro and no cell apoptosis was observed. The molecule was able to induce a parasitic process after significant elimination of amastigotes in leishmania-infected dog macrophages. We propose that marbofloxacin, compared to standard chemotherapeutic agents (meglumine antimoniate and sodium stibogluconate), could be an effective and pragmatic oral route alternative to treat canine leishmaniasis.     

Sample size for diversity studies in tetraploid alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>) based on codominantly coded SSR markers

The number of genotypes investigated per population is important for the reliability of diversity studies. The objective of this study was to determine the sample size for the identification of differences among populations of an outcrossing autotetraploid species, alfalfa (Medicago sativa), using codominantly coded SSR markers. One hundred and twenty genotypes from each of two closely related populations were analysed with two markers. Twenty random subsamples for each of three sample sizes (10, 20 and 40 genotypes) were built. Compared to the populations with 120 genotypes, alleles that were no longer present in subsamples with 40 genotypes were mainly rare, whereas abundant alleles were also excluded in subsamples with 10 genotypes. F _ST values for pairs of subsamples between the two populations were always significantly different based on 40 genotypes, whereas for 10 genotypes more than half of the pairs were not significantly different. We concluded that 40 genotypes are a reasonable sample size for diversity studies with closely related populations of tetraploid alfalfa investigated with SSR markers. Twenty genotypes may be an economical alternative for large scale studies, but 10 genotypes were a too low number for reliable results.     

Integrating functional diversity into tropical forest plantation designs to study ecosystem processes

? The elucidation of relationships between biodiversity and ecosystem processes has been limited by the definition of metrics of biodiversity and their integration into experimental design. Functional trait screening can strengthen the performance of these designs.

? We suggest the use of Rao’s quadratic entropy to measure both functional diversity and phylogenetic diversity of species mixtures proposed for an experimental design, and demonstrate how they can provide complementary information.

? We also present an index assessing the statistical performance of these independent variables in different experimental designs. Measurement of independent variables as continuous vs. discrete variables reduces statistical performance, but improves the model by quantifying species differences masked by group assignments.

? To illustrate these advances, we present an example from a tropical forest tree community in which we screened 38 species for nine functional traits. The proposed TropiDEP design is based on the relative orthogonality of two multivariate trait axes defined using principal component analysis.

? We propose that independent variables describing functional diversity might be grouped to calculate independent variables describing suites of different traits with potentially different effects on particular ecosystem processes. In other systems these axes may differ from those reported here, yet the methods of analysis integrating functional and phylogenetic diversity into experimental design could be universal.

     

Effect of Geogenic Lead on Fungal and Collembolan Communities in Garden Topsoil

Geogenic lead (Pb) is considered to be less bioavailable than anthropogenic Pb and exerts less effect on the soil fauna. However, Pb contamination in vegetables has been reported in the case of geogenic anomalies, even at moderate concentrations (around 170 mg kg^–1). In this study, we investigated collembolan communities using both taxonomic- and trait-based approaches and observed fungal communities to assess the effects of a moderate geogenic Pb anomaly on collembolans and fungi in an urban vegetable garden soil. Results indicated that geogenic Pb indeed modified fungi communities and altered the functional structure of collembolan communities in garden soils. Although geogenic Pb presented low bioavailability, it affected soil fauna and vegetables similar to anthropogenic Pb.     

Responses to light in a soil-dwelling springtail

It has been widely assumed that Collembola respond to light, but until now there has been very little experimental proof of this. Field observations allowed to distinguish soil-dwelling species that would escape from light from surface-dwelling species that would be attracted to light. However, the supposed effect of light could be due to other factors such as temperature or dryness. We demonstrated that individuals of the collembolan species Heteromurus nitidus (Entomobryidae), when placed in a light gradient (temperature and moisture being homogeneous), clustered in the darker area. This effect occurred rapidly and changes in the distribution of animals persisted after illumination ceased. This shows light to act as a strong repellent for this soil-dwelling collembolan species.     

Relationships between soil fauna communities and humus forms: Response to forest dynamics and solar radiation

This study investigated the responses of soil animal communities, soil functioning and humus forms to forest dynamics and solar radiation. We examined changes in invertebrate communities and soil features in two subalpine spruce forests (Eastern Italian Alps, Trento) growing on a calcareous bedrock, with different sun exposures (north and south), each forming a chronosequence of three developmental phases: clearing, regeneration stand (25-year-old trees) and mature stand (170-year-old trees). Our results indicate that the two forest sites differed in solar energy input, soil chemical properties and the relationships between forest dynamics and animal communities. In the north-facing site, soil fauna communities were very similar in the three forest developmental phases. Conversely, in the south-facing site, the composition of invertebrate communities and the diversity of zoological groups varied greatly among developmental phases. The highest abundance of total invertebrates, and mites in particular, occurred in the south-facing mature stands while the south-facing regeneration stand was characterised by higher densities of Collembola, Chilopoda, Symphyla, Protura and Aranea. The structure of communities in clearings was the same as in regeneration stands but with lower invertebrate abundance. Humus forms and soil features changed with developmental phases in both the south- and north-facing sites, although variations were more pronounced in the southern exposure. Mature stands were characterised by high levels of soil organic carbon and nitrogen, C/N values and low pH, the clearings and regeneration stands being characterised by a greater release of mineral nitrogen. The diversity of zoological groups (Shannon–Wiener index) was linearly correlated to soil pH, Humus Index, the amount of organic carbon and the species richness of herbaceous plants. Our results about the composition and the diversity of invertebrate communities are consistent with the observations of other authors studying south-exposed forests growing on different bedrock types, indicating that such relationships are widespread. The higher densities of invertebrates in the south-facing site may be attributed to higher solar radiation, and the positive correlation observed between total soil fauna abundance and solar energy supports the “more individuals” hypothesis that assumes a positive relationship between the number of individuals and energy availability. Possible ways by which forest dynamics control soil invertebrate communities are discussed.     

A simplified procedure for terminal restriction fragment length polymorphism analysis of the soil bacterial community to study the effects of pesticides on the soil microflora using 4,6-dinitroorthocresol as a test case

We have studied the structural effects of application to the soil of a potentially detrimental herbicide, 4,6-dinitroorthocresol (DNOC) by analysing amplified ribosomal DNA restriction analysis (ARDRA) and terminal restriction fragment length polymorphism (T-RFLP) signatures of 16S rDNA fragments of culturable bacterial communities isolated from diluted soil suspensions. This approach has the potential to reveal changes induced by stressing the soil microflora with DNOC. This paper shows that, whereas only few changes of the ARDRA and T-RFLP profiles result from ageing of the soil, treatment of the soil with DNOC induces major modifications of these profiles. Therefore, for the practical purpose of pesticide registration, ARDRA and T-RFLP analysis performed on the dominant culturable fraction of the soil bacteria, implemented using conventional gel electrophoresis, offers the means of a routine, simple and meaningful test for detecting some of the changes affecting the structure of the soil microflora in response to pesticide application.     

A comparative study of two antimicrobial/anti-inflammatory formulations in the treatment of canine otitis externa

The efficacy and tolerability of a marbofloxacin-clotrimazole-dexamethasone otic suspension (MCD) was compared with a standard topical treatment using a phase III clinical trial protocol. In a total of 140 dogs with clinical signs of acute or subacute otitis externa, Staphylococcus, Pseudomonas, Enterobacteriaceae and Malassezia were isolated from samples taken at inclusion to identify the causative pathogen; a further sample was collected in the event of failure or relapse, and from dogs (at day 14) for which Pseudomonas species had been isolated at inclusion. One group received MCD (10 drops per affected ear) once daily and a second received Surolan (containing polymyxin B, miconazole and prednisolone) (5 drops per affected ear), twice daily. Each group received treatment for 7 or 14 days according to the clinical outcome on day 7. Efficacy and tolerability were evaluated on days 7, 14 and, if necessary, 28 for dogs treated for 14 days. The trial demonstrated equivalence of both treatments in terms of efficacy, with a cure rate of 58.3% for MCD and 41.2% for Surolan. Both medications were equally well tolerated by dogs, but MCD was superior in terms of pain relief, decrease in pus quantity and smell, response rate and investigator's assessment on day 14.