BackgroundThe two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.MethodsIn this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC50 lower than that of BZ.FindingsThis method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.ConclusionThese methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

Background Trypanosoma cruzi is a protozoan parasite that causes severe disease in millions of habitants of developing countries. Currently there is no vaccine to prevent this disease and the available drugs have the consequences of side effects. Live vaccines are likely to be more effective in inducing protection than recombinant proteins or DNA vaccines; however, safety problems associated to their use have been pointed out. In recent years, increasing knowledge on the molecular genetics of Trypanosomes has allowed the identification and elimination of genes that may be necessary for parasite infectivity and survival. In this sense, targeted deletion or disruption of specific genes in the parasite genome may protect against such reversion to virulent genotypes.Methods and FindingsBy targeted gene disruption we generated monoallelic mutant parasites for the dhfr-ts gene in a T. cruzi strain that has been shown to be naturally attenuated. In comparison to T. cruzi wild type epimastigotes, impairment in growth of dhfr-ts+/− mutant parasites was observed and mutant clones displayed decreased virulence in mice. Also, a lower number of T. cruzi-specific CD8+ T cells, in comparison to those induced by wild type parasites, was detected in mice infected with mutant parasites. However, no remarkable differences in the protective effect of TCC wild type versus TCC mutant parasites were observed. Mice challenged with virulent parasites a year after the original infection with the mutant parasites still displayed a significant control over the secondary infection.ConclusionThis study indicates that it is possible to generate genetically attenuated T. cruzi parasites able to confer protection against further T. cruzi infections.

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Fertiliser application can not only influence plant communities, but also the soil microbial community dynamics, and consequently soil quality. Specifically, mineral fertilisation can directly or indirectly affect soil chemical properties, microbial abundance and, the structure and diversity of soil microbial communities. We investigated the impact of six different mineral fertiliser regimes in a maize/soybean rotation system: control (CK, without fertilisation), PS (application of phosphorus plus sulphur), NS (application of nitrogen plus S), NP (application of N plus P), NPS (application of N, P plus S) and NPSm (application of N, P, S plus micronutrients). Soil samples were collected at the physiological maturity stage of maize and soybean in March of 2013 and 2014, respectively. Overall, mineral fertilisation resulted in significantly decreased soil pH and increased total organic carbon compared with the control (CK). The analysis of terminal restriction fragment length polymorphism (T‐RFLP) revealed that mineral fertilisers caused a shift in the composition of both bacterial and fungal communities. In 2013, the highest value of Shannon diversity of bacterial terminal restriction fragments (TRFs) was found in control soils. In 2014, NPSm treated soils showed the lowest values of diversity for both bacterial and fungal TRFs. In both crop growing seasons, the analysis of phospholipid fatty acid (PLFA) detected the lowest value of total microbial biomass under CK. As PLFA analysis can be used to evaluate total microbial community, this result suggests that fertilisation increased total microbial biomass. When the bacterial and fungal abundance were examined using real time polymerase chain reaction, the results revealed that mineral fertilisation led to decreased bacterial abundance (16S rRNA), while fungal abundance (18S rRNA) was found to be increased in both crop growing seasons. Our results show that mineral fertiliser application has a significant impact on soil properties, bacterial and fungal abundance and microbial diversity. However, further studies are needed to better understand the mechanisms involved in the changes to microbial communities as a consequence of mineral fertilisation.

The aim of this study was to evaluate the response pattern of diazotrophic microbes, denitrifiers and nitrifiers to different types of land use management, such as soybean monoculture (M) during 5 and 24 years (M5 and M24) and soybean-maize rotation (R) during 4 and 15 years (R4 and R15) in two subsequent years at the time point of flowering. Soil samples from a site recently introduced into agriculture (RUA) and a pristine soil under native vegetation (NV) were used as controls. Abundances of different functional groups of microbes were assessed using the direct quantification of marker genes by quantitative real-time PCR using extracted DNA from rhizosphere samples. In addition, soil chemical and physical properties were analysed and correlated with the abundance data from the functional microbial groups under investigation. Overall, the results indicate that the abundance of nifH genes was higher under R treatments compared to M treatments. The abundance of ammonium monooxygenase genes amoA (AOA) was generally higher under rotation systems and decreased under M24. RUA evidenced a negative effect on the establishment and development of AOA communities. The influence of land use on nirS abundance was inconsistent. However, R treatments showed a high abundance of nirK genes compared to M treatments. In both growing seasons, the abundance of nosZ genes was higher under NV compared with the other treatments. Furthermore, M24 treatment was related to strongly changed chemical and physical soil properties compared with the other sites. As expected, soil samples from RUA showed the strong dynamics of measured parameters indicating the high sensitivity of soils under transition to environmental parameters. Our results also indicated that the long-term crop rotation modified the abundance of the investigated microbial groups compared to the monoculture and increased soil chemical and physical quality. Therefore, our results provide evidence for a stimulatory effect of the long-term crop rotation on the abundance of microbes involved in N transformation.

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Intensive agricultural practices have resulted in progressive soil degradation, with consequences on soil ecosystem services. The inclusion of service crops is a promising alternative to support the sustainability of the agricultural system. The aim of this study was to analyze in a six-year field experiment the effect of Brachiaria brizantha (perennial tropical grass) and Zea mays as service crops in a degraded common bean monoculture system in northwest Argentina. After six years, service crop treatments revealed a significant increase in most physical, chemical and biological properties of the soil (enzyme activities, microbial biomass, respiration and glomalin-related soil protein), compared with common bean monoculture. Also, a lower disease incidence was observed under B. brizantha treatments, associated with increased populations of Trichoderma spp. and Gliocladium spp. The phospholipid fatty acid profiles detected higher values of total microbial biomass under service crops. Our results suggest that the inclusion of several cycles of B. brizantha constitutes a promising soil management for recovering degraded agroecosystems.