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Human activities can increase or decrease risks of acquiring a zoonotic disease, notably by affecting the composition and abundance of hosts. This study investigated the links between land use and infectious disease risk in northeast Madagascar, where human subsistence activities and population growth are encroaching on native habitats and the associated biota. We collected new data on pathogenic Leptospira , which are bacteria maintained in small mammal reservoirs. Transmission can occur through close contact, but most frequently through indirect contact with water contaminated by the urine of infected hosts. The probability of infection and prevalence was compared across a gradient of natural moist evergreen forest, nearby forest fragments, flooded rice and other types of agricultural fields, and in homes in a rural village. Using these data, we tested specific hypotheses for how land use alters ecological communities and influences disease transmission. The relative abundance and proportion of exotic species was highest in the anthropogenic habitats, while the relative abundance of native species was highest in the forested habitats. Prevalence of Leptospira was significantly higher in introduced compared to endemic species. Lastly, the probability of infection with Leptospira was highest in introduced small mammal species, and lower in forest fragments compared to other habitat types. Our results highlight how human land use affects the small mammal community composition and in turn disease dynamics. Introduced species likely transmit Leptospira to native species where they co-occur, and may displace the Leptospira species naturally occurring in Madagascar. The frequent spatial overlap of people and introduced species likely also has consequences for public health.

Synthetic DNA barcodes are double-stranded DNA molecules designed to carry recoverable information, information that can be used to represent and track objects and organisms. DNA barcodes offer robust, sensitive detection using standard amplification and sequencing techniques. While numerous research groups have promoted DNA as an information storage medium, less attention has been devoted to the design of economical, scalable DNA barcode libraries. Here, we present an alternative modular approach to sequence design. Barcode sequences were constructed from smaller, interchangeable blocks, allowing for the combinatorial assembly of numerous distinct tags. We demonstrated the design and construction of first-generation (N = 256) and second-generation (N = 512) modular barcode libraries, from fewer than 50 total single-stranded oligonucleotides for each library. To avoid contamination during experimental validation, a liquid-handling robot was employed for oligonucleotide mixing. Generating barcode sequences in-house reduces dependency upon external entities for unique tag generation, increasing flexibility in barcode generation and deployment. Next generation sequencing (NGS) detection of 256 different samples in parallel highlights the multiplexing afforded by the modular barcode design coupled with high-throughput sequencing. Deletion variant analysis of the first-generation library informed sequence design for enhancing barcode assembly specificity in the second-generation library.

Uganda is home to a rich diversity of bats, which carry high ecological and socioeconomic value through the ecosystem services that they provide. However, critical bat habitats, including caves, are facing increasing anthropogenic pressures, and the types and frequencies of disturbances to cave-roosting bats are not well understood in Uganda. Therefore, we examined the role of anthropogenic disturbances in caves to assess the threats posed to bat populations. We used the Bat Cave Vulnerability Index (BCVI) framework to score 14 caves inhabited by bats within the study region. We included qualitative surveys with human communities to better understand various aspects within the BCVI. All bat species recorded were of the IUCN category “Least Concern”. The BCVI indicated 50% of the caves (with insectivorous and frugivorous bats) require urgent conservation interventions due to high bat diversity and anthropogenic disturbances (e.g., guano collection). Most of the caves studied were highly vulnerable to anthropogenic disturbances, as assessed in the study. All the caves we studied (except two) are outside the protected area, and due to their imputed vulnerabilities, interventions ought to be implemented to balance cave conservation and human use in the Mt. Elgon area. Such interventions should integrate human factors.

Small terrestrial mammals are major hosts of infectious agents responsible for zoonotic diseases. Astroviruses (AstVs)–the cause of non-bacterial gastroenteritis mainly affecting young children–have been detected in a wide array of mammalian and avian host species. However, understanding the factors that influence AstV infection within and across hosts is limited. Here, we investigated the impact of land use changes on AstVs in terrestrial small mammals in rural northeastern Madagascar. We sampled 515 small mammals, representing seven endemic and four introduced species. Twenty-two positive samples were identified, all but one of which were found in the introduced species Mus musculus and Rattus rattus (family Muridae), with a positivity rate of 7.7% (6/78) and 5.6% (15/266), respectively. The non-introduced rodent case was from an endemic shrew-tenrec (family Tenrecidae). We found the highest positivity rate of AstVs infection in brushy regrowth (17.5%, 7/40) as compared to flooded rice fields (4.60%, 8/174), secondary forest (4.1%, 3/74), agroforest (3.6%, 1/28), village (2.61%, 3/115), and semi-intact forest (0%, 0/84). A phylogenetic analysis revealed an association between AstVs and their rodent host species. None of the viruses were phylogenetically related to AstVs previously described in Malagasy bats. This study supports AstV circulation in synanthropic animals in agricultural habitats of Madagascar and highlights the need to assess the spillover risk to human populations in rural areas.

Conventional mosquito marking technology for mark–release–recapture (MRR) is quite limited in terms of information capacity and efficacy. To overcome both challenges, we have engineered, lab-tested, and field-evaluated a new class of marker particles, in which synthetic, short DNA oligonucleotides (DNA barcodes) are adsorbed and protected within tough, crosslinked porous protein microcrystals. Mosquitoes self-mark through ingestion of microcrystals in their larval habitat. Barcoded microcrystals persist trans-stadially through mosquito development if ingested by larvae, do not significantly affect adult mosquito survivorship, and individual barcoded mosquitoes are detectable in pools of up to at least 20 mosquitoes. We have also demonstrated crystal persistence following adult mosquito ingestion. Barcode sequences can be recovered by qPCR and next-generation sequencing (NGS) without detectable amplification of native mosquito DNA. These DNA-laden protein microcrystals have the potential to radically increase the amount of information obtained from future MRR studies compared to previous studies employing conventional mosquito marking materials.

Human disturbance of wildlife habitat leads to a change in wildlife community composition of an area, often decreasing species richness and favoring opportunistic or generalist species that thrive in a wide variety of environmental conditions. Such changes in a community have the potential to alter disease transmission dynamics and can affect human health. To make progress in linking ecosystem changes and disease risk, it is essential to develop new techniques to evaluate how wildlife communities interact, and how ecosystem stressors affect disease risk. The construction of social networks based on spatial data will provide information on community structure and disease transmission dynamics in relation to anthropogenic land use change. In the summer of 2017, small mammals were captured for 5 consecutive days at three sites in northeast Madagascar. Spatial data, Leptospira infection status, and morphometric data were collected from small mammals to identify host characteristics important to disease transmission. Data were input into R to construct a spatial network that aimed to model disease transmission. Through this study I found that a spatial network does not adequately model the environmental transmission of Leptospira, and highlights the importance of considering pathogen life cycle during the construction of disease transmission models. Additionally, Mus musculus were found to connect separate communities of small mammals, and thus inhabit an epidemiologically critical position in the spatial network.