Nanopore-Based Surveillance of Zoonotic Bacterial Pathogens in Farm-Dwelling Peridomestic Rodents

The effective control of rodent populations on farms is crucial for food safety, as rodents are reservoirs and vectors for several zoonotic pathogens. Clear links have been identified between rodents and farm-level outbreaks of pathogens throughout Europe and Asia; however, comparatively little research has been devoted to studying the rodent–agricultural interface in the USA. Here, we address this knowledge gap by metabarcoding bacterial communities of rodent pests collected from Minnesota and Wisconsin food animal farms. We leveraged the Oxford Nanopore MinION sequencer to provide a rapid real-time survey of putative zoonotic foodborne pathogens, among others. Rodents were live trapped (n = 90) from three dairy and mixed animal farms. DNA extraction was performed on 63 rodent colons along with 2 shrew colons included as outgroups in the study. Full-length 16S amplicon sequencing was performed. Our farm-level rodent-metabarcoding data indicate the presence of multiple foodborne pathogens, including Salmonella spp., Campylobacter spp., Staphylococcus aureus, and Clostridium spp., along with many mastitis pathogens circulating within five rodent species (Microtus pennsylvanicus, Mus musculus, Peromyscus leucopus, Peromyscus maniculatus, and Rattus norvegicus) and a shrew (Blarina brevicauda). Interestingly, we observed a higher abundance of enteric pathogens (e.g., Salmonella) in shrew feces compared to the rodents analyzed in our study. Knowledge gained from our research efforts will directly inform and improve farm-level biosecurity efforts and public health interventions to reduce future outbreaks of foodborne and zoonotic disease.

Molecular surveillance of zoonotic bacterial pathogens in farm dwelling peridomestic rodents across the upper Midwest, USA

The effective control of rodent populations on farms is a critical component of food-safety, as rodents are reservoirs and vectors for many foodborne pathogens in addition to several zoonotic pathogens. The functional role of rodents in the amplification and transmission of pathogens is likely underappreciated. Clear links have been identified between rodents and outbreaks of pathogens throughout Europe and Asia, however, comparatively little research has been devoted to studying this rodent-agricultural interface in the USA, particularly across the Midwest. Here, we address this existing knowledge gap by characterizing the metagenomic communities of rodent pests collected from Minnesota and Wisconsin food animal farms. We leveraged the Oxford Nanopore MinION sequencer to provide a rapid real-time survey of the putative zoonotic food-borne and other human pathogens. Rodents (mice and rats) were live trapped from three dairy and mixed animal farms. Tissues and fecal samples were collected from all rodents. DNA extraction was performed on 90 rodent colons along with 2 shrew colons included as outgroups in the study. Full-length 16S amplicon sequencing was performed with the MinION. Our data suggests the presence of putative foodborne pathogens including Salmonella spp., Campylobacter spp., Staphylococcus aureus, and Clostridium spp., along with many important mastitis pathogens. A critically important observation is that we discovered these pathogens within all five species of rodents (Microtus pennsylvanicus, Mus musculus, Peromyscus leucopus, Peromyscus maniculatus, and Rattus norvegicus) and shrew (Blarina brevicauda) in varying abundances. Interestingly, we observed a higher abundance of enteric pathogens (e.g. Salmonella) in shrew feces compared to the rodents analyzed in our study, however more data is required to establish that connection. Knowledge gained from our research efforts will directly inform and improve upon farm-level biosecurity efforts and public health interventions to reduce future outbreaks of foodborne and zoonotic disease.

High Prevalence of Borrelia mayonii (Spirochaetales: Spirochaetaceae) in Field-Caught Tamias striatus (Rodentia: Sciuridae) From Northern Wisconsin

Borrelia mayonii is a recently discovered bacterial spirochete that causes Lyme disease and is transmitted by the blacklegged tick, Ixodes scapularis Say (Acari: Ixodidae). To date, B. mayonii has been isolated from two vertebrate host species in Minnesota: field-caught white-footed mice (Peromyscus leucopus Rafinesque; Rodentia: Cricetidae) and American red squirrel (Tamiasciurus hudsonicus Erxleben). Here, we describe the first detection of B. mayonii in field-caught eastern chipmunks (Tamias striatus L. (Rodentia: Cricetidae)) from northern Wisconsin. During our study, we captured 530 unique small mammals and found an infection prevalence of 23.50% in field-caught eastern chipmunks (4/17) and 1.19% in Peromyscus spp. (5/420). Mean larval and nymphal burdens were determined for captured Blarina brevicauda (0, 0), Glaucomys volans (0.29, 0.14), Myodes gapperi (0.27, 0), Napaeozapus insignis (0, 0.25), Peromyscus spp. (1.88, 0.11), T. striatus (1.06, 0.65), and Sorex cinereus (0.09, 0). The high B. mayonii infection prevalence in eastern chipmunks suggests that the species may be an important reservoir for B. mayonii in the Upper Midwest.

A Comprehensive Multi-Omic Approach Reveals a Relatively Simple Venom in a Diet Generalist, the Northern Short-Tailed Shrew, Blarina brevicauda

Animals that use venom to feed on a wide diversity of prey may evolve a complex mixture of toxins to target a variety of physiological processes and prey-defense mechanisms. Blarina brevicauda, the northern short-tailed shrew, is one of few venomous mammals, and is also known to eat evolutionarily divergent prey. Despite their complex diet, earlier proteomic and transcriptomic studies of this shrew’s venom have only identified two venom proteins. Here, we investigated with comprehensive molecular approaches whether B. brevicauda venom is more complex than previously understood. We generated de novo assemblies of a B. brevicauda genome and submaxillary-gland transcriptome, as well as sequenced the salivary proteome. Our findings show that B. brevicauda’s venom composition is simple relative to their broad diet and is likely limited to seven proteins from six gene families. Additionally, we explored expression levels and rate of evolution of these venom genes and the origins of key duplications that led to toxin neofunctionalization. We also found three proteins that may be involved in endogenous self-defense. The possible synergism of the toxins suggests that vertebrate prey may be the main target of the venom. Further functional assays for all venom proteins on both vertebrate and invertebrate prey would provide further insight into the ecological relevance of venom in this species.

The crouching of the shrew: Mechanical consequences of limb posture in small mammals

An important trend in the early evolution of mammals was the shift from a sprawling stance, whereby the legs are held in a more abducted position, to a parasagittal one, in which the legs extend more downward. After that transition, many mammals shifted from a crouching stance to a more upright one. It is hypothesized that one consequence of these transitions was a decrease in the total mechanical power required for locomotion, because side-to-side accelerations of the body have become smaller, and thus less costly with changes in limb orientation. To test this hypothesis we compared the kinetics of locomotion in two mammals of body size close to those of early mammals (< 40 g), both with parasagittally oriented limbs: a crouching shrew (Blarina brevicauda; 5 animals, 17 trials) and a more upright vole (Microtus pennsylvanicus; 4 animals, 22 trials). As predicted, voles used less mechanical power per unit body mass to perform steady locomotion than shrews did (P= 0.03). However, while lateral forces were indeed smaller in voles (15.6 ± 2.0% body weight) than in shrews (26.4 ± 10.9%;P= 0.046), the power used to move the body from side-to-side was negligible, making up less than 5% of total power in both shrews and voles. The most power consumed for both species was that used to accelerate the body in the direction of travel, and this was much larger for shrews than for voles (P= 0.01). We conclude that side-to-side accelerations are negligible for small mammals–whether crouching or more upright–compared to their sprawling ancestors, and that a more upright posture further decreases the cost of locomotion compared to crouching by helping to maintain the body’s momentum in the direction of travel.

Effects of Brush Piles on Small Mammal Abundance and Survival in Central Pennsylvania

Brush piles have long been promoted as a means to enhance wildlife habitat, yet few studies have experimentally tested the perceived benefits of these structures for wildlife or evaluated the efficacy of different arrangements of these piles within landscapes. During summers 2005 and 2006, we used a mark–recapture study to compare small mammal abundance and survival in forested habitats provisioned with brush piles vs. similar habitats without, both at sites located adjacent to agricultural edges and within the interior of forests in central Pennsylvania. Northern short-tailed shrews Blarina brevicauda, mice Peromyscus, and eastern chipmunks Tamias striatus were all frequently captured within brush piles at edge and interior sites. Peromyscus were significantly more abundant at edge vs. interior sites. The presence of brush piles, however, seemed to have little effect on small mammal abundance or survival with the only potential effect being an increased overwinter survival rate experienced by Peromyscus at brush pile sites. We hypothesize that although brush piles provide cover and foraging sites for small mammals, the large quantities of coarse woody debris that exist naturally in many forested systems may already adequately provide these habitat features, minimizing the effects of brush piles on small mammal population dynamics.