The evolutionary genomics of adaptation to stress in wild rhizobium bacteria.

Microbiota comprise the bulk of life's diversity, yet we know little about how populations of microbes accumulate adaptive diversity across natural landscapes. Adaptation to stressful soil conditions in plants provides seminal examples of adaptation in response to natural selection via allelic substitution. For microbes symbiotic with plants however, horizontal gene transfer allows for adaptation via gene gain and loss, which could generate fundamentally different evolutionary dynamics. We use comparative genomics and genetics to elucidate the evolutionary mechanisms of adaptation to physiologically stressful serpentine soils in rhizobial bacteria in western North American grasslands. In vitro experiments demonstrate that the presence of a locus of major effect, the nre operon, is necessary and sufficient to confer adaptation to nickel, a heavy metal enriched to toxic levels in serpentine soil, and a major axis of environmental soil chemistry variation. We find discordance between inferred evolutionary histories of the core genome and nreAXY genes, which often reside in putative genomic islands. This suggests that the evolutionary history of this adaptive variant is marked by frequent losses, and/or gains via horizontal acquisition across divergent rhizobium clades. However, different nre alleles confer distinct levels of nickel resistance, suggesting allelic substitution could also play a role in rhizobium adaptation to serpentine soil. These results illustrate that the interplay between evolution via gene gain and loss and evolution via allelic substitution may underlie adaptation in wild soil microbiota. Both processes are important to consider for understanding adaptive diversity in microbes and improving stress-adapted microbial inocula for human use.

Experimental SARS-CoV-2 Infection of Elk and Mule Deer.

To assess the susceptibility of elk (Cervus canadensis) and mule deer (Odocoileus hemionus) to SARS-CoV-2, we performed experimental infections in both species. Elk did not shed infectious virus but mounted low-level serologic responses. Mule deer shed and transmitted virus and mounted pronounced serologic responses and thus could play a role in SARS-CoV-2 epidemiology.

Disease Progression and Serological Assay Performance in Heritage Breed Pigs following Brucella suis Experimental Challenge as a Model for Naturally Infected Feral Swine.

Invasive feral swine (Sus scrofa) are one of the most important wildlife species for disease surveillance in the United States, serving as a reservoir for various diseases of concern for the health of humans and domestic animals. Brucella suis, the causative agent of swine brucellosis, is one such pathogen carried and transmitted by feral swine. Serology assays are the preferred field diagnostic for B. suis infection, as whole blood can be readily collected and antibodies are highly stable. However, serological assays frequently have lower sensitivity and specificity, and few studies have validated serological assays for B. suis in feral swine. We conducted an experimental infection of Ossabaw Island Hogs (a breed re-domesticated from feral animals) as a disease-free proxy for feral swine to (1) improve understanding of bacterial dissemination and antibody response following B. suis infection and (2) evaluate potential changes in the performance of serological diagnostic assays over the course of infection. Animals were inoculated with B. suis and serially euthanized across a 16-week period, with samples collected at the time of euthanasia. The 8% card agglutination test performed best, whereas the fluorescence polarization assay demonstrated no capacity to differentiate true positive from true negative animals. From a disease surveillance perspective, using the 8% card agglutination test in parallel with either the buffered acidified plate antigen test or the Brucella abortus/suis complement fixation test provided the best performance with the highest probability of a positive assay result. Application of these combinations of diagnostic assays for B. suis surveillance among feral swine would improve understanding of spillover risks at the national level.

H7N9 influenza A virus transmission in a multispecies barnyard model.

Influenza A viruses are a diverse group of pathogens that have been responsible for millions of human and avian deaths throughout history. Here, we illustrate the transmission potential of H7N9 influenza A virus between Coturnix quail (Coturnix sp.), domestic ducks (Anas platyrhynchos domesticus), chickens (Gallus gallus domesticus), and house sparrows (Passer domesticus) co-housed in an artificial barnyard setting. In each of four replicates, individuals from a single species were infected with the virus. Quail shed virus orally and were a source of infection for both chickens and ducks. Infected chickens transmitted the virus to quail but not to ducks or house sparrows. Infected ducks transmitted to chickens, resulting in seroconversion without viral shedding. House sparrows did not shed virus sufficiently to transmit to other species. These results demonstrate that onward transmission varies by index species, and that gallinaceous birds are more likely to maintain H7N9 than ducks or passerines.

Hosts winnow symbionts with multiple layers of absolute and conditional discrimination mechanisms.

In mutualism, hosts select symbionts via partner choice and preferentially direct more resources to symbionts that provide greater benefits via sanctions. At the initiation of symbiosis, prior to resource exchange, it is not known how the presence of multiple symbiont options (i.e. the symbiont social environment) impacts partner choice outcomes. Furthermore, little research addresses whether hosts primarily discriminate among symbionts via sanctions, partner choice or a combination. We inoculated the legume, Acmispon wrangelianus, with 28 pairs of fluorescently labelled Mesorhizobium strains that vary continuously in quality as nitrogen-fixing symbionts. We find that hosts exert robust partner choice, which enhances their fitness. This partner choice is conditional such that a strain's success in initiating nodules is impacted by other strains in the social environment. This social genetic effect is as important as a strain's own genotype in determining nodulation and has both transitive (consistent) and intransitive (idiosyncratic) effects on the probability that a symbiont will form a nodule. Furthermore, both absolute and conditional partner choice act in concert with sanctions, among and within nodules. Thus, multiple forms of host discrimination act as a series of sieves that optimize host benefits and select for costly symbiont cooperation in mixed symbiont populations.

A novel vaccine candidate against rabbit hemorrhagic disease virus 2 (RHDV2) confers protection in domestic rabbits.

OBJECTIVE: To evaluate efficacy of a novel vaccine against rabbit hemorrhagic disease virus 2 (RHDV2) in domestic rabbits. ANIMALS: 40 New Zealand White rabbits obtained from a commercial breeder. PROCEDURES: Rabbits were vaccinated and held at the production facility for the duration of the vaccination phase and transferred to Colorado State University for challenge with RHDV2. Rabbits were challenged with oral suspensions containing infectious virus and monitored for clinical disease for up to 10 days. Rabbits that died or were euthanized following infection were necropsied, and livers were evaluated for viral RNA via RT-PCR. RESULTS: None of the vaccinated animals (0/9) exhibited clinical disease or mortality following infection with RHDV2 while 9/13 (69%) of the control animals succumbed to lethal disease following infection. CLINICAL RELEVANCE: The novel vaccine described herein provided complete protection against lethal infection following RHDV2 challenge. Outside of emergency use, there are currently no licensed vaccines against RHDV2 on the market in the United States; as such, this vaccine candidate would provide an option for control of this disease now that RHDV2 has become established in North America.

Evolution of manipulative microbial behaviors in the rhizosphere.

The rhizosphere has been called "one of the most complex ecosystems on earth" because it is a hotspot for interactions among millions of microbial cells. Many of these are microbes are also participating in a dynamic interplay with host plant tissues, signaling pathways, and metabolites. Historically, breeders have employed a plant-centric perspective when trying to harness the potential of microbiome-derived benefits to improve productivity and resilience of economically important plants. This is potentially problematic because: (i) the evolution of the microbes themselves is often ignored, and (ii) it assumes that the fitness of interacting plants and microbes is strictly aligned. In contrast, a microbe-centric perspective recognizes that putatively beneficial microbes are still under selection to increase their own fitness, even if there are costs to the host. This can lead to the evolution of sophisticated, potentially subtle, ways for microbes to manipulate the phenotype of their hosts, as well as other microbes in the rhizosphere. We illustrate this idea with a review of cases where rhizosphere microbes have been demonstrated to directly manipulate host root growth, architecture and exudation, host nutrient uptake systems, and host immunity and defense. We also discuss indirect effects, whereby fitness outcomes for the plant are a consequence of ecological interactions between rhizosphere microbes. If these consequences are positive for the plant, they can potentially be misconstrued as traits that have evolved to promote host growth, even if they are a result of selection for unrelated functions. The ubiquity of both direct microbial manipulation of hosts and context-dependent, variable indirect effects leads us to argue that an evolutionary perspective on rhizosphere microbial ecology will become increasingly important as we continue to engineer microbial communities for crop production.

Experimental Infection of Brazilian Free-Tailed Bats (Tadarida brasiliensis) with Two Strains of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is presumed to have originated from wildlife and shares homology with other bat coronaviruses. Determining the susceptibility of North American bat species to SARS-CoV-2 is of utmost importance for making decisions regarding wildlife management, public health, and conservation. In this study, Brazilian free-tailed bats (Tadarida brasiliensis) were experimentally infected with two strains of SARS-CoV-2 (parental WA01 and Delta variant), evaluated for clinical disease, sampled for viral shedding and antibody production, and analyzed for pathology. None of the bats (n = 18) developed clinical disease associated with infection, shed infectious virus, or developed histopathological lesions associated with SARS-CoV-2 infection. All bats had low levels of viral RNA in oral swabs, six bats had low levels of viral RNA present in the lungs during acute infection, and one of the four bats that were maintained until 28 days post-infection developed a neutralizing antibody response. These findings suggest that Brazilian free-tailed bats are permissive to infection by SARS-CoV-2, but they are unlikely to contribute to environmental maintenance or transmission.

Susceptibility of Wild Canids to SARS-CoV-2.

We assessed 2 wild canid species, red foxes (Vulpes vulpes) and coyotes (Canis latrans), for susceptibility to SARS-CoV-2. After experimental inoculation, red foxes became infected and shed infectious virus. Conversely, experimentally challenged coyotes did not become infected; therefore, coyotes are unlikely to be competent hosts for SARS-CoV-2.

Transgenic Soybeans Expressing Phosphatidylinositol-3-Phosphate-Binding Proteins Show Enhanced Resistance Against the Oomycete Pathogen Phytophthora sojae.

Oomycete and fungal pathogens cause billions of dollars of damage to crops worldwide annually. Therefore, there remains a need for broad-spectrum resistance genes, especially ones that target pathogens but do not interfere with colonization by beneficial microbes. Motivated by evidence suggesting that phosphatidylinositol-3-phosphate (PI3P) may be involved in the delivery of some oomycete and fungal virulence effector proteins, we created stable transgenic soybean plants that express and secrete two different PI3P-binding proteins, GmPH1 and VAM7, in an effort to interfere with effector delivery and confer resistance. Soybean plants expressing the two PI3P-binding proteins exhibited reduced infection by the oomycete pathogen Phytophthora sojae compared to control lines. Measurements of nodulation by nitrogen-fixing mutualistic bacterium Bradyrhizobium japonicum, which does not produce PI3P, revealed that the two lines with the highest levels of GmPH1 transcripts exhibited reductions in nodulation and in benefits from nodulation. Transcriptome and plant hormone measurements were made of soybean lines with the highest transcript levels of GmPH1 and VAM7, as well as controls, following P. sojae- or mock-inoculation. The results revealed increased levels of infection-associated transcripts in the transgenic lines, compared to controls, even prior to P. sojae infection, suggesting that the plants were primed for increased defense. The lines with reduced nodulation exhibited elevated levels of jasmonate-isoleucine and of transcripts of a JAR1 ortholog encoding jasmonate-isoleucine synthetase. However, lines expressing VAM7 transgenes exhibited normal nodulation and no increases in jasmonate-isoleucine. Overall, together with previously published data from cacao and from P. sojae transformants, the data suggest that secretion of PI3P-binding proteins may confer disease resistance through a variety of mechanisms.

Negotiating mutualism: A locus for exploitation by rhizobia has a broad effect size distribution and context-dependent effects on legume hosts.

In mutualisms, variation at genes determining partner fitness provides the raw material upon which coevolutionary selection acts, setting the dynamics and pace of coevolution. However, we know little about variation in the effects of genes that underlie symbiotic fitness in natural mutualist populations. In some species of legumes that form root nodule symbioses with nitrogen-fixing rhizobial bacteria, hosts secrete nodule-specific cysteine-rich (NCR) peptides that cause rhizobia to differentiate in the nodule environment. However, rhizobia can cleave NCR peptides through the expression of genes like the plasmid-borne Host range restriction peptidase (hrrP), whose product degrades specific NCR peptides. Although hrrP activity can confer host exploitation by depressing host fitness and enhancing symbiont fitness, the effects of hrrP on symbiosis phenotypes depend strongly on the genotypes of the interacting partners. However, the effects of hrrP have yet to be characterised in a natural population context, so its contribution to variation in wild mutualist populations is unknown. To understand the distribution of effects of hrrP in wild rhizobia, we measured mutualism phenotypes conferred by hrrP in 12 wild Ensifer medicae strains. To evaluate context dependency of hrrP effects, we compared hrrP effects across two Medicago polymorpha host genotypes and across two experimental years for five E. medicae strains. We show for the first time in a natural population context that hrrP has a wide distribution of effect sizes for many mutualism traits, ranging from strongly positive to strongly negative. Furthermore, we show that hrrP effect size varies across host genotypes and experiment years, suggesting that researchers should be cautious about extrapolating the role of genes in natural populations from controlled laboratory studies of single genetic variants.

American alligators are capable of West Nile virus amplification, mosquito infection and transmission.

West Nile virus (WNV) overwintering is poorly understood and likely multifactorial. Interest in alligators as a potential amplifying host arose when it was shown that they develop viremias theoretically sufficient to infect mosquitoes. We examined potential ways in which alligators may contribute to the natural ecology of WNV. We experimentally demonstrated that alligators are capable of WNV amplification with subsequent mosquito infection and transmission capability, that WNV-infected mosquitoes readily infect alligators and that water can serve as a source of infection for alligators but does not easily serve as in intermediate means for transmission between birds and alligators. These findings indicate potential mechanisms for maintenance of WNV outside of the primary bird-mosquito transmission cycle.

Susceptibility of livestock to SARS-CoV-2 infection.

We report pilot studies to evaluate the susceptibility of common domestic livestock (cattle, sheep, goat, alpaca, rabbit, and horse) to intranasal infection with SARS-CoV-2. None of the infected animals shed infectious virus via nasal, oral, or faecal routes, although viral RNA was detected in several animals. Further, neutralizing antibody titres were low or non-existent one month following infection. These results suggest that domestic livestock are unlikely to contribute to SARS-CoV-2 epidemiology.

An alphavirus replicon-based vaccine expressing a stabilized Spike antigen induces protective immunity and prevents transmission of SARS-CoV-2 between cats.

Early in the SARS-CoV-2 pandemic concerns were raised regarding infection of new animal hosts and the effect on viral epidemiology. Infection of other animals could be detrimental by causing clinical disease, allowing further mutations, and bares the risk for the establishment of a non-human reservoir. Cats were the first reported animals susceptible to natural and experimental infection with SARS-CoV-2. Given the concerns these findings raised, and the close contact between humans and cats, we aimed to develop a vaccine candidate that could reduce SARS-CoV-2 infection and in addition to prevent spread among cats. Here we report that a Replicon Particle (RP) vaccine based on Venezuelan equine encephalitis virus, known to be safe and efficacious in a variety of animal species, could induce neutralizing antibody responses in guinea pigs and cats. The design of the SARS-CoV-2 spike immunogen was critical in developing a strong neutralizing antibody response. Vaccination of cats was able to induce high neutralizing antibody responses, effective also against the SARS-CoV-2 B.1.1.7 variant. Interestingly, in contrast to control animals, the infectious virus could not be detected in oropharyngeal or nasal swabs of vaccinated cats after SARS-CoV-2 challenge. Correspondingly, the challenged control cats spread the virus to in-contact cats whereas the vaccinated cats did not transmit the virus. The results show that the RP vaccine induces protective immunity preventing SARS-CoV-2 infection and transmission. These data suggest that this RP vaccine could be a multi-species vaccine useful to prevent infection and spread to and between animals should that approach be required.

Δcps1 vaccine protects dogs against experimentally induced coccidioidomycosis.

Coccidioidomycosis is a significant health problem of dogs and humans in endemic regions, especially California and Arizona in the U.S. Both species would greatly benefit from a vaccine to prevent this disease. A live avirulent vaccine candidate, Δcps1, was tested for tolerability and efficacy to prevent pulmonary coccidioidomycosis in a canine challenge model. Vaccine injection-site reactions were transient and there were no systemic effects observed. Six of seven vaccine sites tested and all draining lymph nodes were sterile post-vaccination. Following infection with Coccidioides posadasii, strain Silveira, arthroconidia into the lungs, dogs given primary and booster vaccinations had significantly reduced lung fungal burdens (P = 0.0003) and composite disease scores (P = 0.0002) compared to unvaccinated dogs. Dogs vaccinated once had fungal burdens intermediate between those given two doses or none, but disease scores were not significantly different from unvaccinated (P = 0.675). Δcps1 was well-tolerated in the dogs and it afforded a high level of protection when given as prime and boost. These results drive the Δcps1 vaccine toward a licensed veterinary vaccine and support continued development of this vaccine to prevent coccidioidomycosis in humans.

Evolution: Symbiont switching and environmental adaptation.

Can hosts swap ancient symbionts for new ones? A new study shows that a novel partnership between a protist and an algal symbiont can rapidly evolve to both overcome initial incompatibility and adapt to environmental challenges.

Peridomestic Mammal Susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Wild animals have been implicated as the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but it is largely unknown how the virus affects most wildlife species and if wildlife could ultimately serve as a reservoir for maintaining the virus outside the human population. We show that several common peridomestic species, including deer mice, bushy-tailed woodrats, and striped skunks, are susceptible to infection and can shed the virus in respiratory secretions. In contrast, we demonstrate that cottontail rabbits, fox squirrels, Wyoming ground squirrels, black-tailed prairie dogs, house mice, and racoons are not susceptible to SARS-CoV-2 infection. Our results expand the knowledge base of susceptible species and provide evidence that human-wildlife interactions could result in continued transmission of SARS-CoV-2.

Evolution of specialization in a plant-microbial mutualism is explained by the oscillation theory of speciation.

Specialization in mutualisms is thought to be a major driver of diversification, but few studies have explored how novel specialization evolves, or its relation to the evolution of other niche axes. A fundamental question is whether generalist interactions evolve to become more specialized (i.e., oscillation hypothesis) or if partner switches evolve without any change in niche breadth (i.e., musical chairs hypothesis). We examined alternative models for the evolution of specialization by estimating the mutualistic, climatic, and edaphic niche breadths of sister plant species, combining phylogenetic, environmental, and experimental data on Acmispon strigosus and Acmispon wrangelianus genotypes across their overlapping ranges in California. We found that specialization along all three niche axes was asymmetric across species, such that the species with broader climatic and edaphic niches, Acmispon strigosus, was also able to gain benefit from and invest in associating with a broader set of microbial mutualists. Our data are consistent with the oscillation model of specialization, and a parallel narrowing of the edaphic, climatic, and mutualistic dimensions of the host species niche. Our findings provide novel evidence that the evolution of specialization in mutualism is accompanied by specialization in other niche dimensions.

Decreased coevolutionary potential and increased symbiont fecundity during the biological invasion of a legume-rhizobium mutualism.

Although most invasive species engage in mutualism, we know little about how mutualism evolves as partners colonize novel environments. Selection on cooperation and standing genetic variation for mutualism traits may differ between a mutualism's invaded and native ranges, which could alter cooperation and coevolutionary dynamics. To test for such differences, we compare mutualism traits between invaded- and native-range host-symbiont genotype combinations of the weedy legume, Medicago polymorpha, and its nitrogen-fixing rhizobium symbiont, Ensifer medicae, which have coinvaded North America. We find that mutualism benefits for plants are indistinguishable between invaded- and native-range symbioses. However, rhizobia gain greater fitness from invaded-range mutualisms than from native-range mutualisms, and this enhancement of symbiont fecundity could increase the mutualism's spread by increasing symbiont availability during plant colonization. Furthermore, mutualism traits in invaded-range symbioses show lower genetic variance and a simpler partitioning of genetic variance between host and symbiont sources, compared to native-range symbioses. This suggests that biological invasion has reduced mutualists' potential to respond to coevolutionary selection. Additionally, rhizobia bearing a locus (hrrP) that can enhance symbiotic fitness have more exploitative phenotypes in invaded-range than in native-range symbioses. These findings highlight the impacts of biological invasion on the evolution of mutualistic interactions.

Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached nearly every country in the world with extraordinary person-to-person transmission. The most likely original source of the virus was spillover from an animal reservoir and subsequent adaptation to humans sometime during the winter of 2019 in Wuhan Province, China. Because of its genetic similarity to SARS-CoV-1, it is probable that this novel virus has a similar host range and receptor specificity. Due to concern for human-pet transmission, we investigated the susceptibility of domestic cats and dogs to infection and potential for infected cats to transmit to naive cats. We report that cats are highly susceptible to infection, with a prolonged period of oral and nasal viral shedding that is not accompanied by clinical signs, and are capable of direct contact transmission to other cats. These studies confirm that cats are susceptible to productive SARS-CoV-2 infection, but are unlikely to develop clinical disease. Further, we document that cats developed a robust neutralizing antibody response that prevented reinfection following a second viral challenge. Conversely, we found that dogs do not shed virus following infection but do seroconvert and mount an antiviral neutralizing antibody response. There is currently no evidence that cats or dogs play a significant role in human infection; however, reverse zoonosis is possible if infected owners expose their domestic pets to the virus during acute infection. Resistance to reinfection holds promise that a vaccine strategy may protect cats and, by extension, humans.