Bighorn sheep show similar in‐host responses to the same pathogen strain in two contrasting environments

Ecological context—the biotic and abiotic environment, along with its influence on population mixing dynamics and individual susceptibility—is thought to have major bearing on epidemic outcomes. However, direct comparisons of wildlife disease events in contrasting ecological contexts are often confounded by concurrent differences in host genetics, exposure histories, or pathogen strains. Here, we compare disease dynamics of a Mycoplasma ovipneumoniae spillover event that affected bighorn sheep populations in two contrasting ecological contexts. One event occurred on the herd''s home range near the Rio Grande Gorge in New Mexico, while the other occurred in a captive facility at Hardware Ranch in Utah. While data collection regimens varied, general patterns of antibody signal strength and symptom emergence were conserved between the two sites. Symptoms appeared in the captive setting an average of 12.9 days postexposure, average time to seroconversion was 24.9 days, and clinical signs peaked at approximately 36 days postinfection. These patterns were consistent with serological testing and subsequent declines in symptom intensity in the free‐ranging herd. At the captive site, older animals exhibited more severe declines in body condition and loin thickness, higher symptom burdens, and slower antibody response to the pathogen than younger animals. Younger animals were more likely than older animals to clear infection by the time of sampling at both sites. The patterns presented here suggest that environment may not be a major determinant of epidemiological outcomes in the bighorn sheep—M. ovipneumoniae system, elevating the possibility that host‐ or pathogen‐factors may be responsible for observed variation.     

Natural history of a bighorn sheep pneumonia epizootic: Source of infection,course of disease,and pathogen clearance

A respiratory disease epizootic at the National Bison Range (NBR) in Montana in 2016–2017 caused an 85% decline in the bighorn sheep population, documented by observations of its unmarked but individually identifiable members, the subjects of an ongoing long‐term study. The index case was likely one of a small group of young bighorn sheep on a short‐term exploratory foray in early summer of 2016. Disease subsequently spread through the population, with peak mortality in September and October and continuing signs of respiratory disease and sporadic mortality of all age classes through early July 2017. Body condition scores and clinical signs suggested that the disease affected ewe groups before rams, although by the end of the epizootic, ram mortality (90% of 71) exceeded ewe mortality (79% of 84). Microbiological sampling 10 years to 3 months prior to the epizootic had documented no evidence of infection or exposure to Mycoplasma ovipneumoniae at NBR, but during the epizootic, a single genetic strain of M. ovipneumoniae was detected in affected animals. Retrospective screening of domestic sheep flocks near the NBR identified the same genetic strain in one flock, presumptively the source of the epizootic infection. Evidence of fatal lamb pneumonia was observed during the first two lambing seasons following the epizootic but was absent during the third season following the death of the last identified M. ovipneumoniae carrier ewe. Monitoring of life‐history traits prior to the epizootic provided no evidence that environmentally and/or demographically induced nutritional or other stress contributed to the epizootic. Furthermore, the epizootic occurred despite proactive management actions undertaken to reduce risk of disease and increase resilience in this population. This closely observed bighorn sheep epizootic uniquely illustrates the natural history of the disease including the (presumptive) source of spillover, course, severity, and eventual pathogen clearance.     

Low‐coverage reduced representation sequencing reveals subtle within‐island genetic structure in Aldabra giant tortoises

Aldabrachelys gigantea (Aldabra giant tortoise) is one of only two giant tortoise species left in the world and survives as a single wild population of over 100,000 individuals on Aldabra Atoll, Seychelles. Despite this large current population size, the species faces an uncertain future because of its extremely restricted distribution range and high vulnerability to the projected consequences of climate change. Captive‐bred A. gigantea are increasingly used in rewilding programs across the region, where they are introduced to replace extinct giant tortoises in an attempt to functionally resurrect degraded island ecosystems. However, there has been little consideration of the current levels of genetic variation and differentiation within and among the islands on Aldabra. As previous microsatellite studies were inconclusive, we combined low‐coverage and double‐digest restriction‐associated DNA (ddRAD) sequencing to analyze samples from 33 tortoises (11 from each main island). Using 5426 variant sites within the tortoise genome, we detected patterns of within‐island population structure, but no differentiation between the islands. These unexpected results highlight the importance of using genome‐wide genetic markers to capture higher‐resolution genetic structure to inform future management plans, even in a seemingly panmictic population. We show that low‐coverage ddRAD sequencing provides an affordable alternative approach to conservation genomic projects of non‐model species with large genomes.     

Coinfection with a virus constrains within‐host infection load but increases transmission potential of a highly virulent fungal plant pathogen

The trade‐off between within‐host infection rate and transmission to new hosts is predicted to constrain pathogen evolution, and to maintain polymorphism in pathogen populations. Pathogen life‐history stages and their correlations that underpin infection development may change under coinfection with other parasites as they compete for the same limited host resources. Cross‐kingdom interactions are common among pathogens in both natural and cultivated systems, yet their impacts on disease ecology and evolution are rarely studied. The host plant Plantago lanceolata is naturally infected by both Phomopsis subordinaria, a seed killing fungus, as well as Plantago lanceolata latent virus (PlLV) in the Åland Islands, SW Finland. We performed an inoculation assay to test whether coinfection with PlLV affects performance of two P. subordinaria strains, and the correlation between within‐host infection rate and transmission potential. The strains differed in the measured life‐history traits and their correlations. Moreover, we found that under virus coinfection, within‐host infection rate of P. subordinaria was smaller but transmission potential was higher compared to strains under single infection. The negative correlation between within‐host infection rate and transmission potential detected under single infection became positive under coinfection with PlLV. To understand whether within‐host and between‐host dynamics are correlated in wild populations, we surveyed 260 natural populations of P. lanceolata for P. subordinaria infection occurrence. When infections were found, we estimated between‐hosts dynamics by determining pathogen population size as the proportion of infected individuals, and within‐host dynamics by counting the proportion of infected flower stalks in 10 infected plants. In wild populations, the proportion of infected flower stalks was positively associated with pathogen population size. Jointly, our results suggest that the trade‐off between within‐host infection load and transmission may be strain specific, and that the pathogen life‐history that underpin epidemics may change depending on the diversity of infection, generating variation in disease dynamics.     

A dynamic history of admixture from Mediterranean and Carpathian glacial refugia drives genomic diversity in the bank vole

Understanding the historical contributions of differing glacial refugia is key to evaluating the roles of microevolutionary forces, such as isolation, introgression, and selection in shaping genomic diversity in present‐day populations. In Europe, where both Mediterranean and extra‐Mediterranean (e.g., Carpathian) refugia of the bank vole (Clethrionomys glareolus) have been identified, mtDNA indicates that extra‐Mediterranean refugia were the main source of colonization across the species range, while Mediterranean peninsulas harbor isolated, endemic lineages. Here, we critically evaluate this hypothesis using previously generated genomic data (>6,000 SNPs) for over 800 voles, focusing on genomic contributions to bank voles in central Europe, a key geographic area in considering range‐wide colonization. The results provide clear evidence that both extra‐Mediterranean (Carpathian) and Mediterranean (Spanish, Calabrian, and Balkan) refugia contributed to the ancestry and genomic diversity of bank vole populations across Europe. Few strong barriers to dispersal and frequent admixture events in central Europe have led to a prominent mid‐latitude peak in genomic diversity. Although the genomic contribution of the centrally located Carpathian refugium predominates, populations in different parts of Europe have admixed origins from Mediterranean (28%–47%) and the Carpathian (53%–72%) sources. We suggest that the admixture from Mediterranean refugia may have provisioned adaptive southern alleles to more northern populations, facilitating the end‐glacial spread of the admixed populations and contributing to increased bank vole diversity in central Europe. This study adds critical details to the complex end‐glacial colonization history of this well‐studied organism and underscores the importance of genomic data in phylogeographic interpretation.     

Epizootic Pneumonia of Bighorn Sheep following Experimental Exposure to Mycoplasma ovipneumoniae

Background

Bronchopneumonia is a population limiting disease of bighorn sheep (Ovis canadensis). The cause of this disease has been a subject of debate. Leukotoxin expressing Mannheimia haemolytica and Bibersteinia trehalosi produce acute pneumonia after experimental challenge but are infrequently isolated from animals in natural outbreaks. Mycoplasma ovipneumoniae, epidemiologically implicated in naturally occurring outbreaks, has received little experimental evaluation as a primary agent of bighorn sheep pneumonia.

Methodology/Principal Findings

In two experiments, bighorn sheep housed in multiple pens 7.6 to 12 m apart were exposed to M. ovipneumoniae by introduction of a single infected or challenged animal to a single pen. Respiratory disease was monitored by observation of clinical signs and confirmed by necropsy. Bacterial involvement in the pneumonic lungs was evaluated by conventional aerobic bacteriology and by culture-independent methods. In both experiments the challenge strain of M. ovipneumoniae was transmitted to all animals both within and between pens and all infected bighorn sheep developed bronchopneumonia. In six bighorn sheep in which the disease was allowed to run its course, three died with bronchopneumonia 34, 65, and 109 days after M. ovipneumoniae introduction. Diverse bacterial populations, predominantly including multiple obligate anaerobic species, were present in pneumonic lung tissues at necropsy.

Conclusions/Significance

Exposure to a single M. ovipneumoniae infected animal resulted in transmission of infection to all bighorn sheep both within the pen and in adjacent pens, and all infected sheep developed bronchopneumonia. The epidemiologic, pathologic and microbiologic findings in these experimental animals resembled those seen in naturally occurring pneumonia outbreaks in free ranging bighorn sheep.     

Restoration of a bighorn sheep population impeded by Mycoplasma ovipneumoniae exposure

Bighorn sheep (Ovis canadensis) were once extirpated from the Black Hills region of South Dakota, U.S.A., mirroring declining populations throughout North America. Since the 1960s, several reintroductions have occurred in the Black Hills to reestablish populations, with varying success. We translocated 26 bighorn sheep from Alberta, Canada, to the Black Hills (February 2015) to restore bighorn sheep to their historic range. Due to prior examinations of cause‐specific survival, subsequent genetic diversity and disease prevalence analyses were required to evaluate success of the restoration effort. We measured a mean allelic diversity of 5.23 (SE = 0.44 [mean number of alleles]) and an observed heterozygosity of 0.71 (SE = 0.06; expected = 0.64 ± 0.05) in the translocated individuals. Translocated bighorn sheep tested negative for Mycoplasma ovipneumoniae at capture. An autogenous vaccine was administered prior to release in an attempt to safeguard the translocated bighorn sheep from infection with a strain known to be resident in adjacent bighorn sheep populations. However, the year following the translocation, a different strain of M. ovipneumoniae was associated with a pneumonia outbreak that resulted in 57.9% mortality. Our results suggest that allelic diversity and heterozygosity were sufficient for long‐term herd establishment, reducing the potential for founder effects. However, the overwhelming mortality associated with pneumonia, via the transfer of M. ovipneumoniae from an unknown source, limited the success or our reintroduction efforts. Successful attempts to restore bighorn sheep to their historic ranges must consider and mitigate potential routes for M. ovipneumoniae transmission pre‐ and post‐reintroduction.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

Multiple introductions and overwintering shape the progressive invasion of Aedes albopictus beyond the Alps

Aedes albopictus originates from Southeast Asia and is considered one of the most invasive species globally. This mosquito is a nuisance and a disease vector of significant public health relevance. In Europe, Ae. albopictus is firmly established and widespread south of the Alps, a mountain range that forms a formidable biogeographic barrier to many organisms. Recent reports of Ae. albopictus north of the Alps raise questions of (1) the origins of its recent invasion, and (2) if this mosquito has established overwintering populations north of the Alps. To answer these questions, we analyzed population genomic data from >4000 genome‐wide SNPs obtained through double‐digest restriction site‐associated DNA sequencing. We collected SNP data from specimens from six sites in Switzerland, north and south of the Alps, and analyzed them together with specimens from other 33 European sites, five from the Americas, and five from its Asian native range. At a global level, we detected four genetic clusters with specimens from Indonesia, Brazil, and Japan as the most differentiated, whereas specimens from Europe, Hong Kong, and USA largely overlapped. Across the Alps, we detected a weak genetic structure and high levels of genetic admixture, supporting a scenario of rapid and human‐aided dispersal along transportation routes. While the genetic pattern suggests frequent re‐introductions into Switzerland from Italian sources, the recovery of a pair of full siblings in two consecutive years in Strasbourg, France, suggests the presence of an overwintering population north of the Alps. The suggestion of overwintering populations of Ae. albopictus north of the Alps and the expansion patterns identified points to an increased risk of further northward expansion and the need for increased surveillance of mosquito populations in Northern Europe.     

Engineering a genome‐reduced bacterium to eliminate Staphylococcus aureus biofilms in vivo

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome‐reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm‐associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome‐reduced bacterium that can fight against clinically relevant biofilm‐associated bacterial infections.     

Genomic dynamics of brown trout populations released to a novel environment

Population translocations occur for a variety of reasons, from displacement due to climate change to human‐induced transfers. Such actions have adverse effects on genetic variation and understanding their microevolutionary consequences requires monitoring. Here, we return to an experimental release of brown trout (Salmo trutta) in order to monitor the genomic effects of population translocations. In 1979, fish from each of two genetically (F _ST = 0.16) and ecologically separate populations were simultaneously released, at one point in time, to a lake system previously void of brown trout. Here, whole‐genome sequencing of pooled DNA (Pool‐seq) is used to characterize diversity within and divergence between the introduced populations and fish inhabiting two lakes downstream of the release sites, sampled 30 years later (c. 5 generations). Present results suggest that while extensive hybridization has occurred, the two introduced populations are unequally represented in the lakes downstream of the release sites. One population, which is ecologically resident in its original habitat, mainly contributes to the lake closest to the release site. The other population, migratory in its natal habitat, is genetically more represented in the lake further downstream. Genomic regions putatively under directional selection in the new habitat are identified, where allele frequencies in both established populations are more similar to the introduced population stemming from a resident population than the migratory one. Results suggest that the microevolutionary consequences of population translocations, for example, hybridization and adaptation, can be rapid and that Pool‐seq can be used as an initial tool to monitor genome‐wide effects.     

Genome‐scale metabolic modeling reveals SARS‐CoV‐2‐induced metabolic changes and antiviral targets

Tremendous progress has been made to control the COVID‐19 pandemic caused by the SARS‐CoV‐2 virus. However, effective therapeutic options are still rare. Drug repurposing and combination represent practical strategies to address this urgent unmet medical need. Viruses, including coronaviruses, are known to hijack host metabolism to facilitate viral proliferation, making targeting host metabolism a promising antiviral approach. Here, we describe an integrated analysis of 12 published in vitro and human patient gene expression datasets on SARS‐CoV‐2 infection using genome‐scale metabolic modeling (GEM), revealing complicated host metabolism reprogramming during SARS‐CoV‐2 infection. We next applied the GEM‐based metabolic transformation algorithm to predict anti‐SARS‐CoV‐2 targets that counteract the virus‐induced metabolic changes. We successfully validated these targets using published drug and genetic screen data and by performing an siRNA assay in Caco‐2 cells. Further generating and analyzing RNA‐sequencing data of remdesivir‐treated Vero E6 cell samples, we predicted metabolic targets acting in combination with remdesivir, an approved anti‐SARS‐CoV‐2 drug. Our study provides clinical data‐supported candidate anti‐SARS‐CoV‐2 targets for future evaluation, demonstrating host metabolism targeting as a promising antiviral strategy.     

Gastric stem cells promote inflammation and gland remodeling in response to Helicobacter pylori via Rspo3‐Lgr4 axis

Helicobacter pylori is a pathogen that colonizes the stomach and causes chronic gastritis. Helicobacter pylori can colonize deep inside gastric glands, triggering increased R‐spondin 3 (Rspo3) signaling. This causes an expansion of the “gland base module,” which consists of self‐renewing stem cells and antimicrobial secretory cells and results in gland hyperplasia. The contribution of Rspo3 receptors Lgr4 and Lgr5 is not well explored. Here, we identified that Lgr4 regulates Lgr5 expression and is required for H. pylori‐induced hyperplasia and inflammation, while Lgr5 alone is not. Using conditional knockout mice, we reveal that R‐spondin signaling via Lgr4 drives proliferation of stem cells and also induces NF‐κB activity in the proliferative stem cells. Upon exposure to H. pylori, the Lgr4‐driven NF‐κB activation is responsible for the expansion of the gland base module and simultaneously enables chemokine expression in stem cells, resulting in gland hyperplasia and neutrophil recruitment. This demonstrates a connection between R‐spondin‐Lgr and NF‐κB signaling that links epithelial stem cell behavior and inflammatory responses to gland‐invading H. pylori.     

The synergy of germline C634Y and V292M RET mutations in a northern Chinese family with multiple endocrine neoplasia type 2A

Genetic analysis for germline mutations of RET proto‐oncogene has provided a basis for individual management of medullary thyroid carcinoma (MTC) and pheochromocytoma. Most of compound mutations have more aggressive phenotypes than single point mutations, but the compound C634Y/V292M variant in MTC has never been reported. Thus, we retrospectively investigated synergistic effect of C634Y and V292M RET germline mutations in family members with multiple endocrine neoplasia type 2A. Nine of 14 family members in a northern Chinese family underwent RET mutation screening using next‐generation sequencing and PCR followed by direct bidirectional DNA sequencing. Clinical features of nine individuals were retrospectively carefully reviewed. In vitro, the scratch‐wound assay was used to investigate the difference between the cells carrying different mutations. We find no patients died of MTC. All 3 carriers of the V292M variant were asymptomatic and did not have biochemical or structural evidence of disease (age: 82, 62 and 58). Among 4 C634Y mutation carriers, 2 patients had elevated calcitonin with the highest (156 pg/mL) in an 87‐year‐old male. Two carriers of compound C634Y/V292M trans variant had bilateral MTC with pheochromocytoma or lymph node metastasis (age: 54 and 41 years, respectively). Further, the compound C634Y/V292M variant had a faster migration rate than either single point mutation in vitro (P < .05). In conclusion, the V292M RET variant could be classified as ‘likely benign’ according to ACMG (2015). The compound variant V292M/C634Y was associated with both more aggressive clinical phenotype and faster cell growth in vitro than was either single mutation.     

Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

Aging‐associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world''s population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging‐associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin‐37 (IL‐37) is a potent anti‐inflammatory cytokine, and we present data demonstrating that IL‐37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin‐37 (IL‐37) in aged mice reduces or prevents aging‐associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL‐37 expression decreases the surface expression of programmed cell death protein 1 (PD‐1) and augments cytokine production from aged T‐cells. Improved T‐cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4⁺ T‐cells and Lat in CD8⁺ T‐cells when aged mice were treated with recombinant IL‐37 (rIL‐37) but not control immunoglobin (Control Ig). Importantly, IL‐37‐mediated rejuvenation of aged endogenous T‐cells was also observed in aged chimeric antigen receptor (CAR) T‐cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL‐37 in boosting the function of aged T‐cells and highlight its therapeutic potential to overcome aging‐associated immunosenescence.