Unlike rodents, ferrets have human-like distribution of submucosal glands expressing MUC5B, associated with idiopathic pulmonary fibrosis (IPF). We evaluated ferrets exposed to a single dose of bleomycin (5 U/kg) longitudinally, and found sustained restrictive physiology, increased opacification and fibrotic injury in the lungs through 22 weeks. Notably, these lungs had an aberrant “proximalization” repair phenotype indicated by increased proportion of smaller airways co-expressing club cell secretory protein and alpha-tubulin that was associated with extent of fibrotic injury. We also observed MUC5B-positive cystic structures in the distal lung suggestive of honeycombing, consistent with increase of MUC5B+ airways in combination with a size shift to smaller airways. We conclude that ferrets exhibit aberrant repair and pathologic features characteristic of human IPF, including proximalization of the distal airways that has not been recapitulated in rodents. Heightened MUC5B expression may play a key role in promoting airway remodeling and sustained lung injury in IPF.

Competition from non-native species constitutes a significant threat to numerous native species worldwide. Endangered riparian woodrats (Neotoma fuscipes riparia) may be restricted to a single population occupying approximately 100 ha at Caswell Memorial State Park (CMSP) in central California. This population is vulnerable to a number of threats including from non-native black rats (Rattus rattus) that co-occur at CMSP. Black rats potentially engage in both interference and exploitative competition with woodrats. From September 2001 to September 2004, we investigated interactions between riparian woodrats and black rats to determine whether competitive interactions were reducing woodrat abundance or reproductive success. Two sites with riparian woodrats were identified at CMSP. Between January 2003 and September 2004, 179 black rats were removed from one site. Abundance of both species and woodrat reproductive success were assessed through live-trapping and radio-telemetry. Mean litter size, mean number of young for litters with emerged young, and mean number of young per female were all higher on the black rat removal site compared to the control site. Also, mean number of young for litters with emerged young increased on the removal site from 2003 to 2004 and decreased on the control site between years. Woodrat abundance trends were more equivocal and actually were higher on the control site during most of the period of black rat removal. The results of this investigation suggest that black rats may indeed suppress reproductive success of riparian woodrats, and that black rat removal could benefit woodrats. Black rats have been implicated in the declines and even extinction of other native rodents, including other woodrat species. Thus, black rats may constitute a significant threat to riparian woodrats, particularly in concert with other threats such as flooding, wildfires, and continued habitat loss and degradation. Therefore, we recommend that black rats be removed quarterly from highly suitable woodrat habitat in the CMSP. We also recommend that surveys be conducted to identify additional riparian woodrat populations and that black rat removals be conducted in those populations as well.

Cigarette smoking is associated with COVID-19 prevalence and severity, but the mechanistic basis for how smoking alters SARS-CoV-2 pathogenesis is unknown. A potential explanation is that smoking alters the expression of the SARS-CoV-2 cellular receptor and point of entry, angiotensin-converting enzyme 2 (ACE-2), and its cofactors including transmembrane protease serine 2 (TMPRSS2). We investigated the impact of cigarette smoking on the expression of ACE-2, TMPRSS2, and other known cofactors of SARS-CoV-2 infection and the resultant effects on infection severity in vitro. Cigarette smoke extract (CSE) exposure increased ACE-2 and TMPRSS2 mRNA expression compared to air control in ferret airway cells, Calu-3 cells, and primary human bronchial epithelial (HBE) cells derived from normal and chronic obstructive pulmonary disease (COPD) donors. CSE-exposed ferret airway cells inoculated with SARS-CoV-2 had a significantly higher intracellular viral load versus vehicle-exposed cells. Likewise, CSE exposure increased both SARS-CoV-2 intracellular viral load and viral replication in both normal and COPD HBE cells over vehicle control. Apoptosis was increased in CSE-exposed, SARS-CoV-2-infected HBE cells. Knockdown of ACE-2 via an antisense oligonucleotide (ASO) reduced SARS-CoV-2 viral load and infection in CSE-exposed ferret airway cells that was augmented by co-administration of camostat mesylate to block TMPRSS2 activity. Smoking increases SARS-CoV-2 infection via upregulation of ACE2 and TMPRSS2.

Rationale The role of MUC5B mucin expression in IPF pathogenesis is unknown. Bleomycin-exposed rodent models do not exhibit sustained fibrosis or airway remodeling. Unlike mice, ferrets have human-like distribution of MUC5B expressing cell types and natively express the risk-conferring variant that induces high MUC5B expression in humans. We hypothesized that ferrets would consequently exhibit aberrant repair to propagate fibrosis similar to human IPF. Methods Bleomycin (5U/kg) or saline-control was micro-sprayed intratracheally then wild-type ferrets were evaluated through 22 wks. Clinical phenotype was assessed with lung function. Fibrosis was assessed with µCT imaging and comparative histology with Ashcroft scoring. Airway remodeling was assessed with histology and quantitative immunofluorescence. Results Bleomycin ferrets exhibited sustained restrictive physiology including decreased inspiratory capacity, decreased compliance, and shifted Pressure-Volume loops through 22 wks. Volumetric µCT analysis revealed increased opacification of the lung bleomycin-ferrets. Histology showed extensive fibrotic injury that matured over time and MUC5B-positive cystic structures in the distal lung suggestive of honeycombing. Bleomycin ferrets had increased proportion of small airways that were double-positive for CCSP and alpha-tubulin compared to controls, indicating an aberrant ‘proximalization’ repair phenotype. Notably, this aberrant repair was associated with extent of fibrotic injury at the airway level. Conclusions Bleomycin-exposed ferrets exhibit sustained fibrosis through 22 wks and have pathologic features of IPF not found in rodents. Ferrets exhibited proximalization of the distal airways and other pathologic features characteristic of human IPF. MUC5B expression through native cell types may play a key role in promoting airway remodeling and lung injury in IPF.

Substantial clinical evidence supports the notion that ciliary function in the airways is important in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, the extent or nature of impairment of mucociliary transport (MCT) in in vivo models remains unknown. We hypothesize that SARS-CoV-2 infection results in MCT deficiency in the airways of golden Syrian hamsters that precedes pathological injury in lung parenchyma. Micro-optical coherence tomography was used to quantitate functional changes in the MCT apparatus. Both genomic and subgenomic viral RNA pathological and physiological changes were monitored in parallel. We show that SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 days postinfection (dpi) in hamsters, principally due to 79% diminished airway coverage of motile cilia. Correlating quantitation of physiological, virological, and pathological changes reveals steadily descending infection from the upper airways to lower airways to lung parenchyma within 7 dpi. Our results indicate that functional deficits of the MCT apparatus are a key aspect of COVID-19 pathogenesis, may extend viral retention, and could pose a risk factor for secondary infection. Clinically, monitoring abnormal ciliated cell function may indicate disease progression. Therapies directed toward the MCT apparatus deserve further investigation.

The San Joaquin kit fox ( Vulpes macrotis mutica ; SJKF) is federally endangered due to profound habitat loss. A population of SJKF occurs in the urban environment in the city of Bakersfield, California, and may be important for SJKF conservation. We conducted a systematic survey using automated camera stations and occupancy analysis to identify suitable habitat for SJKF in Bakersfield and to estimate a conceptual carrying capacity in the urban environment. We identified high, medium, and low suitability habitat totaling 121 km 2 , 196 km 2 , and 40 km 2 , respectively. Based on a mean urban kit fox home range size of 0.78 km 2 and an assumption of two adults in high suitability home ranges and one adult in medium suitability ranges, we estimated the adult carrying capacity in Bakersfield to be 561 foxes. This estimate seems plausible as the number of adult SJKF in Bakersfield was estimated to be 381 in 2015, and the number of foxes already had been reduced by a sarcoptic mange epidemic that began two years earlier. A carrying capacity of 561 adults would increase the estimated range-wide carrying capacity by as much as 38%. Adult density estimates derived for the urban SJKF population based on the carrying capacity (1.57/km 2 ) and home range size (2.56/km 2 ) were higher than estimates for foxes in natural habitats. The urban SJKF population in Bakersfield is substantial and therefore could contribute significantly to conservation and recovery efforts for SJKF. Given our results, a potential conservation strategy may be to encourage or even establish additional urban SJKF populations.

Photovoltaic solar power generating facilities are proliferating rapidly in California and elsewhere. While this trend is welcomed for many reasons (e.g., reducing greenhouse gas emissions), these facilities also can have profound environmental impacts, particularly to local species populations. These impacts become more significant when species of conservation concern are affected. In the San Joaquin Desert region in central California, a number of conservation measures have been routinely implemented on solar facilities, and these measures have facilitated continued use of the facilities by a number of species of conservation concern. Some of the more significant measures include permeable security fences, vegetation management, movement corridors, avoiding critical features such as dens and burrows, and vehicle speed limits. Detailed studies have been conducted on San Joaquin kit foxes (Vulpes macrotis mutica) using solar facilities in the San Joaquin Desert. Demographic and ecological attributes of foxes are similar between foxes using the facilities and foxes on nearby reference sites, and values for foxes on solar sites are within the ranges of values for foxes reported from sites within core population areas. Facilitated by the conservation measures, kit foxes are using at least six facilities in the San Joaquin Desert as are a variety of other species of conservation concern. This successful model also potentially could be adapted to other ecosystems and applied to facilities in regions outside of the San Joaquin Desert, such as the Mojave Desert. Determining whether species in other regions can use photovoltaic solar facilities and identifying the most efficacious conservation measures will require time and testing, and these efforts would benefit from collaborative efforts among landowners, solar developers, natural resources agencies, researchers, and others. The San Joaquin Desert facilities and a recent demonstration facility in the Mojave Desert provide strong evidence that solar facilities can be constructed and operated in a manner that also accommodates continued use of the facilities by some species of conservation concern.

The Tipton kangaroo rat (Dipodomys nitratoides nitratoides; TKR) is listed as endangered both Federally and by the state of California due to profound habitat loss throughout its range in the southern San Joaquin Valley of California. Habitat loss is still occurring and critical needs for TKR include identifying occupied sites, quantifying optimal habitat conditions, and conserving habitat. Our objectives were to (1) conduct surveys to identify sites where TKR were extant, (2) assess habitat attributes on all survey sites, (3) generate a GIS-based model of TKR habitat suitability, (4) use the model to determine the quantity and quality of remaining TKR habitat, and (5) use these results to develop conservation recommendations. We surveyed for TKR on 44 sites by live-trapping and detected TKR on 15 sites. Sites with TKR tended to have larger alkali scalds and no obvious sign of past tilling compared to sites without TKR. Also, sites with TKR usually had relatively sparse ground cover and seepweed (Suaeda nigra) was present. The non-protected Heermann’s kangaroo rat (Dipodomys heermanni), a larger competitor, was either absent or present in relatively low numbers at sites with TKR, and when present its abundance was inversely related to that of TKR. Based on our habitat suitability modeling, an estimated 30,000 ha of moderately high or high quality TKR habitat and 60,000 ha of lower or medium quality habitat remain. However, habitat is still being lost and conversion of at least one survey site with TKR occurred during this project. Recommendations for TKR conservation are to (1) conduct additional TKR surveys on unsurveyed but suitable sites, (2) conserve suitable habitat on unprotected lands, (3) manage vegetation on occupied sites if necessary, (4) restore disturbed lands to increase suitability for TKR, and (5) research methods and conduct translocations of TKR to unoccupied sites with suitable habitat.

RATIONALE: SARS-CoV-2 causes COVID-19 disease and infects respiratory epithelial cells, but how it affects ciliated cell function and the mucociliary transport apparatus is unknown. Abnormal mucociliary function could predispose to COVID-19 progression and/or secondary infection. Micro-optical coherence tomography (μOCT) is a novel method to simultaneously visualize and quantify the functional microanatomy of airways. Here, we established a hamster model of COVID-19 and evaluated their tracheas by μOCT. METHODS: Adult golden Syrian hamsters were inoculated intranasally with 3.2 × 105 (high dose, HD, N=4) or 3.2 × 104 (low dose, LD, N=4) plaque-forming units of SARS-CoV-2 (WA/1 strain). Clinical signs were monitored daily, nasal brushes collected intermittently, and hamsters were euthanized seven days (D7) after inoculation. Tracheas were imaged by μOCT, nasal washes and bronchial alveolar lavage fluid (BALF) from right lung lobes were collected for quantitation of viral load by qRT-PCR, and left lungs were inflated with and fixed in 10% neutral buffered formalin for histological analysis. Age-matched hamsters were used as uninfected controls (N=5). RESULTS: SARS-CoV-2 hamsters lost weight through D7 in dose-dependent fashion (-11% in HD vs. -4% in LD, p=0.02) and HD hamsters showed moderate lethargy. Nasal brushes on D4 and nasal washes on D7 contain 105-106 genome copies of virus while BALF on D7 was less than 104 genome copies and intermittently detected in LD. Histology demonstrated patchy and multifocal interstitial pneumonia (type II pneumocyte hyperplasia and mononuclear cell infiltrate), with ∼20% area affected in HD that was more variable in LD. Functional microanatomy of tracheas revealed diminished area of active ciliary beating (control 18 ± 2 vs. LD 7 ± 1%, p=0.0002, control vs. HD 9 ± 1%, p=0.001), reduced ciliary beat frequency (control 10.88 ± 0.70 vs. LD 8.83 ± 0.34 Hz, p=0.01, control vs. HD 8.26 ± 0.33 Hz, p=0.001), and decreased periciliary liquid depth (control 6.41 ± 0.18 vs. HD 5.61± 0.12 μ m, p=0.027). Mucociliary transport rate was diminished (control 0.84 ± 0.19 vs. LD 0.48 ± 0.16 vs. HD 0.37 ± 0.13 mm/min) although not statistically significant. Additional cohorts are in progress. CONCLUSION: SARS-Cov-2 infected hamsters exhibit reduced body weight, high viral load, and histopathological injury through 7 days. SARS-CoV-2 caused functional deficits of the mucociliary transport apparatus, consistent with early findings in COVID-19 patients (see Vijaykumar et al.). Abnormal ciliated cell function is important to SARS-CoV-2 pathogenesis, and may help monitor progression and represent a treatment opportunity for COVID-19.

RATIONALE: Cigarette smoking is associated with COVID-19 prevalence and severity, but the mechanistic basis for how smoking alters SARS-CoV-2 pathogenesis is unknown. A potential explanation is that smoking could alter expression of angiotensin converting enzyme-2 (ACE2), which functions as the cellular receptor and point of entry. Here we investigated the severity of SARS CoV-2 infection ex vivo and in vitro and using tissue samples and primary ferret and human airway epithelial cells with and without antecedent exposure to cigarette smoke. Methods: ACE2 expression measured by Quantitative PCR (Q-PCR) of ferret lungs exposed to 6 months of cigarette smoke, and findings validated by immunofluorescence (IF). Primary airway cells isolated from airways of ferrets or human non-smokers or COPD subjects were grown until terminally differentiated at air liquid interface. Cells were then exposed to cigarette smoke extract (CSE) or vehicle control, then infected with SARS CoV-2 (3 MOI) or mock control. Viral copy was measured by Q-PCR. Viral infection was quantified by foci forming assay (FFU/mL) using VeroE6 cells. Results: Ferret lungs following 6 months of smoke exposure had increased ACE2 as compared to air controls by Q-PCR (>1.5 fold, P 1.5 fold, P 2 fold, P 2 fold, P 2 fold (P<0.01, N=5). Likewise, CSE exposed (105-107 copy/μL, P<0.0001, N=4) and COPD (106-108 copy/μL, P<0.0001, N=4) HBE had increased viral load as compared to controls (103-104 copy/μL, P <0.001, N=4), and 2 to 3-fold increase in viral infection, respectively. TUNEL staining was increased in infected cells, indicating apoptosis. Transcript analysis of HBE cells with and without SARS-CoV-2 by RNASeq to identify differentially expressed genes in CSE exposed cells as compared to controls is in progress. Conclusion: Cigarette smoke and CSE increased ACE2 expression in ferrets, and ferret and human cells respectively. CSE-induction increased viral replication and infection severity, resulting in increased apoptosis. Cigarette smoking likely influences the severity of SARS-CoV-2 infection by altering expression of ACE2, inducing airway cell apoptosis upon infection.

The San Joaquin antelope squirrel (Ammospermophilus nelsoni: SJAS) is listed as Threatened pursuant to the California Endangered Species Act due to profound habitat loss throughout its range in the San Joaquin Desert in California. Habitat loss is still occurring and critical needs for SJAS include identifying occupied sites, quantifying optimal habitat conditions, and conserving habitat. Our objectives were to (1) conduct surveys to identify sites where SJAS were present, (2) assess habitat attributes on all survey sites, (3) generate a GIS-based model of SJAS habitat suitability, (4) use the model to determine the quantity and quality of remaining habitat, and (5) use these results to develop conservation recommendations. SJAS were detected on 160 of the 326 sites we surveyed using automated camera stations. Sites with SJAS typically were in arid upland shrub scrub communities where desert saltbush (Atriplex polycarpa) or jointfir (Ephedra californica) were the dominant shrubs, although shrubs need not be present for SJAS to be present. Sites with SJAS usually had relatively sparse ground cover with >10% bare ground and Arabian grass (Schismus arabicus) was the dominant grass. SJAS were more likely to occur on sites where kangaroo rats (Dipodomys spp.) were present and burrow abundance was greater, but SJAS were less likely to be present on sites with California ground squirrels (Otospermophilus beecheyi). Based on our habitat suitability model, an estimated 5,931 km2 of high or moderately high quality habitat and 4,753 km2 of lower quality habitat remain. To conserve SJAS, we recommend (1) conducting additional SJAS surveys on sites not surveyed but with suitable habitat, (2) conserving unprotected lands with suitable habitat, (3) managing vegetation on occupied sites if necessary, (4) restoring disturbed lands to increase suitability for SJAS, and (5) conducting translocations of SJAS to unoccupied sites with suitable habitat.

We examined geographic patterns of diversification in the highly impacted San Joaquin kangaroo rat, Dipodomys nitratoides, throughout its range in the San Joaquin Valley and adjacent basins in central California. The currently recognized subspecies were distinct by the original set of mensural and color variables used in their formal diagnoses, although the Fresno kangaroo rat (D. n. exilis) is the most strongly differentiated with sharp steps in character clines relative to the adjacent Tipton (D. n. nitratoides) and short-nosed (D. n. brevinasus) races. The latter two grade more smoothly into one another but still exhibit independent, and different, character clines within themselves. At the molecular level, as delineated by mtDNA cytochrome b sequences, most population samples retain high levels of diversity despite significant retraction in the species range and severe fragmentation of local populations in recent decades due primarily to landscape conversion for agriculture and secondarily to increased urbanization. Haplotype apportionment bears no relationship to morphologically defined subspecies boundaries. Rather, a haplotype network is shallow, most haplotypes are single-step variants, and the time to coalescence is substantially more recent than the time of species split between D. nitratoides and its sister taxon, D. merriami. The biogeographic history of the species within the San Joaquin Valley appears tied to mid-late Pleistocene expansion following significant drying of the valley resulting from the rain shadow produced by uplift of the Central Coastal Ranges.