For over two decades, researchers have sought to improve smallpox vaccines and also develop therapies to ensure protection against smallpox or smallpox-like disease. The 2022 human monkeypox pandemic is a reminder that these efforts should persist. Advancing such therapies have involved animal models primarily using surrogate viruses such as monkeypox virus. The intravenous monkeypox model in macaques produces a disease that is clinically similar to the lesional phase of fulminant human monkeypox or smallpox. Two criticisms of the model have been the unnatural route of virus administration and the high dose required to induce severe disease. Here, we purified monkeypox virus with the goal of lowering the challenge dose by removing cellular and viral contaminants within the inoculum. We found that there are advantages to using unpurified material for intravenous exposures. As of the end of June 2022, there were close to over 4900 cases of human monkeypox spanning 50 countries that are non-endemic for the disease. Transmission has extended beyond cases linked to initial exposure in Africa to human-to-human spread in each country . BecauseBecause of the similarity of the diseases in humans and in the absence of an accessible etiological agent, MPXV is thought to be an appropriate surrogate for smallpox. Arguably, one of the premier models for severe human monkeypox and smallpox intervention is the intravenous monkeypox virus model in macaques using the Zaire 79 strain. To date, multiple vaccines and antivirals have been assessed in this model .Delivery of monkeypox virus or variola virus is thought to mimic the secondary viremia in human monkeypox and/or smallpox disease. In macaques, similar to humans, fever is followed by the onset of a centrifugal rash. In classical disease, a maculopapular rash progresses to vesicles, pustules, and then umbilicates before scabbing. The main correlate of the intravenous model is death with secondary correlates being lesion counts and DNA viremia. Despite its popularity and faithfulness to lesional and fulminant orthopoxvirus disease, improvements such as delivery via a more natural route and/or decreasing the amount of virus required for severe disease are coveted. Currently, the only low dosed, nonhuman primate model using monkeypox virus is the common marmoset ,10. The Traditionally, monkeypox virus primarily used for challenge has been propagated and collected from cell culture, sonicated, and freeze/thawed to liberate cell-associated mature virus (MV) and consequently disrupt any extracellular virus particles present, with no additional purification steps. Zwartow, Westwood, and Appleyard showed with sucrose gradient purified vaccinia and rabbitpox virus stocks that impurity, such as viral hemagglutinin and cellular protein, could be dramatically reduced . As theyHere, we directly compare and characterize the disease induced by purified monkeypox virus, Zaire strain. We capture clinical manifestations and correlates of the model using two different doses of the monkeypox virus Zaire administered intravenously. We show there are differences in disease between the high doses of virus, especially in the consistency in clinical presentation.Monkeypox virus strain Zaire was propagated from scab material on chorioallantoic membranes, followed by one passage in LLC-MK2 cells, two passages in BSC-40 cells, and two passages in Vero E6 cells. This material was received from the Center for Disease Control and Prevention and passed in MA104 cells. This virus will be referred to as the master stock and was provided by John Huggins (USAMRIID).The master stock of the virus was thawed, sonicated, and vortexed four times. MA104 cells were inoculated at a multiplicity of infection (MOI) of 0.5 (5 plaque forming units for every 10 cells). The flasks were incubated in a 37 °C incubator and rocked every 15 min for 1 h. DMEM supplemented with 5% fetal bovine serum was added to each flask, after which the flasks were returned to the incubator. After 3 days, the cells were monitored for a cytopathic effect (CPE) daily. After a CPE of 4+ was noted, the supernatant was removed, cells were scraped and pooled, and three freeze-thaw cycles were performed. The material was then subjected to three sonication/vortex cycles and centrifuged to remove cells and larger cell debris. The supernatant was collected, and aliquots were made into pre-labeled tubes. This material will be known as the “crude” or “unpurified” working stock. A portion of this material was purified via a sucrose cushion, followed by a sucrose gradient , and wasMonkeypox virus, to include the purified and crude inoculums, was titrated using a standard plaque assay . One hunMacaca fascicularis) were screened for neutralizing antibodies to monkeypox virus prior to infection as described below. We opted to use cynomolgus macaques, since they tend to exhibit a more severe disease than do rhesus macaques and, as such, are more sensitive to the virus [t-test comparisons . Necropsies were performed by USAMRIID’s Veterinary Pathology Division.Adult, male Mauritius cynomolgus macaques  and serially diluted in MEM alpha with 2% heat-inactivated FBS and HEPES. A target of 100 PFU/100 µL was added to each dilution and allowed to incubate at 4 °C overnight. Both positive (vaccinia immunoglobulin) and negative (media only) controls were concomitantly prepared. Titration of the samples was performed in duplicate wells using plaque assay. Results were reported as a percent reduction of the negative control.t-test (GraphPad Prism).Extractions and QPCR were performed as described previously . BrieflyHematological data was generated on an ACT 10 Beckmann Coulter using whole EDTA blood. Abaxis Piccolos were used to evaluate clinical chemistries using Abaxis Chem12 or Chem13 reagent disks using serum samples.A necropsy was performed on all animals, either as soon as death occurred or after humane euthanasia of terminally ill or moribund animals. All tissues were immersion-fixed in 10% neutral buffered formalin for a minimum of 21 days, according to institute protocol.Formalin-fixed tissues for histologic examination were trimmed, processed, and embedded in paraffin according to established protocols . Histolo7 PFU/mL (high dose) and 5 × 106 PFU/mL (low dose) of sucrose gradient purified MPXV and our “crude” (unpurified or cell lysate) preparation. Historically, the high and low doses represent a near uniformly lethal and sublethal challenge, respectively [We hypothesized that by purifying MPXV, and thus reducing any confounding stimulation of the immune system by a contaminating material, we could effectively lower the lethal dose of the intravenous MPXV macaque model. To test this supposition, we intravenously exposed cynomolgus macaques (Mauritius) with two different target doses, 5 × 10ectively ,17,18,19When the virus inoculums were back titrated A, the pup = 0.3213 and 0.8864, respectively).The high dose of purified virus and crude virus produced a uniformly lethal disease. Animals in the purified group succumbed or were euthanized on days 7 and 17, whereas the crude virus group succumbed or were euthanized on days 10 and 12 A. AnimalDisease progression was similar in all groups with the exception of two animals from the purified high dose group (1HP and 2HP). Animal 1HP had an accelerated disease course, became confluent in a two-day time period, and subsequently died, whereas animal 2HP had a less severe disease state and had a 5 × 6 cm necrotic lesion at the site of exposure, indicating extravascular leakage during exposure. This animal was euthanized on day 13 for humane reasons. Lymphadenopathy was one of the earliest clinical signs of disease that developed between days 2 and 6 and peaked in size between day 6 and 14. During this peak time (days 6–14), a large portion of the animals became recumbent, dehydrated, and had a loss of appetite . Less frAnimals in both high dose groups had elevated temperatures starting between days 2 and 4 A. The loIn general, lesions developed in all animals between days 4 and 6 with the entire crude high dose cohort developing lesions on day 4 and the remainder on day 6 . With thp = 0.0008). There was no difference between the low dose groups (p = 0.7542). The difference and variability of lesions in the high dose groups suggest an advantage for utilizing crude preparations of virus for intravenous nonhuman primate exposures.The numbers of lesions (peak lesions) were quite different between the high dose purified and unpurified groups and quite variable between animals in the low dose groups A,B. The 4 to 7.2 × 104 genomes/mL and 5.6 × 105 to 7.5 × 105 genomes/mL for the crude low and high groups and 1.0 × 105 to 1.5 × 105 genomes/mL for the low dose purified group. The high dose purified group had a little more variability in one animal (2HP) with values of 2.1 × 106, 2.3 × 106, and 1.9 × 104 genomes/mL. This animal had evidence of extravascularization of the virus (see disease course and pathology sections), and this would explain the difference in circulating genomes. The crude and purified high dose groups were approximately 10-fold higher than the respective low dose purified and unpurified groups. Similar to the inoculums, the purified groups were higher than the crude counterparts.After exposure, blood was immediately (within 2 min) sampled to confirm exposure and consistency among animals within a group. Animals in three of the four groups were consistently challenged, with ranges of 4.5 × 10Viral genomes were detected as early as day 2 in all groups, with only one of the samples  being quantifiable  . All hig8 genomes/mL, was from the high dose purified group, animal 1HP, reflecting its accelerated disease course (see disease development and lesion burden). The other two members of this cohort had a peak viral DNA load 2–3 logs lower. The crude, high dose group was much more consistent and ranged from 5 × 106 to 2 × 107 genomes/mL. These quantities were obtained between days 6 and 12 with all crude maximums falling on day 10 . In summary, comparison of QPCR values in animals receiving the high dose of monkeypox virus suggests that consistency might be an advantage for using crude preparations.The peak genome levels for low dose groups ranged from 3 × 1010 or 12 C. All cr10 or 12 A,B. TherIncreases in circulating white blood cells (WBC) were noted for all groups, with most animals peaking between days 8 and 12 A,B. AnimClinical chemistries were also performed. Albumin levels decreased starting between days 2 and 4 in the high dose groups and never recovered A. In comGross necropsy findings included maculopapular rash and necrotizing and proliferative lesions of oral mucosa, lips, nares, tongue, trachea, esophagus, and lung in all groups. Additionally, lymphadenopathy of the axillary, inguinal, and tracheobronchial lymph nodes in both dose groups was present.Histologically, the skin was the most consistently affected tissue among both groups. Epithelium of multiple tissues  exhibited multifocal proliferative and necrotic lesions. These lesions contained epithelial degeneration and necrosis with intracytoplasmic and intercytoplasmic edema and neutrophilic exocytosis as well as proliferation of epithelium at the periphery of the lesions. Syncytial cells and intracytoplasmic inclusion bodies (ICIB) were also present. Perivascular dermatitis of the lips and nares was noted in all animals except 4HU. Hemorrhage, mainly associated with the oral mucosa, was noted in 4 of 8 animals.There was fibrinonecrotic pleuropneumonia in all animals excluding 1HP and 2HP. In affected animals, the upper and lower respiratory tract and pleura  had multifocal necrosis of epithelium, alveolar septae, and pleural mesothelium with multifocal loss of architecture. Lesions contained mixed inflammatory cells, necrotic and cellular debris, fibrin, and hemorrhage. The pulmonary pleura was expanded by mixed inflammatory cells and abundant fibrin and occasional hemorrhage. Hepatocellular lesions were mild, with necrosis and degeneration in 4 of 8 animals. Three animals lacked hepatocellular damage .Focal proliferative and necrotic lesions with occasional hemorrhage were observed in the gastrointestinal tract  of four animals. Animal 1HP also had multifocal epicardial petechial hemorrhage of the right and left ventricle. These lesions are uncommon in cynomolgus macaques intravenously exposed with monkeypox virus.Additional histologic findings included plasmacytosis in the spleen (7/8), lymphoid depletion of multiple lymphoid tissues such as spleen and various lymph nodes , hemorrhage (2/8), and congestion (3/8).Bacteria were thought to play a role in some of the lesions seen. At the extreme, animal 1HP was bacteremic with colonies found in the bone marrow, liver, spleen, lung, and lymph nodes. Other examples of these lesions were associated with the urinary bladder of 11LU, colon of 3HP, and the axillary and inguinal LN of 4HU.The intravenous monkeypox model is the most utilized nonhuman primate model for the evaluation of vaccines and antiviral drugs developed to mitigate the potential reintroduction of variola virus into an immunologically naïve population ,36,37,387 PFU of intravenously administered unpurified monkeypox virus was 83% lethal in mock vaccinated cynomolgus macaques [In these studies, we sought to lower the lethal dose of monkeypox virus intravenously delivered to cynomolgus macaques by manipulating the starting material (inoculums). In our first series of experiments, animals were exposed to sucrose gradient purified monkeypox virus at two different doses and compared to virus preparation that had been historically utilized in this test system . We hypomacaques  and simimacaques  and 80% macaques .The disease in all groups progressed in a fashion consistent with those previously reported and with features consistent with monkeypox or smallpox in humans ,40. A cuIn terms of disease treatment, rash or lesion onset is typically considered the first indicator of poxvirus disease and the trigger to start treatment. In our study, lymphadenopathy was consistently observed in all animals regardless of outcome, was one of the earliest clinical manifestations, and preceded rash in some cases. Peripheral lymphadenopathy is also a feature of the hemorrhagic and lesional variola model in cynomolgus macaque . When teIn this report, chemistry and hematology reflected those reported for human monkeypox and/or smallpox disease . IncreasTwo animals in the high purified dose group exhibited disparate disease manifestations, in the context of the dose administered. Animal 2HP presented a very mild disease course with reflective pathology. As previously stated, this animal most likely did not receive the full inoculum, as evidenced by the necrotic dermal lesion at the injection site and the relatively low circulating genomic load determined immediately after exposure. This animal was humanely euthanized as this lesion would have progressed and caused complications such as a severe secondary bacterial infection. The other animal, 1HP, progressed very rapidly, and succumbed to infection on day 7. Lesions never progressed beyond the vesicular phase. Again, this is also reflective of the pathological findings, where lymph nodes showed no evidence of an immunological response such as hyperplasia and plasmacytosis. This was most likely a consequence of its abbreviated disease course. Lesions not typically seen in the intravenous cynomolgus model were also present, such as those related to the heart. In fact, virally induced cardiac conditions have rarely been reported for monkeypox or smallpox afflicted individuals, but myocarditis, pericarditis, and ischemic cardiac events have been observed with live vaccines that are administered for protection against these diseases ,44,45,46Macaca fascicularis. Although we highlighted some potential differences, more animals would be required to reliably state that these are true differences. If real, these differences could be related to the relative variability between individual doses administered to the NHPs. It is likely that purification of the virus increases aggregation of virions and prevents equal distribution of the virus within the inoculum, and this leads to animals receiving different effective (and infective) doses. Given the sharp changes in disease manifestation based on dose [Consistency in an animal model system is important when evaluating therapeutic interventions. This is amplified when evaluating correlates of disease that are not absolute, as interventions may protect from the lethal aspect of the disease but may less effectively reduce other manifestations such as the onset or magnitude of the rash and viremia. In our study, there were no differences in mortality, although a few parameters had either significant changes or were more variable in nature. For instance, the unpurified high dose group exhibited a statistically higher mean peak lesional burden than the purified group. Because one of the animals (1HP) died early in the disease course, we cannot be certain it reached its “lesional potential”. Statistical analysis of lesion counts when analyzing only the lesion counts from day 6 did not produce any significance, but day 8, where the last lesion count  for 1HP was carried forward, were significant (data not shown). There was also greater variability in time-to-endpoint when comparing the purified high dose group to the unpurified group . Reports in cynomolgus macaques have ranged from as early as 5 days  to as la on dose  and revi5 PFU/mL and 100% when dosed with approximately one and two more logs of virus. Animals succumbed to disease around day 10. The closest crude virus comparison is with the Johnson et al. study where cynomolgus macaques were exposed via a similar route and doses of 5 × 105 and 5 × 106 PFU/mL [Our data suggest that purified monkeypox virus does not increase virulence as measured by mortality in the intravenous macaque model. Whether purification would alter other nonhuman primate models, such as those using a respiratory route of challenge, is still up for debate. Estep et al. exposed rhesus macaques to purified monkeypox virus via the intrabronchial route and compared the immunological response to a recombinant virus lacking the monkeypox inhibitor of the complement enzyme (MOPICE) gene (D14L) . Of the 6 PFU/mL . At thes6 PFU/mL . If trueThere are advantages to using a crude virus, such as a potential decrease in virion aggregation. Continued use of purified stocks will entail characterization that was already conducted for unpurified stocks. In fact, titer calculation for our purified stock tended to change with increasing freeze-thaws and sonication. The final stock values were calculated after consistent values were established independently by two individuals and were later still unreliable as the animals received more virus than originally calculated via plaque assay . An unde


We found that seagrass recovered within 4–6 weeks from the lower grazing intensities (25 and 50%) and 7–19 weeks from the higher grazing intensities (75 and 100%) across the estuary. Increased grazing intensity led to not only longer recovery times (RTs), but also greater variability in the RT among experimental locations. The RT from the higher grazing intensities at one location in particular was more than double other locations. Seagrass recovery was through vegetative mechanisms and not through sexual reproduction. There was a significant grazing treatment effect on seagrass meadow characteristics, particularly belowground biomass which had not recovered 3 months following grazing. As the pressure of climate change on estuarine environments increases, these quantified RTs for seagrass provide a baseline for understanding grazing pressure as a singular disturbance. Future work can now examine how grazing and other potentially interacting pressures in our changing climate could impact seagrass recovery even further.Herbivore distributions and abundance are shifting because of climate change, leading to intensified grazing pressure on foundation species such as seagrasses. This, combined with rapidly increasing magnitudes of change in estuarine ecosystems, may affect seagrass resilience. While the overall resilience of seagrasses is generally well-studied, the timeframes of recovery has received comparatively little attention, particularly in temperate estuaries. We investigated how the recovery time (RT) of seagrass is affected by simulated grazing in a southwestern Australian estuary. Whilst excluding swans, we simulated different grazing intensities (25, 50, 75, and 100% removal from 1 m While trophic interactions between species have evolved over millennia , they caSeagrasses are a foundation species of SAV in many estuaries  as they Understanding recovery time (RT) is particularly important for seagrasses because of the ecosystem services they provide and their vulnerability to ongoing declines . KnowingEqually important is understanding the mechanism for recovery. Seagrasses are known to recover through three primary mechanisms. Firstly, they expand into unoccupied space by vegetative expansion from surrounding, intact meadows . This isOur study investigated if grazing intensity affects the RT of a colonizing species of seagrass. We expected that the RT of seagrass would be slower with increasing grazing intensities; if there is greater removal of seagrass material, there is more area that seagrass needs to replenish to return to the original condition and will subsequently take longer. We also explored which mechanisms explained the patterns in recovery.Cygnus atratus Latham); an iconic herbivorous waterfowl in Australia. Swans are considered “ecosystem engineers” through their impact on the environment which is linked to their behavior, movements, and foraging  Hook. F., a colonizing species, is the dominant seagrass in the lower Swan-Canning Estuary  were chosen within monospecific meadows of H. ovalis in the estuary at Lucky Bay , Pelican Point , Point Resolution , and Rocky Bay   50%: removal of 50%; (4) 75%: removal of 75%; (5) 100%: removal of 100%; and (6) Procedural Control (PControl): a 1 m2 plot marked in the surrounding meadow at least 1 m from the boundary of the block without grazing exclusion and used to test for experimental artifacts in comparison to Control plots inside the structures. Individual swans can devegetate areas with diameters between 0.3 and 1.5 m, with an average size of 0.28 m2  . Each bl 0.28 m2 , represe 0.28 m2 , indicatCygnus olor, Gmelin)  to prevent natural grazing activity . The exc Gmelin)  and whic2 treatment plot into 25 equally sized cells (0.04 m2) and removing seagrass biomass within a random selection of cells from each plot according to the treatment  at the end of the experiment period. Due to this interference, data is unavailable for biomass characteristics and recovery mechanisms in the 25% grazing treatment at RCK.Halophila ovalis cover (%) was measured within each treatment, Control and PControl plot to determine the RT following simulated grazing treatments. Cover was recorded before grazing was simulated (time = 0), immediately following grazing, and then 14, 25, 49, 63, and 77 days following grazing. To measure cover, all 25 cells within each plot were photographed. A single image was taken of each cell and analyzed using SeaGIS TransectMeasure software. A regular grid of 25 measuring points was overlaid on each image and the presence/absence of seagrass at each point was recorded at each point. The points with seagrass present were pooled across all 25 images taken for one plot, and was divided by the total number of points across the plot (625 points measured over 1 m2 plot) to provide an overall cover (%). Due to low tides which prevented photography, data are unavailable for all treatments on Day 0 at Lucky Bay (LUB) and for 25% at RCK. On rare occasions, treatments were unable to be sampled due to equipment failure: the PControl on Day 49, and 75% on Day 49 and 77 at Pelican Point (PPT); and the 25% on Day 49 at Point Resolution (PRS). Five to seven weeks following simulated grazing, a substantial proportion of H. ovalis leaves were shed from the plants as part of regular senescence resulting from reducing temperatures  in a treatment plot to reach the minimum cover observed in the Control plot over the experimental period. Thus, the cover criterion for recovery was set independently at each block, as there was variation in seagrass cover in the Control plots among blocks. RT for each treatment was calculated by plotting the change in cover over time, fitting a curve to this relationship and then using this curve to estimate the earliest time at which the cover of a treatment intercepted the minimum cover of the Control plot at the same block . In someonential . Consequy = cover (%) at a given time, a = cover (%) at Day 0 following simulated grazing, e = Euler’s Number (∼2.7182), b = growth constant or continuous rate of increase in cover, and x = time (days).where 2. Data are unavailable for RCK in the 25% treatment due to natural grazing occurring before sample collection.The biomass characteristics  were assessed 84 days following simulated grazing by destructively sampling within each plot. Three replicate samples were collected using cylindrical cores (9.6 cm diameter × 15 cm depth) in the same three randomly selected cells from each plot. The collected material was placed in calico bags and transported to the laboratory and stored at −5°C until processed. In the laboratory, the plant material was rewashed in estuary water, and the leaves scraped to remove excess sediment and epiphytic material. The leaves and petioles were separated from the rhizomes using a blade, and the leaf density, aboveground (leaf + petiole) and belowground (rhizome + root) biomass  was recorded per 0.007 mThe experimental sites were monitored on day 14 and day 25, to determine whether the mechanism of recruitment of new vegetative material into grazed areas was through: (a) rhizome extension from surrounding seagrass patches, characterized by unbroken rhizomes extending into “grazed” areas from the adjacent meadow; (b) establishment of vegetative fragments, where there was no rhizome connection between the recruited material and the surrounding meadow; or (c) germination of seeds. The presence of each recovery mechanism was recorded for all “grazed” cells within each treatment. Due to the nature of the simulated grazing treatments, with different treatments having a different number of grazed cells, the data were pooled across the two sampling periods and expressed as a proportion of the number of cells measured in each plot at each location.The samples collected for the biomass were further processed to determine the characteristics related to potential mechanisms of recovery. These were: (a) node density, (b) the ratio between above and belowground biomass which can indicate the allocation of resources, (c) branching frequency , and (d) branching potential frequency  which can all indicate the patterns of clonal growth; (e) flowering and fruiting frequency (as a proportion of nodes), which can indicate potential investment in sexual reproduction.p-value = 0.05) with between 822 and 840 unique permutations for each treatment combination. The dispersion of the data was tested on raw data using PERMDISP . The null hypothesis for the analysis was there was no difference in RT across the four grazing intensity treatments. Permutation pair-wise tests were performed, following significant mains-test, to determine which levels within each factor were significantly different. A PERMANOVA was conducted on the meadow characteristics and recovery mechanisms to test for significant changes in each variable for Treatment, Block, and interactions (Treatment × Block) as fixed factors. Main and pair-wise tests were run as described for RT.To test for differences in RT, the data were analyzed using a permutation two-way analysis of variance (using Primer v6+ and PRIMER-E) with two factors: (1) Treatment: 25, 50, 75, and 100% grazing nested in Block; and (2) Block: LUB, PPT, PRS, and RCK. The PERMANOVA analysis was run on the resemblance matrix using Euclidean distance  of seagrass could predict the characteristics of the meadow , we pooled all replicate blocks and treatments and calculated Spearman rank correlation coefficient (ρ) with a me point .Swans were observed at all blocks except PRS, at least once during the experiment . Swans wSeagrass cover in the control plots was variable over the experiment but was consistently higher than in the treatments, and the cover decreased toward the end of the experiment due to senescence . The graF = 12.489, p < 0.05, Post-hoc analysis showed no significant difference in the mean RT between the 25 and 50% grazing treatments , aboveground biomass  and belowground biomass , 84 days after simulated grazing , but there were among the Control plots and the higher grazing treatments , and between aboveground biomass and cover , and significant, moderately positive correlation between the belowground biomass and cover , branching frequency , and above to belowground biomass ratio  between the treatment and control plots, and sometimes there were also differences among blocks , with post-hoc analysis showing a greater proportion of male flowers observed in treatments compared to the control but at LUB only  to achieve recovery than lower intensity grazing (4–5 weeks). These findings are broadly consistent with H. ovalis can develop large seed bank reserves and this has been documented locally  disruption of self-sustaining feedbacks following disturbance at larger scales; or (2) population-specific resilience to grazing. When loss occurs at a larger scale, recovery can become delayed and difficult to predict . In the –1 day–1. Assuming an estimated swan population of ∼300 individuals  and a total biomass of seagrass in the Swan-Canning Estuary of 6 × 109 g DW , it may not accurately predict other seagrass attributes. A combination of recovery indicators can be considered in monitoring programs which aim to track recovery from disturbance. Multiple indicators of seagrass recovery should be incorporated into programs to increase confidence in the conclusion made regarding meadow regeneration. Failure to do so may limit the detection and predictability of seagrass recovery and put these precious ecosystems at risk.Aboveground cover of dx.doi.org/10.25958/fq3d-m857.The datasets generated for this study can be found in the Research Online repository at Edith Cowan University, available at: The animal study was reviewed and approved by Ethics Committee, Edith Cowan University (Project 21327).CO’D: conceptualization, methodology, formal analysis, investigation, data curation, writing – original draft preparation, visualization, project administration, and funding acquisition. PL and KM: conceptualization, methodology, writing – review and editing, and supervision. CW: formal analysis, investigation, data curation, writing – review and editing, and visualization. All authors contributed to the article and approved the submitted version.


At present, few data are available on the prognosis of hypertensive emergencies and urgencies admitted to emergency departments.The aim of our study was to evaluate the incidence of total and cardiovascular events during follow-up in hypertensive patients admitted to the emergency departments of Brescia Hospital  with hypertensive emergencies or urgencies from 1 January to 31 December 2015.Medical records of patients aged more than 18 years, admitted to the emergency department with SBP values at least 180 mmHg (SBP) and/or DBP values at least 120 mmHg (DBP) were collected and analysed (18% of patients were classified as ‘hypertensive emergency’ and 82% as ‘hypertensive urgency’). Data in 895 patients  were analysed; the mean duration of follow-up after admission to the emergency department was 12 ± 5 months.P < 0.0001). Similar results were obtained when the occurrence of cerebrovascular or renal events were considered separately.During the follow-up, 96 cardiovascular events  occurred . In 40 patients (4.5%), a new episode of acute blood pressure rise with referral to the emergency department was recorded. Cardiovascular mortality and morbidity were greater in patients with a previous hypertensive emergency (14.5 vs. 4.5% in patients with hypertensive emergency and urgency, respectively, chi-square, Admission to the emergency department for hypertensive emergencies and urgencies identifies hypertensive patients at increased risk for fatal and nonfatal cardiovascular events. Our findings add some new finding suggesting that further research in this field should be improved aiming to define, prevent, treat and follow hypertensive urgencies and emergencies. Clinicians have to frequently manage acute BP reduction for a critically ill patient, with specific indications suggested by more recent guidelines or consensus papers –3. ConveOn the basis of the above considerations, we considered worthwhile to investigate the occurrence of clinical events and blood pressure control during follow-up, in patients with hypertensive emergencies and hypertensive urgencies referred to the emergency department of ‘ASST Spedali Civili of Brescia’ during a period of 12 months.Consecutive patients admitted to the emergency department of the Spedali Civili of Brescia aged 18 years or older and presenting with an acute increase in BP values were included in the study. The procedure was approved by the Ethics committees. The study was conducted in accordance with the Helsinki Declaration. In addition, written or oral informed consent was obtained by patient or authorized relatives. When patients could not provide the necessary information, their relatives provided the data registered. All patients  had free access to the emergency department of the Spedali Civili of Brescia, as established by the Italian National Health Service. The procedures followed were in accordance with institutional guidelines. An acute increase in BP was defined as SBP at least 180 mmHg and/or DBP at least 120 mmHg ,17. WomeFollow-up data were obtained by telephone contact in all patients. For each patient, the occurrence of cardiovascular events, including cardiac events , cerebrovascular events (ischemic or hemorrhagic stroke or transient ischemic attack) and renal events  were recorded; patients were also asked to report further emergency department admission for acute BP increase. If a new hospitalization occurred, medical records were collected. In addition, the use of antihypertensive treatment and the measurement of blood pressure values less than 135/85 mmHg (i.e. BP control) were recorded.t test for unpaired data. Differences between categorical variables were analysed by the χ2. Kaplan--Meier survival analysis was used to assess the event-free survival in patients with hypertensive emergencies and hypertensive urgencies. The diagnosis of hypertensive emergencies or hypertensive urgencies was tested as independent variable in multivariate Cox proportional hazard model analyses adjusted for the confounders, having all or fatal cardiovascular events as dichotomic-dependent variables.All data were collected and analysed by SPSS 20  software. Data are expressed as mean standard deviation for continuous variables and percentages for categorical variables. The differences between continuous variables were analysed by the Student's n = 187, 15.4%, 0.22% of all emergency department visits) or urgency .In 2015, out of 69 101 patients admitted to emergency department, 1214 (1.76%)  had a hypertensive emergency  and two patients with hypertensive urgency with an advanced neoplastic disease died from nosocomial infection.Out of the remaining 1203 patients, 249 were not able to give information and 59 refused to answer to the interview; at the end 895 patients  were available for the telephone interview  presented as hypertensive urgency and 158 (18%) as hypertensive emergency.Patients with hypertensive emergencies compared with hypertensive urgencies were older, more frequently men (56 vs. 40%), had higher prevalence of previous cardiovascular disease, and had slightly higher values of SBP and DBP at admission in the emergency department Table .Predominant organ damage in the hypertensive group were heart failure (30%), acute coronary syndrome (27%), neurological disorders/stroke (37%) and acute kidney failure (6%).P < 0.001), whereas withdrawal of prescribed treatment was recorded in 3.7 and 1.9% (P < 0.05), respectively (Table During the follow-up (mean duration 12 ± 5 months), 77% of patients with hypertensive urgency and 89% of patients with hypertensive emergency was treated with different antihypertensive drugs. Among patients with hypertensive urgencies and hypertensive emergencies, no treatment during the follow-up was reported in 14.5 and 2.5% of cases  was reported in patients with hypertensive emergencies and hypertensive urgencies; in few patients in both groups (6%) no data on BP measurement were recorded.P = NS).In 3 cases, a diagnosis of new-onset diabetes, and in 15, a new diagnosis of hypertension was reported. In 40 patients (4.5%), a new episode of acute BP rise with referral to the emergency department was recorded, without a significant difference between hypertensive emergencies and hypertensive urgencies , requiring hospitalization, occurred: 96 cardiovascular  and 107 noncardiovascular . Among 96 cardiovascular events, 20 were cardiac ischemic events (4 deaths), 30 cerebrovascular events (16 deaths), 26 hospital admission for heart failure (6 deaths) and 20 cases of renal failure or need for haemodialysis treatment (2 deaths).P < 0.01).Patients with cardiovascular events during the follow-up were older, more frequently men, and with a higher prevalence of hypertension, diabetes, cerebrovascular and cardiac diseases; in addition, BP control during follow-up was higher in patients without CV events as compared to patients with CV events  3.83, 95% CI 7.03–20.9]. Similar results were obtained when the occurrence of heart failure , coronary events , cerebrovascular events  or renal events  were considered separately. No differences were observed between patients with hypertensive emergencies and hypertensive urgencies in the occurrence of hospitalization for all noncardiovascular causes  or non-cardiovascular mortality .Morbidity and mortality for cardiac events (i.e. acute coronary syndromes and/or acute heart failure) were higher in patients with a previous hypertensive emergencies [12.9 and 3.7% in patients with hypertensive emergencies and hypertensive urgencies, respectively, P = 0.001), DBP , previous cerebrovascular disease  and being an hypertensive emergency  were revealed to be independent predictors of subsequent cardiovascular fatal events , age , diabetes , and a previous diagnosis of a cerebrovascular disease  were independent predictors of cardiovascular fatal and nonfatal events, whereas sex and clinical BP did not reach statistical significance.In the Cox regression analysis including hypertensive emergencies and hypertensive urgencies, age, sex, clinical BP values, diabetes, previous diagnosis of coronary artery disease, and previous diagnosis of cerebrovascular disease only age  with hypertensive urgencies or hypertensive emergencies. We have shown a high 1-year cardiovascular event rate and high 1-year death rate for cardiovascular causes in both hypertensive emergencies and hypertensive urgencies.These data expand our previous observation  and otheWe have previously reported the demographic and clinical characteristics of patients examined in two different period of time (2008 and 2015) showing a prevalence of clinical presentation, which is similar to other observational studies .We observed slight differences between hypertensive emergencies and hypertensive urgencies, with higher mean age and men prevalence for hypertensive emergencies. The difference in sex prevalence could be because of the absence of pregnant women with emergencies, whereas the older age of hypertensive emergencies could be the expression of more comorbidities like hypertension, cardiovascular disease and diabetes mellitus leading to higher degree of atherosclerosis ,23 and fIn our study, patients with hypertensive emergencies present a significantly higher cardiovascular event rate, possibly explained by their higher prevalence of previous cardiovascular disease, and to a lesser extent by differences in age or sex (being men at higher cardiovascular risk profile) or other risk factors, including the degree of BP increase during the acute episode. During follow-up, stroke was the leading cardiovascular event in our study population, followed by heart failure, renal failure and acute coronary syndromes, although the higher risk of subsequent events was confirmed in our patients with all different clinical presentation of hypertension-mediated organ damage.et al.[BP control during the follow-up in our study was not different between hypertensive emergencies and hypertensive urgencies, reaching more than 70% among those receiving treatment. This finding is similar to the observation of Vleck et al., showinget al. as the aet al.[et al.[et al.[et al.[A recent consensus document suggests the use of uncontrolled BP as a term in substitution of hypertensive urgency, since the management with oral antihypertensive drug administration and the outcome of these patients does not differ from those with poorly controlled BP. In fact, Patel et al. have obset al.,13–15 thl.[et al. report al.[et al. observedl.[et al. or the Vl.[et al. studies Most patients with a clear clinical phenotype of hypertension-mediated organ damage are admitted to the emergency department for usually mild and aspecific symptoms like dizziness, headache, pain, frequently associated to anxiety . A less In addition, anxiety, psychopathological traits and drug nonadherence have been frequently overstated causes of hypertensive urgencies –28. VeryIn our study, treated hypertensive patients with hypertensive urgencies had a high rate of BP control during the follow-up, although a greater percentage of patients did not receive antihypertensive treatment or decided to withdraw the prescribed treatment during the follow-up. Therefore, we cannot exclude that in some patients, BP values remained elevated in the long-term, justifying, at least in part, the occurrence of events.Our registry has some obvious limitations. First, this is an observational study, with a retrospective analysis of data prospectively collected in 2015 , and theWhen evaluating treatment, we categorize for classes of drugs and did not record the exact doses of all drugs for management of all patients during the follow-up.Thirdly the results cannot be generalized to other settings as these data were collected at a single department. However, several observational available studies have shown a high rate of cardiovascular events not only in patients with hypertensive emergencies but also with hypertensive urgencies.In conclusion, we have underscored the importance of organizing comprehensive research efforts to improve current knowledge in this growing group of high-risk patients , hoping There are no conflicts of interest.


Neovison vison) for tolerance to Aleutian mink disease virus (AMDV) has gained popularity in recent years, but data on the outcomes of this activity are scant. The objectives of this study were to determine the long-term changes in viremia, seroconversion and survival in infected mink. Mink were inoculated intranasally with a local isolate of Aleutian mink disease virus (AMDV) over 4 years (n = 1742). The animals had been selected for tolerance to AMDV for more than 20 years (TG100) or were from herds free of AMDV (TG0). The progenies of TG100 and TG0, and their crosses with 25, 50 and 75% tolerance ancestry were also used. Blood samples were collected from each mink up to 14 times until 1211 days post-inoculation (dpi) and were tested for viremia by PCR and for anti-AMDV antibodies by counter-immunoelectrophoresis (CIEP). Viremia and CIEP status were not considered when selecting replacements. Low-performing animals were pelted and the presence of antibodies in their blood and antibody titer were measured by CIEP, and viremia and viral DNA in seven organs (n = 936) were tested by PCR.Selecting American mink (The peak incidences of viremia (66.7%) and seropositivity (93.5%) were at 35 dpi. The incidence of viremia decreased over time while the incidence of seroconversion increased. The least-squares means of the incidence of PCR positive of lymph node (0.743) and spleen (0.656) were significantly greater than those of bone marrow, liver, kidneys, lungs and small intestine (0.194 to 0.342). Differences in tolerant ancestry were significant for every trait measured. Incidences of viremia over time, terminal viremia, seropositivity over time, AMDV DNA in organs and antibody titer were highest in the susceptible groups (TG0 or TG25) and lowest in the tolerant groups (TG100 or TG75).Previous history of selection for tolerance resulted in mink with reduced viral replication and antibody titer. Viremia had a negative effect and antibody production had a positive effect on survival and productivity.The online version contains supplementary material available at 10.1186/s12917-022-03462-7. Carnivore amdoparvovirus 1), is a species of the genus Amdoparvovirus, family Parvoviridae [Aleutian mink disease virus  with different degrees of susceptibility to AMDV infection (tolerant and susceptible) over a long period (up to 1211 days) makes this study unique. Animals were inoculated intranasally after sedation, avoiding damage to their mucosal barriers, and the quantity of inoculum was chosen to guarantee the establishment of infection without the collapse of the animal’s immune system. Finally, animals were selected solely based on their health, vigor, and reproductive performance with no consideration given to their viremia or serological status, making this herd comparable to a commercial herd. The findings and conclusions of this study are thus useful in designing strategies for selecting mink with tolerance to AMDV infection.2(4 d.f.) = 9.8, P = 0.04) was the result of a linear decrease in the incidence of viremia in females as the percentage of tolerant ancestry increased, whereas the decrease in viremia in males was erratic  = 369.8, P < 0.001), corresponding to an odds ratio of e-0.0991 = 0.905, which is the estimated change in the odds of mink becoming viremic for each month delay in sampling, and suggesting that fewer mink became viremic as time went by.Prior to inoculation at different dates, between 0.8 and 39.4% of the mink were viremic, and the incidences of viremia increased to 21.6 to 86.3% on 35 dpi and then declined over time  were persistently nonviremic for at least 150 days before termination, 30 (3.0%) were persistently viremic (Supplementary Table P < 0.05), and the incidence of persistently nonviremic mink inoculated on other occasions was intermediate. A significantly greater proportion of pelted mink was persistently nonviremic compared with those that died, and a significantly greater proportion of males were persistently nonviremic than females. The change in the log odds for the incidence of persistently nonviremic mink over time was β = 0.0405 ± 0.0112 (ϰ2(1 d.f.) = 13.8, P < 0.001), and the odds ratio of e0.0405 = 1.041 indicated a 4.1% increase in the odds of mink becoming persistently nonviremic for each incremental months of sampling.The effects of tolerant groups, inoculation dates, disposal methods and sexes on the incidences of persistently nonviremic mink, compared with those which were viremic or showed transient viremia, were significant Table . The tol2(4 d.f.) = 10.4, P = 0.03). The incidence of viremia in TG25 males was significantly higher than that of TG25 females, but the reverse was observed for mink in TG50, TG75 and TG100  = 71.0, P < 0.001), showing an odds ratio of 0.911, which is the estimated change in the odds of mink becoming viremic at termination for each month longer that they remained in the herd.In addition to blood samples collected on the scheduled sampling dates, blood samples from 1217 mink were also collected at pelting  from 4 to 40 months pi and were tested by PCR. Pelting was performed up to 40 days after different scheduled samplings. The least-squares means of the incidences of terminal viremia for TG25 (0.287) and TG0 (0.169) were significantly higher than those of TG100 (0.069) and TG75 (0.069), and that of TG50 was intermediate (Table 2(1 d.f.) = 199.5, P < 0.001). The odds ratio e0.045 = 1.046 suggests an expected 4.6% increase in the odds of mink becoming seropositive for each incremental month of sampling.The incidence of seropositive cases of 1742 mink before inoculation ranged from 4.8% (October 2010) to 64.7% (December 2011), and sharply increased to 82.3 to 100% on subsequent tests  = 1.5, P = 0.22, odds ration = 0.969).The effects of tolerant groups, inoculation dates, sex, and disposal methods on the incidence of persistently seropositive mink, when compared with seronegative mink or those with transient CIEP results, were significant Table . The leaThe mean of seropositive mink at pelting was 96.3% over all pelting occasions. The differences between levels of tolerant groups, sex and inoculation dates for the incidences of seropositive mink at pelting were small (96.0 to 100%) and statistical comparison was not possible because the GENMOD models with binomial or Poisson distributions did not converge.Prior to inoculation, the largest number of mink (68.8%) were CIEP- and PCR-negative, and other CIEP and PCR combinations had comparable values (9.1 to 11.4%). Consistent with the frequencies of PCR and CIEP, the greatest proportion of animals was PCR- and CIEP-positive on 35 and 56 dpi, followed by those that were CIEP-positive and PCR-negative, and the smallest number were those that were CIEP-negative/PCR-positive (Table 2(1 d.f.) = 86.6, P < 0.001), corresponding to an odds ratio of e-0.0586 = 0.943, which was the estimated change in the incidence of PCR-positive organs for each month delay in sampling, suggesting that AMDV DNA was detected in fewer mink that remained longer in the herd.AMDV DNA was detected in at least one organ of 79.4% of the 936 mink with PCR data on seven organs. All seven organs of 82 (8.8%) and 193 (20.6%) of the mink were PCR-positive or PCR-negative, respectively. The GEE analysis of the frequency of AMDV DNA in seven organs of the pelted mink showed significant effects of organs, tolerant groups, inoculation dates and the covariance of sampling time, but the difference between males and females was not significant Table . AMDV DN2(1 d.f.) = 41.4, P < 0.001), corresponding to an odds ratio of 1.0592, indicating 5.92% increase in the odds of mink having seven PCR-negative organs for each month longer that they remained in the herd.The GENMOD procedure showed that the effects of tolerant groups, inoculation dates and sex were significant for the incidence of seven PCR-negative organs, compared to those with at least one PCR positive organ Table . The leaOf the 193 mink with seven PCR-negative organs, three (1.6%) were persistently seronegative from the time of inoculation but were viremic during the early periods after inoculation, the other 190 were persistently or sporadically seropositive, of which 70 were persistently nonviremic and 120 had short-lived viremia. The numbers of nonviremic and seronegative mink at termination with seven PCR-negative organs were 98.9 and 7.8%, respectively. Of the 743 mink with at least one PCR-positive organ, 80.2 and 2.3% were nonviremic and seronegative at termination, respectively.n = 57) to 1024 (n = 9). The distribution of antibody titer and its log transformed values significantly deviated from normality. The GENMOD procedure revealed that the effects of tolerant groups, inoculation dates, sex and the covariance of sampling month pi on the log-transformed antibody titer were significant (Table P < 0.01), suggesting that TG100 mink had significantly lower antibody titer than TG0, TG25 and TG50 mink. The declining trend in antibody titer with increased percentage of tolerant ancestry agrees with the raw means of antibody titer for TG0, TG25, TG50, TG75 and TG100 which were 161.8, 43.8, 63.6, 32.0 and 25.1, respectively, with corresponding medians of 64, 32, 16, 8 and 8. Pairwise comparisons of tolerant groups show that each tolerant group had lower odds of falling into the category of low antibody titer than groups with higher tolerant ancestry, and the differences were significant for those groups with two-level difference in tolerant ancestry, i.e. TG0-TG50, TG50-TG75, and so on. Relative differences between tolerant groups with one step difference in percentage ancestry were not significant, except for TG25-TG50.The distribution of antibody titer in 1217 mink at pelting was positively skewed with a mean of 79.7, median of 16 and a range from 0 (P < 0.01), suggesting that animals inoculated at earlier dates had significantly lower antibody titer at pelting. Pairwise comparison of the inoculation dates showed that the odds of mink at each inoculation dates falling into the category of low antibody titer were lesser than those inoculated on subsequent dates, and the differences were often significant when inoculation dates were farther apart.Every inoculation date had significantly less likelihoods of falling into the categories of low antibody titer compared with those inoculated in September 2013 (reference group). The odds of inoculation dates being in the category of low antibody titer ranged between 0.147 and 0.444 times the odds of mink inoculated in September 2013 being in this category (2(antibody titer) for each month delay in sampling was 0.0806 ± 0.0061 (ϰ2(1 df) = 178.1, P < 0.001, odds ratio = 1.084), indicating an 8.4% increase in the odds of animals falling into the category of lower antibody titer for each month delay in sampling, i.e. a significant decreasing trend in antibody titer over the course of the study.Females had a significantly lower likelihood of falling into the category of low antibody titer than males. The odds of females being in the category of low antibody titer was 0.629 times the odds of males being in the category of low antibody titer, i.e., females had significantly higher antibody titer than males. The estimated change in log odds for log2(antibody titer) and seropositive and viremic mink at pelting were 0.37 and 0.32, respectively . Spearman’s rank correlation between log2(antibody titer) and number of PCR positive organs was 0.52 , and those with PCR-positive individual organs ranged from 0.33 for the spleen to 0.41 for the liver . The mean of antibody titer in mink with seven PCR-positive organs was eight times greater than that in mink with seven PCR-negative organs (245.6 vs 29.3), and the median was 32 times greater (256 vs 8).A large proportion (89.5%) of those 57 mink with no detectable antibody titer were nonviremic at pelting. Of the 1160 mink with detectable antibody titers, 84.1% were nonviremic at pelting. Spearman’s rank correlation coefficients between logMink in the current study were housed in a virus contaminated environment and some had become naturally infected by the time of inoculation between five and seven months of age. Large differences in inoculation dates for viremia (0.8 to 39.4%) and seroconversion (4.8 to 64.7%) agree with previous reports for mink herds naturally exposed to the virus , 20, 34,Although AMDV replication peaks around 10 dpi , it takeA decline in the incidence of viremia and the persistently high incidence of seropositivity over time in this and other studies , 41–43 aOne of the notable findings of this study was that most (70.9%) of the 1001 mink that survived beyond 350 dpi were persistently nonviremic for at least 150 days prior to termination, and 32 were continuously nonviremic for over 1000 days  mink which were persistently nonviremic from the time of inoculation were sporadically or persistently seropositive, of which all seven organs of 70 mink were PCR-negative,158 had between 3 and 6 PCR-negative organs, and none had fewer than 3 PCR-negative organs (26 mink had no organ PCR data). These animals were thus infected but had low rates of viral replication and sequestration which was not detectable in their blood and in some organs, but was sufficient to trigger antibody production. Data on antibody titer were available for 236 of these mink, of which 4 and 3 mink had titers of 256 and 128, respectively, and titers in the others were 64 and lower. These results provided evidence that persistently nonviremic mink were healthy and productive, of which a small number were possibly resistant but mostly were infected but either cleared the virus or had low rates of viral replication and sequestration, concomitant with low antibody titer.The findings that of the 943 mink which survived for at least 350 dpi and were persistently seropositive for at least 150 dpi, 101 lived for at least 1000 dpi, 32 of the 33 mink which remained in the herd until 1211 dpi were persistently seropositive, and only one persistently seronegative mink remained in the herd after 470 dpi  were interesting because they likely did not harbor the virus at termination or had very low levels of sequestered viruses. The rather high percentage of mink with seven PCR-negative organs implies that this was not a rare occurrence in inoculated black mink which remained healthy and productive in an AMDV-contaminated environment. Data on viremia and seroconversion over time in mink with seven PCR-negative organs allowed for an exploration of the relationship between these parameters, which has not previously been investigated in any detail. As stated above, three of the 193 mink with seven PCR-negative organs were persistently seronegative from the time of inoculation but were viremic during the early periods after inoculation, and thus likely cleared the virus. The other 190 mink were persistently or sporadically seropositive, of which 70 were persistently nonviremic and 120 had short-lived viremia. These results support the previous suggestion that persistent antibody production reduces viral replication in macrophages, and that the CIEP test does not accurately determine the presence of AMDV DNA in organs.The observation that 65.2% of 471 persistently nonviremic mink with organ PCR data had detectable viral DNA in at least one of their organs, denotes that negative blood PCR test results, even when repeated over a long period of time, are not proof that mink are free of sequestrated viruses. This result is consistent with previous reports that some mink which harbored the virus in their organs were nonviremic , 40, 41,Anti-AMDV antibody titer was measured by CIEP using 11 two-fold serially diluted plasma samples, a method that has been used extensively on mink , 49, 57)Detailed inspection of the data showed that antibody titer of the 33 mink kept for 1211 dpi had a mean of 57.8, a median of 32 and a range between 1 and 512, which support the notion that mink with moderate antibody titers could tolerate the infection and remain healthy and productive for a considerable length of time. It was previously observed that some AMDV inoculated mink with high antibody titers lived for over 5 years with no histopathological sign of AD . The meaA unique feature of this study was the use of mink with different degrees of tolerance to AMDV infection. One novel finding was that mink with high percentage of tolerant ancestry had significantly lower incidence of viremia over time, terminal viremia, persistently seropositivity, PCR-positive organs, and lower antibody titer at termination, and had significantly greater incidence of being persistently nonviremic and having seven PCR-negative organs compared to susceptible mink. It is tempting to speculate that selection for tolerance to AMDV infection was manifested as a significantly greater ability to subdue viral replication and thus lower antibody titer, and as a higher ability to clear the virus  or reducThere are no published reports on the effect of sex on the traits measured in this study. It may be speculated that pregnancy and lactation cause higher stress on females, which could have led to the lower ability of females to suppress virus replication, causing a significantly higher incidence of persistent seropositivity, a higher antibody titer, and a lower incidence of being persistently nonviremic compared to males. The significantly higher incidence of seven PCR-negative organs in females could be attributed to the greater ability of females to prevent virus sequestration, perhaps because such females were healthy and had higher reproductive performance and were thus kept longer in the herd. The significant interactions between sex and tolerant ancestry for the incidence of viremia over time and terminal viremia resulted from significantly greater estimates of these parameters for TG25 males compared to females Figs.  and 2, wThe significantly lower incidence of viremia over time and the higher proportion of persistently nonviremic pelted mink compared to those that died imply that animals which were viremic or showed transient viremia had a higher chance of dying than persistently nonviremic animals. These findings support the above conclusion that sustained viral replication and viremia have a negative effect on animal health and survival rate. On the contrary, the significantly greater incidence of seropositivity over time and higher incidence of persistent seropositivity in pelted rather than dead mink provide supporting evidence for the positive effects of antibody production on health and survival rates.In view of the fact that the selection of replacement adults and kits was based entirely on health and reproductive performance with no consideration given to viremia or antibody production, the significant decline in the incidence of viremia over time, terminal viremia, and PCR-positive organs, and the significant increase in the incidence of seropositivity over time, persistently nonviremic mink, and seven PCR-negative organs suggests that nonviremic and seropositive mink with low levels of sequestered viruses in their organs, had higher survival rates and remained healthy with higher reproductive performance for a longer time compared with seronegative and viremic mink. These results concur with previous reports that the amounts of AMDV DNA in the organs of infected mink declined over time , 42, 58.The decreasing trend in antibody titer over time is supported by previous findings , 43, 50,The effect of inoculation date was significant on every trait analyzed, but did not follow an interpretable pattern, nor was the ranking of the means comparable among traits. The effect of inoculation date on different measurements could partly be related to the life history of animals maintained in a contaminated environment for different lengths of time and selected for health and reproduction. The duration of time between inoculation and sampling was another notable factor, although it was largely taken care of by the regression of duration of time between inoculation and sampling on each trait.The results of this study have implications for the two current approaches to the control of AD: virus eradication and selection for tolerance. For both approaches, there need to be awareness that seroconversion and viremia change in opposite direction over time, and that the relationship between antibody titer, virus replication rate and mink health and productivity is complex. False negative CIEP test results could increase under the continued retention of seronegative mink on infected farms and thereby lead to an increased likelihood of the failure of the test-and-removal strategy. Short-lived viremia and the presence of the virus in the organs of nonviremic mink (80.2%) suggest that blood testing by PCR alone is also not an accurate method for detecting AMDV infection in chronically infected herds. Performing both tests , or perfIt is thus logical to suggest that selection for tolerance is a more logical strategy for combating AD than virus eradication. Resistance is the ability of an animal to limit the pathogen load whereas tolerance is the ability to limit the damage caused by such a load , 62, i.eAlthough a tolerant population developed in a chronically infected herd over the course of more than 20 years using the iodine agglutination test (IAT) , the proNeovison vison) from a farm at Cape Breton, Nova Scotia, Canada (MM farm), which have been selected for tolerance to AMDV for more than 20 years, using IAT [Black American mink . ADRC is an enclosed bio-secure facility designed to minimize the chance of pathogen introduction or escape. The facility contained 320 breeder cages and 1280 grower cages, clean and dirty entrance/exit areas, a shower facility, an office and a laboratory. Strict biosecurity protocols, including disinfecting footbaths, wearing coveralls and shoe covers were implemented. A feed storage was located outside the building, with a small opening through which feed was transferred to a storage bin inside the building. Mink cadavers, feces and other solid wastes were incinerated, ashes were stored in plastic barrels, sealed and disposed of at a location approved by the Nova Scotia Department of Environment. Liquid waste moved through a certified system where it was treated, thus providing a high level of viral containment. The inside of the building was under video surveillance, and inside temperature and humidity were continuously recorded and digitally stored .The mink were kept at the Aleutian Disease Research Centre (ADRC), and managed according to standard industry practices . The diet of the purchased mink was gradually changed from the wet feed used on the farms of origin to dry pellet. The pellet’s nutritional composition changed based on the production cycles of the mink (https://www.nap.edu/read/1114/). Animals had free access to feed, which was added to feeders as needed. The water was supplied through nipple drinkers connected to waterlines that were heated in winter. Water came from an on-site well, and was eradiated with UV light. Animals were monitored daily and date and signs of death and sickness, such as diarrhea, reduced feed intake or reduced movement, were recorded.Mink were kept in wire-meshed cages measuring 61.0 × 30.5 × 45.7 cm  with an inside wooden nest box for growers, and breeder cages were 76.2 × 30.5 X 45.7 cm with a 30.5 × 30.5 cm outside wooden nest box. Cages were separated by solid plastic sheets with anti-tail biter extension to prevent direct physical contact. Aspen shavings were provided for nest building. Animals were not vaccinated because of the tight biosecurity system at the ADRC. Feed was a commercial dry pellet  and time of sampling (35 or 56 dpi) [A total of 1742 mink were inoculated between October 2010 and September 2013 (Table  56 dpi) .Selection of replacement animals was solely based on their reproductive performance and health status without considering their serological test results. Selected adult females were those ranked the highest for the number of kits weaned, and males were those which impregnated a high proportion of females with which they mated. Male and female kits were selected from litters of greater than five at weaning. Adults and kits in poor conditions, with physical defects, evidence of tail-chewing or erratic behaviour (nervousness) were eliminated. Extra animals and those not suitable for breeding were pelted during January and February from 2011 to 2014, inclusive. Each male mated with three to five females in a single-sire mating scheme. The experiment was terminated and most of the remaining animals were pelted during November and December 2014.Blood samples were collected by toenail clipping under anesthesia prior to inoculation (day 0) and at approximately 35 and 56 dpi. Up to 11 additional blood samples were collected from mink retained in the herd until 1211 dpi . Lymph nodes were stripped of fat before transferring to cryovials. Bone marrow was flushed out of the tibia with 0.5 ml of phosphate buffered saline (PBS). Plasma and tissue samples were stored at -80 °C until use.At pelting time, blood was collected into 4 mL plain vacutainer tubes by heart puncture from anesthetized animals. Some blood was transferred into capillary tubes and processed as explained above for CIEP and PCR tests. Animals were then euthanized while under anesthesia by intracardiac injection of sodium pentobarbital  at the dose of 100 mg per kg body weight or in a COThe CIEP test  was perfhttp://www.klentaq.com/) as previously described [Cell-free suspensions were prepared from 0.25 g of each of the seven organs in 750 μl of sterile PBS and centrifuged at 16,000 RCF (Eppendorf 5415C) for 10 minutes. In 2010, 2011 and 2012, DNA was extracted from approximately100 μL of plasma and from 200 μL of cell-free tissue supernatant, using Dynabeads Silane viral nucleic acid extraction kits according to the manufacturer’s protocol , and eluted in 100 μL elution buffer. AMDV DNA was amplified by PCR using three volumes  of plasma or cell-free tissue homogenate in 15 μL PCR reaction and primers 60F/60R as previously described . In 2013escribed , using tData were analyzed using SAS, Version 9.4 for Windows . The incidences of PCR- and CIEP-positive plasma over time were analyzed using a Generalized Estimating Equation (GEE) algorithm in the GENMOD procedure with a binomial distribution and the logit link function. The model included the fixed effects of tolerant groups, inoculation dates, disposal method  and sex. In this and other analyses, all two-way interactions were included in the models, except the interaction between tolerant groups and inoculation dates because of no observation in several subclasses. Non-significant interactions were removed from the final models, and only significant interactions are reported. Number of days pi at the time of samplings were converted to months and used as a covariate in all analyses. The random effect of individual mink was used in the REPEATED statement to take care of the correlations between the same measurements (viremia or seroconversion) within each mink. The appropriate correlation structures were determined after fitting four models with different correlation structures, namely independent (IND), unstructured (UN), first-order autoregressive (AR-1) and exchangeable (CS) and the model with the smallest QIC value was selected, which was obtained by the IND for all traits analyzed. The same model was used to compare the incidence of PCR-positive organs, which included the fixed effects of organs, tolerant groups, inoculation dates and sex. The random effect of individual mink was used in the REPEATED statement to take care of the correlations in the PCR status of organs of the same mink.The incidences of positive plasma PCR and CIEP of the pelted mink  were analyzed by the GENMOD procedure with a binomial distribution, the logit link function and independent correlation structure. The model included the fixed effects of tolerant groups, inoculation dates and sex. Number of months from inoculation to pelting was used as a covariate. Numbers of mink with seven PCR-negative organs were compared with those with at least one PCR-positive organ using the same mode explained above. Data on mink which survived for at least 350 dpi and were persistently nonviremic or persistently seropositive for at least 150 days prior to termination were analyzed using the GENMOD procedure. The model included the fixed effects of tolerant groups, inoculation dates, sex and disposal methods. In every analysis mentioned above, the least-squares means and their standard errors were converted to the original scales by the ilink option, and multiple comparisons of means were performed using Tukey’s adjustment.2(CIEP) + 1 if CIEP> 0 and 0 if CIEP = 0 prior to analyses and were checked for normality using the Shapiro-Wilk test implemented in the UNIVARIATE procedure. The transformed antibody titer  was analyzed by the GENMOD procedure with a multinomial distribution and the cumulative logit link function. The models included the fixed effects of the tolerant groups, inoculation dates, sex, and regression of sampling age (month pi). TG100, September 2013 and males were used as the reference for other classes within each parameter. Lowest order values were antibody titer of 0. Pairwise comparisons were made using the ESTIMATE statement. The odds ratios and their standard errors are reported.Antibody titers were transformed to logAdditional file 1: Supplementary Table 1. Percentage of viremic mink at each sampling occasion by inoculation date.Additional file 2: Supplementary Table 2. The distribution of mink which survived for at least 350 days post-inoculation and were persistently nonviremic or persistently viremic for at least 150 days by the start and termination dates.Additional file 3: Supplementary Table 3. Percentage of seropositive mink at different sampling occasions by inoculation date§.Additional file 4: Supplementary Table 4. The distribution of mink which survived for at least 350 days and were persistently seropositive or persistently seronegative for at least 150 days until pelting.


Mindfulness, defined as nonjudgmental awareness of the present moment, has been associated with an array of mental and physical health benefits. Mindfulness may also represent a protective factor for Alzheimer’s disease (AD). Here, we tested the potential protective effect of trait mindfulness on cognitive decline and AD pathology in older adults at risk for AD dementia.Measures of trait mindfulness, longitudinal cognitive assessments, and amyloid-β (Aβ) and tau positron emission tomography scans were collected in 261 nondemented older adults with a family history of AD dementia from the PREVENT-AD  observational cohort study. Multivariate partial least squares analyses were used to examine relationships between combinations of different facets of trait mindfulness and 1) cognitive decline, 2) Aβ, and 3) tau.Higher levels of mindful nonjudgment, describing, and nonreactivity were associated with less cognitive decline in attention, global cognition, and immediate and delayed memory. Higher levels of mindful nonjudgment and nonreactivity were related to less Aβ positron emission tomography signal in bilateral medial and lateral temporoparietal and frontal regions. Higher levels of mindful acting with awareness, describing, nonjudgment, and nonreactivity were associated with less tau positron emission tomography signal in bilateral medial and lateral temporal regions.Trait mindfulness was associated with less cognitive decline and less Aβ and tau in the brain in older adults at risk for AD dementia. Longitudinal studies examining the temporal relationship between trait mindfulness and AD markers, along with mindfulness intervention studies, will be important for further clarifying the potential protective benefits of mindfulness on AD risk. Mindfulness is defined as the ability to engage in nonjudgmental awareness of the present moment . GrowingIn the absence of an imminent treatment or cure for AD dementia, increasing focus is being placed on early prevention. Encouragingly, up to 40% of the risk for dementia is attributed to modifiable behavioral factors . It is eLeveraging a longitudinal cohort sample, we tested the potential protective effect of trait mindfulness on cognitive decline in older adults at risk of AD dementia. We also assessed the association between trait mindfulness and AD pathology, with the hypothesis that mindfulness could reduce dementia risk by providing resistance against AD pathology . That isN = 261), with 124 of these individuals also completing Aβ and tau PET scans. Further details of the relative timing of measures are contained within A total of 261 participants were included in this study from the PREVENT-AD  cohort. This ongoing longitudinal observational study enrolls older adults who are cognitively normal but have a parent or at least 2 siblings diagnosed with AD-like dementia, placing them at increased risk for sporadic AD . Other eN = 261). The questionnaire was administered via an online platform for most participants, though 10 individuals chose to receive a paper copy in the post and a further 16 participants completed it over the telephone. The FFMQ contains five subscales measuring different facets of trait mindfulness: observing , describing , acting with awareness , nonjudgment (not judging one’s own experiences), and nonreactivity . Participants were instructed to rate each item based on the extent to which the statements applied to them, on a Likert scale of 1 (never or very rarely true) to 5 . Scores were calculated for each mindfulness subscale, with higher scores reflecting higher levels of mindfulness. The nonreactivity subscale consists of seven items, whereas the remaining subscales consist of eight items. Scores for nonreactivity therefore ranged from 7 to 35, while the remaining subscale scores ranged from 8 to 40. Missing values were replaced with the mean of the subject’s responses for each subscale. Three participants with >10% of missing values were excluded from analyses. The FFMQ has demonstrated reliability and validity in clinical as well as in nonclinical populations (The 39-item self-report Five Facet Mindfulness Questionnaire (FFMQ)  was collulations .As part of the PREVENT-AD study, participants underwent annual cognitive testing using the RBANS. The RBANS comprises 12 cognitive subtests, which produce five cognitive domain index scores , along with a total score reflective of global cognition. Scores are standardized to a mean score of 100 with an SD of 15. Longitudinal cognitive assessment (≥2 time points) was available for 257 (98.5%) participants, with a median follow-up time of 5 years . The FFMQ was collected a mean of 1429.29 days  after the baseline cognitive assessment (SD = 620.97 days). For further details of the closest cognitive time point to the FFMQ for each participant, see the n = 124) underwent PET imaging using [18F]NAV4694 to assess Aβ burden and [18F]AV1451 (flortaucipir) for tau deposition. Aβ and tau PET scans were preprocessed using a standard pipeline (https://github.com/villeneuvelab/vlpp) (see A subset of participants (lpp) see . Standarng using FNAV4694 lpp) see . SUVRs flpp) see . For taulpp) see . This inlpp) see .z-scored due to the differing ranges of each subscale. The second matrix contained separate columns for each of the dependent variables, namely each of the six cognitive change scores for PLS 1  longitudinal cognition, 2) Aβ, and 3) tau, three separate multivariate partial least squares (PLS) analyses were employed  using PLp < .05). Then, for each significant latent variable, bootstrap resampling was employed to ascertain the most stable mindfulness subscales and cognitive scores/brain regions contributing to the multivariate relationship. For the mindfulness subscales, the SE of the resampled distribution was calculated. The mindfulness facets for which the SE values did not overlap with zero were considered as significantly contributing to the multivariate relationship with the dependent variable. A bootstrap ratio was produced for each of the dependent variables. Cognitive scores/brain regions with bootstrap values >1.96 were considered as significant contributors to the multivariate relationship with mindfulness. For Aβ and tau PET PLS analyses, bootstrap ratios for each of the Desikan-Killiany regions of interest were projected on 2-dimensional brain representations using the ggseg package v1.6.01 (APOE status (ɛ4 carrier vs. noncarrier), and number of days between completion of the FFMQ and the baseline cognitive assessment/PET scans.The PLS analyses produced sets of latent variables relating the mindfulness facets with each dependent variable . Each latent variable consisted of a singular value, a vector of weights for each mindfulness facet, and a vector of weights for each dependent variable. Permutation tests were used to identify the significant latent variables . No differences in trait mindfulness were apparent between the APOE ɛ4 carriers versus noncarriers in either the full sample or the PET subsample .Participant characteristics are displayed in p < .001, p = .004), explaining 68.03% and 20.32% of the PLS variance, respectively. We report the first latent variable here, given that it explained the majority of the PLS variance and was replicated in the subset of 124 participants who completed Aβ and tau PET scans (p < .001), which accounted for 5.4% of the variance in cognitive change. This relationship between mindfulness and longitudinal cognitive decline remained significant when covariates  were included in the model . The main results from this PLS analysis were also replicated when participants who had MCI at the time of the FFMQ were removed from analyses, with the exception of the immediate and delayed memory scores, which did not reach statistical significance , which explained 97.72% of the PLS variance. Loadings and SEs for the mindfulness facets and bootstrap ratios for the Aβ regional SUVRs that comprise this latent variable are displayed in p = .005), accounting for 5.4% of the variance in Aβ. The relationship between mindfulness and Aβ remained significant when covariates  were included in the model . When participants with MCI at the time of the FFMQ were removed, PLS results remained predominantly similar, though the mindful nonreactivity subscale was no longer significant , explaining 92.58% of the PLS variance. p = .003), accounting for 6.1% of the tau variance. This relationship between mindfulness and tau remained significant when covariates  were included in the model . Results of this PLS analysis remained unchanged when participants with MCI at the time of the FFMQ were excluded  of cognitively unimpaired participants. Mindfulness has previously been associated with better cognitive performance in older adults, particularly in domains of attention and executive function (In older adults at risk of AD dementia, higher levels of trait mindful nonjudgment, describing, and nonreactivity were related to less cognitive decline in global cognition, attention, and immediate and delayed memory over an average of 5 years. The associations between higher levels of mindfulness and less decline in global cognition and attention were also apparent in the subset (function . By defifunction . Our finfunction . FurtherIn order to explore whether mindfulness could potentially influence dementia risk via providing resistance against AD pathology, we also examined associations between trait mindfulness and Aβ and tau deposition in the brain. In support of this hypothesis, higher levels of mindful nonjudgment and nonreactivity were related to less Aβ in the bilateral medial and lateral temporoparietal and frontal regions, including the precuneus, posterior cingulate cortex, and orbitofrontal cortex. These regions are among the first cortical areas to accumulate Aβ pathology early in the disease course ,29,30. IHigher levels of mindful acting with awareness, describing, nonjudgment, and nonreactivity were also related to less tau in regions known to accumulate pathology early in the course of AD, namely the entorhinal cortex; amygdala; and parahippocampal, inferior temporal, and fusiform gyri. Medial temporal lobe regions are believed to play important roles in mindfulness, with both the trait and formal practice of mindfulness associated with gray matter volume of the amygdala  and hippWhile distinct combinations of mindfulness traits were associated with cognitive decline, Aβ, and tau, mindful nonjudgment was the only subscale related to all three markers. The associations between mindful nonjudgment, cognition, and AD pathology were highly robust: remaining significant even with the exclusion of participants with MCI and the inclusion of other psychological variables in the analyses. Broadly, the construct of mindfulness is considered to incorporate both the attention to present-moment experiences and the attitude of acceptance toward these experiences . The minPreliminary work has begun to explore the effect of mindfulness-based interventions in MCI and early-stage AD dementia. The evidence is mixed regarding their effect on cognition, however, with one study reporting significant cognitive improvements in participants with MCI following an 8-week mindfulness intervention , though There are numerous potential psychological, behavioral, and physiological mechanisms by which mindfulness may be associated with AD pathology and cognitive decline. For example, positive impacts of mindfulness on reducing depression, anxiety, and stress  and modiOther mechanisms are therefore likely involved in the relationships between mindfulness and AD markers. Mindfulness has been associated with an array of physical health benefits, involving similar pathways that are proposed to be involved in AD. Poor cardiovascular health is one of the most significant categories of risk factors for the development of AD pathology and its associated dementia syndrome ,59,60. MOverall, higher levels of trait mindfulness were associated with better cognition over time and less Aβ and tau in the brain in older adults at risk for AD dementia. Our multivariate approach permitted in-depth profiling of associations between specific combinations of mindfulness facets and AD markers, though such relationships were also apparent using standard univariate analyses see . Albeit