Variations in healthcare costs by body mass index and obesity-related complications in a UK population: A retrospective open cohort study.

AIMS: To estimate healthcare resource utilization (HCRU) and healthcare costs by body mass index (BMI) in a UK cohort and to explore how this varied by defined BMI strata. MATERIALS AND METHODS: This retrospective open cohort study used Discover, a linked primary and secondary electronic health records database covering 2.7 million individuals. Adults were stratified by BMI as: overweight (25-<30 kg/m2); obesity class I (30-<35 kg/m2); obesity class II (35-<40 kg/m2); or obesity class III (≥40 kg/m2). Cost data, comprising primary care, secondary care (inpatient admissions, outpatient appointments and emergency room visits) and prescriptions, were reported for 2015-2019. RESULTS: Overall, 1 008 101 individuals were overweight, 278 782 had obesity class I; 80 621 had obesity class II, and 42 642 had obesity class III. Healthcare costs and HCRU events per person per year increased over time (2015: £851-£1321 and 10.6-13.4 events; 2019: £1143-£1871 and 11.4-14.9 events), and were higher for each successive BMI group. Groups with chronic kidney disease or cardiovascular disease incurred particularly high costs. In 270 493 individuals with obesity in 2019, more than 72% of total healthcare costs were incurred by the highest cost quintile, which had a higher mean age and more obesity-related complications (ORCs) than lower cost quintiles. CONCLUSIONS: The economic impact of obesity could be alleviated by weight management support based on unmet need, to limit the effects of BMI progression and ORC development.

Ten-year progression of obesity-related complications in a population with overweight and obesity in the UK: A retrospective open cohort study.

AIM: To assess the prevalence of individual obesity-related complications (ORCs) and multimorbidity (≥ 1, ≥ 2 and ≥ 3 ORCs), and multimorbidity-associated healthcare costs, over 10 years. METHODS: This retrospective open cohort study used Discover, a UK database of linked primary and secondary electronic health records. Adults were stratified by body mass index (BMI; overweight: 25-< 30 kg/m2; obesity class I: 30-< 35 kg/m2; obesity class II: 35-< 40 kg/m2; obesity class III: ≥ 40 kg/m2). Outcomes by year since baseline were assessed for serial cross sections across the study period (1 January 2004 to 31 December 2019; the index date was the date of first eligible BMI measurement). RESULTS: Across 1 410 146 individuals (overweight: 1 008 101; obesity class I: 278 782; obesity class II: 80 621; obesity class III: 42 642), ORC prevalence was higher in successive BMI groups, and increases over time were generally greater for obesity relative to overweight. In those with ORC multimorbidity, both higher BMI and the presence of more ORCs were associated with higher annual per-person healthcare costs. Costs increased over time in those individuals with obesity and one or more ORC, as well as in those with obesity and two or more ORCs. CONCLUSIONS: Higher BMI was associated with higher baseline ORC prevalence and a greater increase in ORC prevalence over time, and with higher healthcare costs in those with multimorbidity. To reduce the burden of overweight and obesity on patients and healthcare systems, the presence, number and type of ORCs should be considered in developing effective, targeted prevention and management care pathways.

Diversity, Leishmania detection, and blood meal sources of sand flies from Iguatama, Minas Gerais, Brazil.

This study investigated the sand fly fauna of the municipality Iguatama, in the Midwest Region of Minas Gerais state, Brazil, including Leishmania infection rates and blood meal sources. Sand flies were collected during four periods over the course of a single year, encompassing both dry and rainy seasons, using CDC light traps placed in peridomiciles where dogs were seropositive for visceral leishmaniasis (VL). A total of 762 sand fly specimens, representing 12 species across seven genera, were collected. Lutzomyia longipalpis was the most abundant species, comprising 57.6% of the collected specimens, followed by Nyssomyia neivai (19.6%) and Nyssomyia whitmani (10.5%). Species richness and diversity varied among collection periods, with the highest diversity observed in January 2019. Molecular analysis detected Leishmania DNA in 12.5% of the sand fly specimens, with Le. infantum being the predominant species. Blood meal analysis revealed feeding on multiple vertebrate species, including humans, rats, dogs, and chickens. The presence of Leishmania DNA in sand flies, and the identification of human blood meals, highlight the potential role of these species in VL transmission. These findings underscore the importance of continued surveillance and control measures to prevent the spread of VL and reduce transmission risk in the region.

Epigenetic role of LINE-1 methylation and key genes in pregnancy maintenance.

Spontaneous abortion is a pregnancy complication characterized by complex and multifactorial etiology. About 5% of childbearing women are globally affected by early pregnancy loss (EPL) and most of them experience recurrence (RPL). Epigenetic mechanisms and controlled inflammation are crucial for pregnancy maintenance and genetic predispositions may increase the risk affecting the maternal-fetal crosstalk. Combined analyses of global methylation, inflammation and inherited predispositions may contribute to define pregnancy loss etiopathogenesis. LINE-1 epigenetic regulation plays crucial roles during embryo implantation, and its hypomethylation has been associated with senescence and several complex diseases. By analysing a group of 230 women who have gone through pregnancy interruption and comparing those experiencing spontaneous EPL (n = 123; RPL, 54.5%) with a group of normal pregnant who underwent to voluntary interruption (VPI, n = 107), the single statistical analysis revealed significant lower (P < 0.00001) LINE-1 methylation and higher (P < 0.0001) mean cytokine levels (CKs: IL6, IL10, IL17A, IL23) in EPL. Genotyping of the following SNPs accounted for different EPL/RPL risk odds ratio: F13A1 rs5985 (OR = 0.24; 0.06-0.90); F13B rs6003 (OR = 0.23; 0.047-1.1); FGA rs6050 (OR = 0.58; 0.33-1.0); CRP rs2808635/rs876538 (OR = 0.15; 0.014-0.81); ABO rs657152 (OR = 0.48; 0.22-1.08); TP53 rs1042522 (OR = 0.54; 0.32-0.92); MTHFR rs1801133/rs1801131 (OR = 2.03; 1.2-3.47) and FGB rs1800790 (OR = 1.97; 1.01-3.87), although Bonferroni correction did not reach significant outputs. Principal Component Analysis (PCA) and logistic regression disclosed further SNPs positive/negative associations (e.g. APOE rs7412/rs429358; FGB rs1800790; CFH rs1061170) differently arranged and sorted in four significant PCs: PC1 (F13A, methylation, CKs); PC3 (CRP, MTHFR, age, methylation); PC4 (F13B, FGA, FGB, APOE, TP53, age, methylation); PC6 (F13A, CFH, ABO, MTHFR, TP53, age), yielding further statistical power to the association models. In detail, positive EPL risk association was with PC1 (OR = 1.81; 1.33-2.45; P < 0.0001) and negative associations with PC3 (OR = 0.489; 0.37-0.66; P < 0.0001); PC4 (OR = 0.72; 0.55-0.94; P = 0.018) and PC6 (OR = 0.61; 0.46-0.81; P = 0.001). Moreover, significant inverse associations were detected between methylation and CKs levels in the whole group (rIL10 = - 0.22; rIL17A = - 0.25; rIL23 = - 0.19; rIL6 = - 0.22), and methylation with age in the whole group, EPL and RPL subgroups (r2TOT = 0.147; r2EPL = 0.136; r2 RPL = 0.248), while VPI controls lost significance (r2VPI = 0.011). This study provides a valuable multilayer approach for investigating epigenetic abnormalities in pregnancy loss suggesting genetic-driven dysregulations and anomalous epigenetic mechanisms potentially mediated by LINE-1 hypomethylation. Women with unexplained EPL might benefit of such investigations, providing new insights for predicting the pregnancy outcome and for treating at risk women with novel targeted epidrugs.

Recombination between non-structural and structural genes as a mechanism of selection in lagoviruses: The evolutionary dead-end of an RHDV2 isolated from European hare.

The genus Lagovirus, belonging to the family Caliciviridae, emerged around the 1980s. It includes highly pathogenic species, rabbit hemorrhagic disease virus (RHDV/GI.1) and European brown hare syndrome virus (EBHSV/GII.1), which cause fatal hepatitis, and nonpathogenic viruses with enteric tropism, rabbit calicivirus (RCV/GI.3,4) and hare calicivirus (HaCV/GII.2). Lagoviruses have evolved along two independent genetic lineages: GI (RHDV and RCV) in rabbits and GII (EBHSV and HaCV) in hares. To be emphasized is that genomes of lagoviruses, like other caliciviruses, are highly conserved at RdRp-VP60 junctions, favoring intergenotypic recombination events at this point. The recombination between an RCV (genotype GI.3), donor of non-structural (NS) genes, and an unknown virus, donor of structural (S) genes, likely led to the emergence of a new lagovirus in the European rabbit, called RHDV type 2 (GI.2), identified in Europe in 2010. New RHDV2 intergenotypic recombinants isolated in rabbits in Europe and Australia originated from similar events between RHDV2 (GI.2) and RHDV (GI.1) or RCV (GI.3,4). RHDV2 (GI.2) rapidly spread worldwide, replacing RHDV and showing several lagomorph species as secondary hosts. The recombination events in RHDV2 viruses have led to a number of viruses with very different combinations of NS and S genes. Recombinant RHDV2 with NS genes from hare lineage (GII) was recently identified in the European hare. This study investigated the first RHDV2 (GI.2) identified in Italy in European hare (RHDV2_Bg12), demonstrating that it was a new virus that originated from the recombination between RHDV2, as an S-gene donor and a hare lagovirus, not yet identified but presumably nonpathogenic, as an NS gene donor. When rabbits were inoculated with RHDV2_Bg12, neither deaths nor seroconversions were recorded, demonstrating that RHDV2_Bg12 cannot infect the rabbit. Furthermore, despite intensive and continuous field surveillance, RHDV2_Bg12 has never again been identified in either hares or rabbits in Italy or elsewhere. This result showed that the host specificity of lagoviruses can depend not only on S genes, as expected until today, but potentially also on some species-specific NS gene sequences. Therefore, because RHDV2 (GI.2) infects several lagomorphs, which in turn probably harbor several specific nonpathogenic lagoviruses, the possibility of new speciation, especially in those other than rabbits, is real. RHDV2 Bg_12 demonstrated this, although the attempt apparently failed.

Pathomorphological Findings and Infectious Diseases in Selected European Brown Hare (Lepus europaeus Pallas, 1778) Populations from Schleswig-Holstein, Germany.

In the northernmost German federal state Schleswig-Holstein, populations of European brown hares (Lepus europaeus Pallas, 1778) show diverse densities and varying courses over the years. To examine differences in pathomorphological findings and infectious diseases as possible reasons for varying population dynamics, we assessed 155 hunted hares from three locations in Schleswig-Holstein from 2016 to 2020. We investigated the association of location, year, age, and sex of animals to certain pathomorphological findings and infectious diseases. Frequent pathomorphological findings were intestinal parasites (63.9%), hepatitis (55.5%), nephritis (31.0%), steatitis (23.2%), enteritis (13.5%), and pneumonia (5.2%). Body condition differed significantly between locations, and the prevalence of pneumonia was significantly higher in females. Enteritis was not detected in 2019, when much more juveniles were sampled. Hepatitis and nephritis occurred significantly more often in 2016 and among adults. Additionally, more adults showed hepatitis with concurrent serotitre for European brown hare syndrome virus (EBHSV), while intestinal parasitosis as well as high excretion rates of coccidia were more common in juveniles. Sampled animals showed high infection rates with Eimeria spp. (96.1%), Trichostrongylus spp. (52.0%), Graphidium strigosum (41.2%), and a high seroprevalence (90.9%) for EBHSV, without severe symptoms. This study revealed a low prevalence of infectious pathogens, but a high prevalence of chronic inflammations of unknown origin in the tested brown hare populations. Overall, our results indicate a rather minor importance of infectious diseases for observed population dynamics of analysed hare populations in Schleswig-Holstein.

Use of Oral Anticoagulants in Patients with Atrial Fibrillation: Preliminary Data from the Italian Atrial Fibrillation (ITALY-AF) Registry.

BACKGROUND: Atrial fibrillation (AFIB), the most frequent cardiac arrhythmia, is a major risk factor for stroke, heart failure, and death. Because of the recent advances in AFIB management and the availability of new oral anticoagulants (OACs), there is a need for a systematic and predefined collection of contemporary data regarding its management and treatment. METHODS: The objective of the ongoing ITALY-AFIB registry is to evaluate the long-term morbidity and mortality in patients with AFIB and to verify the implementation of the current guidelines for stroke prevention in these patients. The registry includes consecutive in- and out-patients with first diagnosed, paroxysmal, persistent, or permanent AFIB. In patients in sinus rhythm at entry, the qualifying episode of AFIB, confirmed by ECG diagnosis, had to have occurred within 1 year before entry. The clinical record form is web-based and accessible by personal keyword. RESULTS: Enrolment into the registry started in the year 2013. In a current cohort of 2470 patients (mean age 75 ± 11 years, males 56%), the mean CHA2DS2-VASc score was 3.7 ± 1.8, and the mean HAS-BLED was 1.6 ± 0.9. There were no significant sex differences in the AFIB subtypes. At the end of the inclusion visit and after receiving knowledge of the web-based electronic estimate of risk for stroke and bleeding, the proportion of patients discharged with OACs was 80%. After exclusion of patients with first diagnosed AFIB (n = 397), the proportion of patients with prescription of OACs rose from 66% before the visit to 82% on discharge (p < 0.0001). Prescription of aspirin or other antiplatelet drugs fell from 18% before the visit to 10% on discharge (p < 0.0001). CONCLUSIONS: A web-based management of AFIB with automated estimation of risk profiles appears to favorably affect adherence to AFIB guidelines, based on a high proportion of patients treated with OACs and a substantial decline in the use of antiplatelet drugs.

Correction: HIV-1-neutralizing antibody induced by simian adenovirus- and poxvirus MVA-vectored BG505 native-like envelope trimers.

[This corrects the article DOI: 10.1371/journal.pone.0181886.].

Real-world costs of obesity-related complications over eight years: a US retrospective cohort study in 28,500 individuals.

BACKGROUND: Obesity-related complications (ORCs) are associated with high costs for healthcare systems. We assessed the relationship between comorbidity burden, represented by both number and type of 14 specific ORCs, and total healthcare costs over time in people with obesity in the USA. METHODS: Adults (≥ 18 years old) identified from linked electronic medical records and administrative claims databases, with a body mass index measurement of 30-< 70 kg/m2 between 1 January 2007 and 31 March 2012 (earliest measurement: index date), and with continuous enrolment for ≥ 1 year pre index (baseline year) and ≥ 8 years post index, were included. Individuals were grouped by type and number of ORCs during the pre-index baseline year. The primary outcome was annual total adjusted direct per-person healthcare costs. RESULTS: Of 28,583 included individuals, 12,686 had no ORCs, 7242 had one ORC, 4180 had two ORCs and 4475 had three or more ORCs in the baseline year. Annual adjusted direct healthcare costs increased with the number of ORCs and over the 8-year follow-up. Outpatient costs were the greatest contributor to baseline annual direct costs, irrespective of the number of ORCs. For specific ORCs, costs generally increased gradually over the follow-up; the largest percentage increases from year 1 to year 8 were observed for chronic kidney disease (+ 78.8%) and type 2 diabetes (+ 47.8%). CONCLUSIONS: In a US real-world setting, the number of ORCs appears to be a cost driver in people with obesity, from the time of initial obesity classification and for at least the following 8 years.

Air pollution and out-of-hospital cardiac arrest risk: a 7-year study from a highly polluted area.

AIMS: Globally, nearly 20% of cardiovascular disease deaths were attributable to air pollution. Out-of-hospital cardiac arrest (OHCA) represents a major public health problem; therefore, the identification of novel OHCA triggers is of crucial relevance. The aim of the study was to evaluate the association between air pollution (short-, mid-, and long-term exposures) and OHCA risk, during a 7-year period in a highly polluted urban area in northern Italy, with a high density of automated external defibrillators (AEDs). METHODS AND RESULTS: Out-of-hospital cardiac arrests were prospectively collected from the 'Progetto Vita Database' between 1 January 2010 and 31 December 2017; day-by-day air pollution levels were extracted from the Environmental Protection Agency stations. Electrocardiograms of OHCA interventions were collected from the AED data cards. Day-by-day particulate matter (PM) 2.5 and 10, ozone (O3), carbon monoxide (CO), and nitrogen dioxide (NO2) levels were measured. A total of 880 OHCAs occurred in 748 days. A significant increase in OHCA risk with a progressive increase in PM2.5, PM10, CO, and NO2 levels was found. After adjustment for temperature and seasons, a 9% and 12% increase in OHCA risk for each 10 µg/m3 increase in PM10 (P < 0.0001) and PM2.5 (P < 0.0001) levels was found. Air pollutant levels were associated with both asystole and shockable rhythm risk, while no correlation was found with pulseless electrical activity. CONCLUSION: Short- and mid-term exposures to PM2.5 and PM10 are independently associated with the risk of OHCA due to asystole or shockable rhythm.

Association between weight loss and health care resource utilization in adults living with obesity: Evidence from a UK primary care database.

AIMS: We investigated the impact of intentional weight loss on health care resource utilization (HCRU) and costs among people with obesity. MATERIALS AND METHODS: This retrospective, observational cohort study used data from the Clinical Practice Research Datalink (CPRD) GOLD database. Adults >18 years at index date [first recorded body mass index (BMI) of 30-50 kg/m2 between 2006 and 2015 with a further BMI record 4 years later] were assigned to an intentional weight loss cohort (-25% to -10% BMI change) or a stable weight cohort (-3% to +3%), based on their BMI change during a 4-year baseline period from index date. Evidence of intention to lose weight during the baseline period was required. Linked Hospital Episode Statistics datasets captured HCRU and costs over an 8-year follow-up period. Mixed effects models adjusted for demographics, total costs during baseline and baseline comorbidities were used. RESULTS: Baseline characteristics were similar between cohorts with weight loss (n = 8676) and stable weight (n = 44 519). Over follow-up, the weight loss cohort experienced a significantly lower mean annual increase in total costs [2.1% (95% confidence interval: 1.3-2.8)] than the stable weight cohort [4.3% (95% confidence interval: 4.0-4.6); p < .0001]. Weight loss was associated with a lower mean annual increase in multiple HCRU and cost components compared with maintaining a stable high weight. CONCLUSIONS: Our findings suggest that intentional weight loss of 10-25% is associated with lower HCRU and costs in the long term among individuals living with obesity, relative to stable weight.

Anti-arrhythmic drugs in atrial fibrillation: tailor-made treatments.

During the last decades, many improvements have been made regarding the treatment of atrial fibrillation in terms of risk prevention, anti-coagulation strategies, and gain in quality of life. Among those, anti-arrhythmic drugs (AADs) have progressively fallen behind and overtaken by technological aspects as devices as procedures are now the standards of care for many patients. But is this it? Are AADs doomed to be relegated to an obscure and rarely read paragraph of the European recommendations? Or could they be still employed safely and effectively? In the present paper, we will discuss contemporary evidence in order to define where AADs still play a pivotal role, how should AADs be used, and whether a tailored approach can be the way to propose the right treatment to the right patient.

HeartInsight: from SELENE HF to implementation in clinical practice.

There is strong evidence that remote monitoring in cardiac implantable electronic devices can detect device malfunctions earlier than conventional monitoring and that it can be useful for detecting cardiac arrhythmias, while little data are available for an improved management of heart failure (HF). HeartInsight is a new remote monitoring algorithm developed and validated in the SELENE HF study that combines information from a diverse set of sensors integrated into one alert to detect worsening HF with promising accuracy. However, the shift from detecting technical issues or arrhythmia episodes to early predicting clinical events underscores the need to understand how to properly integrate these tools into the clinical workflow by defining an organizational model and shared guidelines for the management of HF alerts. Here, we critically discuss issues that will be raised by the implementation of this 'enhanced' remote monitoring approach to HF care in daily clinical practice.

Status of Infectious Diseases in Free-Ranging European Brown Hares (Lepus europaeus) Found Dead between 2017 and 2020 in Schleswig-Holstein, Germany.

The European brown hare (Lepus europaeus) is a quite adaptable species, but populations have been decreasing for several decades in different countries, including Germany. To investigate infectious diseases as possible influences on observed population decline in the German federal state Schleswig-Holstein, 118 deceased free-ranging European brown hares were collected between 2017 and 2020 and underwent detailed postmortem examination with extended sampling. Infectious diseases were a major cause of death (34.7%). The number of juveniles found exceeded the adult ones. The main pathomorphological findings were hepatitis (32.8%), pneumonia (22.2%), nephritis (19.1%), liver necrosis (12.9%), and enteritis (40.7%). An unusual main finding was steatitis (20.9%) of unknown origin. Animals were mainly emaciated and showed high infection rates with Eimeria spp. (91.3%) and Trichostrongylus spp. (36.2%). European Brown Hare Syndrome Virus reached an epidemic status with few fatal infections (4.2%) and high seroprevalence (64.9%), whereas the prevalence of Rabbit Haemorrhagic Disease Virus 2 was very low (0.8%) in hares in Schleswig-Holstein. Pathogens such as Yersinia pseudotuberculosis (5.9%), Pasteurella multocida (0.8%), and Staphylococcus aureus (3.4%) only caused sporadic deaths. This study illustrates the wide distribution of various infectious pathogens with high mortality and even zoonotic potential. Infectious diseases need to be considered as an important influence on population dynamics in Schleswig-Holstein.

Pathological changes and viral antigen distribution in tissues of Iberian hare (Lepus granatensis) naturally infected with the emerging recombinant myxoma virus (ha-MYXV).

BACKGROUND: A cross-species jump was confirmed in 2018, when a novel recombinant myxoma virus (MYXV) (ha-MYXV) caused high mortality in Iberian hare (Lepus granatensis) in the Iberian Peninsula. METHOD: The aim of this study was to evaluate the main lesions, tissular distribution and target cells of ha-MYXV in Iberian hare. Gross postmortem examinations and histological and immunohistochemical studies to detect ha-MYXV were carried out in 28 animals that were confirmed as ha-MYXV positive by PCR. RESULTS: The main macroscopic lesions were bilateral blepharoconjunctivitis, epistaxis, intense congestion and oedema in several organs and some internal haemorrhages. Visible myxomas were not found. Histopathological examination revealed hyperplastic epidermis with predominant hyperkeratosis and myxoid matrix in the dermis. ha-MYXV-positive keratinocytes showed hydropic degeneration and cytoplasmic inclusion bodies. Alveolar oedema, interstitial pneumonia, dramatic lymphoid depletion in the spleen and necrosis in the liver and testis were observed. ha-MYXV was mainly detected in epithelial and myxoma cells in the skin, and also in macrophages, lymphocytes, fibroblasts and endothelial cells in several organs, as well as in hepatocytes and Leydig cells. LIMITATIONS: A non-homogeneous number of samples were included in all the animals. Future experimental studies with controlled variables are necessary. CONCLUSION: These findings correspond to an unusual form of myxomatosis, characterised by an acute or hyperacute presentation.

Ecology and natural infection of phlebotomine sand flies in different ecotopes and environments in the municipality of Pains, Minas Gerais, Brazil.

Phlebotomines (Diptera: Psychodidae) are vectors of protozoa of the genus Leishmania and distributed throughout Brazil, formerly restricted to rural areas, have expanded including to periurban and urban regions, been recorded in different habitats. This study aimed to understand the dynamics of sand flies in different ecotopes in the municipality of Pains. Sand flies were captured during thirty samplings using HP light traps installed in seven different ecotopes for two consecutive nights, once a month, from August 2018 to July 2019. A total of 1,352 sand flies were captured, representing 24 species belong to ten genera. Evandromyia edwardsi was the most abundant species, followed by Evandromyia lenti and Micropygomyia quinquefer. Leishmania DNA was detected in seven female sand flies in four ecotopes, for an infection rate of 0.9%. Sand flies were collected in all seven ecotopes, although forest (23.04%), cave (20.88%) and pasture (17.75%) had higher abundance and richness. Similarity was found among ecotopes, indicating that they are all important for the maintenance of the sand fly community. Spatial analysis indicated high densities of sand flies in areas with natural characteristics. All ecotopes in the municipality were evidenced to have an adequate and harmonious epidemiological profile for the transmission and expansion of leishmaniasis throughout the territory. Because of the increasing environmental changes and deforestation in the municipality, the risk of generating ecological imbalance and increased cases of leishmaniasis is imminent, which highlights the importance of developing preventive and control strategies.

Retrospective serological and molecular survey of myxoma or antigenically related virus in the Iberian hare, Lepus granatensis.

The 2018 outbreak of myxomatosis in the Iberian hare (Lepus granatensis) has been hypothesized to originate from a species jump of the rabbit-associated myxoma virus (MYXV), after natural recombination with an unknown poxvirus. Iberian hares were long considered resistant to myxomatosis as no prior outbreaks were reported. To provide insights into the emergence of this recombinant virus (ha-MYXV), we investigated serum samples from 451 Iberian hares collected over two time periods almost two decades apart, 1994-1999 and 2017-2019 for the presence of antibodies and MYXV-DNA. First, we screened all serum samples using a rabbit commercial indirect ELISA (iELISA) and then tested a subset of these samples in parallel using indirect immunofluorescence test (IFT), competitive ELISA (cELISA) and qPCR targeting M000.5L/R gene conserved in MYXV and ha-MYXV. The cut-off of iELISA relative index 10 = 6.1 was selected from a semiparametric finite mixture analysis aiming to minimize the probability of false positive results. Overall, MYXV related-antibodies were detected in 57 hares (12.6%) including 38 apparently healthy hares (n = 10, sampled in 1994-1999, none MYXV-DNA positive, and n = 28 sampled in 2017-2019 of which four were also ha-MYXV-DNA positive) and 19 found-dead and ha-MYXV-DNA-positive sampled in 2018-2019. Interestingly, four seronegative hares sampled in 1997 were MYXV-DNA positive by qPCR, the result being confirmed by sequencing of three of them. For the Iberian hares hunted or live trapped (both apparently health), seroprevalence was significantly higher in 2017-2019 (13.0%, CI95% 9.2-18.2%) than in 1994-1999 (5.4%, CI95% 3.0-9.6%) (p = .009). Within the second period, seroprevalence was significantly higher in 2019 compared to 2017 (24.7 vs 1.7% considering all the sample, p = .007), and lower during the winter than the autumn (p < .001). While our molecular and serological results show that Iberian hares have been in contact with MYXV or an antigenically similar virus at least since 1996, they also show an increase in seroprevalence in 2018-2019. The remote contact with MYXV may have occurred with strains that circulated in rabbits, or with unnoticed strains already circulating in Iberian hare populations. This work strongly suggests the infection of Iberian hares with MYXV or an antigenically related virus, at least 20 years before the severe virus outbreaks were registered in 2018.

Comparative analysis of two genomes of Chlamydia pecorum isolates from an Alpine chamois and a water buffalo.

BACKGROUND: To date, whole genome sequencing has been performed mainly for isolates of Chlamydia trachomatis, C. pneumoniae, C. psittaci and C. abortus, but only a few isolates of C. pecorum have been entirely sequenced and this makes it difficult to understand its diversity and population structure. In this study the genome of two C. pecorum strains isolated from the lung of an Alpine chamois affected with pneumonia (isolate PV7855) and the brain of a water buffalo affected with meningoencephalomyelitis (isolate PV6959), were completely sequenced with MiSeq system (Illumina) and analyzed in their most polymorphic regions. RESULTS: The genome length and GC content of the two isolates were found to be consistent with other C. pecorum isolates and the gene content of polymorphic membrane proteins and plasticity zone was found to be very similar. Some differences were observed in the phospholipase genes for both isolates and in the number of genes in the plasticity zone, such as the presence of some hypothetical proteins in PV6959, not present in any other genomes analyzed in this study. Interestingly, PV6959 possesses an extra pmp and has an incomplete tryptophan biosynthesis operon. Plasmids were detected in both isolates. CONCLUSIONS: Genome sequencing of the two C. pecorum strains did not reveal differences in length and GC content despite the origin from different animal species with different clinical disease. In the plasticity zone, the differences in the genes pattern might be related to the onset of specific symptoms or infection of specific hosts. The absence of a tryptophan biosynthesis pathway in PV6959 may suggest a strict relationship between C. pecorum and its host.