Introgression from local cultivars is a driver of agricultural adaptation in Argentinian weedy rice.

Weedy rice, a pervasive and troublesome weed found across the globe, has often evolved through fertilization of rice cultivars with little importance of crop-weed gene flow. In Argentina, weedy rice has been reported as an important constraint since the early 1970s, and, in the last few years, strains with herbicide-resistance are suspected to evolve. Despite their importance, the origin and genetic composition of Argentinian weedy rice as well its adaptation to agricultural environments has not been explored so far. To study this, we conducted genotyping-by-sequencing on samples of Argentinian weedy and cultivated rice and compared them with published data from weedy, cultivated and wild rice accessions distributed worldwide. In addition, we conducted a phenotypic characterization for weedy-related traits, a herbicide resistance screening and genotyped accessions for known mutations in the acetolactate synthase (ALS) gene, which confers herbicide resistance. Our results revealed large phenotypic variability in Argentinian weedy rice. Most strains were resistant to ALS-inhibiting herbicides with a high frequency of the ALS mutation (A122T) present in Argentinian rice cultivars. Argentinian cultivars belonged to the three major genetic groups of rice: japonica, indica and aus while weeds were mostly aus or aus-indica admixed, resembling weedy rice strains from the Southern Cone region. Phylogenetic analysis supports a single origin for aus-like South American weeds, likely as seed contaminants from the United States, and then admixture with local indica cultivars. Our findings demonstrate that crop to weed introgression can facilitate rapid adaptation to agriculture environments.

Assessment of potential maladaptive pain in dogs with elbow osteoarthritis using a von Frey aesthesiometer.

BACKGROUND: This study aimed to investigate the possible presence of maladaptive pain in the thoracic limbs of dogs with elbow osteoarthritis (OA) using an electronic von Frey aesthesiometer (eVFA). METHODS: Twenty-eight client- and staff-owned dogs (OA, n = 14; controls, n = 14) were enrolled in the study. Every dog underwent a full orthopaedic examination, and then five von Frey measurements were obtained from each carpal pad of each dog. A maximum test threshold of 400 g was set and approved by an ethics committee. RESULTS: eVFA thresholds were significantly lower (p < 0.001) in dogs with OA (median 248 g, range 128-369 g) than in control dogs (median 390 g, range 371-400 g). In the OA group, the sensory threshold was significantly lower (p = 0.048) in the more severely affected limb than the less severely affected limb. LIMITATION: The low maximum threshold required for ethical approval may influence the variability in the control group. CONCLUSIONS: Dogs with elbow OA had significantly lower sensory thresholds than control dogs, which is compatible with the presence of maladaptive pain, potentially due to central sensitisation. Further research is required to evaluate the potential use of the eVFA for monitoring clinical progression and treatment response in dogs with elbow OA.

First evidence of widespread positivity to anticoagulant rodenticides in grey wolves (Canis lupus).

Second-generation Anticoagulant Rodenticides (ARs) can be critical for carnivores, due to their widespread use and impacts. However, although many studies explored the impacts of ARs on small and mesocarnivores, none assessed the extent to which they could contaminate large carnivores in anthropized landscapes. We filled this gap by exploring spatiotemporal trends in grey wolf (Canis lupus) exposure to ARs in central and northern Italy, by subjecting a large sample of dead wolves (n = 186) to the LC-MS/MS method. Most wolves (n = 115/186, 61.8 %) tested positive for ARs (1 compound, n = 36; 2 compounds, n = 47; 3 compounds, n = 16; 4 or more compounds, n = 16). Bromadiolone, brodifacoum and difenacoum, were the most common compounds, with brodifacoum and bromadiolone being the ARs that co-occurred the most (n = 61). Both the probability of testing positive for multiple ARs and the concentration of brodifacoum, and bromadiolone in the liver, systematically increased in wolves that were found at more anthropized sites. Moreover, wolves became more likely to test positive for ARs through time, particularly after 2020. Our results underline that rodent control, based on ARs, increases the risks of unintentional poisoning of non-target wildlife. However, this risk does not only involve small and mesocarnivores, but also large carnivores at the top of the food chain, such as wolves. Therefore, rodent control is adding one further conservation threat to endangered large carnivores in anthropized landscapes of Europe, whose severity could increase over time and be far higher than previously thought. Large-scale monitoring schemes for ARs in European large carnivores should be devised as soon as possible.

Eleven Years of Health Monitoring in Wild Boars (Sus scrofa) in the Emilia-Romagna Region (Italy).

In recent years, the growth of wild ungulates has increased the focus on their health monitoring. In particular, the health status of wild boars is relevant for the economic impact on the pig industry. The Emilia-Romagna region activated a wildlife monitoring plan to better evaluate the health status of the wild boar population. Between 2011 and 2021, samples of found dead and hunted wild boar have been examined for trichinellosis, tuberculosis, brucellosis, african swine fever, classical swine fever, Aujeszky's disease, swine vesicular disease, and swine influenza A. Trichinella britovi was identified in 0.001% of the examined wild boars; neither M. bovis nor M. tuberculosis were found in M. tuberculosis complex positive samples; 2.3% were positive for Brucella suis; 29.4% of the sera were positive for Aujeszky's disease virus; and 0.9% of the samples were positive for swine influenza A virus. With an uncertain population estimate, the number of animals tested, the number of positives, and the sampling method do not allow us to make many inferences but suggest the need to implement and strengthen the existing surveillance activity, as it seems to be the only viable alternative for safeguarding animal and human health.

Food exchange list based on macronutrients: adapted for the Ecuadorian population.

Background: Food exchange lists allow health professionals to generate healthy eating plans adapted to individual or population needs. The objective of this study was to develop the first food exchange list based on the macronutrients and energy provided by the various food groups of the Ecuadorian diet. Methods: The list of Ecuadorian food exchanges was constructed by going through the following phases: (1) Selection of household measurements; (2) Selection of tables and databases of the nutritional composition of food items; (3) Definition of food groups and quantities; (4) Determination of the average energy and macronutrient values of each group; and (5) Photographic record. For the definition of food quantities, statistical criteria were applied according to a standard deviation of ±2SD; thus, for carbohydrates: ±5 g, total fat: ±2 g, and protein: ±3 g. To ensure the inclusion of the food items in the groups, a coefficient of variation of less than 30% and a Z value of ±2 were also considered. Results: The list of food exchanges is presented with eight general groups according to the predominant nutrient (carbohydrates, proteins, or fats), and, where necessary, subgroups are included according to the second predominant nutrient. The list includes 404 food items with their photographic record, represented by their net weights and corresponding household measurement. All food items met the statistical criteria that help to reduce the variability of the nutritional composition of the food items in each group. Conclusion: This is the first list of Ecuadorian food exchanges based on statistical criteria. It represents a novel tool for public health professionals as well as researchers. Resulting healthier eating plans may improve daily dietetic practice, facilitate better clinical trial designs and help establish guidelines according to Ecuador's cultural and dietary patterns. The described methodology can further be used to develop other food exchanges lists for patients with specific nutritional requirements.

A human-neutral large carnivore? No patterns in the body mass of gray wolves across a gradient of anthropization.

The gray wolf (Canis lupus) expanded its distribution in Europe over the last few decades. To better understand the extent to which wolves could re-occupy their historical range, it is important to test if anthropization can affect their fitness-related traits. After having accounted for ecologically relevant confounders, we assessed how anthropization influenced i) the growth of wolves during their first year of age (n = 53), ii) sexual dimorphism between male and female adult wolves (n = 121), in a sample of individuals that had been found dead in Italy between 1999 and 2021. Wolves in anthropized areas have a smaller overall variation in their body mass, during their first year of age. Because they already have slightly higher body weight at 3-5 months, possibly due to the availability of human-derived food sources. The difference in the body weight of adult females and males slightly increases with anthropization. However, this happens because of an increase in the body mass of males only, possibly due to sex-specific differences in dispersal and/or to "dispersal phenotypes". Anthropization in Italy does not seem to have any clear, nor large, effect on the body mass of wolves. As body mass is in turn linked to important processes, like survival and reproduction, our findings indicates that wolves could potentially re-occupy most of their historical range in Europe, as anthropized landscapes do not seem to constrain such of an important life-history trait. Wolf management could therefore be needed across vast spatial scales and in anthropized areas prone to social conflicts.

Antimicrobial Resistance and Virulence Factors Assessment in Escherichia coli Isolated from Swine in Italy from 2017 to 2021

Prudent antibiotic use in pigs is critical to ensuring animal health and preventing the development of critical resistance. We evaluated the antimicrobial resistance (AMR) pattern in commensal and enterotoxigenic Escherichia coli (ETEC) isolates obtained in 2017−2021 from pigs suffering from enteric disorders. Overall, the selected 826 E. coli isolates showed the highest level of resistance to ampicillin (95.9%), tetracycline (89.7%), cefazolin (79.3%), and trimethoprim/sulfamethoxazole (74.8%). The resistance rates of the isolates to ampicillin increased (p < 0.05), reaching 99.2% of resistant strains in 2021. Regarding isolates harboring virulence genes, ETEC F18+ were significantly more resistant to florfenicol, gentamicin, kanamycin, and trimethoprim/sulfamethoxazole than ETEC F4+ strains. E. coli lacking virulence factor genes were more resistant to amoxicillin with clavulanic acid and cefazolin, but less resistant to gentamicin (p < 0.01) than isolates harboring virulence factors. Throughout the study period, a significant number of ETEC F18+ isolates developed resistance to florfenicol, gentamicin, and kanamycin. Finally, ETEC 18+ significantly (p < 0.05) increased resistance to all the tested antibiotics. In conclusion, AMR varied for E. coli over time and showed high levels for molecules widely administered in the swine industry, emphasizing the need for continuous surveillance. The observed differences in AMR between commensal and ETEC isolates may lead to the hypothesis that plasmids carrying virulence genes are also responsible for AMR in E. coli, suggesting more research on genetic variation between pathogenic and nonpathogenic E. coli.

Exploring the ATR-CHK1 pathway in the response of doxorubicin-induced DNA damages in acute lymphoblastic leukemia cells.

Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B-/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B-/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses. • Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells. • ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway. • The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells. • The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.

Wildlife Hosts of Leishmania infantum in a Re-Emerging Focus of Human Leishmaniasis, in Emilia-Romagna, Northeast Italy.

In the last decade, an upsurge of human leishmaniasis has been reported in the Emilia-Romagna region, Northeast Italy. Epidemiologic data have raised doubts about the role of dogs as the main reservoirs for Leishmania infantum. In the present study, a total of 1,077 wild animals were screened for L. infantum DNA in earlobe and spleen samples from 2019 to 2022. The lymph nodes were tested only in 23 animals already positive in the earlobe and/or spleen. A total of 71 (6.6%) animals resulted positive in at least one of the sampled tissues, including 3/18 (16.7%) wolves, 6/39 (15.4%) European hares, 38/309 (12.3%) roe deer, 1/11 (9.1%) red deer, 8/146 (4.9%) wild boars, 13/319 (4.1%) red foxes, 1/54 (1.9%) porcupine, and 1/59 (1.7%) European badger. Most of the infected animals (62/71) tested positive only in the earlobe tissue, only four animals (two roe deer and two wild boars) tested positive only in the spleen, and five animals (three roe deer and two red foxes) resulted positive for both tissues. L. infantum DNA was detected in the lymph nodes of 6/23 animals. L. infantum detection occurred in all seasons associated with low real-time PCR Ct values. Further research is needed in order to clarify the role of wildlife in the re-emerging focus of leishmaniasis in Northeast Italy.

Delayed union, non-union and mal-union in 442 dogs.

OBJECTIVES: (1) To estimate the prevalence of delayed union, non-union and mal-union in canine fractures; (2) to describe fracture, demographic, and treatment characteristics for these outcomes; (3) to identify risk factors for delayed or non-union. STUDY DESIGN: Retrospective study. SAMPLE POPULATION: Four hundred and forty two dogs (461 fractures). METHODS: A review was conducted of clinical records and radiographs from 2 teaching hospitals. "Union," "delayed union," "non-union" and "mal-union" were defined, and fracture, demographic, treatment, and outcome variables described. Differences in proportions or medians between "union," "delayed union" and "non-union" were tested using χ2 and Mann-Whitney U-tests for categorical and continuous variables respectively. Potential explanatory variables for "delayed or non-union" were tested using logistic regression to identify risk factors. RESULTS: Median radiographic follow up was 53 days (14-282). Delayed union occurred in 13.9% of fractures (64/461), non-union in 4.6% (21/461), and mal-union in 0.7% (3/461). Risk factors for delayed or non-union were age (OR 1.21, 95% CI 1.12-1.31); comminuted fracture (OR 4.24, 95% CI 2.4-7.5); treatment with bone graft (all types) (OR 3.32, 95% CI 1.3-8.5); surgical site infection (OR 3.24, 95% CI 1.17-8.97), and major implant failure (OR 12.94, 95% CI 5.06-33.1). CONCLUSION: Older dogs, dogs with comminuted fractures, surgical site infection, or major implant failure were at increased odds of delayed or non-union. Radius and ulna fractures in toy breed dogs were not at increased odds of delayed or non-union. CLINICAL SIGNIFICANCE: The identified risk factors should inform fracture planning and prognosticating. The prognosis for radial fractures in toy breeds appears better than historically believed.

Influenza A in Wild Boars: Viral Circulation in the Emilia-Romagna Region (Northern Italy) between 2017 and 2022.

A systematic surveillance against influenza A viruses (IAVs) in the Suidae population is essential, considering their role as IAV mixing vessels. However, the viral circulation in wild Sus scrofa species is poorly investigated in comparison to the knowledge of IAV infection dynamics in domestic pigs. This study investigated the circulation and the genetic diversity of wild boars' IAVs detected in the Emilia-Romagna region (2017-2022). A total of 4605 lung samples were screened via an M gene real-time RT-PCR for SwIAV; positive samples were subtyped by multiplex RT-PCR, and viral isolation was attempted. Isolated strains (3 out of the 17 positives) were fully sequenced to evaluate viral genotypic diversity. H1N1 was the most frequently detected subtype, with identification of H1pdm09N1 and H1avN1. Whole-genome phylogenetic analysis revealed SwIAVs belonging to different genotypes, with different genetic combinations, and highlighted the simultaneous circulation of the same genotypes in both pigs and wild boars, supporting the hypothesis of SwIAV spillover events at the wildlife-livestock interface. This study represents an update on the wild boar SwIAV Italian situation, and the strains' complete genome analysis showed an evolving and interesting situation that deserves further investigation.

Release of IFNγ by Acute Myeloid Leukemia Cells Remodels Bone Marrow Immune Microenvironment by Inducing Regulatory T Cells.

PURPOSE: The stromal and immune bone marrow (BM) landscape is emerging as a crucial determinant for acute myeloid leukemia (AML). Regulatory T cells (Treg) are enriched in the AML microenvironment, but the underlying mechanisms are poorly elucidated. Here, we addressed the effect of IFNγ released by AML cells in BM Treg induction and its impact on AML prognosis. EXPERIMENTAL DESIGN: BM aspirates from patients with AML were subdivided according to IFNG expression. Gene expression profiles in INFγhigh and IFNγlow samples were compared by microarray and NanoString analysis and used to compute a prognostic index. The IFNγ release effect on the BM microenvironment was investigated in mesenchymal stromal cell (MSC)/AML cell cocultures. In mice, AML cells silenced for ifng expression were injected intrabone. RESULTS: IFNγhigh AML samples showed an upregulation of inflammatory genes, usually correlated with a good prognosis in cancer. In contrast, in patients with AML, high IFNG expression was associated with poor overall survival. Notably, IFNγ release by AML cells positively correlated with a higher BM suppressive Treg frequency. In coculture experiments, IFNγhigh AML cells modified MSC transcriptome by upregulating IFNγ-dependent genes related to Treg induction, including indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 inhibitor abrogated the effect of IFNγ release by AML cells on MSC-derived Treg induction. In vivo, the genetic ablation of IFNγ production by AML cells reduced MSC IDO1 expression and Treg infiltration, hindering AML engraftment. CONCLUSIONS: IFNγ release by AML cells induces an immune-regulatory program in MSCs and remodels BM immunologic landscape toward Treg induction, contributing to an immunotolerant microenvironment. See related commentary by Ferrell and Kordasti, p. 2986.

Integrated Use of Molecular Techniques to Detect and Genetically Characterise DNA Viruses in Italian Wolves (Canis lupus italicus).

In this study, internal organs (tongue, intestine, and spleen) of 23 free-ranging Italian wolves (Canis lupus italicus) found dead between 2017 and 2019 were tested for Carnivore protoparvovirus 1, Canine adenovirus (CAdV), and Canine circovirus (CanineCV) using real-time PCR assays. Genetic characterisation of the identified viruses was carried out by amplification, sequencing, and analysis of the complete viral genome or informative viral genes. All the wolves tested positive for at least one of the DNA viruses screened, and 11/23 were coinfected. Carnivore protoparvoviruses were the most frequently detected viruses (21/23), followed by CanineCV (11/23) and CAdV (4/23). From the analysis of the partial VP2 gene of 13 carnivore protoparvoviruses, 12 were canine parvovirus type 2b, closely related to the strains detected in dogs and wild carnivores from Italy, and one was a feline panleukopenia-like virus. Of the four CAdV identified, two were CAdV-1 and two were CAdV-2. The complete genome of seven CanineCVs was sequenced and related to the CanineCV identified in dogs, wolves, and foxes worldwide. Close correlations emerged between the viruses identified in wolves and those circulating in domestic dogs. Further studies are needed to investigate if these pathogens may be potentially cross-transmitted between the two species.

Illegal fishing with electrofishing devices in the Po river basin, Emilia Romagna, Italy.

Electric fishing is an illegal hunting method, unfortunately widely used by poachers to paralyze fish and to catch many animals in a short time. In Italy, it is authorized only for scientific and conservative purposes. Between 2014 and 2018, the Ferrara section of the Experimental Zooprophylactic Institute of Lombardy and Emilia Romagna, Italy, received nine cases of potentially illegal electric fishing in Po river and its tributary rivers. Necropsies were performed following standard protocols and samples of different tissues were collected and examined using histochemical and immunohistochemical techniques. Gross lesions frequently observed were circulatory alteration phenomena (i.e. multi-organ hyperemia, hemorrhages and congestion, hemopericardium), also found histologically, in addition to multifocal degenerative and necrotic muscular processes that could be attributed to injuries from electric current, as already reported in literature. Immunohistochemical investigations confirmed degenerative and necrotic lesions with myoglobin depletion and a corresponding fibrinogen accumulation. Myoglobin globules were also detected in the renal parenchyma, as consequent of rhabdomyolysis. The results of this study allowed to correlate electric fishing to gross, histologic and immunohistochemical lesions, which together constitute a pathognomonic picture to be considered a reference standard in this type of illegal controversy.

Integrated genomic-metabolic classification of acute myeloid leukemia defines a subgroup with NPM1 and cohesin/DNA damage mutations.

Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.

Natural distemper infection in stone martens (Martes foina): From infection to neutralizing antibodies.

We report an outbreak of canine distemper virus (CDV) among stone martens (Martes foina) in Italy. After being rescued in Northern Italy between April and June 2018, six subjects were kept in a wildlife and exotic animal rescue center in Bologna province. Subjects have been monitored for 15 months in captivity. Within this time-lapse, two subjects died, while among the remaining four, only one showed clinical symptoms referable to distemper. Surviving subjects have been regularly tested for CDV by means of reverse transcriptase-PCR from conjunctival and oropharyngeal swabs for eleven months. The identified viruses belonged to the Wildlife-Europe CDV genetic subgroup. Neutralizing antibodies were detected at the end of the eleven months, when all subjects tested reverse transcriptase-PCR negative. Our findings confirm the circulation of the Wildlife-Europe CDV genetic subgroup (Europe 1/South America 1 lineage) within the Italian wildlife, and improve knowledge on viral infection in stone martens.

Carbapenemase IncF-borne blaNDM-5 gene in the E. coli ST167 high-risk clone from canine clinical infection, Italy.

The blaNDM-5-producing E. coli Sequence Type (ST)167 high-risk clone is emerging worldwide in human clinical cases, while its presence in companion animals is sporadic and has never been described in Italy. Using a combined Oxford Nanopore (ONT) long-reads and Illumina short-reads sequencing approach, an E. coli ST167 isolated from a hospitalized dog, was in-depth characterized by WGS and the plasmid containing blaNDM-5 was fully reconstructed. The complete sequence of the pMOL008 mosaic plasmid (F36:F31:A4:B1; pMOL008) harbouring blaNDM-5, was resolved and characterized. Moreover, a (pro)phage and IncFII, containing blaCMY-2 and ermB, and IncI2 plasmid types were also identified. pMOL008 was almost identical to blaNDM-5-containing plasmids from E. coli ST167 isolated from Italian human clinical cases and from a Swiss dog and colonized humans. blaNDM-5 was located in a class 1 integron together with aadA2, aac(3)-IIa, mph(A), sul1, tet(A) and dfrA12. The risk of spill-over and spill-back transmission of carbapenem-resistance genes, related plasmids and strains between humans and dogs, represents a Public Health threat and highlights the importance of the One Health approach for the AMR surveillance.