Concomitant Infections With Canine Parvovirus Type 2 and Intracellular Tick-Borne Pathogens in Two Puppy Dogs

In this report the concomitant infection with canine parvovirus type 2 (CPV-2), Hepatozoon canis and Ehrlichia canis in two puppy dogs from Southern Italy is described. Dogs were referred to a veterinary university hospital for the acute onset of lethargy and gastrointestinal signs. A complete clinical and clinicopathological evaluation was carried out and the multiple infection was confirmed by microscopic detection of inclusion bodies in peripheral blood smear, rapid immunoenzymatic tests, indirect fluorescent antibody tests, and molecular assays. Sequence analysis revealed that the CPV-2 identified belonged to the 2c variant and had amino acid residues in the predicted VP2 protein typical of “Asian-like” strains widespread in Asia and occasionally reported in Romania, Nigeria and Italy, particularly in the region of Sicily. Numerous monocytes were infected by both H. canis gamonts and E. canis morulae, suggesting that this co-infection is not accidental and that E. canis preferably infects those cells parasitized by H. canis. The clinical presentation of these animals was severe but supportive cares associated with early etiological therapy allowed a good prognosis. Movement of puppies from geographic areas where vector-borne pathogens are endemic must be carefully evaluated and core vaccinations and ectoparasite prevention treatments must be rigorously adopted

a metaproteomic pipeline to identify newborn mouse gut phylotypes

Abstract In order to characterize newborn mouse gut microbiota phylotypes in very early-life stages, an original metaproteomic pipeline, based on LC–MS 2 -spectra and Mascot driven NCBI non-redundant repository database interrogation was developed. An original computational analysis assisted in the generation of a taxonomic gut architecture from protein hits to operational taxonomic units (OTUs) and related functional categories. Regardless of the mouse's genetic background, a prevalence of Firmicutes (Lactobacillaceae) and Proteobacteria (Enterobacteriaceae) was observed among the entire Eubacteria taxonomic node. However, a higher abundance of Firmicutes was retrieved for Balb/c gut microbiota compared to Rag2 ko mice, the latter was mainly characterized by a Proteobacteria enriched microbiota. The metaproteomic-obtained OTUs were supported, for the identification (ID) of the cultivable bacteria fraction, corroborated by axenic culture-based MALDI-TOF MS IDs. Particularly, functional analysis of Rag2 ko mice gut microbiota proteins revealed the presence of abundant glutathione, riboflavin metabolism and pentose phosphate pathway components, possibly related to genetic background. The metaproteomic pipeline herein presented may represent a useful tool to investigate the highly debated onset of the human gut microbiota in the first days of life, when the bacterial composition, despite its very low diversity (complexity), is still very far from an exhaustive description and other complex microbial consortia. Biological significance The manuscript deals with a "frontier" topic regarding the study of the gut microbiota and the application of a metaproteomic pipeline to unveil the complexity of this fascinating ecosystem at the very early stages of life. Indeed during these phases, its diversity is very low but the bacterial content is highly "instable", and the relative balance between mucosal and fecal bacteria starts its dynamics of "fight" to get homeostasis. However, in the neonatal period, especially immediately after birth, a comprehensive description of this microbial eco-organ is still lacking, while it should be mandatory to highlight its first mechanisms of homeostasis and perturbation, while it co-develops with and within the host species. In order to unravel its low but almost unknown microbial community multiplicity, the newborn mouse gut, characterized by a "very" low complexity, was herein selected as model to design a LC–MS 2 -based shotgun metaproteomic approach, potentially suitable to study onset and shaping in human newborns. A microbiological semi-automatic computational analysis was performed to infer gut phylotypes; such as proof of evidence, related OTUs were compared to axenic-culture-based MALDI-TOF MS IDs showing consistency at family and phyla levels for the bacterial cultivable fraction. This article is part of a Special Issue entitled: Trends in Microbial Proteomics

Mesenchymal Stem Cell-Based Immunomodulation in Allogeneic Heterotopic Heart-Lung Transplantation

Mesenchymal stem cells are able to differentiate in various cell lineages and they have shown immunomodulatory properties in vitro, altering the cytokine secretion profile of T helper, T effector and dendritic cells and stimulating natural killer cells towards an anti-inflammatory and tolerant phenotype. In vivo they prolong skin allograft survival and may decrease graft-versus-host disease after hematopoietic stem cell transplants. In this work we studied the effects of mesenchymal stem cell treatment in an allogeneic heterotopic heart-lung transplant model. The following experimental groups were formed: A) Control B) Immunosuppressive therapy (Cyclosporine A) C) Mesenchymal stem-cell intravenous infusion D) Mesenchymal stem-cell infusion plus immunosuppressive treatment. The infusion of mesenchymal stem cells improved the mean graft survival up to 14.5±3.7 days with respect to the control group (3±0.6 days). Treatment with Cyclosporine A plus mesenchymal stem cells (group D) produced a mean survival time of 18.25±4.9 days, and was not significantly different to the results for group B (21.75±3.5 days). Furthermore, in the immunosuppressive treatment and the mesenchymal stem cell treatment, histological analysis revealed a reduction in the grade of rejection in heart and lung grafts. This decrease was most significant in group D. In conclusion, mesenchymal stem cells alone or in combination with Cyclosporine A were able to prolong graft survival time. These data suggest that, in vivo, mesenchymal stem cells retain their ability, already shown in vitro, to suppress lymphocyte activation and proliferation

Mechanisms of antibiotic resistance to enrofloxacin in uropathogenic Escherichia coli in dog

Escherichia coli (E. coli) urinary tract infections (UTIs) are becoming a serious problem both for pets and humans (zoonosis) due to the close contact and to the increasing resistance to antibiotics. This study has been performed in order to unravel the mechanism of induced enrofloxacin resistance in canine E. coli isolates that represent a good tool to study this pathology. The isolated E. coli has been induced with enrofloxacin and studied through 2D DIGE and shotgun MS. Discovered differentially expressed proteins are principally involved in antibiotic resistance and linked to oxidative stress response, to DNA protection and to membrane permeability. Moreover, since enrofloxacin is an inhibitor of DNA gyrase, the overexpression of DNA starvation/stationary phase protection protein (Dsp) could be a central point to discover themechanismof this clone to counteract the effects of enrofloxacin. In parallel, the dramatic decrease of the synthesis of the outer membrane proteinW, which represents one of the main gates for enrofloxacin entrance, could explain additional mechanismof E. coli defense against this antibiotic

The impact of carotid plaque presence and morphology on mortality outcome in cardiological patients

BACKGROUND: Carotid plaque severity and morphology can affect cardiovascular prognosis. We evaluate both the importance of echographically assessed carotid artery plaque geometry and morphology as predictors of death in hospitalised cardiological patients. METHODS: 541 hospitalised patients admitted in a cardiological division (age = 66 ± 11 years, 411 men), have been studied through ultrasound Duplex carotid scan and successively followed-up for a median of 34 months. Echo evaluation assessed plaque severity and morphology (presence of heterogeneity and profile). RESULTS: 361 patients showed carotid stenosis (67% with <50% stenosis, 18% with 50–69% stenosis, 9% with >70% stenosis, 4% with near occlusion and 2% with total occlusion). During the follow-up period, there were 83 all-cause deaths (15% of the total population). Using Cox's proportional hazard model, age (RR 1.06, 95% CI 1.03–1.09, p = 0.000), ejection fraction > 50% (RR = 0.62, 95% CI 0.4–0.96, p = 0.03), treatment with statins (RR = 0.52, 95% CI 0.29–0.95, p = 0.34) and the presence of a heterogeneous plaque (RR 1.6; 95% CI, 1.2 to 2.14, p = 0.002) were independent predictors of death. Kaplan – Meier survival estimates have shown the best outcome in patients without plaque, intermediate in patients with homogeneous plaques and the worst outcome in patients with heterogeneous plaques (90% vs 79% vs 73%, p = 0.0001). CONCLUSION: In hospitalised cardiological patients, carotid plaque presence and morphology assessed by ultrasound are independent predictors of death

Integrated use of molecular techniques for the detection and genetic characterization of infectious agents in domestic and wild carnivores

Nel periodo compreso tra il 2019 e il 2022 sono state testate differenti matrici biologiche di carnivori domestici e selvatici provenienti dall’Italia e da altri Paesi europei (Norvegia, Romania). Diversi saggi molecolari, tra cui real-time PCR, end-point PCR, semi-nested PCR, retrotrascrizione e rolling circle amplification, sono stati utilizzati per ricercare il DNA o l’RNA genomico di virus e batteri. Il sequenziamento dell’intero genoma o di geni informativi dei patogeni identificati ne ha inoltre consentito la caratterizzazione genetica e l’analisi filogenetica. Gli studi, svolti presso il Dipartimento di Scienze Mediche Veterinarie dell’Università di Bologna, erano focalizzati nei confronti di alcuni virus a DNA, come Carnivore protoparvovirus 1 in lupi dall’appennino italiano e cani dalla Romania, adenovirus canino di tipo 1 e 2 in cani e lupi provenienti dal territorio nazionale, circovirus canino in cani e lupi italiani e volpi rosse e artiche della Norvegia; virus a RNA, come il canine distemper virus in faine recuperate nel territorio italiano e il calicivirus felino in gatti con diagnosi di poliartrite; e batteri appartenenti alla specie Anaplasma phagocytophilum in gatti deceduti e sottoposti a necroscopia in Italia. Dai risultati ottenuti è emerso che gli agenti infettivi indagati circolano nelle popolazioni di carnivori domestici e selvatici in forma asintomatica o determinando talvolta sintomatologia clinica. In alcuni animali testati è stata rilevata la coinfezione con diversi agenti patogeni, condizione che può predisporre ad un aggravamento della sintomatologia clinica. Dall’analisi filogenetica sono emerse relazioni tra gli agenti infettivi rilevati nelle differenti specie animali suggerendone la trasmissione tra ospiti domestici e selvatici e confermando il ruolo epidemiologico svolto dei carnivori selvatici nel mantenimento dei patogeni nel territorio. Alla luce dei dati ottenuti, è importante sottolineare l’importanza delle misure di profilassi, in particolare la vaccinazione degli animali da compagnia, per ridurre la trasmissione e la diffusione degli agenti infettivi.Different biological samples of domestic and wild carnivores from Italy and other European countries (Norway, Romania) were tested between 2019 and 2022. Several molecular assays, including real-time PCR, end-point PCR, semi-nested PCR, reverse transcription and rolling circle amplification, have been used to detect the genomic DNA or RNA of viruses and bacteria. Sequencing of the whole genome or of informative genes of the identified pathogens also allowed their genetic characterization and phylogenetic analysis. The studies, carried out at the Department of Veterinary Medical Sciences of the University of Bologna, were focused on some DNA viruses, such as Carnivore protoparvovirus 1 in wolves from the Italian Apennines and dogs from Romania, canine adenovirus types 1 and 2 in dogs and wolves from Italy, canine circovirus in dogs and wolves from Italy and red and arctic foxes from Norway; RNA viruses, such as the canine distemper virus in stone martens recovered in Italy and the feline calicivirus in cats with polyarthritis; and bacteria belonging to the species Anaplasma phagocytophilum in deceased cats subjected to necropsy in Italy. The results obtained showed that the infectious agents investigated circulate in the populations of domestic and wild carnivores asymptomatically or sometimes causing clinical symptoms. Coinfection with different pathogens was found in some animals tested, a condition which may predispose to a worsening of the clinical picture. From the phylogenetic analysis, relationships between the infectious agents detected in the different animal species emerged, suggesting their transmission between domestic and wild hosts and confirming the epidemiological role of wild carnivores in maintaining pathogens in the territory. In the light of the data obtained, it is important to underline the importance of prophylactic measures, in particular the vaccination of pets, to reduce the transmission and spread of infectious agents

Ancient origin and genetic segregation of canine circovirus infecting arctic foxes (Vulpes lagopus) in Svalbard and red foxes (Vulpes vulpes) in Northern Norway

This is the pre-peer reviewed version of the following article: Urbani, Tryland, Ehrich, Fuglei, Battilani, Balboni. Ancient origin and genetic segregation of canine circovirus infecting arctic foxes (Vulpes lagopus) in Svalbard and red foxes (Vulpes vulpes) in Northern Norway. Transboundary and Emerging Diseases. 2020;00:1-11, which has been published in final form at https://doi.org/10.1111/tbed.13783. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.Canine circovirus (CanineCV) is a relatively new viral species, belonging to the family Circoviridae, whose pathogenic role is still uncertain. Since its first description in one domestic dog in 2011 from the USA, several reports have been documenting its distribution worldwide. Recently, CanineCV was also detected in wild animals such as wolves, foxes and badgers. In order to investigate the presence and the genetic characteristics of CanineCV in foxes of Arctic and Sub‐Arctic regions, the presence of CanineCV DNA in internal organs (liver and spleen) of 51 arctic foxes (Vulpes lagopus) from Svalbard archipelago and 59 red foxes (Vulpes vulpes) from Northern Norway, sampled from 1996 to 2001 and from 2014 to 2018, respectively, was screened by real‐time PCR. CanineCV was detected in 11/51 arctic foxes and in 10/59 red foxes, backdating the circulation of the virus at least to 1996 in the arctic fox population. The complete genome of 14 identified CanineCV was sequenced and analysed showing an identity higher than 80.8% with the reference strains available to date. According to the species demarcation threshold of 80% genome‐wide nucleotide sequence identity for members of the family Circoviridae provided by International Committee on Taxonomy of Viruses (ICTV), all the CanineCV belong to a single species. Phylogenetic analysis revealed that all the CanineCV were subdivided into five main clusters with one including only CanineCV identified in foxes. Furthermore, CanineCV identified in arctic foxes and red foxes formed two distinct lineages. From these data, we hypothesize that the viral transmission did not occur between the two species of foxes as a consequence of the lack of contact between the two hosts or that the virus acquired mutations in the time elapsed between the samplings

Genomic Characterization of Canine Circovirus Detected in Red Foxes (Vulpes vulpes) from Italy using a New Real-time PCR Assay

Data on canine circovirus circulation among red foxes (Vulpes vulpes) are limited. We report the detection of canine circovirus in a red fox from Italy. The virus was closely related to strains from dogs (Canis lupus familiaris) rather than those from foxes, suggesting a possible transmission between the two species

Monitoring Perinatal Gut Microbiota in Mouse Models by Mass Spectrometry Approaches: Parental Genetic Background and Breastfeeding Effects

At birth, contact with external stimuli, such as nutrients derived from food, is necessary to modulate the symbiotic balance between commensal and pathogenic bacteria, protect against bacterial dysbiosis, and initiate the development of the mucosal immune response. Among a variety of different feeding patterns, breastfeeding represents the best modality. In fact, the capacity of breast milk to modulate the composition of infants' gut microbiota leads to beneficial effects on their health. In this study, we used newborn mice as a model to evaluate the effect of parental genetic background (i.e., IgA-producing mice and IgA-deficient mice) and feeding modulation (i.e., maternal feeding and cross-feeding) on the onset and shaping of gut microbiota after birth. To investigate these topics, we used either a culturomic approach that employed Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MS), or bottom-up Liquid Chromatography, with subsequent MSMS shotgun metaproteomic analysis that compared and assembled results of the two techniques. We found that the microbial community was enriched by lactic acid bacteria when pups were breastfed by wild-type (WT) mothers, while IgA-deficient milk led to an increase in the opportunistic bacterial pathogen (OBP) population. Cross-feeding results suggested that IgA supplementation promoted the exclusion of some OBPs and the temporary appearance of beneficial species in pups fed by WT foster mothers. Our results show that both techniques yield a picture of microbiota from different angles and with varying depths. In particular, our metaproteomic pipeline was found to be a reliable tool in the description of microbiota. Data from these studies are available via ProteomeXchange, with identifier PXD004033

A food safety control low mass-range proteomics platform for the detection of illicit treatments in veal calves by MALDI-TOF-MS serum profiling.

International audiencePerformance enhancing agents (PEAs) are illegally used in cattle and other meat producing species to increase food conversion and lean meat production. Due to the very short breeding cycle, veal calves represent the meat producing bovine category mostly subjected to illicit treatments. These chemical agents are difficult to detect by conventional analytical approaches due to the employment of synergistic formulations at very low dosage and given the use of uncharacterised novel compounds. Such a scenario has fostered a strong interest in the discovery of functional molecular biomarkers for the detection of growth promoting agents in meat producing species. A multivariate MALDI-TOF-MS proteomics platform has been developed using bovine serum samples. Analytical performances have been thoroughly evaluated in order to enable reproducible profiles from 10 μl sera samples. We propose univariate and multivariate discrimination models capable to identify calves undergoing illicit treatments. In particular, we found a strong discrimination power associated with a polypeptide fragment from β2-glycoprotein-I. We provide a fundamental proof of concept in the potential application of MALDI-TOF-MS proteomics profiling in the food safety control