Untargeted fecal metabolome analysis in obese dogs after weight loss achieved by feeding a high-fiber-high-protein diet.

INTRODUCTION: In humans and companion animals, obesity is accompanied by metabolic derangements. Studies have revealed differences in the composition of the fecal microbiome between obese dogs and those with an ideal body weight. OBJECTIVES: We have previously reported that the fecal microbiome in obese dogs changes after controlled weight reduction, induced by feeding a diet high in fiber and protein. Despite these findings, it is unclear if taxonomic differences infer differences at the functional level between obese dogs and those with an ideal body weight. METHODOLOGY: Untargeted fecal metabolome analysis was performed on dogs with obesity before and after weight loss achieved by feeding a high-fiber-high-protein diet. RESULTS: Fecal metabolome analysis revealed a total of 13 compounds that changed in concentration in obese dogs after weight loss. Of these compounds, metabolites associated with bacterial metabolism decreased after weight loss including purine, L-(-)-methionine, coumestrol, and the alkaloids 1-methylxanthine and trigonelline. Conversely, the polyphenols (-)-epicatechin and matairesinol and the quinoline derivatives 1,5-isoquinolinediol and 2-hydroxiquinoline increased after weight loss. CONCLUSION: These results suggest differences in intestinal microbiome at the functional level after weight loss, but further studies are needed to determine the role of these compounds in the etiology of obesity and weight loss.

Evidence of avian poxvirus and papillomavirus infection in Gyps fulvus in Italy.

The identification of avian poxvirus and avian papillomavirus associated with cutaneous lesions in griffon vultures (Gyps fulvus) by histopathology, electron microscopy and PCR analysis is reported. Sequence analysis of the fpv140 gene revealed 99% identity to two poxviruses obtained from a white-tailed sea eagle (Haliaeetus albicilla) and a common buzzard (Buteo buteo). Partial sequence of the papillomavirus L1 gene showed sequence similarity to papillomavirus LI genes from African grey parrot (Psittacus erithacus) (69% identity), duck (Anas platyrhynchos) (68% identity), and yellow-necked francolin (Francolinus leucoscepus) (66% identity). To date, this is the first identification of avian poxvirus and papillomavirus in griffon vultures and the first evidence of infection of both viruses in live wild birds.

Molecular detection of canine bufaviruses in wild canids.

Novel protoparvoviruses genetically related to human and non-human primate bufaviruses (BuVs) have been detected recently in respiratory and enteric specimens collected from dogs and cats. In this study, by molecular screening of archival collections of faecal samples from wolves and foxes, we detected BuVs with a rate of 17.1% (7/41) and 10.5% (9/86), respectively. Sequence analysis of a portion of the ORF2 gene region of nine positive samples showed that the viruses in these samples were closely related to BuVs (97.5-99.0% nucleotide sequence identity) found in domestic carnivores.

Detection of selected pathogens in Apennine wolf (Canis lupus italicus) by a non-invasive GPS-based telemetry sampling of two packs from Majella National Park, Italy.

In this study, a multi-pathogens survey was conducted to verify the sanitary status of two Italian wolf packs of Majella National Park. Twenty fecal samples (10/pack) were collected using a sampling protocol, based on the combining data from radio-collared wolves with geographic information system (GIS) analysis, allowing to mark off the home range of packs and to recover group-specific and high-quality specimens. Virological screening against the most prevalent canine viruses (protoparvovirus, distemper virus, adenoviruses, and coronaviruses) was carried out by molecular methods, while parasites were detected by means of copromicroscopic and molecular analysis. Canine parvovirus type 2b (CPV-2b) is the most prevalent virus in both packs (7/20), followed by canine adenovirus type 2 (CAdV-2), while no sequences of canine distemper virus and coronaviruses were detected. The sequence analysis of the viruses demonstrated the domestic origin of the infection, highlighting the importance of vaccination of local dogs in order to reduce the risk of exposure of wildlife to these pathogens. Fourteen samples resulted positive for parasites. Capillaria aerophila (sin. Eucoleus aerophilus), Ancylostoma/Uncinaria, Trichuris vulpis eggs, Sarcocystis spp., Cystoisospora canis, and Angiostrongylus vasorum larvae were identified. Echinococcus granulosus sensu stricto (ovine genotype G1) and Giardia duodenalis(canid-specific Assemblage C) were also characterized, providing insights into the wolves' diet and their effects on environmental contamination. The sampling protocol applied in this study, based on a multidisciplinary approach, represents an innovative tool for the survey of Apennine wolf, able to integrate sanitary data with the ecological and demographic features of this population.

Serological and molecular investigation of 2117-like vesiviruses in cats.

Vesivirus 2117 was first discovered as a contaminant in Chinese hamster ovary (CHO) cell cultures used for human drug production. Similar vesiviruses (VeVs) have been detected recently in dogs. In order to address the hypothesis that cats may also be exposed to 2117-like VeVs, in this study, we screened 236 feline sera using an enzyme-linked immunosorbent assay (ELISA) based on a recombinant VP1 protein from the canine VeV Bari/212/07/ITA. IgG antibodies against the 2117-like VeV were detected in 37.3% of the sera tested. Also, by screening cat faecal specimens, the RNA of a 2117-like VeV was detected in a clinically healthy cat.

First molecular identification of kobuviruses in wolves (Canis lupus) in Italy.

Canine kobuviruses (CaKoVs) were first identified in diarrhoeic and asymptomatic dogs in 2011 in the USA. Subsequent studies have demonstrated a worldwide distribution of these viruses, but it is not clear if CaKoVs play a role as enteric pathogens of dogs. More recently, CaKoV RNA has been detected in wild carnivores, including red fox, golden jackal, side-striped jackal and spotted hyena. In this study, we addressed the hypothesis that wolves are susceptible to CaKoV infections. A total of 185 wolf stool samples were collected from necropsied animals and from transects in the Liguria, Piemonte and Valle D'Aosta regions of Italy, and CaKoV RNA was identified in two of these specimens. Both samples were obtained from necropsied wolves, with a prevalence rate of 4.9% (2/41). Sequence analysis of the full-length VP1 region showed that these strains displayed the highest nucleotide (nt) sequence identity (86.3-98.5%) to canine strains identified in the UK and Africa, and to kobuviruses that were previously detected in other African wild carnivores. This suggests that genetically related CaKoV strains circulate in domestic and wild carnivores, with interspecies transmission being not uncommon among carnivores of different ecosystems.

First molecular evidence of kobuviruses in goats in Italy.

By screening 139 rectal swabs collected from either asymptomatic or diarrhoeic goats in Italy, we identified kobuvirus RNA in eight samples (5.8 %). Higher positivity rates were observed in diarrhoeic goats (6.5 %, 3/46) than in asymptomatic animals (5.4 %, 5/93), although the difference was not statistically significant. Based on the analysis of a portion of the 3D gene, four strains were found to share the highest nucleotide (nt) sequence identity with bovine kobuviruses (95.0-98.0 %), which had been detected previously in calves in the UK and Korea. Interestingly, two strains were genetically related to the newly discovered caprine kobuviruses (83.0-97.0 % nt sequence identity), which had been identified in black goats in Korea and in roe deer in Italy. Taken together, these findings demonstrate that kobuviruses are common enteric viruses of goats, although their clinical relevance remains to be investigated.

Detection and genetic characterization of hepatitis E virus (HEV) genotype 3 subtype c in wild boars in Italy.

Hepatitis E virus (HEV) was detected in stools collected from wild boars in Italy, with an overall prevalence of 1.5 % (3/196). The sequence of a ~3.0-kb portion at the 3' end of the genome of one such strain, HEV/WB/P6-15/ITA, was determined. In the full-length ORF2, which encodes the capsid protein, the virus was genetically closest to wild boar and human HEV strains currently classified as genotype 3 subtype c. Interestingly, the 3' end of ORF2 of the WB/P6-15/ITA matched the 340-nucleotide (nt) sequence (94.0 % nt identity) of the human strain PeGe, identified in 2015 from a patient with acute hepatitis E in Genoa, Italy, suggesting that similar HEV strains are circulating in the same geographical setting in humans and animals.

Molecular detection of kobuviruses in European roe deer (Capreolus capreolus) in Italy.

Kobuvirus RNA was found in 6.6 % (13/198) of stool specimens from roe deer (Capreolus capreolus) captured during the regular hunting season. Upon sequence analysis of a fragment of the 3D gene, nine strains displayed the highest nucleotide sequence identity (91.2-97.4 %) to bovine kobuviruses previously detected in either diarrhoeic or asymptomatic calves. Interestingly, four strains were genetically related to the newly discovered caprine kobuviruses (84.2-87.6 % nucleotide identity) identified in black goats in Korea.

Seroprevalence of norovirus genogroup IV antibodies among humans, Italy, 2010-2011.

Noroviruses (NoVs) of genogroup IV (GIV) (Alphatron-like) cause infections in humans and in carnivorous animals such as dogs and cats. We screened an age-stratified collection of serum samples from 535 humans in Italy, using virus-like particles of genotypes GIV.1, circulating in humans, and GIV.2, identified in animals, in ELISA, in order to investigate the prevalence of GIV NoV-specific IgG antibodies. Antibodies specific for both genotypes were detected, ranging from a prevalence of 6.6% to 44.8% for GIV.1 and from 6.8% to 15.1% for GIV.2 among different age groups. These data are consistent with a higher prevalence of GIV.1 strains in the human population. Analysis of antibodies against GIV.2 suggests zoonotic transmission of animal NoVs, likely attributable to interaction between humans and domestic pets. This finding, and recent documentation of human transmission of NoVs to dogs, indicate the possibility of an evolutionary relationship between human and animal NoVs.

Molecular evidence of kobuviruses in free-ranging red foxes (Vulpes vulpes).

Red foxes (Vulpes vulpes) are susceptible to viral diseases of domestic carnivores. In this study, by screening rectal swabs collected from 34 red foxes in Italy, we identified kobuvirus RNA in five samples. Based on analysis of partial RdRp and full-length VP1 genes, all of the strains shared the highest identity with canine kobuviruses (CaKVs) recently detected in the US, the UK and Italy. These findings provide the first evidence of the circulation of these novel viruses in foxes.

Genetic heterogeneity of bovine noroviruses in Italy.

By screening 104 faecal samples from asymptomatic calves in Italy, bovine norovirus RNA was detected with a prevalence rate of 10.5 % (11/104). A continuous sequence spanning the RdRp region and the 5' end of the capsid gene was generated for 7 of the 11 strains. Upon phylogenetic analysis, five strains were grouped with GIII.2 Newbury2-like viruses, and one strain was grouped with GIII.1 Jena-like noroviruses. Interestingly, one strain (80TE/IT) was genetically related to the GIII.1/Jena/80/De in the RdRp but resembled the GIII.2/Newbury2/76/UK in the capsid gene, suggesting a recombination event occurring in the ORF1/ORF2 junction region.

Circoviridae Survey in Captive Non-Human Primates, Italy.

Circoviruses (CVs) and cycloviruses (CyVs), members of the family Circoviridae, have been identified only occasionally in non-human primates (NHPs). In this study, we investigated the presence and genetic features of these viruses in 48 NHPs housed in the Bioparco-Rome Zoological Garden (Italy) and in the Anima Natura Wild Sanctuary Semproniano (Grosseto, Italy), testing fecal, saliva, and serum samples with a broadly reactive consensus nested PCR able of amplifying a partial region of the replicase (Rep) gene of members of the family Circoviridae. Viral DNA was detected in a total of 10 samples, including a saliva swab and 9 fecal samples collected, respectively from five Japanese macaques (Macaca fuscata) and four mandrills (Mandrillus sphinx), with an overall prevalence of 18.7% (9/48). On genome sequencing, five strains revealed the highest nucleotide identity (98.3-98.6%) to a CyV strain (RI196/ITA) detected in the intestinal content of a Maltese wall lizard (Podarcis filfolensis) in Italy. Although the origin of the Italian NHP strains, genetically distant from previously detected NHP CyVs, is uncertain, our results also highlight that the virome of captive animals is modulated by the different dietary and environmental sources of exposure.

Molecular detection of Aichi virus in raw sewage in Italy.

Aichi virus (AiV) is suspected to play a role in viral gastroenteritis in humans. In this study, we assessed the presence of AiV in untreated influent sewage samples collected at four wastewater treatment plants in central Italy. AiV was detected in 6 (12.5 %) of the 48 specimens and in all plants. All of the Italian strains showed the highest nucleotide and amino acid sequence identity to genotype B AiV detected recently in Asia, especially in China.

Microbial Community and Abundance of Selected Antimicrobial Resistance Genes in Poultry Litter from Conventional and Antibiotic-Free Farms.

In this study, a culture-independent approach was applied to compare the microbiome composition and the abundance of the antimicrobial resistance genes (ARGs) aadA2 for aminoglycosides, tet(A), tet(B), tet(K), and tet(M) for tetracyclines, and mcr-1 for colistin in broiler litter samples collected from conventional and antibiotic-free flocks located in Central Italy. A total of 13 flocks and 26 litter samples, collected at the beginning and at the end of each rearing cycle, were submitted to 16s rRNA sequence analysis and quantitative PCR for targeted ARGs. Firmicutes resulted in the dominant phylum in both groups of flocks, and within it, the Clostridia and Bacilli classes showed a similar distribution. Conversely, in antibiotic-free flocks, a higher frequency of Actinobacteria class and Clostridiaceae, Lactobacillaceae, Corynebacteriaceae families were reported, while in the conventional group, routinely treated with antibiotics for therapeutic purposes, the Bacteroidia class and the Enterobacteriaceae and Bacillaceae families were predominant. All investigated samples were found to be positive for at least one ARG, with the mean values of aadA2 and tet(A) the highest in conventional flocks by a significant margin. The results suggest that antibiotic use can influence the frequency of resistance determinants and the microbial community in poultry flocks, even though other environmental factors should also be investigated more deeply in order to identify additional drivers of antimicrobial resistance.

Antibiotic Resistance in the Apennine Wolf (Canis lupus italicus): Implications for Wildlife and Human Health.

The Apennine wolf (Canis lupus italicus) is a subspecies of gray wolf that is widespread throughout Italy. Due to hunting and habitat loss, their population declined dramatically in the late 19th and early 20th centuries, but conservation efforts improved to restore the species to an estimated population of 3300 individuals. The presence of antibiotic-resistant bacteria in Apennine Wolf may pose a risk to its health and survival, as well as the health of other animals in its environment. In this study, we investigated the antibiotic resistance profiles of bacteria collected from Apennine wolves admitted to the Wildlife Research Center of Maiella National Park (Italy) in 2022. A total of 12 bacteria collected from four wolves were isolated and tested for susceptibility to antibiotics used in veterinary medicine and to critically important antibiotics for human health by means of the Vitek 2 system. All isolates were resistant to at least one antibiotic, and six bacteria were multidrug resistant to critically important antibiotics (third-generation cephalosporins, carbapenems and fluoroquinolones). The results of this pilot study have allowed for the characterization of resistant profiles in Escherichia coli, Enterococcus faecalis and other bacterial species not previously reported in Apennine wolves. Our findings provide important insights into antibiotic resistance in wildlife and its potential implications for the conservation of biodiversity and public health.

Whole Genome Sequencing of Escherichia coli and Enterococcus spp. in wildlife-livestock interface: a pilot study.

OBJECTIVES: This pilot study provides a multidisciplinary investigation to monitor livestock-wildlife interface. Ecological data, microbiological investigations, and whole genome sequencing were used to characterize eight bacterial isolates obtained from sympatric domestic and wild ruminants in Maiella National Park (Italy) in terms of genetic patterns of antimicrobial resistance. METHODS: Using selective culturing of fresh fecal samples of monitored and georeferenced populations of Apennine chamois, goats, red deer, and sheep, Escherichia coli, Enterococcus faecium, and Enterococcus faecalis isolates were isolated and subjected to minimum inhibitory concentration determination and whole genome sequencing. RESULTS: The analyzed isolates showed phenotypic and genotypic resistance to tetracycline and critically important antibiotics such as linezolid and carbapenems. Virulence genes related to biofilm regulation and Shiga toxins were also detected. Furthermore, serotypes related to nosocomial infections, harbouring plasmids recognized as important mobile resistance gene transmitters, were identified. CONCLUSIONS: This multidisciplinary pilot study represents a promising initial step to identify the environmental drivers and the transmission routes of antimicrobial resistance and virulence factors, providing new data on bacteria from rare and endangered species such as Apennine chamois.

Antibiotic-Resistant Bacteria Dissemination in the Wildlife, Livestock, and Water of Maiella National Park, Italy.

Antimicrobial resistance (AMR) is a global health concern that has been linked to humans, animals, and the environment. The One Health approach highlights the connection between humans, animals, and the environment and suggests that a multidisciplinary approached be used in studies investigating AMR. The present study was carried out to identify and characterize the antimicrobial resistance profiles of bacteria isolated from wildlife and livestock feces as well as from surface water samples in Maiella National Park, Italy. Ecological and georeferenced data were used to select two sampling locations, one where wildlife was caught within livestock grazing areas (sympatric group) and one where wildlife was caught outside of livestock grazing areas (non-sympatric group). Ninety-nine bacterial isolates from 132 feces samples and seven isolates from five water samples were collected between October and December 2019. The specimens were examined for species identification, antibiotic susceptibility and molecular detection of antibiotic resistance. Forty isolates were identified as Escherichia coli, forty-eight as Enterococcus spp., eight as Streptococcus spp. and ten as other gram-negative bacteria. Phenotypic antibiotic resistance to at least one antimicrobial agent, including some antibiotics that play a critical role in human medicine, was detected in 36/106 (33.9%, 95% CI: 25-43) isolates and multidrug resistance was detected in 9/106 isolates (8.49%, 95% CI: 3.9-15.5). In addition, genes associated with antibiotic resistance were identified in 61/106 (57.55%, 95% CI: 47.5-67) isolates. The samples from sympatric areas were 2.11 (95% CI: 1.2-3.5) times more likely to contain resistant bacterial isolates than the samples from non-sympatric areas. These data suggest that drug resistant bacteria may be transmitted in areas where wildlife and livestock cohabitate. This emphasizes the need for further investigations focusing on the interactions between humans, wildlife, and the environment, the results of which can aid in the early detection of emerging AMR profiles and possible transmission routes.

Feline Infectious Peritonitis: European Advisory Board on Cat Diseases Guidelines.

Feline coronavirus (FCoV) is a ubiquitous RNA virus of cats, which is transmitted faeco-orally. In these guidelines, the European Advisory Board on Cat Diseases (ABCD) presents a comprehensive review of feline infectious peritonitis (FIP). FCoV is primarily an enteric virus and most infections do not cause clinical signs, or result in only enteritis, but a small proportion of FCoV-infected cats develop FIP. The pathology in FIP comprises a perivascular phlebitis that can affect any organ. Cats under two years old are most frequently affected by FIP. Most cats present with fever, anorexia, and weight loss; many have effusions, and some have ocular and/or neurological signs. Making a diagnosis is complex and ABCD FIP Diagnostic Approach Tools are available to aid veterinarians. Sampling an effusion, when present, for cytology, biochemistry, and FCoV RNA or FCoV antigen detection is very useful diagnostically. In the absence of an effusion, fine-needle aspirates from affected organs for cytology and FCoV RNA or FCoV antigen detection are helpful. Definitive diagnosis usually requires histopathology with FCoV antigen detection. Antiviral treatments now enable recovery in many cases from this previously fatal disease; nucleoside analogues (e.g., oral GS-441524) are very effective, although they are not available in all countries.

Feline Injection-Site Sarcoma and Other Adverse Reactions to Vaccination in Cats.

Vaccine-associated adverse events (VAAEs), including feline injection-site sarcomas (FISSs), occur only rarely but can be severe. Understanding potential VAAEs is an important part of informed owner consent for vaccination. In this review, the European Advisory Board on Cat Diseases (ABCD), a scientifically independent board of feline medicine experts, presents the current knowledge on VAAEs in cats, summarizing the literature and filling the gaps where scientific studies are missing with expert opinion to assist veterinarians in adopting the best vaccination practice. VAAEs are caused by an aberrant innate or adaptive immune reaction, excessive local reactions at the inoculation site, an error in administration, or failure in the manufacturing process. FISS, the most severe VAAE, can develop after vaccinations or injection of other substances. Although the most widely accepted hypothesis is that chronic inflammation triggers malignant transformation, the pathogenesis of FISS is not yet fully understood. No injectable vaccine is risk-free, and therefore, vaccination should be performed as often as necessary, but as infrequently as possible. Vaccines should be brought to room temperature prior to administration and injected at sites in which FISS surgery would likely be curative; the interscapular region should be avoided. Post-vaccinal monitoring is essential.