Monitoring parasitic infections in wildlife is essential for assessing ecosystem health and pathogen dynamics, particularly in apex predators like the wolf (Canis lupus). As top-level carnivores with wide-ranging habitats and diverse interactions with prey, wolves can serve as effective sentinels for the circulation of parasitic agents within ecosystems. The present study aimed at monitoring parasitic infections, particularly those of zoonotic importance, in wolves from central Italy. The study was conducted on 169 wolf carcasses recovered between 2018 and 2024. Leishmania spp. and Trichinella spp. infections were evaluated using molecular techniques (on 169 spleen and 150 striated muscle samples, respectively), while intestinal and extra-intestinal parasites were investigated using flotation and Baermann tests (147 fecal samples). Of the 169 wolves included in the study, 89 (52.66

IntroductionDefinitive guidelines for the classification of cases in Veterinary Forensic Medicine (VFM) are currently unavailable. An attempt to classify lesions observed in forensic necropsies was proposed in 2022 following the codes of the ICD-11. The archives of the Istituti Zooprofilattici Sperimentali (IIZZSS) are structured to identify forensic pathology cases using pre-coded information only on wild animals' mortality and suspected animal poisonings at the time of acceptance. In contrast, other forensic documentation sources, including non-pre-coded necropsy diagnoses, require interpretation based on laboratory test results and accompanying expert opinions.MethodsThis study proposes the classification of previously unclassified VFM cases through a three-step approach: (1) data analysis, (2) review and classification of expert opinions by assigning labels corresponding to the main forensic topics, and (3) organization of diagnoses into categories and subcategories. Cases recorded in the database of the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise between 2013 and 2023 were retrospectively evaluated.ResultsA total of 1,221 non-coded cases with an expert opinion were identified. Among these, 405 cases (33.16%) were classified as potential Veterinary Forensic Cases and categorized into five macro-categories and 19 subcategories. The main cause of mortality differed according to animal type: injury was the most frequent cause in pets, attack in livestock, and unlawful killing in wildlife.DiscussionThis classification approach enabled the identification and organization of previously unclassified VFM cases within the institutional database. Poisoning cases and wild animal deaths caused by vehicular collisions were excluded as they were already pre-classified. Therefore, the reported percentages refer only to this subset of cases and do not reflect the overall mortality rates in the region.

Both domestic and wild animals are frequently affected by accidental, intentional, or illegal poisoning globally, but data from some regions, including Italy, remain scarce. This study aimed to characterize animal poisoning events in a central Italian region over the period 2011-2020. A total of 2722 suspected cases were analyzed, of which 51.3% were confirmed positive for at least one poison. Among the 1397 confirmed cases, 863 animal carcasses and 534 poisoned baits were recorded, corresponding to 762 distinct poisoning events. The most frequently involved toxicants were metaldehyde, carbamates, and organophosphorus pesticides, followed by anticoagulant rodenticides and other compounds. Spatial analysis revealed distinct patterns in the use of toxicants. Sub-regional areas encompassing national and regional parks were primarily associated with agricultural pesticides, whereas urban areas were dominated by metaldehyde and anticoagulant rodenticides. These differences suggest that, in addition to their illegal nature, poisoning events may reflect underlying socio-environmental conflicts between humans and animals. The results highlight the value of an interdisciplinary approach to mitigate poisoning, involving coordinated efforts among scientific, veterinary, enforcement, and public stakeholders. Such strategies could reduce both the frequency and ecological impact of this illegal practice.

Vagococcus lutrae is an emerging pathogen that can cause severe disease, especially in immunocompromised patients. Unlike Vagococcus fluvialis, which is recognized as a human and animal pathogen, there are few reports of V. lutrae from human and animal infections. In humans, it has been reported in patients with severe skin lesions and bloodstream infections. In veterinary medicine, V. lutrae was accidentally isolated from a Eurasian otter and a largemouth bass, and only once from the genitourinary tract of a pig with a urinary tract infection. However, the prevalence may be underestimated due to difficulties in identification using traditional methods. In addition, V. lutrae could be a carrier of resistance genes and contribute to the spread of AMR. A neutered male cat with feline urological syndrome underwent urethrostomy surgery due to serious problems with dysuria and urolithiasis that could not be resolved through catheterizations. Urine culture revealed the presence of Vagococcus lutrae. The strain showed resistance genes against aminoglycoside, lincosamide, streptogramin a and b, pleuromutilin, macrolide, tetracycline, oxazolidinone, and amphenicol classes. We report the first isolation of V. lutrae from the urinary tract of a cat.

ABSTRACT Members of the RNA virus order Nidovirales infect hosts ranging from marine invertebrates to terrestrial mammals. As such, understanding the determinants of host range in this group of viruses, as well as their patterns of emergence and disease potential, is of clear importance. The Mesoniviridae are a recently documented family within the Nidovirales . To date, mesoniviruses have only been associated with the infection of arthropod species, particularly mosquitoes, and hence are regarded as insect-specific viruses (ISVs). Herein, we report the first detection of a mesonivirus—Alphamesonivirus-1 —in mammals. Specifically, we utilized genomic and histological techniques to identify Alphamesonivirus-1 in lung and lymph node tissues of two horses (a mare and its foal) from Italy that succumbed to an acute respiratory syndrome. The genome sequences of Alphamesonivirus-1 obtained from the two horses were closely related to each other and to those from a local Culex mosquito pool and an Alphamesonivirus-1 previously identified in Italy, indicative of ongoing local transmission. The discovery of Alphamesonivirus-1 in horse tissues prompts further investigation into the host range of mesoniviruses, the possible role of insect-specific viruses in mammalian disease processes, the determinants of and barriers to cross-species virus transmission, and the potential epizootic threats posed by understudied viral families. IMPORTANCE Alphamesoniviruses, members of the family Mesoniviridaeare, are considered insect-specific RNA viruses with no known association with vertebrate hosts. Herein, we report the identification of Alphamesonivirus-1 in mammals. Using detailed molecular and histological analyses, we identified Alphamesonivirus-1 in lung and lymph node tissues of two horses that presented with an acute respiratory syndrome and that was phylogenetically related to virus sequences found in local Culex mosquitoes. Hence, Alphamesoniviruses may possess a broader host range than previously believed, prompting the investigation of their possible role in mammalian disease. This work highlights the need for increased surveillance of atypical viruses in association with unexplained respiratory illness, including those commonly assumed to be insect-specific, and may have implications for epizootic disease emergence.

Streptococcus equi subsp. zooepidemicus (SEZ) is a bacterium that primarily affects horses but can also infect other animals and humans. In 2021-2022, two SEZ outbreaks occurred in Abruzzo, Italy: one in humans linked to unpasteurized cheese (37 cases) and another in donkeys (4 deaths). These events led researchers to investigate SEZ in horses, donkeys, and a mule in the regions of Abruzzo and Molise, focusing on antibiotic resistance and genetic traits. A total of 490 nasal and genital swabs were collected from equids and analyzed for SEZ presence, with 61 positive samples. Isolated strains underwent antimicrobial susceptibility testing to assess their resistance to various antibiotics. Genetic analysis was performed to detect antibiotic resistance and virulence genes. Multi-locus sequence typing (MLST) was used to compare SEZ strains and identify potential epidemiological links. The study found that 37.7% of SEZ strains were resistant to at least one antibiotic, with tetracycline and trimethoprim/sulfamethoxazole being the most common. Additionally, 8.2% of strains showed MDR. Genetic analysis identified resistance genes such as tet(W) and erm(B), along with virulence genes like Fbp54. No major genetic clusters were detected, but ST61 was identified, similar to the human outbreak. The presence of antibiotic-resistant SEZ highlights the importance of monitoring and controlling its spread under a One Health approach, considering human, animal, and environmental health.

Streptococcus equi subsp. zooepidemicus (SEZ) is a major problem in equine veterinary medicine. Typically, a commensal in horses, SEZ can cause severe disease including respiratory infections, septicaemia and reproductive tract infections under certain conditions. Recent evidence suggests that humans can also develop severe disease infection through direct contact with infected animals or the consumption of contaminated unpasteurised milk and milk products. This study investigates SEZ strains isolated from nasal swabs of equidae in central Italy in 2023 to describe the epidemiology and genomic characteristics of circulating strains. A sampling plan was implemented to randomly collect nasal swabs from equid farms in the Abruzzo and Molise regions. In addition, a sampling form was designed to collect information on risk factors related to the presence of the bacterium and the potential development of respiratory disease. Relative risk was used to measure the association between the presence of SEZ in the samples and various variables. The swabs were analysed by real-time PCR and isolation. To confirm the identification and characterise the strains, the isolates were fully sequenced by next-generation sequencing (NGS) using the Illumina platform. A total of 478 animals from 99 farms were sampled and 30% of the animals tested positive for SEZ (79% horses, 20% donkeys, 1% mules). Forty-five percent of the farms tested positive for SEZ. Monitoring the clonal spread of SEZ is essential to understand the ecology of this emerging zoonotic pathogen, to assess the risk, and to implement effective control measures. In addition, genomic assessments are recommended to investigate the pathogenicity of circulating strains. This study provides a comprehensive understanding of the epidemiology and genomic diversity of SEZ strains isolated in central Italy.

As members of the RNA virus order Nidovirales include those that infect hosts ranging from marine invertebrates to terrestrial mammals, understanding their emergence, host range and disease potential is of clear importance. The Mesoniviridae are a recently documented family of viruses within the Nidovirales. To date, mesoniviruses have only been associated with the infection of arthropods, particularly mosquitoes. Herein, we report the first detection of a mesonivirus – Alphamesonivirus-1 – in mammals. Specifically, we utilized genomic and histological techniques to identify the presence of Alphamesonivirus-1 in lung and lymph node tissues of two horses that succumbed to an acute respiratory syndrome. Notably, no other pathogens typically associated with respiratory disease in horses were detected in these samples. Counter to the previous contention that mesoniviruses only infect insects, our findings suggest a potentially broader host range and cross-species transmission of these viruses. The genome sequences of Alphamesonivirus-1 obtained from the two horses were closely related to those from a local Culex mosquito pool as well as an Alphamesonivirus-1 previously in identified Italy, suggestive of ongoing local transmission. The discovery of Alphamesonivirus-1 in tissues from diseased horses not only challenges current understandings of mesonivirus host range, but prompts further investigation into the role of insect-specific viruses in mammalian disease processes. Our results emphasize the importance of considering atypical pathogens in cases of unexplained animal deaths and suggest a potential zoonotic threat posed by previously overlooked viral families.

Coxiella burnetii is the causative agent of Q fever. The main reservoirs for this bacterium, which can lead to human infection, in our region are typically cattle, goats, and sheep. In animals, C. burnetii infection is often detected due to reproductive problems. European Member States are required to report confirmed cases annually, but the lack of uniform reporting methods makes the data rather inconsistent. The Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise is involved in official controls to identify the causes of abortions, monitor suspected or positive herds, evaluate suspected infections in pets and humans, monitor the spread in wildlife, etc. In this paper, we summarize the presence of C. burnetii over the last five years (2019–2023). Additionally, a detailed overview of C. burnetii infection in wild and domestic animals is provided. Five hundred sixty animals—including cattle; goats; sheep; wild animals, such as deer, boars, wolves, roe deer, owls, and otters; buffalo; dogs; horses; cats; and a donkey—and six human samples were tested by real-time PCR on the transposase gene IS1111 to detect C. burnetii. The MST profile was identified in some of the samples. Outbreaks of C. burnetii occurred in four herds. In one of them, it was possible to follow the outbreak from inception to eradication by evaluating the effect of vaccination on real-time PCR Ct values. A total of 116 animals tested positive for C. burnetii, including 73 goats, 42 sheep, and one bovine. None of the other samples tested positive. The strains for which the ST was performed were identified as ST79, a strain that has been present in the area for more than ten years. The effect of vaccination on the reduction of positive samples and the variation of real-time PCR Ct values was evaluated in strict correlation.

Streptococcus equi sub. zooepidemicus (SEZ) is described as a commensal bacterium of several animal species, including humans. Growing evidence supports the potential role of SEZ in the onset and progression of severe clinical manifestations of diseases in horses and other animals. In the present communication, we describe the diagnostic procedure applied to characterize the streptococcal infections caused by a novel SEZ sequence type (ST525) in donkeys raised on a farm in Abruzzo, Italy. The diagnostic process began with anamnesis and anatomopathological analysis, which revealed a severe bacterial suppurative bronchopneumonia associated with systemic vascular damage and haemorrhages. Then, SEZ infection was confirmed by applying an integrative diagnostic strategy that included standard bacterial isolation techniques, analytical tools for bacteria identification (MALDI-TOF MS), and molecular analysis (qPCR). Furthermore, the application of the whole-genome sequencing approach helped us to identify the bacterial strains and the virulence factors involved in animal diseases. The novel SEZ-ST525 was identified in two cases of the disease. This new sequence type was isolated from the lung, liver, and spleen in Case 1, and from retropharyngeal lymph nodes in Case 2. Moreover, the presence of the virulence gene mf2, a virulence factor carried by prophages in Streptococcus pyogenes, was also found for the first time in an SEZ strain. The results of the present study highlight the need to apply an integrated diagnostic approach for the identification and tracking of pathogenic strains of SEZ, shedding new light on the re-evaluation of these bacteria as a causative agent of disease in animals and humans.

Welfare conditions in shelters, where dogs might be housed for a long period of time, may have a possible correlation with the occurrence of bacterial pathogens and their antimicrobial resistance (AMR). In this study, we assessed the occurrence of AMR in 54 strains of Escherichia coli isolated from dogs housed in 15 Italian shelters and we correlated the resistance patterns to animal welfare. We also aimed to evaluate the presence of specific pathogens with zoonotic potential in sheltered dogs. Thus, nasopharyngeal, rectal, and oral swabs were collected from a group of 20 dogs in each shelter and totaled 758 swabs. We identified 9 Staphylococcus pseudointermedius, 1 Pasteurella multocida, 9 Staphylococcus aureus, 12 Campylobacter spp., 54 Escherichia coli, 2 Salmonella enterica, and 246 Capnocytophaga spp. The antimicrobial susceptibility was assessed for the E. coli isolates using a panel of 14 antibiotics. The highest level of relative AMR was recorded for ampicillin and sulfamethoxazole. The association found between AMR and the levels of animal welfare scores in shelters was evident although not statistically significant. These results support the hypothesis that the good management of shelters can increase the level of animal welfare, thus reducing the use of antibiotics and, as a consequence, the AMR occurrence found in dogs that share their domestic environment with humans.

Brucella RB51 is a live modified vaccine. Its use in water buffalo has been proposed using a vaccination protocol different to that used for cattle, but knowledge of the long-term effects of RB51 vaccination in this species remains incomplete. The aim of the study was to evaluate the safety and kinetics of antibody responses in water buffaloes vaccinated according to the protocol described for the bovine species in the WOAH Manual, modified with the use of a triple dose. Water buffaloes were vaccinated with the vaccine RB51. A booster vaccination was administered at 12 months of age. When turning 23-25 months old, female animals were induced to pregnancy. RB51-specific antibodies were detected and quantified using a CFT based on the RB51 antigen. Vaccinated animals showed a positive serological reaction following each vaccine injection, but titers and the duration of the antibody differed among animals. For 36 weeks after booster vaccination, the comparison of CFT values between vaccinated and control groups remained constantly significant. Afterwards, antibody titers decreased. No relevant changes in antibody response were recorded during pregnancy or lactation. In conclusion, results indicated that the vaccination schedule applied is safe and allows for vaccinated and unvaccinated controls to be discriminated between for up to 8 months after booster vaccination.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

During November 2021-May 2022, we identified 37 clinical cases of Streptococcus equi subspecies zooepidemicus infections in central Italy. Epidemiologic investigations and whole-genome sequencing showed unpasteurized fresh dairy products were the outbreak source. Early diagnosis by using sequencing technology prevented the spread of life-threatening S. equi subsp. zooepidemicus infections.

The isolation of B. abortus RB51 vaccine strain from a milk sample in a water buffalo farm in southern Italy emphasizes the risk to public health of consuming contaminated milk or milk products following illegal vaccination.

Although there are increasing reports on the prevalence of Listeria monocytogenes in wild species, this is the first case of listeriosis in sea turtle. An adult female Caretta caretta was rescued after being stranded alive along the coast of the Abruzzo region (Italy) in summer 2021. The turtle died in 6 days due to respiratory failure. The necropsy showed widespread organ lesions, such as yellow foci of necrosis in many organs, gastrointestinal erosions, pericarditis, and granulomatous pneumonia. Microbiological and histological analyses were performed on several organs. Listeria monocytogenes was isolated from multiple organs, indicating a case of septicaemic listeriosis, and the genome was sequenced and characterized. All the colonies analysed belonged to the same strain serogroup IVb, ST388, and CC388.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are emerging worldwide. Here, we report the complete genome sequences of 13 severe acute SARS-CoV-2 strains belonging to lineage B.1.525 (variant η).

BACKGROUND:Few cases of scedosporiosis have been reported in animals, but the true prevalence is probably underestimated due to a lack of awareness. Scedosporiosis in dogs has often been associated with localized infection (i.e., nasal infection, eumycetoma, or keratomycosis) or, in rare cases, disseminated infections.CASE PRESENTATION:This case report describes the clinical and pathological features and the diagnostic process of a rare systemic and fatal fungal infection in a dog caused by Scedosporium apiospermum. A 10-month-old female Maremmano-Abruzzese sheepdog showing weakness, lethargy, lateral decubitus, miosis and muscular rigidity was presented. Rodenticide poisoning was clinically suspected for the differential diagnosis. However, postmortem examinations revealed the presence of a swollen and soft subcutaneous nodule located near the right inguinal breast, which was associated with massive enlargement of the inguinal lymph nodes and small disseminated, cream-colored nodules in the kidneys and mesentery. Multiple fungal pyogranulomas were observed upon histological examination. Fungal isolation from the kidneys, breast and inguinal lymph nodes was performed. The internal transcribed spacer (ITS) sequences from the fungal colony DNA were searched in BLAST in the NCBI GenBank for species identification. The sequences of the fungi isolated from the kidney and breast cultures showed 100% sequence identity with sequences from Scedosporium apiospermum.CONCLUSIONS:This report shows that Scedosporium apiospermum may act as a primary pathogen in young and apparently healthy dogs and represents an important pathogen that should be considered during the diagnostic process, particularly when a fungal infection is suspected.

Rickettsiae have worldwide occurrence and rickettsiosis are widely recognized as emerging infections in several parts of the world. For decades, it was thought that a single pathogenic tick-borne spotted fever group (SFG), Rickettsia, occurred in each continent. Nowadays, thanks to molecular biology, new species of Rickettsia responsible for disease in humans are continuously identified worldwide. In a framework of diagnostic activities of the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Gaporale" and considering some reports of suspected human clinical cases of rickettsiosis, a survey on ticks collected form animals and humans was carried out with the aim to identify the Rickettsia species circulating in Abruzzi and Molise regions. A total of 603 ticks, previously identified at species level by morphology, pooled into 178 tick samples, were tested by pan-Rickettsia RealTime PCR. DNA from specimens positive for Rickettsia spp. was then sequenced in order to identify the Rickettsia species involved. The highest infection rate was detected in Dermacentor marginatus followed by Ixodes ricinus. The selected targets for this purpose were OmpA and gltA. Rickettsia slovaca, Rickettsia monacensis, Rickettsia massiliae, Rickettsia conorii, Rickettsia aeschlimannii, Rickettsia helvetica, Rickettsia raoultii, and Rickettsia felis - like organisms were identified in this study. These are the first data available in the literature for the circulation of SFG Rickettsia species in the selected geographical area. Results made evidence of high rate of infection in ticks. All Rickettsia species detected have been previously involved in human infection. The diversity of Rickettsia detected, and tick species collected reflects the biodiversity in term of wildlife and environment of the area. An association between Rickettsia species, ticks, and the relationships with vertebrate host species are discussed. Due to the peculiar eco-biology of each Rickettsia species, the use of diagnostic tools able to identify Rickettsia at the species level is thus recommended in order to assess the risk for humans and to elucidate more precise etiological diagnosis in clinical cases.

A case of Vibrio parahaemolyticus- and V. alginolyticus-associated meningo-encephalitis in a bottlenose dolphin (Tursiops truncatus) found stranded along the Adriatic coast of Italy in 2016 is herein reported, along with a minireview on V. parahaemolyticus and V. alginolyticus infections in aquatic mammals. Macroscopically, two abscesses were found in the dolphin's forebrain, along with an extensive, bilateral, parasitic broncho-pneumonia. Histologically, a suppurative-to-pyogranulomatous meningo-encephalitis involved the brain but not the cerebellum. Microbiological investigations yielded isolation of V. parahaemolyticus and V. alginolyticus from the aforementioned abscesses and from the brain parenchyma, respectively, with simultaneous recovery of Shewanella algae from the heart and of Photobacterium damselae from a blowhole swab. Although V. parahaemolyticus and V. alginolyticus, which are widely distributed across marine ecosystems worldwide, likely played a role in the development of the suppurative meningo-encephalitis in this dolphin, we are not aware of previous isolations of any of these two bacteria neither from cetacean brain lesions, nor from abscesses in aquatic mammals.