Comparative analysis of rare earth elements concentrations in domestic dogs and Apennine wolves of Central Italy: Influence of biological, nutritional, and lifestyle factors.

Rare Earth Elements (REEs) are strategical elements playing a crucial role in the industry, especially in producing high-tech materials. Therefore, REEs are new contaminants of emerging concerns. However, due to the lack of exposure data on REE occurrence in environmental matrices, especially in European countries, it is still tricky to establish environmental background levels to assess the ecotoxicological risk related to REEs exposure. The present study aimed to evaluate the liver concentrations of REEs in domestic dogs (Canis lupus familiaris) and Apennine wolves (Canis lupus italicus) living in the Abruzzo region, Italy. Moreover, for the scope of the present study, the dog's group was divided according to their sex, age, lifestyle, and diet. Wolves were categorized concerning their sex and genetic characteristics. Liver samples from dogs and wolves were collected during diagnostic necropsies from carcasses, sample mineralization was performed by a microwave digestion system with a single reaction chamber, and simultaneous determination of the presence of REEs was performed by Inductively Coupled Plasma Mass Spectrometer (Q-ICP-MS) using standard mode for all rare earth elements except scandium (Sc) which was acquired in kinetic energy discrimination (KED) mode. Hepatic concentrations of REEs were statistically significantly higher in wolves compared to dogs. Moreover, significant differences in REEs concentrations arose also from the genetic type of wolf, since "pure wolves" had higher liver concentrations of REEs compared to wolf-dog hybrids. Female and adult dogs also showed elevated REEs compared to male and juvenile dogs, while no significant differences were demonstrated for dogs' diet and lifestyle. The results of the present study confirm the exposure of domestic and wild carnivores to REEs, showing also the ability of REEs to accumulate in carnivore livers, suggesting the potential role of this species as an alternative bioindicator.

Brucella abortus Strain RB51 Administered to Prepubescent Water Buffaloes, from Vaccination to Lactation: Kinetics of Antibody Response and Vaccine Safety.

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.

Identification of the Novel Streptococcus equi subsp. zooepidemicus Sequence Type 525 in Donkeys of Abruzzo Region, Italy.

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.

Severe Streptococcus equi Subspecies zooepidemicus Outbreak from Unpasteurized Dairy Product Consumption, Italy.

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.

Morphological and Immunohistochemical Characterization of an Oral Metastatic Carcinosarcoma in a Cat.

A 15-year-old neutered male mixed breed Domestic Shorthair cat was presented for a rapidly growing, intraoral soft gingival mass on the left mandibular region. The neoplastic tissue consisted histologically of two distinct malignant cell populations: spindle cells arranged in bands and epithelioid cells arranged in cords. A few multinucleated giant cells were scattered among the neoplastic cells. Spindle cells and multinucleated giant cells strongly expressed vimentin while epithelial cells strongly expressed pancytokeratins. On the basis of the histological and immunohistochemical results, a diagnosis of oral carcinosarcoma was made. After 2 months, due to the extent of disease and poor prognosis, the cat was euthanized. Necropsy revealed a markedly enlarged, multilobulated white-pink neoplastic mass that had originated from the left side of the sublingual region and involved the coronoid process of the left mandibular bone. The cut surface of the enlarged left submandibular lymph node was glistening, whitish-tan in colour with a multinodular appearance, suggestive of metastasis and confirmed by histological examination. Oral carcinosarcoma is uncommonly recorded in humans and dogs and, to the best of our knowledge, this is the first reported case in a cat.

The Italian Network of Laboratories for Veterinary Oncology (NILOV) 2.0: Improving Knowledge on Canine Tumours.

Advances in tumour research are crucial, and comparative oncology can improve the knowledge in several ways. Dogs are not only models of specific naturally occurring tumours but can also be sentinels of environmental exposures to carcinogens, as they share the same environment with their owners. The purpose of this work was to describe the data collected by The Italian Network of Laboratories for Veterinary Oncology in the first 9 years of activity (2013-2021) and to evaluate their potential epidemiological significance. Frequencies of tumour topographies and main morphologies in dogs were described, analysed and compared, calculating age-adjusted proportional morbidity ratios and considering several risk factors (breed, sex, period and region of residence). These observations allowed us to highlight differences not only in morphology and topography of some tumours but also to formulate hypotheses on the potential role of some risk factors, e.g., neutering/spaying or geographical location. In our opinion, the results of this case series confirm the importance of initiating and consolidating animal cancer registration initiatives that would facilitate the possibility of conducting multicentric collaborative studies to deepen the knowledge of the epidemiology of tumours in dogs from a comparative perspective.

Detection of Brucella abortus Vaccine Strain RB51 in Water Buffalo (Bubalus bubalis) Milk.

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.

Validation of AmpliSeq NGS Panel for BRCA1 and BRCA2 Variant Detection in Canine Formalin-Fixed Paraffin-Embedded Mammary Tumors.

Mammary carcinomas are the most common neoplasms observed in women and in female dogs. Canine mammary tumors show epidemiological, clinical, genetic, and prognostic characteristics comparable to human breast cancers. The recent introduction of next generation sequencing (NGS) technologies has greatly improved research and diagnostics for humans, while these new tools still need to be implemented in animal models. In this study we developed and validated an AmpliSeq Panel assay for the identification of BRCA variants in twenty-two different dogs. The amplicon mean coverage was 5499× and uniformity was higher than 98% in all samples. The results of germline single nucleotide variants (SNVs) and insertions/deletions (INDELs) were fully concordant regardless of the types of samples considered (blood, fresh and FFPE tissues). Moreover, despite the high DNA degradation observed in older FFPE blocks (>5 years), the assay allowed full coverage of all amplicons for downstream analyses. We consider the NGS panel developed in this study as a useful tool for expanding information on BRCA genes in the veterinary field and for human health from a comparative oncology perspective.

Phenotypic and Genetic Characterization of Klebsiella pneumoniae Isolates from Wild Animals in Central Italy.

Despite Klebsiella pneumoniae being widely recognized as a nosocomial pathogen, there is a critical lack in defining its reservoirs and sources of infections. Most studies on risk factors have focused on multidrug-resistant (MDR) isolates and clinically-oriented questions. Over a two-year period, we sampled 131 wild animals including mammal and bird species from three regions of Central Italy. All typical colonies isolated from the analytical portions were confirmed by real-time PCR and identified by MALDI-TOF mass spectrometry (MALDI-TOF MS). All confirmed K. pneumoniae isolates were tested for antimicrobial susceptibility to 29 antimicrobials and subjected to whole genome sequencing. Typical colonies were detected in 17 samples (13%), which were identified as K. pneumoniae (n = 16) and as K. quasipneumoniae (n = 1) by MALDI-TOF MS. The antimicrobial susceptibility profile showed that all the isolates were resistant to ß-lactams (ceftobiprole, cloxacillin, cefazolin) and tetracycline; resistance to ertapenem and trimethoprim was observed and nine out of 16 K. pneumoniae isolates (56.2%) were classified as MDR. Genomic characterization allowed the detection of fluoroquinolone resistance-associated efflux pumps, fosfomycin and ß-lactamase resistance genes, and virulence genes in the overall dataset. The cluster analysis of two isolates detected from wild boar with available clinical genomes showed the closest similarity. This study highlights the link between humans, domestic animals, and wildlife, showing that the current knowledge on this ecological context is lacking and that the potential health risks are underestimated.

Canine brucellosis due to Brucella canis: description of the disease and control measures.

Brucellosis is a contagious disease caused by bacteria of the genus Brucella, which can affect different animal species. Dogs may occasionally be infected with B. abortus, B. melitensis or B. suis, or by the endemic form of the disease, caused by B. canis. Among the brucellosis­affecting domestic animals, that of the dog is certainly the least frequent, but also the least studied. Canine brucellosis due to B. canis represents the dog­specific brucellosis, both because it is the main susceptible animal species, and because it constitutes its fundamental reservoir of infection. The disease can also affect humans, although its course does not assume the characteristics of severity typical of the infection determined by the 'classical' species of the genus Brucella. In Italy, there are frequent imports of dogs from countries where the disease is present, often with non­controlled movements and without sanitary controls. Considering that the zoonotic potential of the disease can be favored by the close cohabitation between man and dog, which occurs especially in urban environments, canine brucellosis has to be regarded as a public health problem susceptible to introduction and spread in the Italian territory.

First Isolation of Brucella canis from a breeding kennel in Italy.

Brucella canis has been isolated for the first time in Italy in a commercial breeding kennel. It was diagnosed after a deep investigation related to the onset of reproductive disorders. Animals were tested with direct and indirect techniques. The agent was first detected in two Chihuahua aborted foetuses by direct culture. Further, it was also isolated from blood samples of dogs hosted in the kennel, which also showed reaction to conventional serological tests (microplate serum agglutination test). The isolates were identified as B. canis by standard microbiological methods and a Bruce­ladder multiplex PCR. To investigate the genomic diversity, whole genome sequencing was used, applying the core genome Multilocus Sequence Typing (cgMLST ). In a first round of serological testing performed on 598 animals, 269 (46.1%) tested positive. In the second round of laboratory testing carried out 4­5 weeks apart, the number of serologically positive dogs was 241 out of 683 tested (35.3%), while the number of dogs positive to isolation was 68 out of 683 tested (10.0%). The PCR showed a lack of sensitivity when compared to direct isolation. The epidemiological investigation did not identify the source of the infection, given the time elapsed from the onset of abortions to the definitive diagnosis of B. canis infection in the kennel. The genomic analyses featured the strains as ST21 and, according to the cgMLST, revealed the presence of a tight cluster with a maximum diversity of four allelic differences. The observed limited genomic variation, largely within the known outbreak cut­offs, suggests that the outbreak herein described was likely caused by a single introduction. Moreover, in a broader scale comparison using the public available genomes, we found that the closest genome, isolated in China, differed by more than 50 alleles making not possible to find out the likely origin of the outbreak. The lack of updated data on B. canis genome sequences in the public databases, together with the limited information retrieved from the epidemiological investigations on the outbreak, hampered identification of the source of B. canis infection.

First report of Trichinella pseudospiralis in a wolf (Canis lupus italicus).

Within the genus Trichinella, Trichinella pseudospiralis is the only recognized non-encapsulated species known to infect mammals and birds. In October 2020, larvae recovered from muscle tissues of a wolf (Canis lupus italicus) originating from Molise Region, Central Italy, were molecularly confirmed as those of Trichinella britovi and T. pseudospiralis. This is the first detection of T. pseudospiralis from a wolf. In Italy, this zoonotic nematode was detected in a red fox (Vulpes vulpes), three birds (Strix aluco, Athene noctua, Milvus milvus) and five wild boars (Sus scrofa), and was also identified as the etiological agent of a human outbreak of trichinellosis in 2015. Since T. pseudospiralis is rarely reported from carnivore mammals in comparison to the encapsulated species frequently detected in these hosts, this finding opens the question of the role of carnivores as reservoirs for this parasite.

SARS-CoV-2 replicates in respiratory ex vivo organ cultures of domestic ruminant species.

There is strong evidence that severe acute respiratory syndrome 2 virus (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, originated from an animal reservoir. However, the exact mechanisms of emergence, the host species involved, and the risk to domestic and agricultural animals are largely unknown. Some domestic animal species, including cats, ferrets, and minks, have been demonstrated to be susceptible to SARS-CoV-2 infection, while others, such as pigs and chickens, are not. Importantly, the susceptibility of ruminants to SARS-CoV-2 is unknown, even though they often live in close proximity to humans. We investigated the replication and tissue tropism of two different SARS-CoV-2 isolates in the respiratory tract of three farm animal species - cattle, sheep, and pigs - using respiratory ex vivo organ cultures (EVOCs). We demonstrate that the respiratory tissues of cattle and sheep, but not of pigs, sustain viral replication in vitro of both isolates and that SARS-CoV-2 is associated to ACE2-expressing cells of the respiratory tract of both ruminant species. Intriguingly, a SARS-CoV-2 isolate containing an amino acid substitution at site 614 of the spike protein (mutation D614G) replicated at higher magnitude in ex vivo tissues of both ruminant species, supporting previous results obtained using human cells. These results suggest that additional in vivo experiments involving several ruminant species are warranted to determine their potential role in the epidemiology of this virus.

A COVID-19 Hotspot Area: Activities and Epidemiological Findings.

By late March 2020, Villa Caldari, a small village of the municipality of Ortona (Abruzzo region), was registering an incidence rate of COVID-19 cases ten times greater than the overall municipality and was declared a hotspot area. Twenty-two days later, epidemiological investigation and sampling were performed, to evaluate SARS-CoV-2 circulation and the presence of SARS-CoV-2 antibodies. Overall, 681 nasopharyngeal swabs and 667 blood samples were collected. Only one resident of the village resulted in being positive for RNA viral shedding, while 73 were positive for SARS-CoV-2 antibodies. The overall seroprevalence was 10.9%. The difference between the seroprevalence of infection in asymptomatic and symptomatic individuals was significant (χ2 = 14.50 p-value = 0.0001). Amongst the residents positive for antibodies, fatigue and/or muscle pain, fever and anosmia were the most experienced symptoms, whose most frequent onset was observed during the first two weeks of March. Familial and habit-related clusters were highlighted. Nevertheless, the investigations showed a low SARS-CoV-2 circulation in the village at the time of the sampling, demonstrating virus transmission could be limited when strict emergency measures are followed. Given the favorable results, the emergency measures were then lifted.

Disseminated Scedosporium apiospermum infection in a Maremmano-Abruzzese sheepdog.

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.

Detection of Astrovirus in a Cow with Neurological Signs by Nanopore Technology, Italy.

In this study, starting from nucleic acids purified from the brain tissue, Nanopore technology was used to identify the etiological agent of severe neurological signs observed in a cow which was immediately slaughtered. Histological examination revealed acute non-suppurative encephalomyelitis affecting the brainstem, cerebrum, cerebellum, and medulla oblongata, while by using PCR-based assays, the nucleic acids of major agents for neurological signs were not detected. By using Nanopore technology, 151 sequence reads were assigned to Bovine Astrovirus (BoAstV). Real-time RT-PCR and in situ hybridization (ISH) confirmed the presence of viral RNA in the brain. Moreover, using the combination of fluorescent ISH and immunofluorescence (IF) techniques, it was possible to detect BoAstV RNA and antigens in the same cells, suggesting the active replication of the virus in infected neurons. The nearly whole genome of the occurring strain (BoAstV PE3373/2019/Italy), obtained by Illumina NextSeq 500, showed the highest nucleotide sequence identity (94.11%) with BoAstV CH13/NeuroS1 26,730 strain, an encephalitis-associated bovine astrovirus. Here, we provide further evidence of the role of AstV as a neurotropic agent. Considering that in a high proportion of non-suppurative encephalitis cases, which are mostly indicative of a viral infection, the etiologic agent remains unknown, our result underscores the value and versatility of Nanopore technology for a rapid diagnosis when the PCR-based algorithm gives negative results.

Replication kinetics and cellular tropism of emerging reoviruses in sheep and swine respiratory ex vivo organ cultures.

Ex vivo organ cultures (EVOCs) are extensively used to study the cellular tropism and infectivity of different pathogens. In this study, we used ovine and porcine respiratory EVOCs to investigate the replication kinetics and cellular tropism of selected emerging reoviruses namely Pteropine orthoreovirus, an emerging bat-borne zoonotic respiratory virus, and atypical Bluetongue virus (BTV) serotypes which, unlike classical serotypes, do not cause Bluetongue, a major OIE-listed disease of ruminants. BTV failed to replicate in ovine EVOCs. Instead, PRV showed slight replication in porcine lower respiratory EVOCs and a more sustained replication in all ovine respiratory tissues. By confocal laser scanning microscopy, PRV was demonstrated to infect bronchiolar and type I pneumocytes of ovine tissues. Overall, respiratory EVOCs from different animal species, eventually obtained at slaughterhouse, are a useful tool for testing and preliminarily characterize novel and emerging viruses addressing the essential in vivo animal work. Further experiments are, indeed, warranted in order to characterize the pathogenesis and transmission of these emerging reoviruses.

Immunohistochemical investigations on Brucella ceti-infected, neurobrucellosis-affected striped dolphins (Stenella coeruleoalba).

Bacteria of the genus Brucella cause brucellosis, an infectious disease common to humans as well as to terrestrial and aquatic mammals. Since 1994 several cases of Brucella spp. infection have been reported in marine mammals worldwide. While sero-epidemiological data suggest that Brucella spp. infection is widespread globally, detecting Brucella spp.-associated antigens by immunohistochemistry (IHC) in tissues from infected animals is often troublesome. The present study was aimed at investigating, by means of IHC based upon the utilization of an anti-Brucella LPS monoclonal antibody (MAb), the central nervous system (CNS) immunoreactivity shown by B. ceti-infected, neurobrucellosis-affected striped dolphins. The aforementioned MAb, previously characterized by means of ELISA and Western Blotting techniques, was able to immunohistochemically detect smooth brucellae both within the CNS from B. ceti-infected striped dolphins and within a range of tissues from Brucella spp.-infected domestic ruminants. In conclusion, the results of the present study are of relevance both from the B. ceti infection's diagnostic and pathogenetic standpoints.

Peste des Petits Ruminants outbreaks in Tunisia in 2016.

In Tunisia, 86 outbreaks of Peste des Petits Ruminants (PPR) were reported in ovine and caprine herds in 2016. Molecular characterization of PPRV strains was carried out by partial sequencing of nucleoprotein (Np) gene from diagnostic specimens. The results showed that disease outbreaks were caused by virus strains closely related to PPRV strains collected in Egypt in 2014 and 2015.