Systemic tuberculosis by MYCOBACTERIUM BOVIS in a free-ranging MARSICAN brown bear (URSUS ARCTOS MARSICANUS): a Case report.

BACKGROUND: Mycobacterium bovis is known to have a wide host range and has been isolated from numerous free-ranging wildlife species, carnivores included. In bears, M. bovis has been previously reported only from a culture of pooled lymph nodes of a black bear (Ursus americanus) in the absence of lesions. The aims of this study were to describe gross and microscopic pathological findings of M. bovis tuberculosis in a deceased Marsican brown bear (Ursus arctos marsicanus). CASE PRESENTATION: In March 2014, an adult female Marsican brown bear was found in the Abruzzo, Lazio and Molise National Park (Italy) showing severe non-specific clinical signs. The animal died soon after its discovery and the carcass was submitted to post-mortem examination to identify the cause of death. The bear was diagnosed with a severe Mycobacterium bovis infection, with both pathological and microbiological aspects suggesting ongoing generalization. A presumptive diagnosis of mycobacterial infection was initially made based on gross findings. Histopathology showed the presence of acid-fast bacilli in all sampled tissues along with poorly organized granulomatous lesions. Slow-growing Mycobacterium sp. was isolated from multiple organs (intestine, mesenteric lymph nodes, liver, spleen, lung and kidneys). The PCR and sequencing algorithm identified the Mycobacterium sp. isolate as M. bovis. Spoligotyping demonstrated that the M. bovis isolate belonged to spoligotype SB0120. CONCLUSIONS: This is the first report of lethal M. bovis tuberculosis infection in a free-ranging brown bear. This pathogen could have serious adverse effects in an endangered relic population such as the Marsican brown bear. Stricter application of health regulations in force, surveillance of M. bovis infections in wild ungulates and carnivore scavengers, along with dismissal of supplementary feeding points intended for cattle or wildlife, are warranted to control the presence of bovine tuberculosis in wild and domestic animals in protected areas.

Fatal Outbreak in Tonkean Macaques Caused by Possibly Novel Orthopoxvirus, Italy, January 2015 1.

In January 2015, during a 3-week period, 12 captive Tonkean macacques at a sanctuary in Italy died. An orthopoxvirus infection was suspected because of negative-staining electron microscopy results. The diagnosis was confirmed by histology, virus isolation, and molecular analysis performed on different organs from all animals. An epidemiologic investigation was unable to define the infection source in the surrounding area. Trapped rodents were negative by virologic testing, but specific IgG was detected in 27.27% of small rodents and 14.28% of rats. An attenuated live vaccine was administered to the susceptible monkey population, and no adverse reactions were observed; a detectable humoral immune response was induced in most of the vaccinated animals. We performed molecular characterization of the orthopoxvirus isolate by next-generation sequencing. According to the phylogenetic analysis of the 9 conserved genes, the virus could be part of a novel clade, lying between cowpox and ectromelia viruses.

Encephalomyocarditis virus infection in Macaca sylvanus and Hystrix cristata from an Italian rescue centre for wild and exotic animals.

BACKGROUND: The Encephalomyocarditis virus (EMCV) is a small, non enveloped, positive sense single-stranded RNA virus in the genus Cardiovirus, family Picornaviridae, with two known serotypes. It is spread worldwide and infects a huge range of vertebrate hosts with zoonotic potential for humans. The pig is the mammal most likely to be impacted on with the disease, but EMCV occurrence has also been reported in non-human primates and in a variety of domestic, captive and wild animals. Until now, human cases have been very rare and the risk appears to be almost negligible in spite of human susceptibility to the infection. CASE PRESENTATION: Between September and November 2012 a fatal Encephalomyocarditis virus outbreak involving four Barbary macaques and 24 crested porcupines occurred at a rescue centre for wild and exotic animals in Central Italy. In this open-field zoo park located near Grosseto, Tuscany about 1000 animals belonging to different species, including various non-human primates were hosted at that time. Sudden deaths were generally observed without any evident symptoms or only with mild nonspecific clinical signs. The major gross change was characterised by grey-white necrotic foci in the myocardium and the same EMCV strain was isolated both in macaques and crested porcupines. Phylogenetic analysis has confirmed that only one EMCV strain is circulating in Italy, capable of infecting different animal species. CONCLUSIONS: This report confirms the susceptibility of non-human primates to the EMCV infection and describes the disease in porcupine, a common wild Italian and African species. No human cases were observed, but given the zoonotic potential of EMCV these findings are of importance in the context of animal-human interface.

Cetacean strandings in Italy: an unusual mortality event along the Tyrrhenian Sea coast in 2013.

An unusual mortality event involving cetaceans, mainly striped dolphins Stenella coeruleoalba (Meyen, 1833), occurred along the Tyrrhenian Sea coast of Italy during the first 3 mo of 2013. Based on post-mortem analyses carried out according to body condition on 66 dolphins (54% of stranded animals), several hypotheses to explain the causes of this mortality event were proposed. Although no definitive conclusions can be drawn, dolphin morbillivirus was deemed the most likely cause, although other infectious agents (including Photobacterium damselae damselae and herpesvirus) or environmental factors may also have contributed to this recent mortality event.

Pathological findings of Angiostrongylus vasorum infection in red foxes (Vulpes vulpes) from Central Italy, with the first report of a disseminated infection in this host species.

In Europe, the red fox (Vulpes vulpes) is considered the reservoir of Angiostrongylus vasorum, nematode residing in the pulmonary arteries and right heart of dogs and many species of wild carnivores. Italy is considered one of the European countries where this nematode is actually spreading. Between May 2007 and November 2013, 62 foxes found dead in Central Italy were necropsied. Right heart and pulmonary arteries were opened and checked for the presence of adult parasites. Impression smears from sectioned lungs were examined for the presence of first-stage larvae, and samples of lungs were processed for histological examination. In order to detect eventual disseminated infections, samples of heart, pulmonary lymph nodes, liver, kidneys, and brain of foxes positive for A. vasorum at necropsy or lungs histological examination were processed for histological examination. An overall prevalence of 43.5% was recorded. Light, mild, and severe lung lesions were detected in 33.3, 22.2, and 25.9% of infected animals, respectively. Severe lesions were more frequent in animals younger than 12 months. In five infected foxes (18.5%), no gross lesions were observed, while for three animals, angiostrongylosis was considered the cause of death. A case of disseminated angiostrongylosis was detected and another one was suspected. This is the firs report of disseminated angiostrongylosis in the fox.

Ectopic Pregnancy and T-Cell Lymphoma in a Eurasian Red Squirrel (Sciurus vulgaris): Possible Comorbidity and a Comparative Pathology Perspective.

Ectopic pregnancy (EP) is a life-threatening disease that affects humans and other mammals. Tumors causing ruptures of the reproductive tract have been identified as possible predisposing factors in human and veterinary medicine. We here describe a case of concomitant ectopic pregnancy and lymphoma in a Eurasian red squirrel found deceased in Italy and submitted to the public health laboratory Istituto Zooprofilattico Sperimentale del Lazio e della Toscana (IZSLT) for post-mortem examination. A full-term partially mummified ectopic fetus in the abdomen and a large fibrinonecrotic tubal scar adjacent to the right ovary were observed at necropsy. The tubal scar is likely the point of tubal rupture through which the fetus displaced. Histology revealed the presence of neoplastic cells referable to lymphoma infiltrating the ovary, spleen, small intestine, heart and peripancreatic adipose tissue. The lymphoma was further characterized as T-cell-type using immunohistochemistry. We suggest that the lymphoma, by involving the ovary, played a pathogenetic role in the development of a secondary EP by altering the genital tract at the structural and hormonal levels. To the best of our knowledge, this is the first report of concomitant ovarian lymphoma and EP in animals and humans in the literature.

Comparison of Direct and Indirect Detection of Toxoplasma gondii in Ovine Using Real-Time PCR, Serological and Histological Techniques.

Toxoplasma gondii is a zoonotic pathogen and the ingestion of tissue cysts by consumption of lamb or mutton has been identified as a possible cause of infection in humans. Many serological surveys in sheep have been performed, showing relevant serological rates; however, while the detection of antibodies indicates an exposure to T. gondii, this does not necessarily imply the presence of tissue cysts in edible tissue. The current study aims to provide further understanding on the occurrence of T. gondii in sheep muscles and the strength of correlation between serological positivity and presence of the parasite in sheep. From 349 sheep, samples (i.e., blood, heart and diaphragm) were collected and subjected to ELISA tests, real-time PCR and histological tests. Despite the high seroprevalence, T. gondii DNA was detected in the heart and/or the diaphragm from 13 out of the 349 tested sheep (3.7%); all were adults (13/191). Furthermore, the histological tests did not reveal the presence of T. gondii tissue cysts in any of the examined portions of interventricular septum. It should be considered that the likelihood of detecting genetic material of the parasite is probably influenced by the uneven distribution of the tissue cysts in the carcass as well as the methodology applied. The findings of this study support the importance of describing the uncertainty associated with the data used for risk assessment to reduce inaccurate estimation or risk overestimation.

Histiocytic Sarcoma in a Captive Hybrid Orangutan (Pongo sp.): Morphological and Immunohistochemical Features.

Histiocytic sarcoma (HS), an infrequent highly aggressive hematopoietic tumor, has been observed in diverse animal species, with isolated occurrences in non-human primates. This study describes the first case of disseminated HS in a 45-year-old female hybrid captive orangutan. The clinical profile mirrored symptoms seen in human HS cases, encompassing anorexia and ascites. Detailed histopathological examination demonstrated characteristic features of this tumor and immunohistochemistry, using markers such as Iba-1 and HLA-DR, confirmed the diagnosis. Significantly, the absence of CD163 and CD204 expression challenges their diagnostic utility in non-human primates. This investigation enhances our understanding of HS diagnosis in non-human primates, underscoring the necessity for standardized markers and diagnostic protocols.

Detection of Endoparasites in Non-Native Raccoons from Central Italy.

The raccoon (Procyon lotor) is a carnivore native to North and Central America, gradually introduced into Asia and Europe, including Italy. It is an important carrier of multiple endoparasites, both Protozoa and Helminths, some of them being zoonotic. The aim of this study was to investigate the endoparasites of the non-native raccoon population of Central Italy. Sixty-two raccoons were collected by local competent authorities (sixty trapped and euthanized, two found dead) and subjected to necroscopic examination. Carcasses underwent a broad parasitological investigation, including coprological techniques (macroscopic examination of the gastrointestinal tract, lungs, trachea, and heart, Flotac®, Baermann test, and immunofluorescence for Giardia duodenalis and Cryptosporidium spp.), research on respiratory/urinary capillariosis and artificial digestion for Trichinella spp. larvae, and a histopathological examination of the ileum. Ascarid parasites were further identified at the species level using a next-generation sequencing-based amplicon sequencing approach. The results showed the presence of different Protozoa and Nematodes: Baylisascaris procyonis (26/62; 41.9%), Pearsonema sp. (6/62; 9.6%), Capillariidae (6/62; 9.6%), Eimeria sp. (2/62; 3.2%), Cryptosporidium sp. (2/62; 3.2%), and Ancylostomatidae (2/62; 3.2%). B. procyonis is an emerging helminthic zoonotic agent considered a serious concern for public and animal health, given the possibility of its transmission to paratenic hosts, including humans and pets. The demonstrated role of the raccoon as a multi-parasite carrier should be an incentive to continuing the eradication/control of this alien species, and supports the need to implement related disease surveillance programs.

Genomics insights into a Mycobacterium pinnipedii isolate causing tuberculosis in a captive South American sea lion (Otaria flavescens) from Italy.

Tuberculosis (TB) affects humans and other animals, and it is caused by bacteria within the Mycobacterium tuberculosis complex (MTBC). In this study, we report the characterisation of Mycobacterium pinnipedii that caused a TB case in a sea lion (Otaria flavescens) kept in an Italian zoo. The animal died due to severe, progressive disorders involving the respiratory and gastro-enteric systems and the skin. At necropsy, typical gross lesions referable to a TB generalised form were found. In particular, nodular granulomatous lesions were detected in the lungs and several lymph nodes, and colonies referable to Mycobacterium spp. were isolated from lung, mesenteric, and mediastinal lymph nodes. The isolate was identified by PCR as a MTBC, had a spoligotype SB 1480 ("seal lineage"), and was characterised and characterised by whole-genome sequencing analysis confirming that the MTBC involved was M. pinnipedii. The analysis of the resistome and virulome indicated the presence of macrolide and aminoglycoside resistance genes intrinsic in M. tuberculosis [erm-37 and aac(2')-Ic] and confirmed the presence of the region of difference 1 (RD1), harbouring the esxA and esxB virulence genes, differently from its closest taxon, M. microti. As for other MTCB members, M. pinnipedii infection can spill over into non-pinniped mammalian species; therefore, zoological gardens, veterinary practitioners, and public health officers should be aware of the hazard posed by tuberculosis from marine mammals. Since the isolate under study, as well as all available genomes of M. pinnipedii investigated in this study retains almost all the M. tuberculosis virulence genes, it could indeed cause infection, lesions, and disease in other animal species, including humans.

Pathological and virological insights from an outbreak of European brown hare syndrome in the Italian hare (Lepus corsicanus).

European brown hare syndrome (EBHS) is a highly contagious and fatal viral disease, mainly affecting European brown hares (Lepus europaeus). The etiological agent, EBHS virus (EBHSV), belongs to the Lagovirus genus within the Caliciviridae family. The Italian hare (Lepus corsicanus) is endemic to Central-Southern Italy and Sicily and is classified as a vulnerable species. L. corsicanus is known to be susceptible to EBHS, but virological data available is scarce due to the few cases detected so far. In this study, we describe the occurrence of EBHS in two free-ranging L. corsicanus, found dead in a protected area of Central Italy. The two hares were identified as L. corsicanus using phenotypic criteria and confirmed through mitochondrial DNA analysis. Distinctive EBHS gross lesions were observed at necropsy and confirmed by subsequent histological examination. EBHSV was detected in the livers of the two animals initially using an antigen detection ELISA, followed by an EBHSV-specific reverse transcription-PCR, thus confirming the viral infection as the probable cause of death. The EBHS viruses detected in the two hares were identical, as based on blast analysis performed for the VP60 sequences and showed 98.86% nucleotide identity and 100% amino acid identity with strain EBHSV/GER-BY/EI97.L03477/2019, isolated in Germany in 2019. Phylogenetic analysis places our virus in group B, which includes strains that emerged after the mid-1980s. This study supports previous reports of EBHS in L. corsicanus and further expands the knowledge of the pathological and virological characteristics of the etiological agent. The ability of EBHSV to cause a fatal disease in the Italian hare represents a serious threat to the conservation of this vulnerable species, especially in populations kept in enclosed protected areas.

Investigation of multiple Felis catus papillomavirus types (-1/-2/-3/-4/-5/-6) DNAs in feline oral squamous cell carcinoma: a multicentric study.

Recent evidence suggests a possible association of Felis catus papillomavirus type 2 (FcaPV-2) DNA with feline oral squamous cell carcinoma (FOSCC). In this study, type-specific PCR targeting two genes (L1/E6 or E1/E6) of FcaPV-1/-2/-3/-4/-5/-6 was performed to detect viral DNA in a large amount of FOSCC samples collected in Italy and Austria. FcaPV-1/-2/-3/-4/-5 were detected in 7/113 (6.2%), 7/93 (7.5%), 6/113 (5.3%), 1/113 (0.9%) and 2/113 (1.8%) specimens, respectively, with different prevalences in Italian vs. Austrian samples, whilst FcaPV-6 went undetected. Our results confirms that FcaPV-2 is the most prevalent in FOSCC, followed by FcaPV-1/-3 and suggest that FcaPVs have variable circulation rates in European countries.

First Isolation of Yarrowia lipolytica in a Granulomatous Pneumonia of a Spectacled Caiman, Caiman crocodilus Linnaeus, 1758.

Contrary to humans, candidiasis is a rare infection in animals. However, in reptiles, candidiasis can cause gastrointestinal, cutaneous, or rarely systemic infections in stressed animals. The infections due to Yarrowia lipolytica have been increasingly described in human medicine, and hundreds of cases are reported, comprised of granulomatous lung lesions. Herein, granulomatous pneumonia of a spectacled caiman, Caiman crocodilus, was described, and the presence of Y. lipolytica in the lesion was confirmed through histopathology, microbiologic cultures, and molecular methods. The cause of death of the spectacled caiman was ascribed to bacterial shock septicemia consequentially to a traumatic lesion. However, in the right lung, several nodules containing white exudate were evidenced. At mycological and molecular analyses, Y. lipolytica was evidenced, and the histological finding confirmed the presence of a Candida infection in the lung granulomatous lesions. The comparison of ITS sequences with 11 Yarrowia spp. isolates, recently described in green sea turtles, and with a human strain was conducted, and the whole genome of a strain isolated in the spectacled caiman was sequenced. Even though Y. lipolytica is considered a non-pathogenic yeast and has been rarely described in animals, it seems to cause granulomatous lesions in reptiles as in humans.

Systemic Infection by Angiostrongylus vasorum in a Fennec (Vulpeszerda) in an Italian Zoological Garden.

This paper reported a case of a metastrongyloid nematode Angiostrongylus vasorum infection in a fennec (Vulpes zerda) kept in a zoo in central Italy. The fennec had shown paralysis of the hind limbs, anorexia, weakness and respiratory signs before death. Cardiomegaly and granulomatous pneumonia were the major anatomopathological findings. Inflammatory lesions associated with parasitic larvae were observed in the lungs, brain, liver, heart, spinal cord and kidney of the fennec at histology. A. vasorum diagnosis was confirmed by both morphological and molecular identification of adult worms recovered at necropsy. Fennecs are active predators and maintain their hunting behaviour in captivity. Hence, it is likely that the animal was exposed to infection by preying on parasitised gastropods, intermediate hosts of A. vasorum, entering zoo enclosures from the surrounding environment. This is the first report of A. vasorum systemic infection in a captive fennec (V. zerda) in a zoo in Italy.

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.

Cowpox virus in llama, Italy.

Cowpox virus (CPXV) was isolated from skin lesions of a llama on a farm in Italy. Transmission electron microscopy showed brick-shaped particles consistent with orthopoxviruses. CPXV-antibodies were detected in llama and human serum samples; a CPXV isolate had a hemagglutinin sequence identical to CPXV-MonKre08/1-2-3 strains isolated from banded mongooses in Germany.

Morbilliviral encephalitis in a striped dolphin Stenella coeruleoalba calf from Italy.

A free-living, striped dolphin Stenella coeruleoalba calf was stranded on the Latium coast of Italy in November 2009. Significant neuropathological findings included non-suppurative meningoencephalitis, microgliosis, neuronal degeneration, astrocytosis, and occasional multinucleate syncytia. Immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR) for Morbillivirus were positive exclusively from the brain, with morbilliviral antigen and nucleic acid being detected in neurons and, to a lesser extent, in astrocytes. A low neutralizing antibody titer (1:10) against Morbillivirus (Canine distemper virus, CDV) was also found in blood serum, with no simultaneous presence of serum antibodies to Brucella spp. or Toxoplasma gondii. Furthermore, no pathogenic bacteria were isolated from any tissue or biological sample. This is the second report of morbilliviral encephalitis in a striped dolphin stranded along the Italian coastline in a 16 yr period (1993 to 2009). The neurohistopathological, IHC, and biomolecular features of this case are of additional interest, as antigenic and genomic positivity were exclusively confined to the brain of this dolphin, which may have acquired morbilliviral infection either postnatally or transplacentally.

Endoparasite Infections of the European Hedgehog (Erinaceus europaeus) in Central Italy.

The European hedgehog is a synanthropic mammal, widely distributed in Europe. This species usually inhabits the edges of deciduous or mixed woods, but it is also very common in private gardens and public parks. Despite its popularity and frequency of contacts both with humans and with wild and domestic animals, few studies have examined the endoparasitic fauna of the hedgehog in Italy. In the present study, endoparasites of naturally deceased hedgehogs (n = 40) from central Italy (Latium and Tuscany regions) were investigated, along with concurrent gross and histopathological lesions. The most prevalent identified endoparasites were Crenosoma striatum (45%), Capillaria erinacei (42.5%) and Brachylaemus erinacei (22.5%), in accordance with previous reports from hedgehogs in southern Italy. In few subjects, Physaloptera clausa, Acanthocephalans and Cystoisospora rastegaeivae coccidia were also identified. The infection by the lungworm C. striatum was found to be significantly associated (p < 0.01) with bronchial hyperplasia and peribronchiolitis upon histopathological examination. Awareness of the most common parasitic infections in the hedgehog and of their effects on the health of these animals is extremely important, especially in wildlife rescue centers, where European hedgehog represents the most frequently hospitalized mammal species.

Pathology of Urinary Bladder in Pearsonema spp. Infected Wildlife from Central Italy.

The genus Pearsonema, in the nematode family Capillariidae, includes several species that parasitize the urinary bladders of wild and domestic carnivores. The infection has been reported worldwide from several wildlife species, including canids, mustelids, and felids, but the pathological aspects have seldom been investigated. In order to assess the presence and severity of the lesions in Pearsonema-infected wildlife, we performed a parasitological and pathological examination of urinary bladders from 72 animals, belonging to the families Canidae (red fox Vulpes vulpes, n = 28, and wolf Canis lupus, n = 29) and Mustelidae (beech marten Martes foina, n = 3; pine marten Martes martes, n = 2; and European badger Meles meles, n = 10). A greater prevalence of infection for canids (64.91%; 95% confidence interval (95% CI), 52.52-77.30%) than for mustelids (13.33%) (p < 0.001) was recorded. The prevalence of infection in red foxes was 75.0% (95% CI, 58.96-91.04%), in accordance with other reports from European countries, supporting the role of this species as a reservoir for infection. Eosinophilic cystitis was observed in 34 out of the 72 examined animals (47.22%). The influence of Pearsonema sp. infection on the occurrence of eosinophilic cystitis was statistically significant in wolves (p < 0.01), which were also affected by more severe histological lesions compared to foxes.

A Canine Distemper Virus Retrospective Study Conducted from 2011 to 2019 in Central Italy (Latium and Tuscany Regions).

Canine distemper virus (CDV) is a highly lethal contagious viral pathogen mainly found in domestic and wild canids and mustelids. Although, in Italy, circulating strains of Europe 1, Europe wildlife and Arctic type are reported, data relating to Latium and Tuscany regions are limited. In view of this, through passive surveillance, we investigated the presence of CDV and which strains were circulating in these Regions. From March 2017 to October 2019, a group of 122 subjects were tested for CDV using a PCR protocol described in the literature, with 12 detected positive; analyses were carried out on a set of target samples (brain and lung, conjunctival, nasal and rectal swabs, urine or swab from bladder and intracardiac clot) that was defined for the detection of CDV in both live and dead animals. The rectal swab, easily collected also from live animals, represented the most suitable sample for CDV diagnosis, with 9 positive of the 11 (81.82%) tested. In addition, brain and lung of 15 subjects out of 181 susceptible animals collected between 2011 and 2018, during post mortem investigations in routine diagnostic activity, were CDV positive. Molecular analyses of all positive samples, using a 287 bp fragment located within the conserved N terminus of the morbillivirus nucleoprotein gene, detected the circulation of strain CDV599/2016 (KX545421.1) belonging to the "Europe wildlife" lineage, and of strain CDV12254/2015 (KX024709.1), belonging to the Arctic-lineage, thus confirming the co-circulation of the two lineages, as already noted in previous studies.