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The article presents the result of examinations on striped dolphins (stenella coeruleoalba) for cerebral toxoplasmosis stranded in the Ligurian sea coast in Italy. The authors reveal that severe, nonsuppurative meningoencephalitis was detected in 4 striped dolphins. They mention that other findings included severe bronchointerstitial pneumonia and pulmonary atelectasis, consolidation and emphysema.

Abstract Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP). Breeding programs have been implemented to increase scrapie resistance in sheep populations; though desirable, a similar approach has not yet been applied in goats. European studies have now suggested that several polymorphisms can modulate scrapie susceptibility in goats: in particular, PRNP variant K222 has been associated with resistance in case-control studies in Italy, France and Greece. In this study we investigated the resistance conferred by this variant using a natural Italian goat scrapie isolate to intracerebrally challenge five goats carrying genotype Q/Q 222 (wild type) and five goats carrying genotype Q/K 222. By the end of the study, all five Q/Q 222 goats had died of scrapie after a mean incubation period of 19 months; one of the five Q/K 222 goats died after 24 months, while the other four were alive and apparently healthy up to the end of the study at 4.5 years post-challenge. All five of these animals were found to be scrapie negative. Statistical analysis showed that the probability of survival of the Q/K 222 goats versus the Q/Q 222 goats was significantly higher (p = 0.002). Our study shows that PRNP gene mutation K222 is strongly associated with resistance to classical scrapie also in experimental conditions, making it a potentially positive target for selection in the frame of breeding programs for resistance to classical scrapie in goats.

Abstract Background Three distinct forms of bovine spongiform encephalopathy (BSE), defined as classical (C-), low (L-) or high (H-) type, have been detected through ongoing active and passive surveillance systems for the disease. The aim of the present study was to compare the ability of two sets of immunohistochemical (IHC) and Western blot (WB) BSE confirmatory protocols to detect C- and atypical (L- and H-type) BSE forms. Obex samples from cases of United States and Italian C-type BSE, a U.S. H-type and an Italian L-type BSE case were tested in parallel using the two IHC sets and WB methods. Results The two IHC techniques proved equivalent in identifying and differentiating between C-type, L-type and H-type BSE. The IHC protocols appeared consistent in the identification of PrPSc distribution and deposition patterns in relation to the BSE type examined. Both IHC methods evidenced three distinct PrPSc phenotypes for each type of BSE: prevailing granular and linear tracts pattern in the C-type; intraglial and intraneuronal deposits in the H-type; plaques in the L-type. Also, the two techniques gave comparable results for PrPSc staining intensity on the C- and L-type BSE samples, whereas a higher amount of intraglial and intraneuronal PrPSc deposition on the H-type BSE case was revealed by the method based on a stronger demasking step. Both WB methods were consistent in identifying classical and atypical BSE forms and in differentiating the specific PrPSc molecular weight and glycoform ratios of each form. Conclusions The study showed that the IHC and WB BSE confirmatory methods were equally able to recognize C-, L- and H-type BSE forms and to discriminate between their different immunohistochemical and molecular phenotypes. Of note is that for the first time one of the two sets of BSE confirmatory protocols proved effective in identifying the L-type BSE form. This finding helps to validate the suitability of the BSE confirmatory tests for BSE surveillance currently in place.

Herpesvirus (HV) is widely distributed among cetacean populations, with the highest prevalence reported in the Mediterranean Sea. In this study, a comprehensive analysis was conducted, including epidemiological, phylogenetic, and pathological aspects, with particular emphasis on neuropathology, to better understand the impact of HV in these animals. Our results show a higher presence of HV in males compared to females, with males exhibiting a greater number of positive tissues. Additionally, adults were more frequently affected by HV infection than juveniles, with no infections detected in calves or neonates. The affected species were striped (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus). The highest positivity rates were observed in the genital system, cerebrum, and skin tissues. Phylogenetic analysis indicated a higher occurrence of Gammaherpesvirus (GHV) sequences but increased genetic diversity within Alphaherpesvirus (AHV). Key neuropathological features included astro-microgliosis (n = 4) and meningitis with minimal to mild perivascular cuffing (n = 2). The presence of concurrent infections with other pathogens, particularly cetacean morbillivirus (CeMV), underscores the complex nature of infectious diseases in cetaceans. However, the presence of lesions at the Central Nervous System (CNS) with molecular positivity for GHV, excluding the involvement of other potential neurotropic agents, would confirm the potential of this HV subfamily to induce neurological damage. Pathological examination identified lesions in other organs that could potentially be associated with HV, characterized by lymphoid depletion and tissue inflammation. These findings enhance our understanding of HV in odontocetes and highlight the need for ongoing research into the factors driving these infections and their broader implications., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Vargas-Castro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Bacterial infection of the central nervous system (CNS) poses a clinical challenge and is a leading cause of neurological disorders in cattle. Human studies have demonstrated an increase in cerebrospinal fluid (CSF) L-lactate levels in bacterial meningitis. The aims of this study were to establish a Reference Interval (RI) for L-lactate in bovine CSF and assess its potential as a biomarker for detecting infectious-inflammatory disorders., Methods: CSF L-lactate was measured in the field using a commercially available lactate monitor. The RI for CSF L-lactate was calculated on healthy animals; univariate and receiver operating characteristic (ROC) analyses were performed to disclose an association between CSF L-lactate levels and interpretation of CSF in sick animals., Results: Twenty-seven healthy cattle and 86 sick cattle with either CNS infectious-inflammatory disorders (47/86) or CNS disorders of other etiology (39/86) were included in this prospective study. The RI for CSF L-lactate was 1.1-2.4 mmol/L. The concentration was higher in the cattle with neutrophilic pleocytosis and the area under the ROC curve was 0.92 compared to other animals. Based on a cut-off of 3.15 mmol/L, CSF L-lactate had diagnostic sensitivity and specificity for neutrophilic pleocytosis of 93 and 80%, respectively., Discussion: This is the first study to determine a RI for CSF L-lactate in cattle. Elevated CSF L-lactate levels indicated neutrophilic pleocytosis, which is often manifested in acute bacterial infection. The present findings may aid in diagnosis and correct use of antimicrobial drugs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ferrini, Cagnotti, Ala, Avilii, Bellino, Biasibetti, Borriello, Corona, Di Muro, Iamone, Iulini, Pezzolato, Bozzetta and D’Angelo.)

Background: Halicephalobus gingivalis is a nematode with zoonotic potential which can cause fatal opportunistic infections in various mammals. The parasite has never been diagnosed in Sweden, in any species, prior to the presented case., Case Presentation: An imported 21-year-old Icelandic mare developed severe neurological signs. The horse was eventually euthanized and submitted for post-mortem examination where severe lesions in the kidneys were noted. Histopathology revealed the presence of H. gingivalis in both kidneys and the brain. Phylogenetic analysis of the parasite determined it to belong to Lineage 1., Conclusions: With the occurrence of H. gingivalis in Sweden, the disease should be added to the list of differential diagnoses in cases with acute onset of neurological disease in both horses and other mammals including humans., (© 2023. The Author(s).)

Brucella ceti infections have been increasingly reported in cetaceans. In this study, we analyzed all cases of B. ceti infection detected in striped dolphins stranded along the Italian coastline between 2012 and 2021 ( N = 24). We focused on the pathogenic role of B. ceti through detailed pathological studies, and ad hoc microbiological, biomolecular, and serological investigations, coupled with a comparative genomic analysis of the strains. Neurobrucellosis was observed in 20 animals. The primary histopathologic features included non-suppurative meningoencephalitis ( N = 9), meningitis ( N = 6), and meningoencephalomyelitis ( N = 5), which was also associated with typical lesions in other tissues ( N = 8). Co-infections were detected in more than half of the cases, mostly involving Cetacean Morbillivirus (CeMV). The 24 B. ceti isolates were assigned primarily to sequence type 26 (ST26) ( N = 21) and, in a few cases, ST49 ( N = 3). The multilocus sequence typing (cgMLST) based on whole genome sequencing (WGS) data showed that strains from Italy clustered into four genetically distinct clades. Plotting these clades onto a geographic map suggests a link between their phylogeny and the topographical distribution. These results support the role of B. ceti as a primary neurotropic pathogen for striped dolphins and highlight the utility of WGS data in understanding the evolution of this emerging pathogen.

Background: Central nervous system (CNS) infections in cattle are a major cause of economic loss and mortality. Machine learning (ML) techniques are gaining widespread application in solving predictive tasks in both human and veterinary medicine., Objectives: Our primary aim was to develop and compare ML models that could predict the likelihood of a CNS disorder of infectious or inflammatory origin in neurologically-impaired cattle. Our secondary aim was to create a user-friendly web application based on the ML model for the diagnosis of infection and inflammation of the CNS., Animals: Ninety-eight cattle with CNS infection and 86 with CNS disorders of other origin., Methods: Retrospective observational study. Six different ML methods (logistic regression [LR]; support vector machine [SVM]; random forest [RF]; multilayer perceptron [MLP]; K-nearest neighbors [KNN]; gradient boosting [GB]) were compared for their ability to predict whether an infectious or inflammatory disease was present based on demographics, neurological examination findings, and cerebrospinal fluid (CSF) analysis., Results: All 6 methods had high prediction accuracy (≥80%). The accuracy of the LR model was significantly higher (0.843 ± 0.005; receiver operating characteristic [ROC] curve 0.907 ± 0.005 ) than the other models and was selected for implementation in a web application., Conclusion and Clinical Importance: Our findings support the use of ML algorithms as promising tools for veterinarians to improve diagnosis. The open-access web application may aid clinicians in achieving correct diagnosis of infectious and inflammatory neurological disorders in livestock, with the added benefit of promoting appropriate use of antimicrobials., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Introduction: Few cases of myelomonocytic leukemia associated with neurological signs have been described in dogs; none have been related to intraparenchymal spinal cord infiltration by neoplastic cells. This short communication describes a case of acute myelomonocytic leukemia subtype M4 in a dog with spinal cord infiltration. A 3-year-old male Golden Retriever was presented with a history of hyperthermia, lymphadenomegaly, leukocytosis with circulating blast cells, anemia and thrombocytopenia, and acute onset paraplegia. Immunophenotyping of peripheral blood by flow cytometry was consistent with acute myelomonocytic leukemia subtype M4. The dog was euthanized because of clinical deterioration and unfavourable prognosis. Postmortem examination revealed multi-organ neoplastic infiltration, including the spinal cord. To our knowledge, this is the first case of acute myelomonocytic leukemia subtype M4 in a dog with spinal cord infiltration. Our findings hold importance for including myelomonocytic leukemia in the differential diagnosis of patients with neurological signs due to spinal cord localisation.

Chronic wasting disease (CWD) is a prion disease that affects cervids; it is classified under transmissible spongiform encephalopathies (TSEs). CWD is particularly contagious, making its eradication in endemic areas very difficult and creating serious problems for cervid conservation and breeding. It has recently become an emerging public health risk to be managed by health authorities. Starting in 2017, active CWD surveillance in Italy has intensified with the monitoring of wild and farmed cervids. The present study summarizes findings from a histopathological survey of the brains from wild ruminants collected via CWD monitoring between 2017 and 2019. A total of 113 brains from 62 red deer ( Cervus elaphus ) and 51 roe deer ( Capreolus capreolus ) were submitted for analysis at the National Reference Center for Animal Encephalopathies (CEA) to determine major patterns of neuropathological lesions and correlated pathogens. Brain lesions were detected in 20 animals, 10 brain samples were unsuitable for examination, and 83 presented no lesions. Neuropathological examination revealed non-suppurative encephalitis or meningoencephalitis in most cases (15/20). This brain study revealed evidence for the absence of CWD in Italy and provided a reference spectrum of neuropathological lesions for differential diagnosis in cervids.

Cetacean morbillivirus (CeMV) is responsible for epidemic and endemic fatalities in free-ranging cetaceans. Neuro-inflammation sustained by CeMV is a leading cause of death in stranded cetaceans. A novel dolphin morbillivirus (DMV) strain of Atlantic origin circulating in Italian waters since early 2016 has caused acute/subacute lesions associated with positive immunolabelling of the virus. To date, myelin damage has not been fully documented and investigated in cetaceans. This study describes neuropathological findings in the brain tissue of 31 cetaceans found stranded along the Italian coastline and positive for DMV infection on molecular testing. Cell changes in the areas of myelinopathy were revealed by double indirect immunofluorescence. The most frequent DMV-associated lesions were astro-microgliosis, neuronal necrosis, spongiosis, malacia, and non-suppurative meningoencephalitis. Myelin reduction and areas of demyelination were revealed by means of a specific myelin biomarker. Morbilliviral antigen immunolabelling was mainly observed in neurons and microglial cells, in association with a marked activation of microglia and astrocytes. These findings extend our knowledge of DMV-associated brain lesions and shed light on their pathogenesis.

Two striped dolphins (SD1, SD2), stranded along the Ligurian coast of Italy, were diagnosed with a nonsuppurative meningoencephalitis associated with previously undescribed protozoan tissue cysts. As tissue cysts were morphologically different from those of Toxoplasma gondii , additional histopathological, immunohistochemical, ultrastructural, and biomolecular investigations were performed, aiming to fully characterize the organism. Histopathology revealed the presence of large Sarcocystis -like tissue cysts, associated with limited inflammatory lesions in all CNS areas studied. IHC was inconclusive, as positive staining with polyclonal antisera did not preclude cross-reaction with other Sarcocystidae coccidia. Applied to each animal, 11 different PCR protocols precluded a neural infection by Sarcocystis neurona, Sarcocystis falcatula, Hammondia hammondi , and Neospora caninum . T. gondii coinfection was confirmed only in dolphin SD2. Sarcocystis sp. sequences, showing the highest homology to species infecting the Bovidae family, were amplified from SD1 myocardium and SD2 skeletal muscle. The present study represents the first report of Sarcocystis -like tissue cysts in the brain of stranded cetaceans along with the first description of Sarcocystis sp. infection in muscle tissue of dolphins from the Mediterranean basin.

Background: Tick-borne diseases are common throughout Europe. Ticks transmit pathogens to the host while feeding and together with mosquitoes, they are major vectors of infectious agents worldwide. In recent years, there has been a marked increase in the incidence of tick-bite events and tick-borne disease in northwest Italy, but information on the prevalence of tick-borne pathogens in ticks removed from humans remains scarce. To fill this gap, we report here the prevalence of tick bites and tick-borne pathogens documented for humans in Piedmont, northwest Italy, in the 3-year period 2017-2019., Methods: Ticks attached to humans during 2017-2019 were collected from residents of urban and rural area by physicians and veterinarians working with local veterinary agencies. All ticks (n = 1290) were morphologically identified to the species level. A subset of ticks removed from children (age 0-18 years) and the elderly (> 70 years), both age groups considered to be at-risk populations, was screened by biomolecular analysis to detect pathogens (e.g. Rickettsia spp., Borrelia spp., Anaplasma spp.). Pathogen identity was confirmed by Sanger sequencing., Results: Ticks were taxonomically assigned to ten species of six genera (Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus). Most belonged to the genus Ixodes: 1009 ticks (78.22%) were classified as Ixodes ricinus. A subset of 500 ticks collected from the two at-risk populations were subjected to PCR assay to determine the presence of Rickettsia spp., Borrelia spp., and Anaplasma spp. The overall prevalence of infection was 22.8% (n = 114; 95% confidence interval [CI]: 19.19-26.73%), meaning that at least one pathogen was detected: Rickettsia spp. (prevalence 15%, n = 76; 95% CI 12.17-18.65%); Borrelia spp. (prevalence 6.4%, n = 32; 95% CI 4.42-8.92%); and Anaplasma spp. (prevalence 1.2%, n = 6; 95% CI 0.44-2.6%)., Conclusions: Our data underline the importance of surveillance in the epidemiology of tick-borne diseases and the implementation of strategies to control tick infestation and associated pathogens.

Status epileptics (SE) and cluster seizures (CS) are serious neurological emergencies associated with poor outcome in epileptic patients. Data on risk factors associated with outcome in epileptic patients affected by CS and SE have not been studied extensively to date. In the present retrospective study potential risk factors predictive of negative outcome in a population of dogs affected by CS and SE were analyzed. Ninety-three dogs were included in the study: 21/93 patients (23%) presented with SE and 72/93 (77%) with CS. Based on multivariate statistical analysis, factors statistically associated with a poor outcome were the occurrence of the first epileptic seizure outside the defined idiopathic interval (6 months-6 years), a condition of hyperthermia at presentation and the absence of previous antiepileptic drugs (AEDs) in case of previous history of seizures. The results of the present study implement data on risk factors associated with poor outcome in dogs affected by CS or SE and can aid in the creation of an ad hoc scoring system, similar to systems currently applied in human medicine upon hospital admission to benchmark performances and establish protocols for triage and therapeutic management., (Copyright © 2020 Cagnotti, Ferrini, Ala, Bellino, Corona, Dappiano, Di Muro, Iulini, Pepe, Roncone and D'Angelo.)

Brucella ceti infections have been increasingly reported in cetaceans, although a very limited characterization of Mediterranean Brucella spp. isolates has been previously reported and relatively few data exist about brucellosis among cetaceans in Italy. To address this gap, we studied 8 cases of B. ceti infection in striped dolphins (Stenella coeruleoalba) stranded along the Italian coastline from 2012 to 2018, investigated thanks to the Italian surveillance activity on stranded cetaceans. We focused on cases of stranding in eastern and western Italian seas, occurred along the Apulia (N = 6), Liguria (N = 1) and Calabria (N = 1) coastlines, through the analysis of gross and microscopic findings, the results of microbiological, biomolecular and serological investigations, as well as the detection of other relevant pathogens. The comparative genomic analysis used whole genome sequences of B. ceti from Italy paired with the publicly available complete genomes. Pathological changes consistent with B. ceti infection were detected in the central nervous system of 7 animals, showing non-suppurative meningoencephalitis. In 4 cases severe coinfections were detected, mostly involving Dolphin Morbillivirus (DMV). The severity of B. ceti-associated lesions supports the role of this microbial agent as a primary neurotropic pathogen for striped dolphins. We classified the 8 isolates into the common sequence type 26 (ST-26). Whole genome SNP analysis showed that the strains from Italy clustered into two genetically distinct clades. The first clade comprised exclusively the isolates from Ionian and Adriatic Seas, while the second one included the strain from the Ligurian Sea and those from the Catalonian coast. Plotting these clades onto the geographic map suggests a link between their phylogeny and topographical distribution. These results represent the first extensive characterization of B. ceti isolated from Italian waters reported to date and show the usefulness of WGS for understanding of the evolution of this emerging pathogen., Competing Interests: The authors have declared that no competing interests exist.

Squamous cell carcinoma (SCC) is a malignant mucoepithelial tumor that affects pets and farm animals. Common sites are dorsal areas and/or areas of poor skin pigmentation exposed to mutagenic ultraviolet (UV) radiation. Novel ovine papillomavirus (OaPV3) was recently described in SCC lesions in Sardinia breed ovines. In 2017, a 7‑year‑old half‑breed aries was presented with symptoms compatible with a vestibular syndrome. The animal was euthanized 1 month after the onset of clinical signs due to a lack of response to treatment and poor prognosis. A complete postmortem examination was performed. Necropsy revealed only a loss of incisors, associated with alveolar necrotic osteomyelitis, and left unilateral purulent nasal discharge. No other thoracic or abdominal lesions were observed. Opening of the skull revealed a cauliflower‑like space‑occupying mass. Histological examination showed trabecules and islands of squamous, neoplastic epithelial cells with the formation of concentric keratin layers. This raised the suspicion of SCC, which was confirmed with cytokeratin‑positive immunostaining. Simplex PCR on the frozen tissue mass was negative for OaPV1, OaPV2, and OaPV3. This case report suggests that SCC, although rare, should be included in the differential diagnosis of cases of vestibular disorder.

Background: Neuron glial antigen 2 or chondroitin sulphate proteoglycan 4 (NG2/CSPG4) is expressed by immature precursors/progenitor cells and is possibly involved in malignant cell transformation. The aim of this study was to investigate its role on the progression and survival of sixty-one adult gliomas and nine glioblastoma (GB)-derived cell lines., Methods: NG2/CSPG4 protein expression was assessed by immunohistochemistry and immunofluorescence. Genetic and epigenetic alterations were detected by molecular genetic techniques., Results: NG2/CSPG4 was frequently expressed in IDH-mutant/1p19q-codel oligodendrogliomas (59.1%) and IDH-wild type GBs (40%) and rarely expressed in IDH-mutant or IDH-wild type astrocytomas (14.3%). Besides tumor cells, NG2/CSPG4 immunoreactivity was found in the cytoplasm and/or cell membranes of reactive astrocytes and vascular pericytes/endothelial cells. In GB-derived neurospheres, it was variably detected according to the number of passages of the in vitro culture. In GB-derived adherent cells, a diffuse positivity was found in most cells. NG2/CSPG4 expression was significantly associated with EGFR gene amplification ( p = 0.0005) and poor prognosis ( p = 0.016) in astrocytic tumors., Conclusion: The immunoreactivity of NG2/CSPG4 provides information on the timing of the neoplastic transformation and could have prognostic and therapeutic relevance as a promising tumor-associated antigen for antibody-based immunotherapy in patients with malignant gliomas.

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.

Canine distemper (CD) may pose a serious threat to Alpine wild carnivores and affect their population dynamics. Since 2006, the strain Europe Wildlife 2006-09, a distinct CD virus subgroup within viral lineage Europe 1 (EU1) characterized by increased virulence and host range expansion, has been linked to multiple CD outbreaks in Alpine wild carnivores. The aim of this study was to fill knowledge gaps about ongoing Alpine outbreaks of CD. To do this, we report on the circulation of canine distemper virus (CDV) and outbreaks of CD in Alpine wild carnivores in northwest Italy. A specific diagnostic protocol applied to a sample of 548 wild carnivores collected between January 2013 and December 2015 revealed the circulation of CDV belonging to the EU1 lineage. All isolates were carriers of amino-acid mutations defining the cluster Europe Wildlife 2006-09. A self-maintained multihost pathogen system may have developed in northwest Italy in which interspecies transmission from red foxes ( Vulpes vulpes ) to other noncanid species enhanced pathogen maintenance in the system.

Background: Treatment options for at-home management of cluster seizures (CS) and status epilepticus (SE) are limited. The pharmacokinetics of levetiracetam (LEV) after rectal administration in both healthy and epileptic dogs has been investigated recently., Hypothesis/objectives: To investigate the clinical efficacy of rectally administered LEV in preventing additional seizures in dogs presented for CS and SE. We hypothesized that rectal administration of LEV in addition to a standard treatment protocol would provide better control of seizure activity as compared with the standard treatment protocol alone., Animals: Fifty-seven client-owned dogs with CS or SE., Methods: Prospective open-label clinical trial. Patients included in the study were assigned to receive either a standard treatment protocol comprising IV/rectal diazepam and IV phenobarbital q8h (control group) or a standard treatment protocol in association with a single dose of 40 mg/kg LEV rectally (rectal LEV group). Dogs that experienced no additional seizures were defined as responders, whereas those that showed additional seizure activity were classified as nonresponders., Results: Twenty-one dogs were assigned to the rectal LEV group, and 36 to control group. Given the small number of cases of SE, statistical analysis was performed only on patients with CS. The response rate was 94% in the rectal LEV group and 48% in the control group (P < .001)., Conclusions and Clinical Importance: Rectally administered LEV combined with a standard treatment protocol provided good control of seizure activity in patients with CS. The validity of these results should be confirmed in a double-blinded, placebo-controlled clinical trial., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Monitoring of small ruminants for transmissible spongiform encephalopathies (TSEs) has recently become more relevant after two natural scrapie suspected cases of goats were found to be positive for classical BSE (C-BSE). C-BSE probably established itself in this species unrecognized, undermining disease control measures. This opens the possibility that TSEs in goats may remain an animal source for human prion diseases. Currently, there are no data regarding the natural presence of the atypical BSE in caprines. Here we report that C-BSE and L-type atypical BSE (L-BSE) isolates from bovine species are intracerebrally transmissible to goats, with a 100% attack rate and a significantly shorter incubation period and survival time after C-BSE than after L-BSE experimental infection, suggesting a lower species barrier for classical agentin goat. All animals showed nearly the same clinical features of disease characterized by skin lesions, including broken hair and alopecia, and abnormal mental status. Histology and immunohistochemistry showed several differences between C-BSE and L-BSE infection, allowing discrimination between the two different strains. The lymphoreticular involvement we observed in the C-BSE positive goats argues in favour of a peripheral distribution of PrPSc similar to classical scrapie. Western blot and other currently approved screening tests detected both strains in the goats and were able to classify negative control animals. These data demonstrate that active surveillance of small ruminants, as applied to fallen stock and/or healthy slaughter populations in European countries, is able to correctly identify and classify classical and L-BSE and ultimately protect public health., Competing Interests: The authors have declared that no competing interests exist.

We summarized the neuropathologic findings in 60 cetaceans stranded along the Italian coastline from 2002 to 2014. The following neuropathologic changes were detected in 45% (27/60) of animals: nonsuppurative meningo-encephalitides (30%, 18/60), nonspecific lesions (12%, 7/60), suppurative encephalitis (2%, 1/60), and neoplasm (2%, 1/60). No histologic lesions were found in 47% (28/60) of the specimens. Five (8%, 5/60) samples were unsuitable for analysis. Analysis with PCR detected Brucella spp., morbillivirus, and Toxoplasma gondii infection in one, six, and seven individuals, respectively. Immunohistochemical analysis confirmed positivity for morbillivirus and for T. gondii infection in three cases each. No evidence of the scrapie-associated prion protein PrPSc was detected. Our findings underscore the importance of an adequate surveillance system for monitoring aquatic mammal pathologies and for protecting both animal and human health.

Between 2007 and 2014, 83 cetaceans were found stranded along the Ligurian coast of Italy, in the Pelagos Sanctuary, the largest marine protected area in the Mediterranean basin. Forty-nine (59%) were submitted to complete or partial necropsy, depending on the conservation status of the carcass. Based on gross and histological pathology and ancillary testing, the cause of death was determined and categorized as anthropogenic or natural (i.e., nonanthropogenic) in origin for 33 animals (67%) and of undetermined origin in the remaining 16 (33%). Natural causes of death, accompanied by either poor or good nutritional status, were attributed to 29 animals (59%), whereas four (8%) were diagnosed with an anthropogenic cause of death, consisting of interaction with fishing activities. Infectious and noninfectious disease was the most common cause of death, involving 29 cetaceans (59%). These data are valuable for understanding health and mortality trends in cetacean populations and can provide information for establishing policies for cetacean conservation and management in such an important protected area of the Mediterranean basin.

Background: A fatal case of meningoencephalitis was reported in a 13-year-old Koninklijk Warmbloed Paard Nederland stallion, suspected of West Nile virus (WNV) infection, in the Piedmont region of Italy. Clinical signs included right head tilt and circling, depression alternated with excitability, fever and lateral strabismus. Combined treatment consisting of dimethylsulfoxide, dexamethasone, sulphonamides and sedative was administered, but because of the poor conditions the horse was euthanatized and submitted for necropsy., Results: At post-mortem examination no skin lesions were observed, all organs appeared normal on gross evaluation and only head and blood samples were further investigated. Neuropathological findings consisted of granulomatous meningoencephalitis and larvae and adult females of Halicephalobus gingivalis were isolated and identified from the digested brain. Frozen brain was submitted to PCR amplification and 220 bp multiple sequence alignment was analysed by Bayesian phylogenetic analysis., Conclusions: Phylogenetic inference revealed that the isolate belongs to H. gingivalis Lineage 3. WN surveillance can help to deepen our knowledge of horse neurological disorders investigating their causes and incidence. Moreover, it can help to understand the geographic distribution of the H. gingivalis, to unravel epidemiological information, and to estimate risk for humans.

Many mammalian species can be affected by prion diseases, also known as transmissible spongiform encephalopathies (TSEs). "Classical" bovine spongiform encephalopathy (C-BSE) was the first prion disease recognized in cattle and it is the only known zoonotic prion disease, having caused variant Creutzfeldt-Jakob disease (vCJD) in humans. Based on the biochemical signatures of disease-associated prion protein (PrP Sc ), two distinct forms of atypical bovine spongiform encephalopathies (H-BSE and L-BSE) have been distinguished from C-BSE since 2004. To date there is no comprehensive information about the origin of atypical BSEs (sporadic vs. acquired) and this has an influence on the interpretation of the knowledge gathered from experimental studies, regarding how well such models may represent the real distribution of the agent in the body of naturally affected animals. Moreover, there are only very limited data available concerning the pathogenesis of both atypical BSE forms, as compared to C-BSE. Thus, precautions that are presently taken to minimize the risk of prion contamination of the food supply might not be as effective at preventing the spread of these recently recognized strains. In the last few years a wide range of experimental transmission studies of atypical strains in different animal hosts have been performed. The most recent data on classical and atypical BSE studies concerning characteristics, pathogenesis and transmissions in cattle will be summarized in this review., Competing Interests: Conflict of interest statement: The authors had no conflicts of interest to declare in this article., (©2016 Food Safety Commission, Cabinet Office, Government of Japan.)

In 2008, a 2 months-old male German shepherd was presented with fever, depression, and evident organic wasting. The puppy died within 48 hours after the onset of clinical signs. A complete necropsy was performed. Bacteriological examination of samples from the brain, lung, liver, spleen, and bone marrow tested positive for Pasteurella pneumotropica. Histopathology demonstrated inflammatory and vascular lesions in the central nervous system and internal organs. Canine adenovirus type 1 nucleic acid was detected by polymerase chain reaction in the frozen brain but not in the formalin-fixed, paraffin-embedded liver and lung samples. The positive PCR was subsequently confirmed by indirect fluorescent antibody testing of the paraffin-embedded brain and liver sections. Although the liver is the primary site of viral damage, these laboratory findings suggest that Canine adenovirus type 1 infection should be included in the differential diagnosis of neuropathological diseases in dogs and that adenoviral infections could promote septicaemia caused by opportunistic pathogens.

West Nile virus (WNV) is a recently re-emerged health problem in Europe. In Italy, an increasing number of outbreaks of West Nile disease, with occurrences of human cases, have been reported since 2008. This is particularly true in northern Italy, where entomological surveillance systems have been implemented at a regional level. The aim of this study was to use, for the first time, all the entomological data collected in the five regions undergoing surveillance for WNV in northern Italy to characterize the viral circulation (at a spatial and temporal scale), identify potential mosquito vectors, and specify relationships between virus circulation and meteorological conditions. In 2013, 286 sites covering the entire Pianura Padana area were monitored. A total of 757,461 mosquitoes were sampled. Of these, 562,079 were tested by real-time PCR in 9,268 pools, of which 180 (1.9%) were positive for WNV. The largest part of the detected WNV sequences belonged to lineage II, demonstrating that, unlike those in the past, the 2013 outbreak was mainly sustained by this WNV lineage. This surveillance also detected the Usutu virus, a WNV-related flavivirus, in 241 (2.6%) pools. The WNV surveillance systems precisely identified the area affected by the virus and detected the viral circulation approximately two weeks before the occurrence of onset of human cases. Ninety percent of the sampled mosquitoes were Culex pipiens, and 178/180 WNV-positive pools were composed of only this species, suggesting this mosquito is the main WNV vector in northern Italy. A significantly higher abundance of the vector was recorded in the WNV circulation area, which was characterized by warmer and less rainy conditions and greater evapotranspiration compared to the rest of the Pianura Padana, suggesting that areas exposed to these conditions are more suitable for WNV circulation. This observation highlights warmer and less rainy conditions as factors able to enhance WNV circulation and cause virus spillover outside the sylvatic cycle.

Listeriosis is a disease that causes significant economic losses at the farm level because of high morbidity and mortality in ruminants. This study was performed to investigate the role of ruminants in the epidemiology of listeriosis in northern Italy and the possible association of animal-adapted strains of Listeria monocytogenes with strains associated with human disease. Twenty ruminant rhombencephalitis isolates previously confirmed as L. monocytogenes by bacteriology and PCR were characterized by serotyping, pulsed-field gel electrophoresis, multi-virulence-locus sequence typing (MVLST), and multiplex single nucleotide polymorphism (mSNP) typing for the detection of epidemic clones. Subtyping results were subsequently compared with those obtained from human, food, and environmental isolates of L. monocytogenes, including 311 isolates from the University of Turin, Grugliasco, Italy, and 165 isolates representing major human listeriosis outbreaks worldwide, in addition to other unrelated isolates. Both mSNP typing and MVLST showed that 60% of the isolates analyzed belonged to epidemic clone I (ECI), which has been epidemiologically linked to several human outbreaks of listeriosis. In particular, the 1981 Canada outbreak was linked to the use of sheep manure and the 1985 California outbreak was linked to the use of raw cow's milk. In our study, ECI isolates were collected from different ruminant species on geographically and temporally distinct occasions for the last 13 years. Our results support the hypothesis that ruminants represent possible natural reservoirs of L. monocytogenes strains capable of causing epidemics of listeriosis in humans.

In Transmissible Spongiform Encephalopathies (TSEs) and Alzheimer disease (AD) both misfolding and aggregation of specific proteins represent key features. Recently, it was observed that PrP (c) is a mediator of a synaptic dysfunction induced by Aβ oligomers. We tested a novel γ secretase modulator (CHF5074) in a murine model of prion disease. Groups of female mice were intracerebrally or intraperitoneally infected with the mouse-adapted Rocky Mountain Laboratory prions. Two weeks prior infection, the animals were provided with a CHF5074-medicated diet (375 ppm) or a standard diet (vehicle) until they showed neurological signs and eventually died. In intracerebrally infected mice, oral administration of CHF5074 did not prolong survival of the animals. In intraperitoneally-infected mice, CHF5074-treated animals showed a median survival time of 21 days longer than vehicle-treated mice (p < 0.001). In these animals, immunohistochemistry analyses showed that deposition of PrP (Sc) in the cerebellum, hippocampus and parietal cortex in CHF5074-treated mice was significantly lower than in vehicle-treated animals. Immunostaining of glial fibrillary acidic protein (GFAP) in parietal cortex revealed a significantly higher reactive gliosis in CHF5074-treated mice compared to the control group of infected animals. Although the mechanism underlying the beneficial effects of CHF5074 in this murine model of human prion disease is unclear, it could be hypothesized that the drug counteracts PrP (Sc ) toxicity through astrocyte-mediated neuroprotection. CHF5074 shows a pharmacological potential in murine models of both AD and TSEs thus suggesting a link between these degenerative pathologies.

The amyloidotic form of bovine spongiform encephalopathy (BSE) termed BASE is caused by a prion strain whose biological properties differ from those of typical BSE, resulting in a clinically and pathologically distinct phenotype. Whether peripheral tissues of BASE-affected cattle contain infectivity is unknown. This is a critical issue since the BASE prion is readily transmissible to a variety of hosts including primates, suggesting that humans may be susceptible. We carried out bioassays in transgenic mice overexpressing bovine PrP (Tgbov XV) and found infectivity in a variety of skeletal muscles from cattle with natural and experimental BASE. Noteworthy, all BASE muscles used for inoculation transmitted disease, although the attack rate differed between experimental and natural cases (∼70% versus ∼10%, respectively). This difference was likely related to different prion titers, possibly due to different stages of disease in the two conditions, i.e. terminal stage in experimental BASE and pre-symptomatic stage in natural BASE. The neuropathological phenotype and PrP(res) type were consistent in all affected mice and matched those of Tgbov XV mice infected with brain homogenate from natural BASE. The immunohistochemical analysis of skeletal muscles from cattle with natural and experimental BASE showed the presence of abnormal prion protein deposits within muscle fibers. Conversely, Tgbov XV mice challenged with lymphoid tissue and kidney from natural and experimental BASE did not develop disease. The novel information on the neuromuscular tropism of the BASE strain, efficiently overcoming species barriers, underlines the relevance of maintaining an active surveillance.

Background: Three distinct forms of bovine spongiform encephalopathy (BSE), defined as classical (C-), low (L-) or high (H-) type, have been detected through ongoing active and passive surveillance systems for the disease.The aim of the present study was to compare the ability of two sets of immunohistochemical (IHC) and Western blot (WB) BSE confirmatory protocols to detect C- and atypical (L- and H-type) BSE forms.Obex samples from cases of United States and Italian C-type BSE, a U.S. H-type and an Italian L-type BSE case were tested in parallel using the two IHC sets and WB methods., Results: The two IHC techniques proved equivalent in identifying and differentiating between C-type, L-type and H-type BSE. The IHC protocols appeared consistent in the identification of PrPSc distribution and deposition patterns in relation to the BSE type examined. Both IHC methods evidenced three distinct PrPSc phenotypes for each type of BSE: prevailing granular and linear tracts pattern in the C-type; intraglial and intraneuronal deposits in the H-type; plaques in the L-type.Also, the two techniques gave comparable results for PrPSc staining intensity on the C- and L-type BSE samples, whereas a higher amount of intraglial and intraneuronal PrPSc deposition on the H-type BSE case was revealed by the method based on a stronger demasking step.Both WB methods were consistent in identifying classical and atypical BSE forms and in differentiating the specific PrPSc molecular weight and glycoform ratios of each form., Conclusions: The study showed that the IHC and WB BSE confirmatory methods were equally able to recognize C-, L- and H-type BSE forms and to discriminate between their different immunohistochemical and molecular phenotypes. Of note is that for the first time one of the two sets of BSE confirmatory protocols proved effective in identifying the L-type BSE form. This finding helps to validate the suitability of the BSE confirmatory tests for BSE surveillance currently in place.

The olfactory system (OS) is involved in many infectious and neurodegenerative diseases, both human and animal, and it has recently been investigated in regard to transmissible spongiform encephalopathies. Previous assessments of nasal mucosa infection by prions following intracerebral challenge suggested a potential centrifugal spread along the olfactory nerve fibers of the pathological prion protein (PrP(Sc)). Whether the nasal cavity may be a route for centripetal prion infection to the brain has also been experimentally studied. With the present study, we wanted to determine whether prion deposition in the OS occurs also under field conditions and what type of anatomical localization PrP(Sc) might display there. We report here on detection by different techniques of PrP(Sc) in the nasal mucosa and in the OS-related brain areas of sheep affected by natural scrapie. PrP(Sc) was detected in the perineurium of the olfactory nerve bundles in the medial nasal concha and in nasal-associated lymphoid tissue. Olfactory receptor neurons did not show PrP(Sc) immunostaining. PrP(Sc) deposition was found in the brain areas of olfactory fiber projection, chiefly in the olfactory bulb and the olfactory cortex. The prevalent PrP(Sc) deposition patterns were subependymal, perivascular, and submeningeal. This finding, together with the discovery of an intense PrP(Sc) immunostaining in the meningeal layer of the olfactory nerve perineurium, at the border with the subdural space extension surrounding the nerve rootlets, strongly suggests a probable role of cerebrospinal fluid in conveying prion infectivity to the nasal submucosa.

The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrP(TSE)), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrP(TSE) type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle.

Bovine spongiform encephalopathy (BSE), the prion disease in cattle, was widely believed to be caused by only one strain, BSE-C. BSE-C causes the fatal prion disease named new variant Creutzfeldt-Jacob disease in humans. Two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H, have been discovered in several countries since 2004; their transmissibility and phenotypes in humans are unknown. We investigated the infectivity and human phenotype of BASE strains by inoculating transgenic (Tg) mice expressing the human prion protein with brain homogenates from two BASE strain-infected cattle. Sixty percent of the inoculated Tg mice became infected after 20 to 22 months of incubation, a transmission rate higher than those reported for BSE-C. A quarter of BASE strain-infected Tg mice, but none of the Tg mice infected with prions causing a sporadic human prion disease, showed the presence of pathogenic prion protein isoforms in the spleen, indicating that the BASE prion is intrinsically lymphotropic. The pathological prion protein isoforms in BASE strain-infected humanized Tg mouse brains are different from those from the original cattle BASE or sporadic human prion disease. Minimal brain spongiosis and long incubation times are observed for the BASE strain-infected Tg mice. These results suggest that in humans, the BASE strain is a more virulent BSE strain and likely lymphotropic.