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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.

Neurofilament light chain (Nf-L) is a biomarker for axonal damage in human neurology but is understudied in cattle. With this study we wanted to determine Nf-L stability at two different storage temperatures and Nf-L levels in healthy cattle and the relationship with age, evaluate whether Nf-L holds diagnostic potential for neurological disorders, and whether an association exists between Nf-L in serum and in cerebrospinal fluid (CSF). To do this, we measured Nf-L levels in CSF and serum samples from 49 healthy and 75 sick cattle. Storage at −80 °C or −20 °C had no impact on Nf-L concentration. Physiological median Nf-L levels were 6.3 pg/mL (serum) and 414 pg/mL (CSF) in calves and 5.5 pg/mL (serum) and 828 pg/mL (CSF) in adult cattle. There was no association between Nf-L levels in CSF and calf age (r 2 0.07, p  = 0.13), while a weak association was found for Nf-L in serum (r 2 0.26, p  = 0.01), and a significant association in adult cattle (CSF, r 2 0.69, p  = 0.0001; serum, r 2 0.68, p  = 0.0003). CSF Nf-L levels were higher in samples from animals with degenerative (median Nf-L 49971 pg/mL) and infectious central nervous system (CNS) disorders (median Nf-L, age < 2 months 8863 pg/mL; age 2–12 months 17474 pg/mL; age 1–6 years 3546 pg/mL), CNS anomalies and metabolic/toxic disorders. There was a significant association between CSF Nf-L and serum Nf-L in cattle with neurological disorders (r 2 0.2, p  = 0.009). Taken together, these findings suggest the potential of Nf-L as a diagnostic tool in cattle neurology.

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.

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. Graphical Abstract

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.

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.

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.

CWD surveillance and diagnosis are important issues in Europe since its detection in Norway, as some of its strains, like that of classical scrapie, are contagious. In addition, there are concerns as several matters about CWD are not yet known. Although diagnostic methods for the active surveillance in bovine and small ruminants have been able to detect the European CWD strains, a retrospective study on Italian wild red deer (Cervus elaphus) samples was performed to compare the results obtained from rapid screening tests, authorized according to EU Regulation 999/2001, and the RT-QuIC, a highly sensitive method in the detection of prion disease infection. A total of one hundred brainstems and medial retropharyngeal lymph nodes were selected out of those received from the CWD Italian surveillance system. Confirmed CWD-positive and -negative samples were included in the study as controls. All of the samples were first tested with the HerdChek BSE–Scrapie Antigen Test and then using the RT-QuIC. The rapid test was negative in all brainstem and lymph node samples. RT-QuIC analyses showed only one red deer brainstem sample positive for seeding activity, while all lymph nodes were negative, including the one from this case. This positive brainstem sample was then re-extracted and retested using two different recombinant prion protein substrates (Ha90-231; BV23-231) and their different batches from the first analyses. Seeding activity was consistently confirmed across both substrates and extractions, with positive signals detected down to dilutions of 10−4 using rPrP Ha90-231 and as low as 10−6 with rPrP BV23-231. The additional diagnostic investigations performed on this red deer using the alternative rapid test (TeSeE SAP Combi), Western blot, and immunohistochemistry showed negative results both in the brainstem and lymph nodes. This study showed that overall, the results obtained with the HerdChek BSE–Scrapie Antigen Test and RT-QuIC agree except in one case. Our findings highlight the potential of the RT-QuIC method to detect very low levels of PrPSc-associated seeding activity that may escape detection using classical methods. While seeding activity does not always equate to infectivity, only a bioassay will confirm the real disease status of this Italian case. These findings support the integration of RT-QuIC as a powerful complementary tool within existing surveillance frameworks to strengthen early detection and diagnostic accuracy.

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. 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. 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. 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.

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.