Hepatitis E Virus (HEV): Identification of Subtypes 3b and 3m in Wild Boar Population in Liguria Region, Italy.

The wild boar is an important natural reservoir for the zoonotic transmission of the hepatitis E virus (HEV) around the world. In particular, HEV genotypes 3 and 4 are an emerging problem in industrialized countries, as the number of wild boars has increased, and their territory is ever closer to farms and populated areas. This study describes the HEV prevalence and geographic circulation among wild boar populations in the Ligurian region (Italy) during the period 2019-2022. Liver samples from 849 wild boars were analyzed for HEV RNA using real-time RT-PCR; positive samples were then subjected to sequencing and phylogenetic analysis. Overall, 6.7% of the wild boars were positive for HEV RNA; however, in the last two years, the percentage of positive animals almost doubled. Phylogenetic analysis showed that wild boar HEV sequences belonged to genotype 3 and clustered within subtypes 3a and 3c, and, for the first time in Italy, subtypes 3b and 3m were identified. Interestingly, 13 sequences could not be assigned to a recognized subtype. Furthermore, the results showed different circulations of identified subtypes across the territory. These findings increase the knowledge of HEV-3 heterogeneity in Italy and describe the role of wild boars in maintaining an active viral circulation in the environment.

Evaluation of monkeypox virus infection of black-tailed prairie dogs (Cynomys ludovicianus) using in vivo bioluminescent imaging.

Monkeypox (MPX) is a re-emerging zoonotic disease that is endemic in Central and West Africa, where it can cause a smallpox-like disease in humans. Despite many epidemiologic and field investigations of MPX, no definitive reservoir species has been identified. Using recombinant viruses expressing the firefly luciferase (luc) gene, we previously demonstrated the suitability of in vivo bioluminescent imaging (BLI) to study the pathogenesis of MPX in animal models. Here, we evaluated BLI as a novel approach for tracking MPX virus infection in black-tailed prairie dogs (Cynomys ludovicianus). Prairie dogs were affected during a multistate outbreak of MPX in the US in 2003 and have since been used as an animal model of this disease. Our BLI results were compared with PCR and virus isolation from tissues collected postmortem. Virus was easily detected and quantified in skin and superficial tissues by BLI before and during clinical phases, as well as in subclinical secondary cases, but was not reliably detected in deep tissues such as the lung. Although there are limitations to viral detection in larger wild rodent species, BLI can enhance the use of prairie dogs as an animal model of MPX and can be used for the study of infection, disease progression, and transmission in potential wild rodent reservoirs.

Pharmacokinetics of ceftiofur crystalline-free acid (EXCEDE sterile suspension) administered via intramuscular injection in wild California sea lions (Zalophus californianus).

The pharmacokinetics of ceftiofur crystalline-free acid (EXCEDE Sterile Suspension, 200 mg ceftiofur equivalents/ml) were determined for the California sea lion (Zalophus californianus). A single dose of EXCEDE was administered intramuscularly at 6.6 mg/kg to 12 wild California sea lions during rehabilitation. The first 10 animals were each assigned to two blood collection time points, with a total of 10 time points at: 6, 12, 24, 48, 72, 96, 120, 144, 168, and 192 hr after administration of the drug. An additional two animals were sampled 1, 2, 3, 4, 5, and 6 hr postinjection. Plasma was separated within 10 min of blood collection and stored at -20 degrees C until analysis. Plasma concentrations of ceftiofur, desfuroylceftiofur, and related metabolites, were determined using liquid chromatography with tandem mass spectrometry (MS). Maximum plasma concentrations of ceftiofur and related metabolites were observed 24 hr postdosing with a mean concentration of 3.6 microg/ml. The half life (60 hr) and area under the curve (270 microg x hr/ml) were also determined. These data indicate that a single dose of EXCEDE at 6.6 mg/kg i.m. would likely maintain a mean plasma drug level >0.6 microg/ml for 5 days and >0.5 microg/ml for 8 days.

Antemortem diagnosis of a ventricular septal defect in a California sea lion Zalophus californianus.

A yearling California sea lion Zalophus californianus stranded in poor body condition, and on physical examination a heart murmur was audible bilaterally. The sea lion was diagnosed with a left-to-right shunting membranous ventricular septal defect (VSD) using B-mode, color-flow Doppler and continuous-wave Doppler echocardiography. A left-to-right intracardiac shunting lesion was confirmed during cardiac angiographic computed tomography. The VSD defect was verified during the necropsy examination. On histologic examination concurrent mild multifocal myocarditis with focal mild ventricular free-wall myocardial necrosis were identified. A specific cause for the myocarditis and myocardial necrosis was not found, and association with the VSD and resultant myocardial dysfunction was presumed. This is the first report of the antemortem diagnosis of a VSD in a marine mammal and the first report of a VSD in a California sea lion.

Magnetic resonance imaging quality and volumes of brain structures from live and postmortem imaging of California sea lions with clinical signs of domoic acid toxicosis.

Our goal in this study was to compare magnetic resonance images and volumes of brain structures obtained alive versus postmortem of California sea lions Zalophus californianus exhibiting clinical signs of domoic acid (DA) toxicosis and those exhibiting normal behavior. Proton density-(PD) and T2-weighted images of postmortem-intact brains, up to 48 h after death, provided similar quality to images acquired from live sea lions. Volumes of gray matter (GM) and white matter (WM) of the cerebral hemispheres were similar to volumes calculated from images acquired when the sea lions were alive. However, cerebrospinal fluid (CSF) volumes decreased due to leakage. Hippocampal volumes from postmortem-intact images were useful for diagnosing unilateral and bilateral atrophy, consequences of DA toxicosis. These volumes were similar to the volumes in the live sea lion studies, up to 48 h postmortem. Imaging formalin-fixed brains provided some information on brain structure; however, images of the hippocampus and surrounding structures were of poorer quality compared to the images acquired alive and postmortem-intact. Despite these issues, volumes of cerebral GM and WM, as well as the hippocampus, were similar to volumes calculated from images of live sea lions and sufficient to diagnose hippocampal atrophy. Thus, postmortem MRI scanning (either intact or formalin-fixed) with volumetric analysis can be used to investigate the acute, chronic and possible developmental effects of DA on the brain of California sea lions.

Consumption of baits containing raccoon pox-based plague vaccines protects black-tailed prairie dogs (Cynomys ludovicianus).

Baits containing recombinant raccoon poxvirus (RCN) expressing plague antigens (fraction 1 [F1] and a truncated form of the V protein-V307) were offered for voluntary consumption several times over the course of several months to a group of 16 black-tailed prairie dogs (Cynomys ludovicianus). For comparison, another group of prairie dogs (n = 12) was injected subcutaneously (SC) (prime and boost) with 40 microg of F1-V fusion protein absorbed to alum, a vaccine-adjuvant combination demonstrated to elicit immunity to plague in mice and other mammals. Control animals received baits containing RCN without the inserted antigen (n = 8) or injected diluent (n = 7), and as there was no difference in their survival rates by Kaplan-Meier analysis, all of them were combined into one group in the final analysis. Mean antibody titers to Yersinia pestis F1 and V antigen increased (p < 0.05) in the vaccinated groups compared to controls, but titers were significantly higher (p < 0.0001) in those receiving injections of F1-V fusion protein than in those orally vaccinated with RCN-based vaccine. Interestingly, upon challenge with approximately 70,000 cfu of virulent Y. pestis, oral vaccination resulted in survival rates that were significantly higher (p = 0.025) than the group vaccinated by injection with F1-V fusion protein and substantially higher (p < 0.0001) than the control group. These results demonstrate that oral vaccination of prairie dogs using RCN-based plague vaccines provides significant protection against challenge at dosages that simulate simultaneous delivery of the plague bacterium by numerous flea bites.

Neuroanatomy and volumes of brain structures of a live California sea lion (Zalophus californianus) from magnetic resonance images.

The California sea lion (Zalophus californianus) has been a focal point for sensory, communication, cognition, and neurological disease studies in marine mammals. However, as a scientific community, we lack a noninvasive approach to investigate the anatomy and size of brain structures in this species and other free-ranging, live marine mammals. In this article, we provide the first anatomically labeled, magnetic resonance imaging-based atlas derived from a live marine mammal, the California sea lion. The brain of the California seal lion contained more secondary gyri and sulci than the brains of terrestrial carnivores. The olfactory bulb was present but small. The hippocampus of the California sea lion was found mostly in the ventral position with very little extension dorsally, quite unlike the canids and the mustelids, in which the hippocampus is present in the ventral position but extends dorsally above the thalamus. In contrast to the canids and the mustelids, the pineal gland of the California sea lion was strikingly large. In addition, we report three-dimensional reconstructions and volumes of cerebrospinal fluid, cerebral ventricles, total white matter (WM), total gray matter (GM), cerebral hemispheres (WM and GM), cerebellum and brainstem combined (WM and GM), and hippocampal structures all derived from magnetic resonance images. These measurements are the first to be determined for any pinniped species. In California sea lions, this approach can be used not only to relate cognitive and sensory capabilities to brain size but also to investigate the neurological effects of exposure to neurotoxins such as domoic acid.