Epidemiological and genetic analysis of Cetacean Morbillivirus circulating on the Italian coast between 2018 and 2021.

Cetacean morbillivirus (CeMV) has caused several outbreaks, unusual mortality events, and interepidemic single-lethal disease episodes in the Mediterranean Sea. Since 2012, a new strain with a northeast (NE) Atlantic origin has been circulating among Mediterranean cetaceans, causing numerous deaths. The objective of this study was to determine the prevalence of CeMV in cetaceans stranded in Italy between 2018 and 2021 and characterize the strain of CeMV circulating. Out of the 354 stranded cetaceans along the Italian coastlines, 113 were CeMV-positive. This prevalence (31.9%) is one of the highest reported without an associated outbreak. All marine sectors along the Italian coastlines, except for the northern Adriatic coast, reported a positive molecular diagnosis of CeMV. In one-third of the CeMV-positive cetaceans submitted to a histological evaluation, a chronic form of the infection (detectable viral antigen, the absence of associated lesions, and concomitant coinfections) was suspected. Tissues from 24 animals were used to characterize the strain, obtaining 57 sequences from phosphoprotein, nucleocapsid, and fusion protein genes, which were submitted to GenBank. Our sequences showed the highest identity with NE-Atlantic strain sequences, and in the phylogenetic study, they clustered together with them. Regarding age and species, most of these individuals were adults (17/24, 70.83%) and striped dolphins (19/24, 79.16%). This study improves our understanding on the NE-Atlantic CeMV strain in the Italian waters, supporting the hypothesis of an endemic circulation of the virus in this area; however, additional studies are necessary to deeply comprehend the epidemiology of this strain in the Mediterranean Sea.

A multidisciplinary approach to estimating wolf population size for long-term conservation.

The wolf (Canis lupus) is among the most controversial of wildlife species. Abundance estimates are required to inform public debate and policy decisions, but obtaining them at biologically relevant scales is challenging. We developed a system for comprehensive population estimation across the Italian alpine region (100,000 km2 ), involving 1513 trained operators representing 160 institutions. This extensive network allowed for coordinated genetic sample collection and landscape-level spatial capture-recapture analyses that transcended administrative boundaries to produce the first estimates of key parameters for wolf population status assessment. Wolf abundance was estimated at 952 individuals (95% credible interval 816-1120) and 135 reproductive units (i.e., packs) (95% credible interval 112-165). We also estimated that mature individuals accounted for 33-45% of the entire population. The monitoring effort was spatially estimated thereby overcoming an important limitation of citizen science data. This is an important approach for promoting wolf-human coexistence based on wolf abundance monitoring and an endorsement of large-scale harmonized conservation practices.

Una estrategia multidisciplinaria para la estimación del tamaño poblacional de los lobos para la conservación a largo plazo Resumen El lobo (Canis lupus) está entre las especies de fauna más controversiales. Se requieren estimaciones de abundancia para informar al debate público y las decisiones políticas, pero es un reto obtenerlos en escalas con relevancia biológica. Desarrollamos un sistema para la estimación completa de la población en la región alpina de Italia (100,000 km2 ), con la participación de 1,513 operadores entrenados que representan a 160 instituciones. Esta red extensa permitió una colecta coordinada de muestras genéticas y análisis de captura-recaptura espacial que trascendieron las fronteras administrativas para así producir las primeras estimaciones de los parámetros clave para la evaluación del estado de la población de los lobos. Se estimó la abundancia en 952 individuos (95% intervalo de confianza 816-1120) y 135 unidades reproductivas (es decir, manadas) (95% intervalo de confianza 112-165). También estimamos que los individuos maduros representaban el 33-45% de toda la población. El esfuerzo de monitoreo se estimó espacialmente, por lo que sobrepasó una limitación importante de la ciencia ciudadana. Esta estrategia es importante para promover la coexistencia entre lobos y humanos con base en el monitoreo de la abundancia y el apoyo a las prácticas armonizadas de conservación a gran escala.

First Report of Swinepox in a Wild Boar in Italy: Pathologic and Molecular Findings.

Swinepox virus (SWPV) is responsible for sporadic acute poxvirus infections in swine worldwide, causing a pathognomonic eruptive proliferative dermatitis. Beside direct and congenital transmission, the pig louse Haematopinus suis acts as a mechanical vector and favors virus infection through skin lesions. Infections are generally described in domestic pigs, while only a few cases have been reported in wild boars, in Austria and Germany. In September 2022, SWPV infection was suspected at post-mortem examination of a wild boar piglet with characteristic lesions in Liguria, Northwest Italy. The piglet was heavily parasitized by swine lice (H. suis). SWPV was then confirmed by histological and molecular analyses. Possible viral co-infections were also investigated (African swine fever virus, classical swine fever virus, parvovirus, circovirus, Aujeszky's disease virus and hepatitis E virus). This article describes gross and histopathologic features of SWPV infection, differential diagnosis, and potential vector-borne transmission to domestic pigs, presenting a brief review of the literature on the topic. SWPV infection is reported in wild boars in Italy for the first time. The finding of SWPV in a wild boar in an area with a very limited pig population may suggest the existence of a "wildlife cycle" in the area. Further investigations are needed to understand the real risk of transmission of SWPV to domestic pigs as well as the role of other arthropod vectors.

First detection of an Italian human-to-cat outbreak of SARS-CoV-2 Alpha variant - lineage B.1.1.7.

The emergence of new SARS-CoV-2 variants and their rapid spread pose a threat to both human and animal health and may conceal unknown risks. This report describes an Italian human-to-cat outbreak of SARS-CoV-2 lineage B.1.1.7 (the Alpha variant) . On March 7th, 2021, approximately ten days after COVID-19 appeared in the family, the onset of respiratory signs in a cat by COVID-19-affected owners led to an in-depth diagnostic investigation, combining clinical and serological data with rt-qPCR-based virus detection and whole genome sequencing. The Alpha variant was confirmed first in the owners and a few days later in the cat that was then monitored weekly: the course was similar with one-week lag time in the cat. In addition, based on comparative analysis of genome sequences from our study and from 200 random Italian cases of Alpha variant, the familial cluster was confirmed. The temporal sequence along with the genomic data support a human-to-animal transmission. Such an event emphasizes the importance of studying the circulation and dynamics of SARS-CoV-2 variants in humans and animals to better understand and prevent potential spillover risks or unwarranted alerts involving our pet populations.

Four types of scrapie in goats differentiated from each other and bovine spongiform encephalopathy by biochemical methods.

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrPSc) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrPSc (PrPres) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants-CS-1 and CS-2 (mainly Italy)-which differed in proteolytic resistance of the PrPres N-terminus. Suitable PrPres markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters.

CANINE DISTEMPER VIRUS AS AN EMERGING MULTIHOST PATHOGEN IN WILD CARNIVORES IN NORTHWEST ITALY.

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.

Predicting the impact of selection for scrapie resistance on PRNP genotype frequencies in goats.

The European Union has implemented breeding programmes to increase scrapie resistance in sheep. A similar approach can be applied also in goats since the K222 allele provides a level of resistance equivalent to that of ARR in sheep. The European Food Safety Authority stated that breeding for resistance could be offered as an option for Member States to control classical scrapie in goats. We assessed the impact of different breeding strategies on PRNP genotype frequencies using a mathematical model that describes in detail the evolution of K222 in two goat breeds, Chamois Coloured and Saanen. Different patterns of age structure and replacement rate were modelled as factors affecting response to selection. Breeding for scrapie resistance can be implemented in goats, even though the initial K222 frequencies in these breeds are not particularly favourable and the rate at which the resistant animals increase, both breeding and slaughtered for meat production, is slow. If the goal is not to achieve the fixation of resistance allele, it is advisable to carry out selection only until a desired frequency of K222-carriers has been attained. Nucleus selection vs. selection on the overall populations is less expensive but takes longer to reach the desired output. The programme performed on the two goat breeds serves as a model of the response the selection could have in other breeds that show different initial frequencies and population structure. In this respect, the model has a general applicability.

Genetic resistance to transmissible spongiform encephalopathies (TSE) in goats.

Breeding programmes to promote resistance to classical scrapie, similar to those for sheep in existing transmissible spongiform encephalopathies (TSE) regulations, have not been established in goats. The European Commission requested a scientific opinion from EFSA on the current knowledge of genetic resistance to TSE in goats. An evaluation tool, which considers both the weight of evidence and strength of resistance to classical scrapie of alleles in the goat PRNP gene, was developed and applied to nine selected alleles of interest. Using the tool, the quality and certainty of the field and experimental data are considered robust enough to conclude that the K222, D146 and S146 alleles both confer genetic resistance against classical scrapie strains known to occur naturally in the EU goat population, with which they have been challenged both experimentally and under field conditions. The weight of evidence for K222 is greater than that currently available for the D146 and S146 alleles and for the ARR allele in sheep in 2001. Breeding for resistance can be an effective tool for controlling classical scrapie in goats and it could be an option available to member states, both at herd and population levels. There is insufficient evidence to assess the impact of K222, D146 and S146 alleles on susceptibility to atypical scrapie and bovine spongiform encephalopathy (BSE), or on health and production traits. These alleles are heterogeneously distributed across the EU Member States and goat breeds, but often at low frequencies (< 10%). Given these low frequencies, high selection pressure may have an adverse effect on genetic diversity so any breeding for resistance programmes should be developed at Member States, rather than EU level and their impact monitored, with particular attention to the potential for any negative impact in rare or small population breeds.

Characterization of Amyloid-ß Deposits in Bovine Brains.

Amyloid-ß (Aß) deposits are seen in aged individuals of many mammalian species that possess the same aminoacid sequence as humans. This study describes Aß deposition in 102 clinically characterized cattle brains from animals aged 0 to 20 years. Extracellular and intracellular Aß deposition was detected with 4G8 antibody in the cortex, hippocampus, and cerebellum. X-34 staining failed to stain Aß deposits, indicating the non ß-pleated nature of these deposits. Western blot analysis and surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry revealed in Tris, Triton, and formic acid fractions the presence of different Aß peptides, characterized mainly by C-terminally truncated forms. Exploration of the genetic variability of APOE, PSEN1, and PSEN2 genes involved in Alzheimer's disease pathogenesis revealed several previously unreported polymorphisms. This study demonstrates certain similarities between Aß deposition patterns exhibited in cattle brains and those in the human brain in early stages of aging. Furthermore, the identification of the same Aß peptides reported in humans, but unable to form aggregates, supports the hypothesis that cattle may be protected against amyloid plaque formation.

Pathogenesis and Transmission of Classical and Atypical BSE in Cattle.

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 (PrPSc), 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.

Sarcocystis Spp. Prevalence in Bovine Minced Meat: A Histological and Molecular Study.

Sarcosporidiosis is caused by ingestion of contaminated raw or undercooked bovine meat and, although considered a minor zoonosis, it can represent a threath for immunocompromised people. Aim of this study was to determine the prevalence of Sarcocystis spp. in bovine minced meat intended for raw consumption collected from butcher shops and retail stores in Turin's province (Piedmont region, Northwest Italy). Twenty-five samples were examined in parallel by histology and polymerase chain reaction (PCR). The prevalence of infestation of Sarcocystis spp. resulted to be 64% [confidence interval (CI) 95% 42-82] and 88% (CI 95% 69-97) respectively by histology and PCR. In detail, the prevalence resulted 80% for S. cruzi (CI 95% 59-93), 68% for S. hominis (CI 95% 46-85) and 4% for S. hirsuta (CI 95% 0.10-20). The high prevalence of S. hominis highlights that sarcosporidiosis may constitute a public health problem in Italy, particularly in regions like Piedmont, that has traditional dishes prepared from raw or undercooked bovine meat.

Molecular characterization of flaviviruses from field-collected mosquitoes in northwestern Italy, 2011-2012.

BACKGROUND: The genus Flavivirus comprises several mosquito-borne species, including the zoonotic pathogens West Nile and Usutu virus, circulating in animals and humans in Italy since 1998. Due to its ecological and geographical features, Piedmont is considered a risk area for flavivirus transmission. Here we report the results of a flavivirus survey (detection and genetic characterization) of mosquitoes collected in Piedmont in 2012 and the genetic characterization of three strains detected in 2011. METHODS: Pools of 1-203 mosquitoes, upon RNA extraction with TRIzol, were screened by a PCR assay for a 263 bp fragment of the Flavivirus NS5 gene. All positive samples were tested with a specific PCR for the E protein gene of Usutu virus and a generic Flavivirus RT-nested-PCR for a larger tract of the NS5 gene before sequencing. Phylogenetic trees were built with both NS5 fragments of representative Flavivirus species. DNA extracts of part of the positive pools were tested to detect sequences integrated in the host genome. RESULTS: Thirty-four mosquito pools resulted positive for flaviviruses, and twenty-five flavivirus sequences underwent phylogenetic analysis for the short NS5 fragment. Among the 19 sequences correlating with the insect-specific flavivirus group, ten samples, retrieved from Aedes albopictus, clustered within Aedes flavivirus, while the other nine aggregated in a separate clade composed of strains from various mosquito species (mainly Aedes vexans) from Piedmont and the Czech Republic. Six out of these nine also presented a DNA form of the sequence. The remaining sequences belonged to the mosquito-borne group: four, all from Culex pipiens, correlated to Italian Usutu virus strains, whereas two, from Ochlerotatus caspius, were highly similar to Marisma mosquito virus (MMV). CONCLUSIONS: Our findings confirm the circulation of Usutu virus and of the potentially zoonotic Marisma mosquito virus in Piedmont. This is the first detection of Aedes flavivirus in Piedmont. Finally, further evidence for the integration of Flavivirus nucleic acid into the host genome has been shown. These results underline the importance of continuing intense mosquito-based surveillance in Piedmont, supported by a mosquito control program in areas at high risk for human exposure.

Therapeutic effect of CHF5074, a new γ-secretase modulator, in a mouse model of scrapie.

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 Aß 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.

Evaluation of two sets of immunohistochemical and Western blot confirmatory methods in the detection of typical and atypical BSE cases.

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.

State-of-the-art review of goat TSE in the European Union, with special emphasis on PRNP genetics and epidemiology.

Scrapie is a fatal, neurodegenerative disease of sheep and goats. It is also the earliest known member in the family of diseases classified as transmissible spongiform encephalopathies (TSE) or prion diseases, which includes Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE), and chronic wasting disease in cervids. The recent revelation of naturally occurring BSE in a goat has brought the issue of TSE in goats to the attention of the public. In contrast to scrapie, BSE presents a proven risk to humans. The risk of goat BSE, however, is difficult to evaluate, as our knowledge of TSE in goats is limited. Natural caprine scrapie has been discovered throughout Europe, with reported cases generally being greatest in countries with the highest goat populations. As with sheep scrapie, susceptibility and incubation period duration of goat scrapie are most likely controlled by the prion protein (PrP) gene (PRNP). Like the PRNP of sheep, the caprine PRNP shows significantly greater variability than that of cattle and humans. Although PRNP variability in goats differs from that observed in sheep, the two species share several identical alleles. Moreover, while the ARR allele associated with enhancing resistance in sheep is not present in the goat PRNP, there is evidence for the existence of other PrP variants related to resistance. This review presents the current knowledge of the epidemiology of caprine scrapie within the major European goat populations, and compiles the current data on genetic variability of PRNP.

Detection of typical and atypical bovine spongiform encephalopathy and scrapie prion strains by prion protein motif-grafted antibodies.

To evaluate further the reactivity of prion-specific monoclonal antibodies containing the 89-112 or 136-158 prion protein (PrP) polypeptides, immunoprecipitations were performed on brain extracts from Italian bovines, sheep and goats with transmissible spongiform encephalopathies. No binding of IgG 89-112 or IgG 136-158 to PrP in normal brain extracts was detected. Conversely, both reagents immunoprecipitated PrP from bovine and bovine amyloidotic spongiform encephalopathies, and from typical and atypical scrapie brain extracts. The immunoprecipitated PrP bands mirrored the Western blot (WB) profile of the different prion strains, indicating universal affinity of two independent PrP regions for disease-associated PrP conformers regardless of species source and strain properties. Immunoprecipitation with motif-grafted antibodies increased the sensitivity of conventional detection methods based on centrifugation followed by WB, which was confirmed by assay of diluted samples using both methods. These reagents or derivative molecules may thus find broad applications in prion detection and research.

Olfactory system involvement in natural scrapie disease.

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.

Evaluation of the human transmission risk of an atypical bovine spongiform encephalopathy prion strain.

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.

Molecular discrimination of atypical bovine spongiform encephalopathy strains from a geographical region spanning a wide area in Europe.

Transmissible spongiform encephalopathy strains can be differentiated by their behavior in bioassays and by molecular analyses of the disease-associated prion protein (PrP) in a posttranslationally transformed conformation (PrPSc). Until recently, isolates from cases of bovine spongiform encephalopathy (BSE) appeared to be very homogeneous. However, a limited number of atypical BSE isolates have recently been identified upon analyses of the disease-associated proteinase K (PK) resistance-associated moiety of PrPSc (PrPres), suggesting the existence of at least two additional BSE PrPres variants. These are defined here as the H type and the L type, according to the higher and lower positions of the nonglycosylated PrPres band in Western blots, respectively, compared to the position of the band in classical BSE (C-type) isolates. These molecular PrPres variants, which originated from six different European countries, were investigated together. In addition to the migration properties and glycosylation profiles (glycoprofiles), the H- and L-type isolates exhibited enhanced PK sensitivities at pH 8 compared to those of the C-type isolates. Moreover, H-type BSE isolates exhibited differences in the binding of antibodies specific for N- and more C-terminal PrP regions and principally contained two aglycosylated PrPres moieties which can both be glycosylated and which is thus indicative of the existence of two PrPres populations or intermediate cleavage sites. These properties appear to be consistent within each BSE type and independent of the geographical origin, suggesting the existence of different BSE strains in cattle. The choice of three antibodies and the application of two pHs during the digestion of brain homogenates provide practical and diverse tools for the discriminative detection of these three molecular BSE types and might assist with the recognition of other variants.

Conversion of the BASE prion strain into the BSE strain: the origin of BSE?

Atypical neuropathological and molecular phenotypes of bovine spongiform encephalopathy (BSE) have recently been identified in different countries. One of these phenotypes, named bovine "amyloidotic" spongiform encephalopathy (BASE), differs from classical BSE for the occurrence of a distinct type of the disease-associated prion protein (PrP), termed PrP(Sc), and the presence of PrP amyloid plaques. Here, we show that the agents responsible for BSE and BASE possess different biological properties upon transmission to transgenic mice expressing bovine PrP and inbred lines of nontransgenic mice. Strikingly, serial passages of the BASE strain to nontransgenic mice induced a neuropathological and molecular disease phenotype indistinguishable from that of BSE-infected mice. The existence of more than one agent associated with prion disease in cattle and the ability of the BASE strain to convert into the BSE strain may have important implications with respect to the origin of BSE and spongiform encephalopathies in other species, including humans.