Strengthening preparedness against global health threats: A paradigm shift based on One Health approaches.

The implementation of preparedness strategies to prevent and mitigate the impact of global health threats poses several challenges. It should promptly identify cross-cutting drivers of pandemic threats, assess context-specific risks, engage multiple stakeholders, and translate complex data from multiple sources into accessible information for action. This requires a coordinated, multidisciplinary and multisectoral effort engaging systems that, most of the time, work in isolation. The One Health (OH) approach promotes the collaboration and communication among different disciplines and sectors, and could be applied across the preparedness phases at national and international level. We discuss here gaps and needs in preparedness strategies, which can benefit from the OH approach, and a set of actionable recommendations, as shared with the G20-2021 with a dedicated Policy Brief. The discussion adds to the current debate about OH operationalization and promotes a paradigm shift towards coordinated prevention and preparedness strategies for early assessment and management of global health threats.

Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy.

SARS-CoV-2 emerged from animals and is now easily transmitted between people. Sporadic detection of natural cases in animals alongside successful experimental infections of pets, such as cats, ferrets and dogs, raises questions about the susceptibility of animals under natural conditions of pet ownership. Here, we report a large-scale study to assess SARS-CoV-2 infection in 919 companion animals living in northern Italy, sampled at a time of frequent human infection. No animals tested PCR positive. However, 3.3% of dogs and 5.8% of cats had measurable SARS-CoV-2 neutralizing antibody titers, with dogs from COVID-19 positive households being significantly more likely to test positive than those from COVID-19 negative households. Understanding risk factors associated with this and their potential to infect other species requires urgent investigation.

Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy.

SARS-CoV-2 originated in animals and is now easily transmitted between people. Sporadic detection of natural cases in animals alongside successful experimental infections of pets, such as cats, ferrets and dogs, raises questions about the susceptibility of animals under natural conditions of pet ownership. Here we report a large-scale study to assess SARS-CoV-2 infection in 817 companion animals living in northern Italy, sampled at a time of frequent human infection. No animals tested PCR positive. However, 3.4% of dogs and 3.9% of cats had measurable SARS-CoV-2 neutralizing antibody titers, with dogs from COVID-19 positive households being significantly more likely to test positive than those from COVID-19 negative households. Understanding risk factors associated with this and their potential to infect other species requires urgent investigation. ONE SENTENCE SUMMARY: SARS-CoV-2 antibodies in pets from Italy.

PrPC Governs Susceptibility to Prion Strains in Bank Vole, While Other Host Factors Modulate Strain Features.

Bank vole is a rodent species that shows differential susceptibility to the experimental transmission of different prion strains. In this work, the transmission features of a panel of diverse prions with distinct origins were assayed both in bank vole expressing methionine at codon 109 (Bv109M) and in transgenic mice expressing physiological levels of bank vole PrPC (the BvPrP-Tg407 mouse line). This work is the first systematic comparison of the transmission features of a collection of prion isolates, representing a panel of diverse prion strains, in a transgenic-mouse model and in its natural counterpart. The results showed very similar transmission properties in both the natural species and the transgenic-mouse model, demonstrating the key role of the PrP amino acid sequence in prion transmission susceptibility. However, differences in the PrPSc types propagated by Bv109M and BvPrP-Tg407 suggest that host factors other than PrPC modulate prion strain features. IMPORTANCE: The differential susceptibility of bank voles to prion strains can be modeled in transgenic mice, suggesting that this selective susceptibility is controlled by the vole PrP sequence alone rather than by other species-specific factors. Differences in the phenotypes observed after prion transmissions in bank voles and in the transgenic mice suggest that host factors other than the PrPC sequence may affect the selection of the substrain replicating in the animal model.

Neurochemistry of myenteric plexus neurons of bank vole (Myodes glareolus) ileum.

The neurochemistry of enteric neurons differs among species of small laboratory rodents (guinea-pig, mouse, rat). In this study we characterized the phenotype of ileal myenteric plexus (MP) neuronal cells and fibers of the bank vole (Myodes glareolus), a common rodent living in Europe and in Northern Asia which is also employed in prion experimental transmission studies. Six neuronal markers were tested: choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), calbindin (CALB), calcitonin gene-related peptide (CGRP) and substance P (SP), along with HuC/D as a pan-neuronal marker. Neurons expressing ChAT- and nNOS-immunoreactivity (IR) were 36 ± 12% and 24 ± 5%, respectively. Those expressing CGRP-, SP- and CALB-IR were 3 ± 3%, 21 ± 5% and 6 ± 2%, respectively. Therefore, bank vole MPs differ consistently from murine MPs in neurons expressing CGRP-, SP- and CALB-IR. These data may contribute to define the prion susceptibility of neuron cell populations residing within ileal MPs from bank voles, along with their morpho-functional alterations following oral experimental prion challenge.

Co-existence of classical scrapie and Nor98 in a sheep from an Italian outbreak.

Nor98 is an atypical scrapie strain characterized by a molecular pattern and brain distribution of the pathological prion protein (PrP(Sc)) different from classical scrapie. In Italy, 69 atypical cases have been identified so far and all were characterized as Nor98 strain. In this paper we report an unusual case in a sheep which showed immunohistochemical and molecular features of PrP(Sc) different from the other atypical cases. The sheep was from an outbreak where the index and the other four cases were affected by classical scrapie. Histopathological, immunohistochemical and Western blot analyses on the brain of the unusual case revealed the simultaneous presence of pathological features characteristic of Nor98 and classical scrapie. Interestingly, the prevalent disease phenotype in the brainstem was classical scrapie-like, while in the cerebral cortex and cerebellum the Nor98 phenotype was dominant. The sub-mandibular lymph node was positive and showed a PrP(Sc) molecular pattern referable to classical scrapie. The PrP genotype was AL(141)RQ/AF(141)RQ. Taken together, the occurrence of classical scrapie in the outbreak, the PrP genotype, the involvement of different cellular targets in the brain and the pathological and molecular PrP(Sc) features observed suggest that this unusual case may result from the co-existence of Nor98 and classical scrapie.

A cell line infectible by prion strains from different species.

It has been shown previously that ovine prion protein (PrP(C)) renders rabbit epithelial RK13 cells permissive to the multiplication of ovine prions, thus providing evidence that species barriers can be crossed in cultured cells through the expression of a relevant PrP(C). The present study significantly extended this observation by showing that mouse and bank vole prions can be propagated in RK13 cells that express the corresponding PrP(C). Importantly, the respective molecular patterns of abnormal PrP (PrP(res)) and, where examined, the neuropathological features of the infecting strains appeared to be maintained during the propagation in cell culture. These findings indicate that RK13 cells can be genetically engineered to replicate prion strains faithfully from different species. Such an approach may facilitate investigations of the molecular basis of strain identity and prion diversity.

Quantitative profiling of the pathological prion protein allotypes in bank voles by liquid chromatography-mass spectrometry.

The conversion of the cellular prion protein (PrP(C)) into a misfolded isoform (PrP(TSE)) that accumulates in the brain of affected individuals is the key feature of transmissible spongiform encephalopaties (TSEs). Susceptibility to TSEs is influenced by polymorphisms of the prion gene suggesting that the presence of certain amino acid residues may facilitate the pathological conversion. In this work, we describe a quantitative, fast and reliable HPLC-MS method that allowed to demonstrate that in the brain of 109(Met/Ile) heterozygous bank voles infected with the mouse adapted scrapie strain 139A, there are comparable amounts of PrP(TSE) with methionine or isoleucine in position 109, suggesting that in this TSE model the two allotypes have similar rates of accumulation. This method can be easily adapted for the quantitative determination of PrP allotypes in the brain of other natural or experimental TSE models.

Advances in scrapie research.

The bovine spongiform encephalopathy (BSE) crisis clearly demonstrated the need to keep animal transmissible spongiform encephalopathies (TSE) under control in order to protect animal and human health. Scrapie is the most widespread TSE of livestock in the world. For this reason, health authorities in different countries have elaborated plans that aim towards scrapie eradication. The unusual nature of the scrapie agent and the fragmented status of scientific knowledge about it, along with the limitations of currently available diagnostic tools, make it unlikely that the objective of eradication will be achieved in the near future. Scientific research is focused on acquiring the knowledge that will improve the efficiency of these efforts.

PrP(Sc) deposition in nervous tissues without lymphoid tissue involvement is frequently found in ARQ/ARQ Sarda breed sheep preclinically affected with natural scrapie.

The pathogenesis of natural scrapie in Sarda breed sheep was investigated in 1050 asymptomatic and 49 sick sheep from scrapie-affected flocks. Central and peripheral nervous system, along with lymphoreticular system (LRS) tissues, were subjected to immunohistochemistry (IHC) and Western-blotting (WB) for detection of pathological isoform of the prion protein (PrP(Sc)). A total of 69 of the 1050 clinically healthy sheep were found to be infected with scrapie, with PrP(Sc) being detected in both the central nervous system (CNS) and enteric nervous system (ENS) plexuses of 60 of the sheep, while IHC and WB yielded evidence of (PrP(Sc)) deposition only in lymphoid tissues of the remaining 9 clinically healthy sheep. PrP(Sc) was also detected in the CNS, as well as in ENS plexuses from all of the 49 clinically affected sheep. Nevertheless, 18 of the 69 clinically healthy animals (26%, 17 ARQ/ARQ and 1 ARQ/AHQ sheep), along with 3 ARQ/ARQ sheep (6%) of the clinically affected group, showed no IHC or WB evidence of PrP(Sc) in lymphoid tissues, but PrP(Sc) could be still detected in their CNS and ENS plexuses. The study demonstrates dual CNS and ENS PrP(Sc) deposition in Sarda sheep with scrapie, in spite of an apparent lack of lymphoid tissue involvement in a number of cases.

Identification of the pathological prion protein allotypes in scrapie-infected heterozygous bank voles (Clethrionomys glareolus) by high-performance liquid chromatography-mass spectrometry.

Cerebral formation of the pathological isoform of the prion protein (PrP) is a crucial molecular event in prion diseases. The bank vole (Clethrionomys glareolus) is a rodent species highly susceptible to natural scrapie. The PrP gene of bank vole is polymorphic (Met/Ile) at codon 109. Here we show that homozygous 109Met/Met voles have incubation times shorter than heterozygous 109Met/Ile voles after experimental challenge with three different scrapie isolates. An HPLC-MS/MS method was optimized and applied to investigate whether in heterozygous animals both PrP allotypes are able to undergo pathological conversion. The results demonstrate that both allotypes of the prion protein participate to pathological deposition.

Diagnosis and PrP genotype target of scrapie in clinically healthy sheep of Massese breed in the framework of a scrapie eradication programme.

The application of a selective culling programme in two scrapie affected flocks of Massese breed sheep is described. The genetic susceptibility of this breed and the sensitivity of different diagnostic methods in the pre-clinical diagnosis of scrapie were also investigated. Overall, 2,068 clinically healthy sheep underwent PrP genotyping, providing the basis for selective culling. The prevalence of scrapie infection was investigated in susceptible sheep by two independent diagnostic methods. All the sheep older than 18 months (n = 620) were tested by Prionics Check Western rapid test on the obex, with a prevalence of infection of 3.9%. Furthermore, 385 sheep underwent immunohistochemistry (IHC) on retropharyngeal lymph node (RPLN), with a prevalence of infection of 5.2%. Overall, 32 sheep were diagnosed with pre-clinical scrapie. Of these, 31 were positive by Western blot on the spleen, 29 by IHC on the RPLN and tonsil, 28 by IHC on the obex, 24 by rapid test, and only 18 by IHC on the third eyelid. All the scrapie positive sheep were of the ARQ/ARQ, ARQ/AHQ or ARQ/VRQ genotypes. No significant differences in scrapie prevalence were observed among these genotypes. The estimated risk of the three targeted alleles was also similar, suggesting that in this breed the VRQ allele was not at higher risk for scrapie, compared to the ARQ and AHQ alleles.

Morbillivirus infections in aquatic mammals: a brief overview.

Since 1987, at least eight morbillivirus infection (MI) epidemics have caused mass mortality of several free-living pinniped and cetacean populations around the world. The responsible agents, all belonging to the genus Morbillivirus (family Paramyxoviridae), have been characterized as either "canine distemper virus" strains, infecting pinnipeds, or as three new morbilliviruses, namely "phocid (phocine) distemper virus" , "porpoise morbillivirus" and "dolphin morbillivirus" . The last two agents are currently gathered under the common denomination of "cetacean morbillivirus". At post-mortem examination, a commonly occurring macroscopic lesion is represented by more or less severe bilateral pneumonia, with consolidation, congestion and oedema of both lungs, which fail to collapse. Histologically, a non-suppurative broncho-interstitial pneumonia, characterized by type II pneumocyte hyperplasia and by formation of endobronchial, endobronchiolar and endoalveolar "Warthin-Finkeldey type" syncytia, as well as a multifocal, non-suppurative encephalitis, associated with a severe and generalized lymphoid tissue depletion, are common pathological findings. Furthermore, eosinophilic viral inclusions are often detected, at both the intracytoplasmic and intranuclear level, within bronchial and bronchiolar epithelial, pulmonary syncytial, neuronal and other cell types. These inclusions, along with lymphoid and other cellular elements, are often found to be immunohistochemically positive for morbillivirus antigen. Among the still debated, or even controversial issues regarding MI in sea mammals, the one related to the origin of their causative agents is of particular concern. Another intriguing issue regards the synergistic effects, if any, associated with chronic exposure to a number of environmental pollutants, such as organochlorines and heavy metals. In fact, it is also unknown whether and how these chemicals contribute towards modulating the pathogenic and pathogenetic activity primarily displayed by sea mammal morbilliviruses.

Clinicopathological findings in sheep from Sardinia showing neurological signs of disease.

Histopathological and bacteriological examinations were performed on 178 brains from Sardinian sheep which were showing neurological signs. The sheep represented the total number of sheep with neurological syndromes submitted for diagnostic investigations over a three-year period in Sardinia. Scrapie was detected in 57 cases, cerebrocortical necrosis in 25, intoxication by a typical Mediterranean plant (Cistus species) was suspected in 25, coenurosis was detected in 11 cases, Listeria monocytogenes in eight cases and focal symmetrical encephalomalacia in six cases. Non-suppurative inflammatory changes were observed in three of the brains and suppurative changes were noted in two. Lesions restricted to the spinal cord were found in three cases. In the remaining 38 cases there were no significant neuropathological changes.

Primer extension assay for prion protein genotype determination in sheep.

Scrapie is a transmissible spongiform encephalopathy (TSE) which affects sheep and goats. TSEs are characterised by the conversion of the cellular prion protein (PrP(C)) into the pathological form PrP(Sc). The occurrence of scrapie in sheep is influenced by polymorphisms in the PrP gene; in particular, three codons (136, 154 and 171) are important in conditioning the susceptibility/resistance of sheep to the disease, with the Val/Val(136) Arg/Arg(154) Gln/Gln(171) genotype being the most susceptible and the Ala/Ala(136) Arg/Arg(154) Arg/Arg(171), the most resistant one. The latter genotype seems to confer, in sheep, resistance to the oral infection with bovine spongiform encephalopathy, as well. The selection of genetically resistant sheep populations represents the basis of the recent strategies against ovine TSE in the European Union (EU). Herein, we describe a rapid and simple method, based on the primer extension technique, for PrP genotype determination at codons 136, 154 and 171. Intra-laboratory validation of the method showed accuracy levels comparable to those of sequencing analysis. Such method could be used for both the application of the EU policies requiring PrP genotype analysis in all ovine TSE cases, and the large-scale genotyping claimed by the implementation of breeding programmes for genetic resistance to TSE in sheep.

Animal transmissible spongiform encephalopathies and genetics.

The genotype of the host plays a crucial role in the pathogenesis of transmissible spongiform encephalopathies (TSEs). In this respect, the most important factor is represented by the gene of the prion protein (PrP). The present work summarizes the currently available knowledge on the genetic basis of TSEs focusing, in particular, on sheep scrapie. Interest in this disease has grown markedly following the discovery of bovine spongiform encephalopathy, both for scientific and health reasons. In Italy, specific research grants from the Ministry of Health and the National Research Council (CNR), together with cooperation between the Istituto Superiore di Sanità and the Istituti Zooprofilattici Sperimentali, have allowed us to study the PrP genotype and to investigate the genetic susceptibility to scrapie in the most important Italian sheep breeds, with special reference to Sarda, Comisana and Massese. The PrP genotype in relation to scrapie susceptibility was also studied in goats of Ionica breed.

PrP genotype in Sarda breed sheep and its relevance to scrapie. Brief report.

Several PrP gene polymorphisms modulate sheep scrapie susceptibility. Recently, an increase of scrapie outbreaks has been reported in Italy. A vaccine containing sheep brain homogenate was used in most of the outbreaks. We investigated PrP gene polymorphisms in scrapie-affected and clinically healthy Sarda breed sheep from a flock exposed to the aforementioned vaccine, and in affected Sarda sheep from unexposed flocks. All affected animals were (Gln/Gln)171 homozygous. Moreover, we observed no variation for Ala136 and a new polymorphism (Lys to Asn) at codon 176. Our findings confirm the correlation between scrapie and (Gln/Gln)171 in breeds with no variation for Ala136.

Detection and characterization of Shiga toxin-producing Escherichia coli in feral pigeons.

Escherichia coli strains producing a variant of Shiga toxin 2 (Stx2), designated Stx2f, have been recently described in the stools of feral pigeons. During 1997-1998, 649 pigeons were trapped and examined in three different squares of Rome. Stool samples were collected from each bird and enrichment cultures were examined for the presence of Stx by the vero cell assay. Stx-producing E. coli (STEC) were isolated from the positive cultures and characterized by serotyping and PCR analysis of stx and other virulence-related genes. Stx was detected in 10.8% of the stool enrichment cultures. The percentage of positive birds did not differ significantly for the three flocks considered and the season of sample collection. Conversely, STEC carriage was significantly more frequent in young than in adult birds (17.9 versus 8.2%). None of the birds examined showed signs of disease. STEC strains were isolated from 30 of 42 Stx-positive cultures examined. All the strains produced Stx2f, and most of them possessed genes encoding for intimin and the cytolethal distending toxin (CLDT). Six serogroups were identified, but most of the isolates belonged to O45, O18ab, and O75. Molecular typing indicated that most of the isolates within a flock were clonally-related. This work confirms that pigeons represent a natural reservoir of STEC strains characterized by the production of the toxin variant Stx2f, and by the frequent presence of eae and cldt genes. Further work is needed to clarify whether these STEC may represent a cause of avian disease or even a potential health hazard for humans.