ORF virus causes tumor-promoting inflammation in sheep and goats.

ORF virus (ORFV) causes contagious ecthyma ("ORF"), a disease of sheep and goats characterized by lesions ranging from vesicles and pustules to atypical papilloma-like and angiomatous lesions in the skin and mucosae. The authors investigated the molecular factors leading to the ORF-associated atypical tumor-like changes. Fifteen lambs, 15 kids, and an adult ram clinically affected by natural ORFV infection were enrolled in the study and examined by several methods. ORFV was detected by viral culture or real-time polymerase chain reaction (RT-PCR) in the lesioned tissues and in the blood of the clinically affected sheep and goats. Surprisingly, ORFV was also detected in the blood of healthy goats from an affected herd. Microscopically, they found a pseudo-papillomatous proliferation of the epithelium, while the dermis and lamina propria were expanded by a proliferating neovascular component that highly expressed the viral vascular endothelial growth factor (vVEGF) and its host receptor vascular endothelial growth factor receptor 2 (VEGFR2). Immunohistochemistry, immunofluorescence, and in situ hybridization for mRNA showed that epidermal growth factor receptor (EGFR) was expressed in the fibrovascular component, in the infiltrating CD163+ macrophages, and in the basal stratum of the epidermis. Confocal immunofluorescence microscopy demonstrated that CD163+ macrophages were associated with VEGF and VEGFR2. Finally, they found by quantitative RT-PCR the overexpression of the interleukin-6 and VEGFR2 genes in the lesioned tissues. These findings suggest that ORFV activates an inflammatory reaction characterized by CD163+ macrophages expressing EGFR and VEGFR2, which might play an oncogenic role through synergistic action with vVEGF signaling.

Whole-Genome Sequencing of Two Canine Herpesvirus 1 (CaHV-1) Isolates and Clinicopathological Outcomes of Infection in French Bulldog Puppies.

Canine herpesvirus 1 (CaHV-1) infects dogs, causing neonatal death and ocular, neurological, respiratory, and reproductive problems in adults. Although CaHV-1 is widespread in canine populations, only four studies have focused on the CaHV-1 whole genome. In such context, two CaHV-1 strains from both the kidney and spleen of 20-day-old deceased French Bulldog puppies were recently isolated in Sardinia, Italy. The extracted viral DNA underwent whole-genome sequencing using the Illumina MiSeq platform. The Italian CaHV-1 genomes were nearly identical (>99%), shared the same tree branch, and clustered near the ELAL-1 (MW353125) and BTU-1 (KX828242) strains, enlarging the completely separated clade discussed by Lewin et al., in 2020. This study aims to provide new insights on the evolution of the CaHV-1, based on high-resolution whole-genome phylogenetic analysis, and on its clinicopathological characterization during a fatal outbreak in puppies.

Advances in understanding the genetic architecture of antibody response to paratuberculosis in sheep by heritability estimate and LDLA mapping analyses and investigation of candidate regions using sequence-based data.

BACKGROUND: Paratuberculosis is a contagious and incurable disease that is caused by Mycobacterium avium subsp. paratuberculosis (MAP) with significant negative effects on animal welfare and farm profitability. Based on a large naturally infected flock over 12 years, we analyzed repeated enzyme-linked immunosorbent assay tests (ELISA), OvineSNP50 BeadChip genotypes and whole-genome sequences imputed from 56 influential animals. The main goals were to estimate the genetic parameters of proxy traits for resistance to MAP, identify genomic regions associated with the host's immune response against MAP and search for candidate genes and causative mutations through association and functional annotation analyses of polymorphisms identified by sequencing. RESULTS: Two variables were derived from ELISA tests. The first, a binary variable, assessed the infection status of each animal over the entire productive life, while the second considered the level of antibody recorded over time. Very similar results were obtained for both variables. Heritability estimates of about 0.20 were found and a significant region capturing 18% and 13% of the genetic variance was detected on ovine chromosome 20 by linkage disequilibrium and linkage analysis on OvineSNP50 positions. Functional annotation and association analyses on the imputed sequence polymorphisms that were identified in this region were carried out. No significant variants showed a functional effect on the genes that mapped to this region, most of which belong to the major histocompatibility complex class II (MHC II). However, the conditional analysis led to the identification of two significant polymorphisms that can explain the genetic variance associated with the investigated genomic region. CONCLUSIONS: Our results confirm the involvement of the host's genetics in susceptibility to MAP in sheep and suggest that selective breeding may be an option to limit the infection. The estimated heritability is moderate with a relevant portion being due to a highly significant region on ovine chromosome 20. The results of the combined use of sequence-based data and functional analyses suggest several genes belonging to the MHC II as the most likely candidates, although no mutations in their coding regions showed a significant association. Nevertheless, information from genotypes of two highly significant polymorphisms in the region can enhance the efficiency of selective breeding programs.

The Global Evolutionary History of Orf Virus in Sheep and Goats Revealed by Whole Genomes Data.

Orf virus (ORFV) belongs to the genus Parapoxvirus (Poxviridae family). It is the causative agent of contagious ecthyma (CE) that is an economically detrimental disease affecting small ruminants globally. Contagious ecthyma outbreaks are usually reported in intensive breeding of sheep and goats but they have also been reported in wildlife species. Notably, ORFV can infect humans, leading to a zoonotic disease. This study aims to elucidate the global evolutionary history of ORFV genomes in sheep and goats, including the first genomes from Central America in the analyses. In comparison to the last study on ORFV whole genomes, the database now includes 11 more sheep and goat genomes, representing an increase of 42%. The analysis of such a broader database made it possible to obtain a fine molecular dating of the coalescent time for ORFV S and G genomes, further highlighting the genetic structuring between sheep and goat genomes and corroborating their emergence in the latter half of 20th century.

Origin, Genetic Variation and Molecular Epidemiology of SARS-CoV-2 Strains Circulating in Sardinia (Italy) during the First and Second COVID-19 Epidemic Waves.

Understanding how geography and human mobility shape the patterns and spread of infectious diseases such as COVID-19 is key to control future epidemics. An interesting example is provided by the second wave of the COVID-19 epidemic in Europe, which was facilitated by the intense movement of tourists around the Mediterranean coast in summer 2020. The Italian island of Sardinia is a major tourist destination and is widely believed to be the origin of the second Italian wave. In this study, we characterize the genetic variation among SARS-CoV-2 strains circulating in northern Sardinia during the first and second Italian waves using both Illumina and Oxford Nanopore Technologies Next Generation Sequencing methods. Most viruses were placed into a single clade, implying that despite substantial virus inflow, most outbreaks did not spread widely. The second epidemic wave on the island was actually driven by local transmission of a single B.1.177 subclade. Phylogeographic analyses further suggest that those viral strains circulating on the island were not a relevant source for the second epidemic wave in Italy. This result, however, does not rule out the possibility of intense mixing and transmission of the virus among tourists as a major contributor to the second Italian wave.

Malignant Catarrhal Fever in Sardinia (Italy): A Case Report.

Using a multidisciplinary approach, this report describes a clinical case of malignant catarrhal fever (MCF) occurring in a calf, which shared the pasture with sheep on a farm located in the island of Sardinia (Italy). We confirmed the conventional clinico-histopathological features of MCF, as well was the presence of Ovine herpesvirus type 2 (OvHV-2) DNA in several tissues, employing histological and virological investigations. The phylogenetic analysis revealed that this Sardinian OvHV-2 strain is genetically similar to all the other Italian strains. By Real Time PCR examinations of blood samples collected across Sardinia's sheep population, which is considered the most important reservoir species, we discovered an OvHV-2 prevalence ranging from 20 to 30 percent. Despite the high prevalence of OvHV-2 in the Sardinian sheep population, clinical disease in bovine remains sporadic; further investigations are needed to understand the risk factors that regulate this epidemiological aspect.

A Deeper Insight into Evolutionary Patterns and Phylogenetic History of ORF Virus through the Whole Genome Sequencing of the First Italian Strains.

Orf virus (ORFV) is distributed worldwide and is the causative agent of contagious ecthyma that mainly occurs in sheep and goats. This disease was reported for the first time at the end of 18th century in Europe but very little is currently known about the temporal and geographic origins of this virus. In the present study, the use of new Italian whole genomes allowed for better inference on the evolutionary history of ORFV. In accordance with previous studies, two genome types (S and G) were described for infection of sheep and goats, respectively. These two well-differentiated groups of genomes originated for evolutive convergence in the late 1800s in two different areas of the world (Europe for S type and Asia for G type), but it was only in the early 1900s that the effective size of ORFV increased among hosts and the virus spread across the whole European continent. The Italian strains which were sequenced in the present study were isolated on the Mediterranean island of Sardinian and showed to be exclusive to this geographic area. One of them is likely representative of the early European forms of ORFV which infected sheep and became extinct about one century ago. Such an ancient Sardinian strain may have reached the island simple by chance, where it quickly adapted to the new habitat.

Molecular Insights into the Genetic Variability of ORF Virus in a Mediterranean Region (Sardinia, Italy).

Orf virus (ORFV) represents the causative agent of contagious ecthyma, clinically characterized by mild papular and pustular to severe proliferative lesions, mainly occurring in sheep and goats. In order to provide hints on the evolutionary history of this virus, we carried out a study aimed to assess the genetic variation of ORFV in Sardinia that hosts a large affected small ruminant population. We also found a high worldwide mutational viral evolutionary rate, which resulted, in turn, higher than the rate we detected for the strains isolated in Sardinia. In addition, a well-supported genetic divergence was found between the viral strains isolated from sheep and those from goats, but no relevant connection was evidenced between the severity of lesions produced by ORFV and specific polymorphic patterns in the two species of hosts. Such a finding suggests that ORFV infection-related lesions are not necessarily linked to the expression of one of the three genes here analyzed and could rather be the effect of the expression of other genes or rather represents a multifactorial character.

"Frozen evolution" of an RNA virus suggests accidental release as a potential cause of arbovirus re-emergence.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-borne virus of ruminants, emerged in livestock in northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of euros. Although the outbreak was successfully controlled through vaccination by early 2010, puzzlingly, a closely related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this long period without replication, we hypothesise that the second outbreak could have been initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence.

Dicrocoeliosis in extensive sheep farms: a survey.

BACKGROUND: This study investigated the epidemiological and molecular aspects of dicrocoeliosis in extensive sheep farms. METHODS: From 2013 to 2014, copromicroscopical analyses in 190 dairy sheep farms and anatomo-pathological inspections in six slaughterhouses were carried in Sardinia, Italy. Rectal faecal samples were analyzed using the FLOTAC® method, and anatomo-pathological examinations were based on detecting thickened terminal bile ducts (TTBDs). In addition, genetic analyses were conducted on representative DNA samples of adult Dicrocoelium spp. RESULTS: Ninety-seven (51.1%) out of 190 sheep farms were coprologically positive for Dicrocoelium spp. In the liver, on the surface and cut surface, TTBDs were reported in 40.1% (309/770) and 15.3% (118/770) of the animals examined, respectively, with an overall prevalence of 25.5% (196/770). No intraspecific genetic variation was observed among the Dicrocoelium dendriticum isolates. CONCLUSIONS: Our survey reveals the widespread presence of D. dendriticum in Sardinia, although seasonal, geographical and climatic conditions might be key factors in modulating the infection prevalence. Examining typical lesions due to D. dendriticum in the liver in abattoirs can be used as a marker for tracking chronic dicrocoeliosis infection.

Testicular Degeneration and Infertility following Arbovirus Infection.

Arboviruses can cause a variety of clinical signs, including febrile illness, arthritis, encephalitis, and hemorrhagic fever. The recent Zika epidemic highlighted the possibility that arboviruses may also negatively affect the male reproductive tract. In this study, we focused on bluetongue virus (BTV), the causative agent of bluetongue and one of the major arboviruses of ruminants. We show that rams that recovered from bluetongue displayed signs of testicular degeneration and azoospermia up to 100 days after the initial infection. Importantly, testicular degeneration was induced in rams experimentally infected with either a high (BTV-1IT2006)- or a low (BTV-1IT2013)-virulence strain of BTV. Rams infected with the low-virulence BTV strain displayed testicular lesions in the absence of other major clinical signs. Testicular lesions in BTV-infected rams were due to viral replication in the endothelial cells of the peritubular areas of the testes, resulting in stimulation of a type I interferon response, reduction of testosterone biosynthesis by Leydig cells and destruction of Sertoli cells and the blood-testis barrier in more severe cases. Hence, BTV induces testicular degeneration and disruption of spermatogenesis by replicating solely in the endothelial cells of the peritubular areas unlike other gonadotropic viruses. This study shows that a naturally occurring arboviral disease can cause testicular degeneration and affect male fertility at least temporarily.IMPORTANCE During the recent Zika epidemic, it has become apparent that arboviruses could potentially cause reproductive health problems in male patients. Little is known regarding the effects that arboviruses have on the male reproductive tract. Here, we studied bluetongue virus (BTV), an arbovirus of ruminants, and its effects on the testes of rams. We show that BTV was able to induce testicular degeneration in naturally and experimentally infected rams. Testicular degeneration was caused by BTV replication in the endothelial cells of the peritubular area surrounding the seminiferous tubules (the functional unit of the testes) and was associated with a localized type I interferon response, destruction of the cells supporting the developing germinal cells (Sertoli cells), and reduction of testosterone synthesis. As a result of BTV infection, rams became azoospermic. This study highlights that problems in the male reproductive tract caused by arboviruses could be more common than previously thought.

Atypical outbreak of acute coenurosis by Taenia multiceps in a sheep flock.

Herein, we examined the brain of adult ewes and lambs less than 30 days old which were found affected by neurological signs in a flock located in Sardinia (Italy). Gross anatomo-pathological examination of all brains of the animals revealed multiple linear reddish-yellow foci of necrotic purulent inflammation due to oncosphere migration. Histologically, we confirmed a multifocal pyo-granulomatous meningo-encephalitis both in ewes and in lambs, confirming acute coenurosis. Morphological examination and DNA sequencing identified the Taenia multiceps we isolated as Tm1 strain. This report describes for the first time a natural acute coenurosis infection in suckling lambs under 30 days of age.

Persistence of Bluetongue virus serotype 1 virulence in sheep blood refrigerated for 10 years.

This paper reports that Bluetongue virus serotype 1 (BTV-1) infected blood collected during the 2006 Sardinia (Italy) epidemic from a ewe with clinical disease and stored at ~ 5°C for 10 years, caused Bluetongue (BT)-like clinical disease and death when inoculated into a susceptible Sarda breed ram. Anatomo-histopathological examination and Real-Time Reverse Transcriptase PCR (Real-Time RT-PCR) confirmed the presence of BTV-1 in several tissues proving that the BTV-1 2006 isolate has maintained its infectivity and virulence.

Depletion of follicular dendritic cells in tonsils collected from PMWS-affected pigs.

Post-weaning multisystemic wasting syndrome (PMWS) is a relevant, worldwide disease caused by porcine circovirus type 2 (PCV2). Microscopically, PMWS is mainly characterized by lymphocytic depletion, macrophage infiltration and syncytia in lymphoid tissues. Some data suggest that follicular dendritic cells (FDCs) could be infected by PCV2, thus likely playing a role in the pathogenesis of PMWS. The present paper aims at assessing, qualitatively and quantitatively, the FDCs' network in the soft palate tonsils of clinically healthy and PMWS-affected pigs. Consecutive tissue sections were tested by immunohistochemistry to detect PCV2, FDCs and macrophages. FDCs and PCV2 antigens were quantitatively assessed by means of the Image J software and results submitted to statistical analysis. Our data demonstrated that FDCs are significantly reduced in PMWS-affected pigs compared with healthy pigs and that FDCs' depletion should be considered among microscopic features of PMWS. It is reasonable to hypothesize that depletion of FDCs further compromises the immune response and enhances the occurrence and the severity of secondary infections, which are relevant for the clinical manifestation of PMWS.

Pathological findings in a Dachshund-cross dog with neuroaxonal dystrophy.

BACKGROUND: Neuroaxonal dystrophy (NAD) is a neurodegenerative condition affecting humans and animals characterized by the widespread presence of swollen axons (spheroids). CASE PRESENTATION: Herein, we report the pathological findings in a case of neuroaxonal dystrophy in a Dachshund-cross puppy, which was euthanized because of a proprioceptive positioning deficits and irreversible ataxia of the hind limbs. Histologically, there was a bilaterally symmetric neuroaxonal dystrophy with eosinophilic axonal spheroids exclusively localized at the level of the ventral posterior lateral nucleus of the thalamus, medial lemniscus, gracilis nucleus, medial cuneatus nucleus in the brain as well as the gracilis and cuneatus fasciculi throughout the spinal cord. CONCLUSION: To the authors' knowledge, this is the first report of canine neuroaxonal dystrophy with this exclusive and specific localization only in the neuronal circuit implicated in the transmission of conscious proprioceptive information.

Glioblastoma with oligodendroglioma component in a ewe.

Herein we describe a glioblastoma partially occupying the telencephalic portion of the left cerebral hemisphere of a Sardinian (syn. Sarda) breed ewe. Microscopically, the mass consisted of a pleomorphic spindle-shaped cell component organized as bundles and numerous small areas of round cells displaying an oligodendroglioma-like aspect. A high number of mitotic figures, large areas of necrosis surrounded by pseudopalisading glial cells, and multiple foci of dystrophic mineralization were also observed. The neoplasm was highly vascularized with glomerular vascular proliferation. Immunohistochemically, neoplastic cells proved to be strongly positive for nestin, vimentin, and olig-2, whereas they were invariably negative for synaptophysin. Few neoplastic cells and reactive astrocytes, mainly located at the edge of necrotic foci, proved to be positive for glial fibrillary acidic protein, whereas glomerular vascular proliferation was clearly positive for factor VIII and vascular endothelial growth factor. Gene sequencing analysis demonstrated homozygous p53 tumor suppressor gene (TP53) point mutations in the DNA-binding domain located in exon 8. The presence of round cells immunoreactive for olig-2 demonstrated that this tumor is a glioblastoma with oligodendroglioma component. Our pathologic, immunohistochemical, and molecular findings largely overlap those previously reported in humans and dogs.

Genetic and Pathological Follow-Up Study of Goats Experimentally and Naturally Exposed to a Sheep Scrapie Isolate.

UNLABELLED: Thirty-seven goats carrying different prion protein genotypes (PRNP) were orally infected with a classical scrapie brain homogenate from wild-type (ARQ/ARQ) sheep and then mated to obtain 2 additional generations of offspring, which were kept in the same environment and allowed to be naturally exposed to scrapie. Occurrence of clinical or subclinical scrapie was observed in the experimentally infected goats (F0) and in only one (F1b) of the naturally exposed offspring groups. In both groups (F0 and F1b), goats carrying the R154H, H154H, R211Q, and P168Q-P240P dimorphisms died of scrapie after a longer incubation period than wild-type, G37V, Q168Q-P240P, and S240P goats. In contrast, D145D and Q222K goats were resistant to infection. The immunobiochemical signature of the scrapie isolate and its pathological aspects observed in the sheep donors were substantially maintained over 2 goat generations, i.e., after experimental and natural transmission. This demonstrates that the prion protein gene sequence, which is shared by sheep and goats, is more powerful than any possible but unknown species-related factors in determining scrapie phenotypes. With regard to genetics, our study confirms that the K222 mutation protects goats even against ovine scrapie isolates, and for the first time, a possible association of D145 mutation with scrapie resistance is shown. In addition, it is possible that the sole diverse frequencies of these genetic variants might, at least in part, shape the prevalence of scrapie among naturally exposed progenies in affected herds. IMPORTANCE: This study was aimed at investigating the genetic and pathological features characterizing sheep-to-goat transmission of scrapie. We show that in goats with different prion protein gene mutations, the K222 genetic variant is associated with scrapie resistance after natural and experimental exposure to ovine prion infectivity. In addition, we observed for the first time a protective effect of the D145 goat variant against scrapie. Importantly, our results demonstrate that the phenotypic characteristic of the wild-type sheep scrapie isolate is substantially preserved in goats carrying different susceptible PRNP gene variants, thus indicating that the prion protein gene sequence, which is shared by sheep and goats, plays a fundamental role in determining scrapie phenotypes.

(1)H NMR brain metabonomics of scrapie exposed sheep.

While neurochemical metabolite modifications, determined by different techniques, have been diffusely reported in human and mice brains affected by transmissible spongiform encephalopathies (TSEs), this aspect has been little studied in the natural animal hosts with the same pathological conditions so far. Herein, we investigated, by high resolution (1)H NMR spectroscopy and multivariate statistical data analysis, the brain metabolite profile of sheep exposed to a scrapie agent in a naturally affected flock. On the basis of clinical examinations and western blotting analysis for the pathological prion protein (PrP(Sc)) in brain tissues, sheep were catalogued as not infected (H), infected with clinical signs (S), and infected without clinical signs (A). By discriminant analysis of spectral data, comparing S vs. H, we found a different metabolite distribution, with inosine, cytosine, creatine, and lactate being higher in S than in H brains, while the branched chain amino acids (leucine, isoleucine, and valine), phenylalanine, uracil, tyrosine, gamma-amino butyric acid, total aspartate (aspartate + N-acetyl aspartate) being lower in S. By a soft independent modelling of class analogy approach, 1 out of 3 A samples was assigned to class H. Furthermore, A brains were found to be higher in choline and choline-containing compounds. By means of partial least squares regression, an excellent correlation was found between the PrP(Sc) amount and the (1)H NMR metabolite profile of infected (S and A) sheep, and the metabolite mostly correlated with PrP(Sc) was alanine. The overall results, obtained using different chemometric tools, were able to describe a brain metabolite profile of infected sheep with and without clinical signs, compared to healthy ones, and indicated alanine as a biomarker for PrP(Sc) amounts in scrapie brains.

Genetic and pathological characteristics of Cryptococcus gattii and Cryptococcus neoformans var. neoformans from meningoencephalitis in autochthonous goats and mouflons, Sardinia, Italy.

In this study, we examined in Sardinia the brain of 555 autochthonous sheep, 50 goats, and 4 mouflons which were found affected by neurological signs. We found 6 goats and one mouflon with meningoencephalitis caused by Cryptococcus sp. There was no evidence of cryptococcal infections in any of the examined sheep. MLST genotyping on Cryptococcus sp. isolates identified Cryptococcus gatti genotype AFLP4/VGI and Cryptococcus neoformans var. neoformans genotype AFLP2/VNIV. Phylogenetically, all Cryptococcus gattii isolates fell within the autochthonous animal, human and environmental Mediterranean isolate cluster, forming a distinct branch along with environmental strains from Alicante, in the southern Mediterranean coast of Spain.

Cetacean strandings in Italy: an unusual mortality event along the Tyrrhenian Sea coast in 2013.

An unusual mortality event involving cetaceans, mainly striped dolphins Stenella coeruleoalba (Meyen, 1833), occurred along the Tyrrhenian Sea coast of Italy during the first 3 mo of 2013. Based on post-mortem analyses carried out according to body condition on 66 dolphins (54% of stranded animals), several hypotheses to explain the causes of this mortality event were proposed. Although no definitive conclusions can be drawn, dolphin morbillivirus was deemed the most likely cause, although other infectious agents (including Photobacterium damselae damselae and herpesvirus) or environmental factors may also have contributed to this recent mortality event.