Search

Biting midges are small nematocerous flies belonging to the Ceratopogonidae family. They are also known as no-see-ums, punkies, biting gnats or sandflies. This chapter offers a concise review of the taxonomy, morphology, biology, ecology, medical and veterinary importance, and management of biting midges. Ceratopogonidae are distributed worldwide with over 6200 species. The most important genus is Culicoides, which counts over 1300 species. Members of this genus are known to bite animals, sucking blood. They can be annoying pests to humans and wild and domestic vertebrates. At least 60 Culicoides species are recognized as vectors of viruses, protozoans and filarial nematodes affecting humans and livestock. Oropouche fever is the most known viral disease that affects humans. Key viruses affecting animals include bluetongue virus, African horse sickness virus, epizootic hemorrhagic disease of deer virus; these pathogens can lead to serious damages, such as mortality, abortions, sterility. Some nematode species transmitted by Culicoides cause mansonellosis and onchocerciasis. In the final section, current control strategies against Culicoides in the framework of Integrated Pest/Vector Management are critically discussed.

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.

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.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an insect-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. Though 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 period without replication, we conclude that the second outbreak was most likely 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.

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.

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.

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.

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.

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.

Atypical and classical scrapie-infected sheep brain tissue was monolaterally injected into the tonsils of lambs to investigate their role as a prion entry point. We first detected classical PrP Sc within the inoculated tonsil and in the ipsilateral retropharyngeal lymph node at 3 months postinoculation (p.i.). At 7 months p.i., PrP Sc colonized other lymphoid tissues bilaterally, including ileal Peyer's patches. The earliest PrP Sc deposition within the brain was ipsilaterally observed at 9 months p.i. in the substantia reticularis of the medulla oblongata. At 12 months p.i., PrP Sc deposition was present bilaterally in the nucleus parasympathicus nervi vagi, as well as in the intermediolateral cell column of the thoracolumbar spinal cord. No PrP Sc was detected in the lambs inoculated with atypical scrapie. These findings suggest that neuroinvasion may naturally occur from the tonsil after a widespread prion replication within the lymphoid tissues during classical scrapie only, thus mimicking the pathogenesis after oral ingestion.

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.

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.

Objective—To determine MRI characteristics of the skulls and brains of sheep with chronic cerebral coenurosis (CC) caused by naturally acquired Taenia multiceps infection. Animals—33 sheep with CC and 10 healthy control sheep. Procedures—Sheep underwent MRI of the head. Volumes of the cranial cavity and rostral and caudal fossas of the cranial cavity were determined. For CC-affected sheep, the number, location, and volume of T multiceps cysts were determined and the percentage volumes of cysts in the cranial cavity and rostral and caudal fossas of the cranial cavity were calculated. Focal and diffuse abnormalities of cranial bones in CC-affected sheep were identified. Brain edema and hemorrhage and signs of increased cranial pressure (ICP) in MRI images were determined. Results—Volumes of the cranial cavity and rostral and caudal fossas of the cranial cavity were significantly larger for CC-affected sheep versus healthy control sheep. Total volumes of cysts ranged from 4.40% to 46.93% in cranial cavities of sheep, 4.12% to 51.53% in rostral fossas of cranial cavities of sheep, and 15.24% to 68.30% in caudal fossas of cranial cavities of sheep. Moderate to severe diffuse cranial bone abnormalities and signs of increased ICP in MRI images were detected in 21 and 24 sheep, respectively, and were positively correlated with cyst volumes. Conclusions and Clinical Relevance—Results suggested that cranial cavity volume and morphological abnormalities can be detected in sheep with CC. These changes may reflect abnormalities in ossification of the cranial bones secondary to chronically increased ICP caused by development of T multiceps cysts.

Follicular dendritic cells (FDCs) residing within ileal Peyer's patches (PPs) are of crucial relevance for sheep scrapie early pathogenesis and subsequent scrapie prion neuroinvasion. In this study, ileal PP follicles were significantly more numerous in lambs than in adult Sarda breed sheep, with significant differences being also found in lymphoid follicle area, perimeter and FDC density. Furthermore, PrPd deposition within ileal PPs and host's PrP genotype did not significantly influence these parameters. We conclude that age significantly affects FDC density in ileal PPs from Sarda breed ovines, independently from host's scrapie status and PrP genotype.

In this study, we investigated the susceptibility to scrapie of Sarda breed sheep carrying the genotype ARQ/ARQ with additional polymorphisms at the PRNP gene. To do this, we examined 256 scrapie-affected sheep and 320 flock-mate negative controls from 24 flocks. Logistic regression analysis demonstrated that sheep carrying the ARQ/ARQ genotype with additional dimorphisms had lower risk of becoming scrapie affected when compared with those with ARQ/ARQ(wildtype) genotype. ARQ/ARQ genotypes that were detected with heterozygous or homozygous p.Asn176Lys and p.Met137Thr dimorphisms were associated with the lowest susceptibility to the disease. A significant lower risk was also associated with the p.Arg154His dimorphism, while p.Leu141Phe had a protective effect that was not statistically significant.

Ileal Peyer's patches (PPs) are involved early during sheep scrapie infection. This study qualitatively and semi-quantitatively evaluated ileal tract and PP innervation in 29 Sarda ovines of different age, PrP genotype and scrapie status. A prominent network of fibres was detected within PPs, mainly located in interfollicular lymphoid and stromal components. Intrafollicular fibres were rarely observed, with no apparent differences between scrapie-free and scrapie-affected animals, or among ovines carrying different PrP genotypes. In adult sheep, independent of their scrapie status, nerve fibres could be detected infrequently, close to the follicle-associated epithelium. Fibres were also detected within newly formed follicles and intrafollicular microgranulomas.

Concerns have been raised about the possibility that the bovine spongiform encephalopathy (BSE) agent could have been transmitted to sheep populations via contaminated feedstuffs. The objective of our study was to investigate the suitability of molecular strain typing methods as a surveillance tool for studying scrapie strain variations and for differentiating PrP Sc from sheep scrapie, BSE, and sheep BSE. We studied 38 Italian sheep scrapie cases from 13 outbreaks, along with a British scrapie case, an experimental ovine BSE, and 3 BSE cases, by analyzing the glycoform patterns and the apparent molecular masses of the nonglycosylated forms of semipurified, proteinase-treated PrP Sc . Both criteria were able to clearly differentiate sheep scrapie from BSE and ovine experimental BSE. PrP Sc from BSE and sheep BSE showed a higher glycoform ratio and a lower molecular mass of the nonglycosylated form compared to scrapie PrP Sc . Scrapie cases displayed homogeneous PrP Sc features regardless of breed, flock, and geographic origin. The glycoform patterns observed varied with the antibody used, but either a monoclonal antibody (MAb) (F99/97.6.1) or a polyclonal antibody (P7-7) was able to distinguish scrapie from BSE PrP Sc . While more extensive surveys are needed to further corroborate these findings, our results suggest that large-scale molecular screening of sheep populations for BSE surveillance may be eventually possible.

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.

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.

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.

The use of feed additives with mycotoxin adsorption capacity is a common strategy for controlling negative effects of mycotoxins in swine production systems. However, adsorbents that may results very effective under experimental conditions, i.e. when feed contamination level is rather high, do not necessarily retain their efficacy when tested under field conditions feed with generally low mycotoxin contamination. In this study, the effects of diets artificially contaminated with aflatoxin B1 or ochratoxin A on fattening performance and serum chemistry of fattening pigs are investigated. Moreover, the ability of a commercial glucomannan polymer (Gm polimer) to reduce or eliminate the effects of the contaminated feeds is tested. Thirty heavy pigs (BW = 110±10.6 kg) were fed 6 diets (n = 5 pigs/diet) for 4 weeks until slaughtering. Diets were: control without toxin added (C); added with 0.02 ppm of aflatoxin B1 (AFB1); added with 0.05 ppm of ochratoxin A (OTA); other three diets as the previous but the addition of 2.0 g/kg of Gm polymer (C-GM, AFB1-GM, OTA-GM). Daily weight gain (ADG) and Feed efficiency ratio (FE) were measured every two weeks. Data were analyzed with a two-way ANOVA that included the fixed effect of diet, time and their interaction. After the first 2 weeks the ADG did not differ significantly between the diets, even if the ADG of AFB1 diet was about 20% lower than AFB1-Gm or C. In the last 2 weeks the ADG of AFB1 diet was significantly lover than the other diets (P

Lagovirus is an emerging genus of Caliciviridae, which includes the Rabbit Hemorrhagic Disease Virus (RHDV) of rabbits and the European brown hare syndrome virus (EBHSV) of hares that cause lethal hepatitis. In 2010, a new RHDV related virus (RHDV2) with a unique genetic and antigenic profile and lower virulence was identified in France in rabbits. Here we report the identification of RHDV2 as the cause in Sardinia of several outbreaks of acute hepatitis in rabbits and Cape hare (Lepus capensis mediterraneus). This is the first account of a lagovirus that causes fatal hepatitis in both rabbits and hares.

Prion diseases are fatal neurodegenerative disorders affecting many mammals, ovine scrapie being the archetypal prion disease. Several independent studies in murine and cell-based models of scrapie have highlighted the presence of a link between prion generation and lipid alterations; yet, no data on natural disease are available. In this study we investigated levels of total lipids and cholesterol as well as profiles of fatty acids in brain homogenates from symptomatic and asymptomatic scrapie-infected sheep vs. healthy sheep, all belonging to the same flock. Lipid extracts were analyzed by means of gas chromatography and high performance liquid chromatography. Data of fatty acids were submitted to multivariate statistical analysis to give a picture of the brain lipid profiles of sheep. Interestingly, results revealed abnormalities in the brain fatty acid unsaturation of infected/symptomatic animals. Significant reduction of monoene 18:1 n-9 was detected in brain lipids from infected/symptomatic sheep, as compared to healthy and infected/asymptomatic animals, and this alteration occurred in combination with a significant increase in 18:0 level. The unsupervised Principal Component Analysis showed that infected/symptomatic and healthy sheep samples lie in two different regions of the plot, infected/asymptomatic lie mostly next to healthy. The increase of cerebral saturated fatty acids provides a rough indication of presumed alterations in lipid raft domains of nervous cells during scrapie, suggesting that they may exist in a notable viscous liquid-ordered state. Such physicochemical alteration would have a profound impact on the raft thermodynamic properties, its spatial organization, and signal transduction, all potentially relevant for prion generation.

In sheep scrapie, pathological prion protein (PrP(Sc)) deposition occurs in the lymphoreticular and central nervous systems. We investigated PrP(Sc) distribution in scrapie-affected sheep showing simultaneous evidence of chronic lymphofollicular, lymphoproliferative/non-lymphofollicular, and/or granulomatous inflammations in their mammary gland, lung, and ileum. To do this, PrP(Sc) detection was carried out via immunohistochemistry and Western Blotting techniques, as well as through inflammatory cell immunophenotyping. Expression studies of gene coding for biological factors modulating the host's inflammatory response were also carried out. We demonstrated that ectopic PrP(Sc) deposition occurs exclusively in the context of lymphofollicular inflammatory sites, inside newly formed and well-organized lymphoid follicles harboring follicular dendritic cells. On the contrary, no PrP(Sc) deposition was detected in granulomas, even when they were closely located to newly formed lymphoid follicles. A significantly more consistent expression of lymphotoxin α and β mRNA was detected in lymphofollicular inflammation compared to the other two types, with lymphotoxin α and β signaling new lymphoid follicles' formation and, likely, the occurrence of ectopic PrP(Sc) deposition inside them. Our findings suggest that, in sheep co-affected by scrapie and chronic inflammatory conditions, only newly formed lymphoid follicles provide a suitable micro-environment that supports the scrapie agent's replication in inflammatory sites, with an increased risk of prion shedding through body secretions/excretions.

Ultrasonography is a fundamental diagnostic imaging tool in everyday clinical practice. Here, we are unique in describing the use of functionalized multiwalled carbon nanotubes (MWCNTs) as hyperechogenic material, suggesting their potential application as ultrasound contrast agents. Initially, we carried out a thorough investigation to assess the echogenic property of the nanotubes in vitro. We demonstrated their long-lasting ultrasound contrast properties. We also showed that ultrasound signal of functionalized MWCNTs is higher than graphene oxide, pristine MWCNTs, and functionalized single-walled CNTs. Qualitatively, the ultrasound signal of CNTs was equal to that of sulfur hexafluoride (SonoVue), a commercially available contrast agent. Then, we found that MWCNTs were highly echogenic in liver and heart through ex vivo experiments using pig as an animal model. In contrast to the majority of ultrasound contrast agents, we observed in a phantom bladder that the tubes can be visualized within a wide variety of frequencies (i.e., 5.5-10 MHz) and 12.5 MHz using tissue harmonic imaging modality. Finally, we demonstrated in vivo in the pig bladder that MWCNTs can be observed at low frequencies, which are appropriate for abdominal organs. Importantly, we did not report any toxicity of CNTs after 7 d from the injection by animal autopsy, organ histology and immunostaining, blood count, and chemical profile. Our results reveal the enormous potential of CNTs as ultrasound contrast agents, giving support for their future applications as theranostic nanoparticles, combining diagnostic and therapeutic modalities. journal article research support, non-u.s. gov't 2012 Oct 09 2012 09 24 imported

Prions are misfolded proteins that are infectious and naturally transmitted, causing a fatal neurological disease in humans and animals. Prion shedding routes have been shown to be modified by inflammation in excretory organs, such as the kidney. Here, we show that sheep with scrapie and lentiviral mastitis secrete prions into the milk and infect nearly 90% of naïve suckling lambs. Thus, lentiviruses may enhance prion transmission, conceivably sustaining prion infections in flocks for generations. This study also indicates a risk of prion spread to sheep and potentially to other animals through dietary exposure to pooled sheep milk or milk products.

The placenta is important in the horizontal transmission of the aetiological agent in scrapie-affected sheep. It has been demonstrated that the placentas of fetuses carrying the dimorphism Q171R of the PRNP gene is resistant to pathological prion protein (PrP(Sc)) accumulation in the placenta. To test whether other PRNP polymorphisms are associated with a lack of placental PrP(Sc) deposition, we carried out a study on 26 naturally and 11 experimentally scrapie-affected ewes with or without clinical signs. PrP(Sc) was detected in the placenta of ARQ/ARQ(wild type) fetuses by Western blot and immunohistochemical analysis, but not in ARQN(176)/ARQK(176) or, as expected, ARQ/ARR samples. Furthermore, three of four AL(141)RQ/AF(141)RQ placentas were also PrP(Sc) negative, suggesting that the dimorphism at codon 141 may also mediate placental deposition of PrP(Sc). This finding demonstrates for the first time that fetal PRNP polymorphisms, other than those at codon 171, are associated with the lack of placental deposition of PrP(Sc).

The pathological isoform of the prion protein (PrP(res)) can serve as a marker for prion diseases, but more practical tests are needed for preclinical diagnosis and sensitive detection of many prion infections. Previously we showed that the quaking-induced conversion (QuIC) assay can detect sub-femtogram levels of PrP(res) in scrapie-infected hamster brain tissue and distinguish cerebral spinal fluid (CSF) samples from normal and scrapie-infected hamsters. We now report the adaptation of the QuIC reaction to prion diseases of medical and agricultural interest: human variant Creutzfeldt-Jakob disease (vCJD) and sheep scrapie. PrP(res)-positive and -negative brain homogenates from humans and sheep were discriminated within 1-2 days with a sensitivity of 10-100 fg PrP(res). More importantly, in as little as 22 h we were able to distinguish CSF samples from scrapie-infected and uninfected sheep. These results suggest the presence of prions in CSF from scrapie-infected sheep. This new method enables the relatively rapid and sensitive detection of human CJD and sheep scrapie PrP(res) and may facilitate the development of practical preclinical diagnostic and high-throughput interference tests.

Palatine tonsils (PTs), together with ileal Peyer's patches, rank among the first colonization sites for infectious prions. After replicating in these lymphoid tissues, prions undertake the process of “neuroinvasion,” which is likely mediated by the peripheral nerves connecting lymphoid tissues to the central nervous system (CNS). To study the connections between the tonsils and the CNS, we injected fluorescent tracers into the PTs of lambs; the highest number of Fast Blue (FB)–labeled neurons was found in cranial cervical ganglia (CCG), whereas a progressively decreasing number of cells were detected in proximal glossopharyngeal, proximal vagal, trigeminal, pterygopalatine, and cervicothoracic ganglia. Immunohistochemistry was carried out on tonsil and ganglia cryosections. Immunoreactivity (IR) for tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP), substance P (SP), and calcium-binding protein S100 (S100), was observed in the fibers around and within PT lymphoid nodules. In the trigeminal, proximal glossopharyngeal and vagal ganglia the retrogradely-labeled neurons showed nNOS-, SP- and CGRP-IR. In all ganglia some retrogradely-labeled neurons showed nNOS-, SP- and CGRP-IR co-localization. It is worth noting that only 66±19% and 75±13% of retrogradely-labeled neurons in CCG showed TH- and DBH-IR, respectively. The present results allow us to attribute PT innervation mainly to the sympathetic component and to the glossopharyngeal, vagal and trigeminal cranial nerves. Furthermore, these data also provide a plausible anatomic route through which infectious agents, such as prions, may access the CNS, i.e. by traveling along several cranial and sympathetic nerves, as well as by migration via glial cells.

This study aimed at identifying genes that could mark scrapie infection in the central nervous system of sheep. We used the subtractive suppressive hybridization (SSH) technique on brain samples from sheep healthy or clinically affected by scrapie. Following subtraction, several discrete differential bands appeared between the two reciprocally subtracted samples. These bands were cloned and sequenced, allowing identifying the genes COX1, CHN1, PPP2CA, LRFN5, CAMK2A and RABEPK. Two of the genes identified, CHN1 and RABEPK, appear to locate inside a QTL region known to modulate prion disease incubation time in mice, and LRFN5 maps inside a QTL region identified in sheep. Furthermore, CHN1 and RABEKP showed new unreported differential splicing.

Prions have been documented in extra-neuronal and extra-lymphatic tissues of humans and various ruminants affected by Transmissible Spongiform Encephalopathy (TSE). The presence of prion infectivity detected in cervid and ovine blood tempted us to reason that kidney, the organ filtrating blood derived proteins, may accumulate disease associated PrPSc. We collected and screened kidneys of experimentally, naturally scrapie-affected and control sheep for renal deposition of PrPSc from distinct, geographically separated flocks. By performing Western blot, PET blot analysis and immunohistochemistry we found intraepithelial (cortex, medulla and papilla) and occasional interstitial (papilla) deposition of PrPSc in kidneys of scrapie-affected sheep. Interestingly, glomerula lacked detectable signals indicative of PrPSc. PrPSc was also detected in kidneys of subclinical sheep, but to significantly lower degree. Depending on the stage of the disease the incidence of PrPSc in kidney varied from approximately 27% (subclinical) to 73.6% (clinical) in naturally scrapie-affected sheep. Kidneys from flocks without scrapie outbreak were devoid of PrPSc. Here we demonstrate unexpectedly frequent deposition of high levels of PrPSc in ovine kidneys of various flocks. Renal deposition of PrPSc is likely to be a pre-requisite enabling prionuria, a possible co-factor of horizontal prion-transmission in sheep.

The enteric nervous system (ENS) probably plays a dominant role in sheep scrapie pathogenesis, but little is known about the cell types involved. We investigated the ileal myenteric and submucosal plexuses of four naturally and four orally experimentally scrapie-affected ARQ/ARQ Sarda sheep, as well as those of 12 healthy-control Sarda sheep carrying different PrP genotypes. All scrapie-affected animals, euthanized at clinical-disease end stage, showed PrPd deposition within enteric glial cells (EGCs) and calbindin-immunoreactive (CALB-IR) and neuronal nitric oxide synthase (nNOS)-IR neurons. Whole-mount investigations revealed no significant differences between the densities of total, CALB-IR and nNOS-IR neurons in scrapie-affected versus healthy sheep, irrespective of PrP genotype. Our results suggest that EGCs and CALB-IR and nNOS-IR neurons are probably involved in the pathogenesis of natural and oral experimental sheep scrapie. Furthermore, the infectious agent may be less pathogenic towards ENS neurons than it is towards central nervous system neurons.