Giuseppe Marruchella

Anatomical evidence for ileal Peyer’s patches innervation by enteric nervous system: a potential route for prion neuroinvasion?

We have examined the innervation of the gut-associated lymphoid system of the sheep ileum, with a view to identifying potential sites for neuroinvasion by pathogens, such as prions (PrPSc). Special attention has been paid to the follicles of Peyer’s patches (PPs), which are major sites of PrPSc accumulation during infection. Evidence exists that the enteric nervous system, together with the parasympathetic and sympathetic pathways projecting to the intestine, are important for PrPSc entry into the central nervous system. Thus, PrPSc might move from PPs to the neurons and nerve fibres that innervate them. We investigated, by immunohistochemistry and retrograde tracing (DiI) from the follicles, the distribution and phenotype of enteric neurons innervating the follicles. Antibodies against protein gene product 9.5, tyrosine hydroxylase, dopamine β hydroxylase, choline acetyltransferase, calbindin (CALB), calcitonin gene-related peptide (CGRP), and nitric oxide synthase were used to characterise the neurons. Immunoreactivity for each of these was observed in fibres around and inside PP follicles. CGRP-immunoreactive fibres were mainly seen at the follicular dome. Retrograde tracing revealed submucosal neurons that contributed to the innervation of PPs, including Dogiel type II neurons and neurons immunoreactive for CALB and CGRP. The major source of the adrenergic fibres are the sympathetic ganglia. Our results thus suggest that enteric and sympathetic neurons are involved during the first stage of neuroinvasion, with neurons connecting to them acting as potential carriers of PrPSc to the central nervous system.

Synthetic bone substitute engineered with amniotic epithelial cells enhances bone regeneration after maxillary sinus augmentation.

Background Evidence has been provided that a cell-based therapy combined with the use of bioactive materials may significantly improve bone regeneration prior to dental implant, although the identification of an ideal source of progenitor/stem cells remains to be determined. Aim In the present research, the bone regenerative property of an emerging source of progenitor cells, the amniotic epithelial cells (AEC), loaded on a calcium-phosphate synthetic bone substitute, made by direct rapid prototyping (rPT) technique, was evaluated in an animal study. Material And Methods Two blocks of synthetic bone substitute (∼0.14 cm3), alone or engineered with 1×106 ovine AEC (oAEC), were grafted bilaterally into maxillary sinuses of six adult sheep, an animal model chosen for its high translational value in dentistry. The sheep were then randomly divided into two groups and sacrificed at 45 and 90 days post implantation (p.i.). Tissue regeneration was evaluated in the sinus explants by micro-computer tomography (micro-CT), morphological, morphometric and biochemical analyses. Results And Conclusions The obtained data suggest that scaffold integration and bone deposition are positively influenced by allotransplantated oAEC. Sinus explants derived from sheep grafted with oAEC engineered scaffolds displayed a reduced fibrotic reaction, a limited inflammatory response and an accelerated process of angiogenesis. In addition, the presence of oAEC significantly stimulated osteogenesis either by enhancing bone deposition or making more extent the foci of bone nucleation. Besides the modulatory role played by oAEC in the crucial events successfully guiding tissue regeneration (angiogenesis, vascular endothelial growth factor expression and inflammation), data provided herein show that oAEC were also able to directly participate in the process of bone deposition, as suggested by the presence of oAEC entrapped within the newly deposited osteoid matrix and by their ability to switch-on the expression of a specific bone-related protein (osteocalcin, OCN) when transplanted into host tissues.

Nitric oxide synthase immunoreactivity and NADPH-d histochemistry in the enteric nervous system of Sarda breed sheep with different PrP genotypes in whole-mount and cryostat preparations.

Until now, significant differences in the neurochemical pattern of enteric neurons have been demonstrated in all species studied; however, some strong similarities also occur across species, such as the occurrence of nitric oxide synthase immunoreactivity (NOS-IR) in inhibitory motor neurons to muscle. In consideration of the insufficient data regarding the enteric nervous system (ENS) of sheep, we investigated the myenteric plexus and submucosal plexus of the ovine ileum. Since the pivotal role of the ENS in the early pathogenesis of sheep scrapie, the “prototype” of prion diseases, has been suggested, we have focused our observations also on the hosts PrP genotype. We have studied the morphology and distribution of NOS-IR neurons and their relationships with the enteric glia in whole-mount preparations and in cryostat sections. NOS-IR neurons, always encircled by glial processes, were located in both plexuses. Many NOS-IR fibers were seen in the circular muscle layer, in the submucosa, and in the mucosa. In the submucosa they were close to the lymphoid tissue. No differences in the distribution and percentage of NOS-IR fibers and neurons were observed among sheep carrying different PrP genotype, thus making unlikely their contribution in the determinism of susceptibility/resistance to scrapie infection.

Retrospective study on the occurrence of the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus spp. in endemic areas of Italy.

Aelurostrongylus abstrusus is a metastrongyloid nematode infesting the respiratory system of domestic cats worldwide. Troglostrongylus brevior and Troglostrongylus subcrenatus, two lungworms thought to infest wild felids, have been found recently in domestic cats from Spain and Italy. These unexpected findings have raised doubts about the assumed past and present occurrence of Troglostrongylus spp., especially T. brevior, in domestic hosts and suggest that there may have been missed detection or misdiagnosis. The present retrospective study evaluated the presence of lungworms in cats from Italy with a diagnosis of respiratory parasitism or with compatible lung lesions from 2002 to 2013. Sixty-eight samples of DNA and larvae from cats with a diagnosis of aelurostrongylosis, and 53 formalin-fixed paraffin-embedded lung samples from cats confirmed as lungworm infested or with compatible lesions, were investigated using two DNA-based assays specific for A. abstrusus or T. brevior. All DNA and larval samples were positive for A. abstrusus and one was additionally positive for T. brevior. Most paraffin-embedded lung tissues were positive only for A. abstrusus, but two samples tested positive for both lungworms and one for T. brevior only. This study supports the major role of A. abstrusus in causing feline respiratory parasitism in endemic areas of Italy.

West Nile virus lineage 2 in Sardinian wild birds in 2012: a further threat to public health.

West Nile virus (WNV) strains belonging to lineage 2 were detected and isolated from the tissues of a goshawk and two carrion crows in Sardinia in August 2012. According to NS3 sequence analysis, the Sardinian isolates shared a high level of similarity with those of Italian lineage 2 strains which circulated in 2011 and with the homologous sequence of the 2004 Hungarian isolate. Following the human fatality reported in 2011 in Olbia, this study is the first to report the spread and enzootic circulation of WNV lineage 2 in Sardinia.

Follicular dendritic cell disruption as a novel mechanism of virus-induced immunosuppression

Significance Arboviruses cause increasingly important human and veterinary diseases. Currently, there is a critical lack of understanding about the nature of arbovirus–host interactions in the lymph nodes (LNs), where the adaptive immune response initiates. We used a hemorrhagic arbovirus of sheep, bluetongue virus (BTV), to unveil the early phases of infection in the natural host. We discovered that BTV modulates the humoral immune response by rapidly infecting and destroying follicular dendritic cells (FDCs) in the host LNs. FDC destruction impairs B-cell activation and antibody production, inducing an immunosuppressive phase associated with virus spread in the animal’s tissues. The novel virus evasion strategy described here provides key insights on the initiation of the immune response and the pathogenesis of arboviral diseases. Arboviruses cause acute diseases that increasingly affect global health. We used bluetongue virus (BTV) and its natural sheep host to reveal a previously uncharacterized mechanism used by an arbovirus to manipulate host immunity. Our study shows that BTV, similarly to other antigens delivered through the skin, is transported rapidly via the lymph to the peripheral lymph nodes. Here, BTV infects and disrupts follicular dendritic cells, hindering B-cell division in germinal centers, which results in a delayed production of high affinity and virus neutralizing antibodies. Moreover, the humoral immune response to a second antigen is also hampered in BTV-infected animals. Thus, an arbovirus can evade the host antiviral response by inducing an acute immunosuppression. Although transient, this immunosuppression occurs at the critical early stages of infection when a delayed host humoral immune response likely affects virus systemic dissemination and the clinical outcome of disease.

A Lympho-Follicular Microenvironment Is Required for Pathological Prion Protein Deposition in Chronically Inflamed Tissues from Scrapie-Affected Sheep

In sheep scrapie, pathological prion protein (PrPSc) deposition occurs in the lymphoreticular and central nervous systems. We investigated PrPSc 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, PrPSc 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 PrPSc 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 PrPSc 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 PrPSc 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.

Fatal bronchopneumonia in a Metastrongylus elongatus and Porcine circovirus type 2 co-infected pig

Porcine circovirus type 2 (PCV2) infection is distributed worldwide and PCV2-associated disease (PCVAD) is considered among the most economically relevant ones to the global swine industry. PCV2 is known to play a causal role in the porcine respiratory disease complex, usually in close association with a large plethora of other biologic agents. We describe herein a case of fatal parasitic bronchopneumonia by Metastrongylus elongatus in a PCV2-infected pig. Metastrongylosis may still represent a major concern for outdoor herds. Our recent experience suggests that a concurrent PCVAD condition may trigger metastrongylosis, which may subsequently result, at its turn, in severe, sometimes fatal, pulmonary disease.

5-lipoxygenase and cyclooxygenase-2 in porcine parasitic bronchopneumonia: immunohistochemical and biochemical investigations.

Eicosanoids are products of arachidonic acid metabolism and have numerous biological roles. The present study aimed to investigate the role of 5-lipoxygenase (5-LOX)- and cyclooxygenase-2 (COX-2)- dependent enzymatic pathways in the pathogenesis of porcine parasitic bronchopneumonia caused by Metastrongylus spp. Pulmonary tissue samples from healthy control and parasitized pigs were processed for histopathological, immunohistochemical and biochemical investigations. In control animals, immunohistochemistry demonstrated that 5-LOX and COX-2 expression was almost exclusively limited to the bronchiolar epithelial cells. Parasitized pigs had greater 5-LOX- and COX-2- specific immunoreactivity, involving a wide range of cell types within foci of granulomatous and eosinophilic bronchopneumonia. Biochemical investigations demonstrated the presence of 5-LOX (and the related product Leukotriene B(4)) and COX-2 (and the related product prostaglandin E(2); PGE(2)) in all tissues under study. COX-2 activity and PGE(2) concentration were significantly higher in diseased lungs compared with normal healthy controls. These findings demonstrate that 5-LOX and COX-2 are differentially expressed in normal versus lungworm-infected lungs and therefore suggest that both biochemical pathways are likely to be involved in the pathogenesis of porcine parasitic bronchopneumonia.

Ileal tract and Peyer's patch innervation in scrapie-free versus scrapie-affected ovines

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.