Testicular tumors in commercial boars with infertility: A gross, histologic, and immunohistochemical study.

Tumors in boars are uncommon, and testicular tumors even rarer. This study describes the pathological and immunohistochemical characteristics of a case series of testicular tumors in commercial boars with fertility problems. Tumors were detected in 19 of 333 animals (19/333, 5.9%). Macroscopically, tumors were observed in 13 (13/19, 68%) boars, while 6 cases (6/19, 32%) were only detected by microscopic examination. Testicular enlargement was observed in 1 boar, while in the others, tumors were only observed after removal of the scrotal skin or after sectioning of the testis. Histologically, tumors were classified as seminomas (16/19, 84%), mixed germ cell-stromal tumors (2/19, 11%), and B-cell lymphoma (1/19, 5%). Seminomas had 3 different growth patterns: intratubular (6/16, 38%), diffuse (4/16, 25%), and intratubular/diffuse (6/16, 38%). All tumors that were not evident on macroscopic examination were intratubular seminomas. Intratesticular metastases were observed in 2 cases and extratesticular metastases, located in the pampiniform plexus, were observed in 1 case. In 1 seminoma, the rete testis was also involved. By immunohistochemistry, all intratubular seminomas were negative for c-kit, cytokeratin, and vimentin. In diffuse seminomas, c-kit and cytokeratin were also negative, while vimentin showed granular or perinuclear cytoplasmic labeling in some areas. PAX-5 and CD-3 antibodies classified the lymphoma as a B-cell lymphoma. This study suggests that testicular tumors in boars may be more common than previously reported, especially when microscopic examination is performed. It also shows that testicular tumors in pigs are predominantly seminomas.

Novel polymorphisms in the prion protein gene (PRNP) and stability of the resultant prion protein in different horse breeds.

Prion diseases are fatal neurodegenerative disorders in which the main pathogenic event is the conversion of the cellular prion protein (PrPC) into an abnormal and misfolded isoform known as PrPSc. Most prion diseases and their susceptibility and pathogenesis are mainly modulated by the PRNP gene that codes for PrP. Mutations and polymorphisms in the PRNP gene can alter PrPC amino acid sequence, leading to a change in transmission efficiency depending on the place where it occurs. Horses are animals that are considered to be highly resistant to prions. Several studies have attempted to identify polymorphisms in the PRNP gene that explain the reason for this high resistance. In this study, we have analysed 207 horses from 20 different breeds, discovering 3 novel PRNP polymorphisms. By using computer programmes such as PolyPhen-2, PROVEAN, PANTHER, Meta-SNP and PredictSNP, we have predicted the possible impact that these new polymorphisms would have on the horse prion protein. In addition, we measured the propensity for amyloid aggregation using AMYCO and analysed the lack of hydrogen bridges that these changes would entail together with their electrostatic potentials using Swiss-PdbViewer software, showing that an increased amyloid propensity could be due to changes at the level of electrostatic potentials.

Susceptibility of Ovine Bone Marrow-Derived Mesenchymal Stem Cell Spheroids to Scrapie Prion Infection.

In neurodegenerative diseases, including prion diseases, cellular in vitro models appear as fundamental tools for the study of pathogenic mechanisms and potential therapeutic compounds. Two-dimensional (2D) monolayer cell culture systems are the most used cell-based assays, but these platforms are not able to reproduce the microenvironment of in vivo cells. This limitation can be surpassed using three-dimensional (3D) culture systems such as spheroids that more effectively mimic in vivo cell interactions. Herein, we evaluated the effect of scrapie prion infection in monolayer-cultured ovine bone marrow-derived mesenchymal stem cells (oBM-MSCs) and oBM-MSC-derived spheroids in growth and neurogenic conditions, analyzing their cell viability and their ability to maintain prion infection. An MTT assay was performed in oBM-MSCs and spheroids subjected to three conditions: inoculated with brain homogenate from scrapie-infected sheep, inoculated with brain homogenate from healthy sheep, and non-inoculated controls. The 3D conditions improved the cell viability in most cases, although in scrapie-infected spheroids in growth conditions, a decrease in cell viability was observed. The levels of pathological prion protein (PrPSc) in scrapie-infected oBM-MSCs and spheroids were measured by ELISA. In neurogenic conditions, monolayer cells and spheroids maintained the levels of PrPSc over time. In growth conditions, however, oBM-MSCs showed decreasing levels of PrPSc throughout time, whereas spheroids were able to maintain stable PrPSc levels. The presence of PrPSc in spheroids was also confirmed by immunocytochemistry. Altogether, these results show that a 3D culture microenvironment improves the permissiveness of oBM-MSCs to scrapie infection in growth conditions and maintains the infection ability in neurogenic conditions, making this model of potential use for prion studies.

Neurogranin and Neurofilament Light Chain as Preclinical Biomarkers in Scrapie.

Prion diseases are diagnosed in the symptomatic stage, when the neuronal damage is spread throughout the central nervous system (CNS). The assessment of biological features that allow the detection of asymptomatic cases is needed, and, in this context, scrapie, where pre-symptomatic infected animals can be detected through rectal biopsy, becomes a good study model. Neurogranin (Ng) and neurofilament light chain (NfL) are proteins that reflect synaptic and axonal damage and have been studied as cerebrospinal fluid (CSF) biomarkers in different neurodegenerative disorders. In this study, we evaluated Ng and NfL both at the protein and transcript levels in the CNS of preclinical and clinical scrapie-affected sheep compared with healthy controls and assessed their levels in ovine CSF. The correlation between these proteins and the main neuropathological events in prion diseases, PrPSc deposition and spongiosis, was also assessed. The results show a decrease in Ng and NfL at the protein and gene expression levels as the disease progresses, and significant changes between the control and preclinical animals. On the contrary, the CSF levels of NfL increased throughout the progression of the disease. Negative correlations between neuropathological markers of prion disease and the concentration of the studied proteins were also found. Although further research is needed, these results suggest that Ng and NfL could act as biomarkers for neurodegeneration onset and intensity in preclinical cases of scrapie.

An assessment of the efficiency of PrPsc detection in rectal mucosa and third-eyelid biopsies from animals infected with scrapie.

In classical scrapie, detection of PrPsc on lymphoreticular system is used for the in vivo and post mortem diagnosis of the disease. However, the sensitivity of this methodology is not well characterised because the magnitude and duration of lymphoid tissue involvement can vary considerably. The aim of the present study was to evaluate the efficiency of detecting PrPsc in rectal mucosa and third-eyelid biopsies. A total of 474 genetically susceptible sheep and 24 goats from three scrapie infected flocks were included in this study. A sample from rectal mucosa and a sample from third-eyelid lymphoid tissue were collected from each animal. Biopsy samples were fixed in formaldehyde and processed for immunohistochemical examination. Animals with negative biopsy results were studied more closely through a post mortem examination of central nervous and lymphoreticular systems and if there was a positive result, additional biopsy sections were further tested. The sensitivity of rectal mucosa and third-eyelid assays were 36% and 40% respectively on initial examination but increased to 48% and 44% respectively after retesting. The results of this field study show a high percentage of infected animals that do not have detectable levels of PrPsc in the biopsied lymphoid tissue, due mainly to the relatively high number of animals with minimal or no involvement of lymphoid tissue in the pathogenesis of the disease.

Detection and clinical evolution of scrapie in sheep by 3rd eyelid biopsy.

The goal of this article was to characterize the clinical evolution of scrapie in naturally affected sheep. Eighteen sheep with scrapie diagnosed by examination of 3rd eyelid biopsy and 12 control ewes were studied throughout the duration of their disease. Diagnosis was confirmed postmortem by histopathologic, immunohistochemical, and Western blot analysis of nervous tissue. Complete clinical examinations were performed every 2 weeks for each animal, of which 3 clinical examinations per animal are reported. Those clinical signs that showed a significant frequency within the corresponding clinical examination were considered representative of each stage of the disease (ie, early, middle, and late). The representative clinical signs for the early stage were hypoesthesia in the limbs, alteration of mental status, and a body condition score <3. Remarkably, hypoesthesia in the limbs was one of the 1st signs appearing during the early clinical stage in the affected animals, even before the appearance of other signs. For the middle stage, representative signs were the same as those for the early stage, together with hyporreflexia in the limbs, cardiac arrhythmia, pruritus/wool loss, and the appearance of the nibbling reflex. Representative clinical signs for the late stage were the same as those for the early and middle stage, together with head tremors, hyperexcitability to external stimuli, ataxia or gait abnormalities, and teeth grinding. On the basis of these results, we propose the calculation of an objective clinical index that allows the differentiation among clinical stages and that could be useful for further studies. The usefulness of 3rd eyelid lymphoid tissue biopsies for sequential clinical studies in naturally scrapie-affected sheep is demonstrated.

Maedi-visna virus infection of ovine mammary epithelial cells.

The aim of this work was to perform a complete study of maedi-visna virus (MVV) infected mammary glands of naturally-infected sheep, and to determine if cells other than macrophages undergo a productive viral infection in this organ. Fifteen seropositive and two seronegative ewes were selected from MVV-infected flocks on the basis of clinical indurative mastitis and three sheep from an MVV-free flock. Within the mammary gland, MVV-positive cells were located by immunohistochemistry in the stroma and the epithelial alveolar barrier, most likely the ovine mammary epithelial cells (OMEC) of the acini. In situ hybridization confirmed these findings. Ultrastructural studies showed the presence of lentivirus-like particles budding off the cell surface in the alveolar barrier and also free in the acinar lumen. The presence of mammary histopathological lesions and MVV together with clear indications of productive infection (demonstration of a cytopathic effect in OMEC cultures and infection of co-cultures) were observed in the 15 seropositive and one of the seronegative sheep from the infected flock. These findings demonstrate that the OMEC were infected in vivo and probably underwent productive infection when studied ex-vivo. The OMEC of MVV-free sheep, which had subsequently been infected in vitro with MVV, also showed productive infection when challenged in vitro, confirming the replication of MVV in OMEC in vitro. The presence of MVV-infected OMEC in the mammary gland from infected animals, the productive infection in these OMEC and the release of lentiviral particles to the acinar lumen may have relevance in the pathogenesis and transmission of MVV infection.