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This study was aimed at assessing the capability of semen experimentally infected with porcine circovirus type 2 (PCV2) to produce porcine blastocysts PCR positive for PCV2. Embryos were obtained from in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes or by parthenogenesis. Sperm suspension was exposed to PCV2b and utilized for IVF. PCV2 spiked semen did not reveal any reduction in sperm viability or motility but its ability to produce infected blastocysts was irrelevant as only one out of 15 blastocysts obtained by IVF were PCV2b; however two blastocysts were PCV2a positive. Furthermore, the presence of PCV2 was demonstrated also in embryos obtained by parthenogenesis (one out of 17 was PCV2b and one PCV2a positive). Even if PCV2 firmly attaches to the surface of spermatozoa, experimentally spiked sperm were not effective in infecting oocytes during IVF and in producing PCR positive embryos. The infected blastocysts we obtained derived most probably from infected oocytes recovered at the abattoir., (© 2015 Japanese Society of Animal Science.)

Intestinal immune response plays an important defensive role for pathogens, particularly for those transmitted by the oro-faecal route or for foecal shedding modulation. This work examined three parts of intestine from twelve gilts experimentally infected with PCV2-spiked semen, six vaccinated (V group) and six unvaccinated (NV group) against PCV2, 29 and 53 days post infection (DPI). An immunohistochemical investigation for IgA-, IgG- and IgM-antibody bearing plasma cells (PCs) was run on intestinal samples coupled with a sandwich immunohistochemical method to reveal anti-PCV2 antibody-secreting PCs. Plasma cell density was compared in the two groups of animals at 29 and 53 DPI. The IgA, IgG and IgM PC density did not differ between groups but displayed an increase from the upper (villus) to the lower part of the crypts while a decreasing trend in PC density was identified from duodenum to ileum. In the NV group, no increase in anti-PCV2 PC density was demonstrable in the two sampling moment: the amounts of lamina propria PCV2-specific antibody-producing PCs remained constant, 10.55 ± 4.24 and 10.06 ± 5.01 at 29 DPI and 53 DPI, respectively. In the V group a significant increase in PCV2-specific antibody-producing PCs was observed over time. The amounts of PCV2-specific antibody-producing PCs increased from 9.37 ± 13.36 at 29 DPI to 18.76 ± 15.83 at 53 DPI. The data on IgA, IgM and IgG PC counts can be considered reference values in a population of adult pigs. The sandwich method can be proposed as a technique able to identify specific antibody-secreting PCs in formalin-fixed paraffin-embedded tissues. A practical application of the sandwich method is the demonstration of a "booster-like" response of the lamina propria in vaccinated compared to unvaccinated animals. After virus challenge, vaccination induced an increase in the number of PCs containing specific anti-PCV2 antibodies at the level of intestinal mucosa., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Since 1999, field evidence of transplacental infection by porcine circovirus type 2 (PCV2) and reproductive failure has been reported in pigs. The objective of this study was to evaluate the clinical and pathological consequences of PCV2 infection in conventional PCV2-seropositive gilts by insemination with PCV2b-spiked semen., Results: Six PCV2 seropositive gilts were inseminated with PCV2b-supplemented semen (infected) and three animals with semen and cell culture medium (controls). Only three out of the six infected animals were pregnant by ultrasonography on day 29 after insemination, while two out of the three controls were pregnant. One control gilt aborted on day 23 after insemination but not due to PVC2. Viraemia was demonstrated in four out of six infected and in one control gilt that became infected with PCV2a. Anti-PCV2 antibody titres showed dynamic variations in the infected group throughout the study. Among infected gilts, the animal with the lowest anti-PCV2 titre (1/100) at the beginning of the experiment and another that reached a similar low value during the experiment showed evident seroconversion over time and had also PCV2 positive foetuses. One placenta displayed mild focal necrosis of the chorionic epithelium positively stained by immunohistochemistry for PCV2 antigen., Conclusions: PCV2-seropositive gilts can be infected with PCV2 after intrauterine exposure and low maternal antibody titre may increase the probability of a foetal infection.

Superficial inguinal lymph nodes from 72 wild boars examined in a previous immunohistochemical (IHC) study on porcine circovirus type 2 (PCV2) were selected for a PCV2 polymerase chain reaction (PCR) analysis. Four of these lymph nodes were PCV2-IHC strongly positive with PMWS histological lesions (outcome 1), 6 weak to mild PCV2-IHC positive without PMWS histological lesions (outcome 2) and 62 PCV2-IHC negative. Considering IHC the gold standard for diagnosis, the aims of the study were to evaluate the suitability of the PCV2-DNA extraction from formalin-fixed and paraffin-embedded (FFPE) tissue and the sensitivity and specificity of PCR under two IHC interpretations criteria: (A) the sample was considered positive if the result was outcome 1; (B) the sample was considered positive if the result was outcome 1 or 2. Under (A) criteria, sensitivity and specificity of PCR were 100% and 89.7%, respectively; the Cohen's Kappa coefficient was 0.49. Under (B) criteria, sensitivity and specificity of PCR were 80.0% and 95.2%, respectively; the Cohen's Kappa coefficient was 0.72. The high Cohen's Kappa coefficient under the (B) interpretative criteria indicates good agreement between the two methods. In conclusion, 1) DNA extracted from FFPE specimens of wild boar is suitable for PCR and further represents a screening test for PCV2/PCVD (PCV2 Diseases) investigations in wild boar as well; 2) routine histological sampling can also be useful for PCV2 virological studies in wild boar.

Onchocercosis is a vector-transmitted parasitic disease involving wild and domestic ungulates, humans, and dogs. Red deer (Cervus elaphus) host numerous Onchocerca spp. which have precise anatomic sites in the host and two species, Onchocerca flexuosa Wedl, 1856 and Onchocerca jakutensis Guba-now, 1964, are found inside subcutaneous nodules. Between September and November 2007, subcutaneous nodules were observed on both thighs in shot red deer of a Tuscany population. We observed cystic structures, surrounded by a fibrous capsule, containing nematodes. Filamentous worms were male and female; microfilariae were also described. Although morphologically we could not distinguish between O. flexuosa and O. jakutensis, genetic studies implicated O. jakutensis. This is the first report of this parasite in Italy.

A well-demarcated mass was found by computed tomography in the left cerebellar hemisphere of a 4-year-old male Boxer with acute onset of progressive central vestibular syndrome. At necropsy, the pink, gelatinous mass was in the flocculonodular lobe. Histologically, neoplastic tissue arose from the granular layer of the cerebellar cortex and consisted of sheets of oval to round hyperchromatic cells, consistent with the diagnosis of medulloblastoma. Synaptophysin and neuron-specific enolase immunoreactivity supported the neuronal origin of the neoplastic cells; furthermore, a weak to moderate c-kit expression was detected, as reported in pediatric medulloblastoma. Telomerase activity of tumor cells was demonstrated by immunohistochemistry and by the telomere repeat amplification protocol, suggesting involvement of this enzymatic pathway., (© The Authors 2011)

The purpose of this study was to validate immunohistochemistry (IHC) as an alternative to telomerase repeat amplification protocol (TRAP) analysis to detect telomerase activity. TRAP-enzyme-linked immunosorbent assay (ELISA) reactivity was compared with telomerase reverse transcription (TERT) IHC staining in 22 feline mammary tissues (6 normal mammary glands, 2 dysplastic mammary glands, 1 fibroadenoma, and 13 malignant neoplasms [6 solid mammary carcinomas, 2 squamous-cell carcinomas, 4 tubulopapillary mammary carcinomas, and 1 mammary carcinosarcoma]). TERT IHC staining revealed enzymatic expression in nuclear, nucleolar, cytoplasmic, and combined nuclear and nucleolar staining patterns that were separately quantified by image analysis and expressed as the absolute number (average) of positive cells or percentage of positive cells with respect to overall cellularity. With TERT IHC staining, the absolute number and percentage of cells with positive nuclei and nucleoli within the same cell were the variables with the greatest discrimination between benign and malignant mammary lesions (analysis of variance [ANOVA], average P < 0.0001; percentage P < 0.001). For TRAP-ELISA-positive versus TRAP-ELISA-negative tissues, a positive test result provided greater differentiation between malignant versus benign mammary lesions (ANOVA, average P = 0.00038; percentage P = 0.0022). The same IHC pattern of expression showed a proportional and significant (average P = 0.004; percentage P = 0.002) but low (average R = 0.60; percentage R = 0.63) correlation with TRAP-ELISA by the Pearson test. The correlation coefficients obtained show that IHC and TRAP cannot be considered interchangeable because the 2 methods are more complementary than exclusive.

In recent decades, the most successful strategy for controlling blood pressure has been inhibition of the angiotensin-converting enzyme (ACE). ACE inhibitors of chemical synthesis (captopril, enalapril, ramipril and trandolapril) have been widely used clinically to reduce mortality in patients with heart failure, and in patients with recent myocardial infarction and heart failure or marked left ventricular dysfunction. In addition to preventive and therapeutic drugs, increased attention has been paid to identifying dietary compounds that may contribute to cardiovascular treatment and prevention. ACE inhibitory peptides, derived from a multitude of plant and animal proteins such as milk, soy or fish, represent sources of health-enhancing components. These ACE inhibitory peptides can be enzymatically released from precursor proteins in vitro and in vivo, respectively during food processing and gastrointestinal digestion. They have shown the ability to lower blood pressure by limiting the vasoconstrictory effects of Angiotensin II and potentiating the vasodilatory effects of Bradykinin. By using specific procedures they may be generated in or incorporated into functional foods for the development of 'natural' beneficial health products. Several products containing peptides with ACE inhibitory properties are currently on the market or in development. This review focuses on the use, application and future perspective of bioactive peptides with properties relevant to cardiovascular health.

Kidney samples with interstitial nephritis from 26 pigs affected by postweaning multisystemic wasting syndrome (PMWS) were selected. A histologic evaluation was carried out to describe the type of inflammation and its relationship with viral load, as assessed by in situ hybridization (ISH). Of 26 cases, 10 revealed a tubulointerstitial, lymphoplasmacytic nephritis, 11 an interstitial granulomatous nephritis, and 5 both types of inflammation (mixed type). In 4 cases of granulomatous inflammation, the pattern was not classically nodular, and a population of macrophages and lymphocytes was present (interstitial lymphohistiocytic nephritis). ISH confirmed the presence of porcine circovirus type 2 (PCV2) nucleic acid in all cases. The epithelium of the renal tubules was the most constantly ISH-positive structure. In tubulointerstitial nephritis, the higher the number of positive inflammatory cells, the more severe the inflammation. The ISH reaction was more heterogeneous and unpredictable in granulomatous nephritis, with some epithelioid and giant cells positive by ISH. To quantify macrophages distributed in the three patterns of nephritis, immunohistochemical methods using anti-major histocompatibility complex II (anti-MHC-II) and anti-lysozyme antibodies were undertaken, and semiquantitative evaluation was carried out. MHC-II was mainly expressed by lymphocytes in tubulointerstitial nephritis, but did not always stain macrophages in cases of granulomatous (including lymphohistiocytic) nephritis; the anti-lysozyme antibody revealed macrophages when present in tissues. The amount of PCV2 nucleic acid was not apparently associated with the pattern of inflammation (tubulointerstitial or granulomatous). PCV2 load seems to reflect the severity of the lymphoplasmacytic inflammation but not that of granulomatous and lympho histiocytic types.

Telomere length maintenance is regarded as a fundamental step in tumorigenesis, as most human brain tumors, including meningiomas, stabilize the ends of their chromosomes using telomerase. This investigation represents an introduction to telomerase expression in canine and feline meningiomas. Twenty-five archived cases (14 dogs and 11 cats) were immunohistochemically tested for human-telomerase reverse transcriptase (h-TERT), scored, and quantified; furthermore, mitoses were counted on sections stained with a modified toluidine blue. The h-TERT antibody immunolabelled the nucleus and nucleolus of meningeal neoplastic cells, with an intensity ranging from mild to strong and a speckled distribution; a significantly higher expression in cats was noted, while no significant association between h-TERT immunolabelling and sex or histotype was evident in dogs or cats. The telomerase enzyme represents a fundamental parameter of potential malignant transformation, which may occur independently of the signal to proliferate, thereby supplying the cells with unlimited growth capabilities. Telomerase expression could be a prognostic indicator independent of the kinetic parameters, although this should be evaluated using a larger dataset with available clinical information.

Immunohistochemical expression of immunocompetent cells bearing major histocompatibility complex (MHC-II) and interleukin 2-R (IL2-R) (CD25) molecules was performed on lymph nodes with spontaneous postweaning multisystemic wasting syndrome (PMWS). Control lymph nodes displayed intense diffuse immunoreactivity to MHC-II in both follicles and interfollicular areas. A marked reduction of follicular MHC-II immunoreactivity and inconsistent staining of histiocytes in interfollicular areas was observed in PMWS cases with a slight lymphoid depletion; in those cases with moderate to severe lymphoid depletion, there was a progressive decrease in MHC-II expression. In controls and in slightly depleted nodes, IL2-R was equally expressed in interfollicular tissue and in follicles, whereas in moderate and severe cases, it was detected in interfollicular remnants only. Immunohistochemical staining was scored semiquantitatively. The mean MHC-II score was significantly reduced in PMWS cases compared with controls (Spearman test), whereas there was no difference in the IL2-R score. The evident reduction of MHC-II immunoreactivity suggests an impairment in MHC-II linked antigen presenting cell expression.

The enzyme telomerase is considered a potential marker for neoplastic tissue and is used as a diagnostic and prognostic tool in clinical medicine and therapeutics. For this reason, the possible role of telomerase activation in the process of malignant transformation is currently the subject of intense research efforts. The focus of the study reported here was to detect telomerase in 37 canine mammary samples, by comparing two methods: immunohistochemical (IHC) analysis for detecting the catalytic subunit of the enzyme, telomerase reverse transcriptase (TERT), and the telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA), a polymerase chain reaction (PCR)-based technique that uses a colorimetric detection method. Using the TRAP-ELISA, samples were considered positive when they yielded a difference of at least 0.2 absorbance units between the readings at 450 nm versus 690 nm wavelength. On the basis of this criterion, 18 negative and 19 positive cases were obtained. Specific immunohistochemical staining was observed mainly in the nucleoli, to a lesser extent in the nuclei, and rarely in the cytoplasm of epithelial cells. A sample was considered positive when at least 10% of the epithelial cells had specific staining. The Pearson correlation between the TRAP-ELISA and IHC results was significant only when IHC nucleolar (r = 0.53, P < 0.01) or nuclear (r = 0.36, P < 0.05) staining or their combination (r = 0.58, P < 0.01) was considered. Thus, IHC staining of nucleoli and nuclei can be considered as an alternative method to the TRAP-ELISA. The detection of telomerase in normal mammary gland and fibrocystic mastopathy using both methods does not support the idea that telomerase may be used as a specific marker of mammary neoplasia in dogs.

Postweaning multisystemic wasting syndrome (PMWS) affects nursery and growing pigs, and is characterized by wasting, failure to thrive, pale skin, respiratory distress, diarrhoea and sometimes jaundice. Macroscopic findings are aspecific, but lymphocyte depletion in lymphoid tissues is one of the histological hallmarks [Vet. Q. 24 (2002) 109]. Spontaneous cases of PMWS were studied to evaluate proliferative activity and apoptosis as mechanisms involved in the pathogenesis of cell depletion in lymph nodes. The presence of Porcine Circovirus type 2 (PCV2) genome in the processed material was confirmed by in situ hybridization (ISH). The lymph node pattern of depletion was graded as initial, intermediate or final stage according to histological criteria in 10 superficial inguinal nodes from piglets with PMWS which died spontaneously or were slaughtered by euthanasia. The apoptotic and proliferative fraction were investigated by monoclonal antibody MIB1 immunohistochemistry and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end labeling) methods, respectively, and compared to three normal cases. One-way analysis of variance (ANOVA) comparison between the MIB1 index (number of positive cells per 100 nuclei) in controls and PMWS cases revealed a decrease of proliferation in both lymphoid and medulla-like tissues in the initial group (respectively, P=0.0017 and 0.024) but not in the intermediate (respectively, P=0.25 and 0.88) or final (respectively, P=0.47 and 0.81) cohorts. The apoptotic index (number of apoptosis/apoptotic bodies in 100 cells) revealed a statistically significant decrease only in the initial group (one-way ANOVA P=0.05). The proliferation/apoptosis ratio (MIB1/APO ratio) assessed to determine cell turnover disclosed a significant decrease of cell turnover from initial to final PMWS cases (Spearman's rank test: P=0.027). Decreased cell proliferation and not increased apoptosis seems to be the most important variable leading to cell depletion in PMWS lymphoid tissues.