Quantitative analysis of telomerase in feline mammary tissues.

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.

Immunohistochemical expression of h-telomerase reverse transcriptase in canine and feline meningiomas.

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.

Evaluation of telomerase in canine mammary tissues by immunohistochemical analysis and a polymerase chain reaction-based enzyme-linked immunosorbent assay.

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.

Porcine circovirus type 2 detection in in vitro produced porcine blastocysts after virus sperm exposure.

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.

PCV2-DNA in formalin-fixed and paraffin embedded lymph nodes of wild boar (Sus scrofa ssp. scrofa): one sampling approach for two laboratory techniques.

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.

Disseminated pulmonary adiaspiromycosis in a crested porcupine (Hystrix cristata Linnaeus, 1758).

Adiaspiromycosis is primarily a necrotizing granulomatous pneumonia caused by a dimorphic fungus of the genus Emmonsia. A young crested porcupine (Hystrix cristata) found dead showed multiple fractures, chronic pleuritis, and granulomatous pneumonia. Microscopically, cystic structures were consistent with adiaspiromycosis by Emmonsia crescens. The diagnosis was confirmed using molecular methods.

Reproduction in porcine circovirus type 2 (PCV2) seropositive gilts inseminated with PCV2b spiked semen.

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.

First description of nodular onchocercosis (Onchocerca jakutensis) in free-ranging Italian red deer (Cervus elaphus).

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.