Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

Sporotrichosis is a chronic, cosmopolitan granulomatous mycosis that affects humans and animals. The infection is caused by the dimorphic fungi Sporothrix sp. The aims of the present study were to evaluate, standardize and validate a nested PCR technique using two DNA purification kits for the extraction of DNA from formalin fixed and paraffin-embedded tissues (FFPE) for Sporothrix sp. detection. FFPE mycological culture pellet samples of different Sporothrix species (S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensis and S. schenckii) were used as positive controls and clinical FFPE tissue samples of animals positive for Cryptococcus sp., Leishmania infantum and Histoplasma sp. were used as negative controls. Ten clinical FFPE skin samples from cats with sporotrichosis were used to validate the nested PCR. These samples were cut into two distinct paraffin sectioning protocols (5 and 16 μm thick). The paraffin sections were subjected to two different DNA extraction kits (chemical and thermal extractions). A nested PCR was performed on the extracted DNA to identify the genus Sporothrix. The chemical extraction protocol with the 5 μm thick paraffin section was more effective in extracting DNA from Sporothrix sp. from FFPE samples and the nested PCR technique showed the highest sensitivities (100% in the positive controls and of 50% in the skin samples of cats) and specificity (100%). Therefore, the nested PCR using this protocol has great potential to be applied in Sporothrix sp. diagnosis in FFPE samples of cats.

Occurrence and relevance of Mycoplasma spp. in free-ranging pheasants from northwestern Germany

Since 2008/2009, the population of free-ranging ring-necked pheasants was recorded to decrease all over Germany. Various Mycoplasma (M.) spp. are causing severe respiratory signs in captive game bird species. Furthermore, M. gallisepticum is responsible for massive die-offs in consequence to severe conjunctivitis in house finches in the USA. Therefore, the prevalence of mycoplasmas in free-ranging pheasants was investigated and a potential impact on the population decline of pheasants discussed. Within this study, 150 free-ranging pheasants were sampled via tracheal swabs and tissue samples of the trachea and the periorbital skin, as the latter displayed inflammatory alterations in previous studies. In total, 177 samples were investigated for the presence of mycoplasmas using cultural and molecular biological methods. In 76 birds, necropsy was performed additionally. In total, 73.7% (51/76) of the examined pheasants had periorbital skin alterations. Furthermore, 64.4% (114/177) of the samples tested positive for mycoplasmas via PCR. Overall, 102/177 samples (57.6%, 78/105 tracheal swabs, 19/51 skin tissue, 5/21 trachea tissue) tested positive for mycoplasmas via culture. Mycoplasma gallinaceum (n = 50), M. pullorum (n = 45), M. glycophilum (n = 43), M. iners (n = 11), and M. gallinarum (n = 5) were frequently isolated. In 45 cases (45.9%), multiple Mycoplasma spp. were isolated from one sample. All examined samples tested negative for M. gallisepticum. Of 51 skin samples investigated for mycoplasmas, 24 (47.1%) showed inflammatory skin alterations in histology, and 58.3% (14/24) of these samples tested positive for Mycoplasma spp. additionally. Overall, there was a significant correlation between inflammatory altered skin samples and the detection of mycoplasmas in periorbital skin samples. Based on the present results, the isolated Mycoplasma spp. may play a role as facultative agents for the observed inflammatory skin alterations. However, additional investigation is needed to confirm this presumption.

Euclidean Distance Versus Manhattan Distance for New Representative SFA Skin Samples for Human Skin Segmentation

The human color skin image database called SFA, specifically designed to assist research in the area of face recognition, constitutes a very important means particularly for the challenging task of skin detection. It has showed high performances comparing to other existing databases. SFA database provides multiple skin and non-skin samples, which in various combinations with each other allow creating new samples that could be useful and more effective. This particular aspect will be investigated, in the present paper, by creating four new representative skin samples according to the four rules of minimum, maximum, mean and median. The obtained samples will be exploited for the purpose of skin segmentation on the basis of the well-known Euclidean and Manhattan distance metrics. Thereafter, performances of the new representative skin samples versus performances of those skin samples, originally provided by SFA, will be illustrated. Simulation results in both SFA and UTD (University of Texas at Dallas) color face databases indicate that detection rates higher than 92% can be achieved with either measure.

A study of the phosphorylation proteomic skin characteristics of Tan sheep during the newborn and er-mao stages

In this experiment, in order to study the formation mechanism of the lamb fur of Tan sheep, skin samples were collected from Tan sheep at the newborn and er-mao stages. Then, the phosphorylated proteomes of the skin samples of Tan sheep at the two different stages were compared and analyzed using a TMT labeled quantitative phosphorylation proteomic technique. A total of 2806 phosphorylated proteins were identified, including 8184 phosphorylation sites. The results of this study’s quantitative analysis showed that when compared with the skin samples at the er-mao stage, the phosphorylation levels of 171 sites had been upregulated in the skin samples at newborn stage. Meanwhile, 125 sites had been downregulated at the same stage. As shown by the results of the functional enrichment analysis of the differentially phosphorylated proteins, they had been mainly enriched in the cysteine and methionine metabolism. In addition, the phosphorylation levels of KAP4.7 and KAP13.1 had also varied during the different skin stages. These results indicated that the cysteine metabolism pathways, as well as the phosphorylation modifications of the keratin associated proteins in the skin, played important roles in the formation of the er-mao stage fur of the Tan sheep. Therefore, the findings of this study provided a new angle for interpreting the formation mechanism of er-mao stage fur properties.

Beclin 1, LC3 and P62 Expression in Equine Sarcoids

Background: It is well known that δ-bovine papillomaviruses (BPV-1, BPV-2 and BPV-13) are one of the major causative agents of equine sarcoids, the most common equine skin tumors. Different viruses, including papillomaviruses, evolved ingenious strategies to modulate autophagy, a complex process involved in degradation and recycling of old and damaged material. Methods: The aim of this study was to evaluate, by immunohistochemistry (IHC) and Western blot (WB) analysis, the expression of the main related autophagy proteins (Beclin 1, protein light chain 3 (LC3) and P62), in 35 BPV1/2 positive equine sarcoids and 5 BPV negative normal skin samples. Results: Sarcoid samples showed from strong-to-moderate cytoplasmic immunostaining, respectively, for Beclin 1 and P62 in >60% of neoplastic fibroblasts, while LC3 immunostaining was weak to moderate in ≤60% of neoplastic fibroblasts. Western blot analysis confirmed the specificity of the antibodies and revealed no activation of autophagic flux despite Beclin 1 overexpression in sarcoid samples. Conclusion: Results could suggest the activation of the initial phase of autophagy in equine sarcoids, and its impairment during the following steps. The impairment of autophagy could lead to a selection of a quiescent population of fibroblasts, which survive longer in a hypoxic microenvironment and produced more and/or altered collagen.

Validation and Application of Skin RT-QuIC to Patients in China with Probable CJD

The definite diagnosis of human sporadic Creutzfeldt–Jakob disease (sCJD) largely depends on postmortem neuropathology and PrPSc detection in the brain. The development of real-time quaking-induced conversion (RT-QuIC) of cerebrospinal fluid (CSF) samples makes it possible for premortem diagnosis for sCJD. To test the diagnostic potential of RT-QuIC of skin specimens for probable sCJD, we collected the paired skin and CSF samples from 51 recruited living patients referred to the Chinese CJD surveillance center, including 34 probable sCJD, 14 non-CJD, and 3 genetic prion disease (gPrD). The samples were subjected to RT-QuIC assays using recombinant hamster PrP protein rHaPrP90-231 as the substrate. Using skin RT-QuIC assay, 91.2% (31/34) probable sCJD patients, and 1 T188K genetic CJD (gCJD) cases showed positive prion-seeding activity, while 85.7% (12/14) non-CJD patients were negative. CSF RT-QuIC positive seeding activity was only observed in 14 probable sCJD patients. Analysis of the reactivity of 38 positive skin RT-QuIC tests revealed that the positive rates in the preparations of 10−2, 10−3 and 10−4 diluted skin samples were 88.6% (39/44), 63.6% (28/44), and 25.0% (11/44), respectively. Eleven probable sCJD patients donated two skin specimens collected at different sites simultaneously. Although 95.5% (21/22) skin RT-QuIC elicited positive reaction, the reactivity varied. Our preliminary data indicate high sensitivity and specificity of skin RT-QuIC in prion detection for Chinese probable sCJD and highlight that skin prion-seeding activity is a reliable biomarker for premortem diagnosis of human prion disease.

Utility of nail enamel for inking of surgical margins in oral biopsy: a comparative study

Background: India Ink is the most commonly used ink in surgical pathology. The main disadvantages of India Ink are longer drying time, monochrome, toxicity and cost. Because of these disadvantages, alternative materials have been suggested to replace India ink. The aim of this study is to evaluate the effectiveness of nail enamel for inking of surgical margin and to compare it with India ink. Materials and methods: N = 20, which included 10 mucosal and 10 skin samples. Each selected margin is divided into 2 equal halves and one is inked with India ink and the other with nail enamel (Vernis A Ongles: Dark green). After routine processing and staining, the effectiveness of nail enamel and India ink were compared based on macroscopic and microscopic parameters. Results: Less drying time and visibility on paraffin wax block were excellent for nail enamel. Microscopic visibility of nail enamel was comparable with that of India ink. However, processing fluids contamination is the main drawback of nail enamel. Conclusion: Nail enamel can be used as an alternative to India ink because of its less drying time, ease of application, good visibility on wax blocks and microscopically, availability in multiple colours, cost effectiveness and non-toxicity.

PLASMA COAGULATING AND NON-COAGULATING STAPHYLOCOCCUS SPP. IN SKIN SAMPLES FROM DOGS AND THEIR RESISTANCE TO ANTIBIOTICS

Staphylococci are human and animal mucosal surface and skin commensals that can cause a variety of infections, such as purulent skin infections, otitis externa, pyoderma, urinary tract infections, and postoperative infections. Dog skin is one of the protective barriers for animals. However, dogs can have and transmit a variety of microorganisms on their skin, including staphylococci. Most studies have compared plasma coagulating and non-coagulating Staphylococcus spp. by dog breeds, sex, and coat length. The aim – to identify plasma coagulating and non-coagulating Staphylococcus spp. in skin samples from dogs and its resistance to antibiotics by place of residence. Staphylococci were detected in more than half of the samples tested, one third of which were plasma coagulating and the remaining two thirds were non-coagulating plasma. Plasma non-coagulating staphylococci were mainly increased among dogs living at home and plasma coagulating – among dogs living outdoors, the difference between these groups is statistically significant. Staphylococcus aureus was predominantly resistant to penicillin and clindamycin, while plasma non-coagulants were resistant to fusidic acid.

Highly divergent herpesviruses in threatened river dolphins from Brazil

River dolphins are a highly threatened polyphyletic group comprised of four odontocete families: Iniidae, Pontoporiidae, Lipotidae, and Platanistidae, the first two endemic to South America. To address the knowledge gap regarding infectious agents in this cetacean group, we surveyed the presence of herpesviruses by PCR in skin and/or blood samples of live-captured Amazon (Inia geoffrensis, n = 25) and Bolivian (Inia boliviensis, n = 22) river dolphins of the Amazon basin and in selected tissue samples of franciscanas (Pontoporia blainvillei, n = 27) stranded or bycaught in southeastern Brazil. Additionally, available franciscana tissue samples were examined by histopathology. Herpesvirus DNA was amplified in 13 Bolivian river dolphins (59.1%, 95% CI 38.5–79.6%) and 14 franciscanas (51.9%, 95% CI 33.0–70.7%). All Amazon river dolphins were herpesvirus-negative. Two different herpesviruses were found in Bolivian river dolphins: a previously known gammaherpesvirus detected in blood and/or skin samples of all positive individuals and a novel alphaherpesvirus in the skin of one animal. A new gammaherpesvirus was found in several franciscana samples—the first herpesvirus recorded in Pontoporiidae. Intranuclear inclusion bodies consistent with herpesvirus were observed in the lymph node of one franciscana. The high divergence among the obtained herpesviruses and those previously described can be explained by viral-host coevolution, and by the fact that these populations are fairly isolated.