Evaluation of an improved Giemsa staining technique for blood borne parasite in thick and thin blood film from dogs

Many a times the use of rapid diagnostic tests for blood borne parasites like trypanosomiasis and Babesiosis is increasingly being used, but the gold standard for its detection is still the use of microscopy both as reference and confirmatory diagnosis. To determine the effectiveness of the adjusted stock giemsa staining technique over the conventional methods. Venous Blood samples were collected from 10 dogs in EDTA and then used for the simultaneous preparation of two thin and thick smear slides, one stained according to Giemsa 1:20 dilution for 30 mins while the other was stained using the Stock Solution for 30seconds the diluted with buffered saline for 20seconds and rinsed. Fixation of the thin smear was done in a covered staining jar containing anhydrous methanol for 1 to 2 min, after which the slides were air-dried. From the result obtained from 10 dogs blood samples gotten from the veterinary clinic, the adjusted giemsa staining technique showed a positive differentiation when compared to the 1:20 dilution, a total of 7 blood samples tested positive for blood borne parasites, Trypanosoma evansi, Babesiosis cani and Heart worm. The highest percentage occurrence was T.evansi (40%), Babesiosis cani(20%) and Heart worm (10%).The adjusted Giemsa staining technique serves as a fast, easy and less complex alternative to the 1:20 dilution, where the solution has to be diluted from the stock solution and then stained, although the only disadvantage to this technique would be easy contamination of the stock solution, but the advantages here is that it saves time, quicker result output and better differentiation microscopically.

Comparative chromosome studies in species of subtribe Orchidinae (Orchidaceae)

In our study, FISH mapping using 18S-5.8S-25S rDNA and 5S rDNA sequences was performed for the first time on Ophrys tenthredinifera Willdenow, 1805, Serapias vomeracea (Burman f., 1770) Briquet, 1910 and Himantoglossum hircinum (Linnaeus, 1753) Sprengel, 1826. A detailed study was also performed on O. tenthredinifera using Giemsa-staining, silver-staining, CMA fluorescence banding and fluorescence in situ hybridisation (FISH) with rDNA probes. We analysed two subspecies, i.e. O. tenthredinifera subsp. neglecta (Parlatore, 1860) E.G. Camus, 1908 and O. tenthredinifera subsp. grandiflora (Tenore, 1819) Kreutz, 2004 by the traditional Feulgen method and constructed the karyotype. The cytotaxonomic implications for both taxa are also discussed. In Himantoglossum hircinum, FISH and silver staining highlighted differences in the number of two rDNA families (35S and 5S) with respect to Barlia robertiana (Loiseleur-Deslongchamps, 1807) Greuter, 1967. In addition, fluorescence in situ hybridisation was also applied to diploid (2n = 2x = 36) and triploid (2n = 3x = 54) Anacamptis morio (Linnaeus, 1753) R.M. Bateman, Pridgeon et M.W. Chase, 1997. As far as we are aware, this is the first case of autotriploidy observed in A. morio.

Cytopathological Findings of Secretory Carcinoma of the Salivary Gland and the Diagnostic Utility of Giemsa Staining

Secretory carcinoma is a salivary gland neoplasm first described as a mammary analogue secretory carcinoma by Skalova et al. in 2010 and redesignated as a secretory carcinoma in the 2017 World Health Organization Classification of Head and Neck Tumors. Secretory carcinoma diagnosis is reliant on specific cytological and histological findings and the detection of an ETV6-NTRK3 fusion gene. Here, we examined the clinical and cytopathological features of four cases of secretory carcinoma occurring in three males and a female, aged between 39 and 74 years. All four tumors involved the parotid gland, and were found to have the ETV6-NTRK3 fusion gene. Fine-needle aspiration-based cytology smears of all tumors displayed papillary and/or dendritic pattern clusters, some of which were associated with blood vessels. The neoplastic cells displayed enlarged nuclei with fine chromatin and small, distinct, single nucleoli. Furthermore, several neoplastic cells with a characteristic vacuolated cytoplasm were identified in each specimen. Giemsa staining revealed cytoplasmic vacuolation, intracytoplasmic metachromatic secretions and/or various sized metachromatic granules, and a background of metachromatic mucin in all four specimens. Given this, we conclude that these cytological findings, especially those of the Giemsa staining, might be helpful in the diagnosis of secretory carcinoma.

Hydroxy-Safflower Yellow A Alleviates Osteoporosis in Ovariectomized Rat Model by Inhibiting Carbonic Anhydrase 2 Activity

Background: To investigate the therapeutic effect of Hydroxy-safflower yellow A (HSYA) on rat’s osteoporosis and explore its potential mechanism of action.Methods: Bilateral ovariectomized female rats (OVX) were used to establish a postmenopausal rat model of osteoporosis. HSYA was given as an intervention, and estradiol was used as a positive control. The levels of serum alkaline phosphatase (ALP), calcium ion (Ca2+), and inorganic phosphorus (IP) were used to detect bone loss. Three months after modeling, the rats were sacrificed and the rat’s ovaries, kidneys, tibia, and femur were used to calculate the organ index. The bone marrow of the femur of the rats was stained with Giemsa staining. The femur strength of rats was measured by INSTRON. The degree of osteoporosis was detected by pathological staining after decalcification of bone tissue. Predicted the main targets of HSYA in combination with bioinformatics, and the proteins related to osteoclast differentiation were detected in combination with western blotting. The effect of HSYA on the differentiation of RAW264.7 cells into osteoclasts was observed.Results: The Giemsa staining and serum test results showed that the operation was successful and affected bone metabolism. In the bone strength test, HSYA significantly increased the maximum threshold of femoral load in rats. Pathological examination showed that tibial cartilage, trabecular bone, and cortex significantly increased after treatment with HYSA. The number of osteoblasts increased while the number of osteoclasts decreased—elevated levels of type I and III collagen. Autodock was used for molecular docking of potential targets of HSYA. qPCR and western blot were used to show that the expression levels of CA2 and osteoclast differentiation-related proteins were significantly decreased after HSYA treatment. Cell level results showed that HSYA could inhibit the activity of osteoclasts and the ability of RAW264.7 cells to differentiate into osteoclasts.Conclusion: HSYA can inhibit the differentiation and formation of osteoclasts by inhibiting the expression of CA2 and relieving osteoporosis symptoms in OVX rats.

Haemocyte variations in 35 species of grasshoppers and locusts

Introduction: Grasshoppers and locusts are widely distributed worldwide, causing significant losses in agriculture. The origin and functions of their haemocytes are not entirely understood. Objectives: Insect haemocytes arbitrate cellular defence and participate in humoral defences. Due to their importance, the haemocytes of 35 species of grasshoppers and locusts from China were morphologically examined in this study. We aim to highlight a simple method for the morphological examination of insect haemocytes. Methods: The haemocytes were observed, counted and compared under a light microscope after Wright-Giemsa staining. Results: High complexity in form and shape were observed in the haemocytes. These include prohaemocytes, plasmatocytes, granulocytes, vermicytes, podocytes and megakaryocytes. No clear relationship was seen between the haemocyte type and their phylogenetic relationship among the three families examined. The high abundance of plasmatocytes and granulocytes suggests their importance in the immunity of grasshoppers and locusts. The minor haemocyte populations including prohaemocytes, vermicytes and podocytes may not be always present in individuals. Conclusion: All examined species shared similarities in their haemocyte types. Wright-Giemsa staining is a simple and efficient method for evaluating haemocytes.

Anti-allergic activities of Umbelliferone against histamine- and Picryl chloride-induced ear edema by targeting Nrf2/iNOS signaling in mice

Background The current study was aimed to investigate the anti-allergic activities of the Umbelliferone (UMB) against the acute Histamine and chronic Picryl chloride (PiCl)-induced allergy in mice. UMB is a coumarin derivative (isolated from Angelica decursiva) found in various parts of the plants such as flowers, roots and, stems isolated from the plants of Umbelliferae family. Methods The UMB (1, 10, 50 mg/kg) was administered intraperitoneally (i.p) half an h before or 2 h after the induction of allergic ear edema. The acute ear edema was induced by histamine (intradermally, i.d), while the chronic ear edema was induced by painting the PiCl (sensitized with the toluene) on the ear. The antioxidants and oxidative stress markers were assessed. The histological changes were assessed using Hematoxylin and eosin (H and E) and giemsa staining. The immunohistochemistry studies were performed to assess the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) and inducible nitric oxide synthase (iNOS). The data was analyzed using one-way ANOVA tests followed by Tukey’s test with p < 0.05 was chosen as criteria for statistical significance. Results UMB treatment markedly reduced the allergic ear edema and ear weight compared to the negative control. Furthermore, the UMB attenuated the oxidative stress markers, while induced the antioxidants enzymes. Similarly, the UMB treatment significantly attenuated the serum immunoglobulin E (IgE) level. The UMB treatment markedly improved the histological parameters using H and E staining and Giemsa staining. The UMB administration induced the Nrf2 expression, while attenuated the iNOS expression. Furthermore, the computational analysis was performed to assess the interaction of the UMB with the various protein targets and to determine the mechanism of interaction with the target proteins. Conclusion In conclusion, the UMB treatment significantly alleviated the allergic symptoms, attenuating the oxidative stress, improved the histological features using in vivo and computational approaches.

Triatominium search and diagnosis of trypanosoma cruzi in vectors captured in cane calk originating from sugarcane industry

The objective of the study was to survey triatomines and diagnose Trypanosoma cruzi in captured vectors and sugarcane juice from sugarcane mills. 100% (3/3) of the active sugar cane mills in the study area were surveyed. An active and passive search of the vectors and research of T. cruzi in the feces of engorged triatomines by compression of the abdomen and for the juice in natura by sedimentation with Giemsa staining technique were carried out, where five samples per mill were collected, totaling 15 researched. As a result, in 100% (3/3) of the mills, 22 hematophagous triatomines were captured, with 41% (9/22) by passive search and 59% (13/22) by active search. 81.8% (18/22) were found in the household environment and 18.2% (04/22) in the intra-household environment of the mills. Two species were identified being 68.18% (15/22) Triatoma brasiliensis and 9.09% (02/22) Triatoma pseudomaculata, with one adult and the four nymphs unable to be identified. T. cruzi diagnosis for triatomines was observed 4.54% (1/22) positive and 95.45% (21/22) negative, while broth, 100% (15/15) negative. However in 33.3% (5/15) broth samples the presence of soiling (microplastics) was observed. Demonstrating the importance of investigation and monitoring of milling sites with the possibility of oral transmission, which currently represent the majority of infection records in Brazil.

Efficacy of immunohistochemical staining in detecting Helicobacter pylori in Saudi patients with minimal and atypical infection

Gastric Helicobacter pylori infection is diagnosed based on histopathological evaluation of gastric mucosal biopsies, urease test, urea breath test, H. pylori culturing, or direct detection using polymerase chain reaction (PCR). This study aimed to evaluate the efficacy of immunohistochemical (IHC) staining in detecting H. pylori in gastric biopsies from patients with chronic gastritis and minimal or atypical infection. Gastric biopsies from 50 patients with chronic gastritis were subjected to routine haematoxylin and eosin (H&E), modified Giemsa, and IHC staining. The results of staining were compared with those of quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis identified 32 (64%) H. pylori-positive cases, whereas IHC, H&E, and modified Giemsa staining identified 29 (58%), 27 (54%), and 21 (42%) positive cases. The sensitivity of IHC staining (87.50%) was higher than that of H&E (59.38%) and modified Giemsa (43.75%) staining. The specificity of H&E, modified Giemsa, and IHC staining was 55.56%, 61.11%, and 94.44%, respectively. IHC staining exhibited the highest diagnostic accuracy (90%), followed by H&E (58%) and modified Giemsa (50%) staining. Active gastritis, intestinal metaplasia, and lymphoid follicles were detected in 32 (64%), 4 (8%), and 22 (44%) cases, respectively, and all of these cases were H. pylori positive. In contrast to routine H&E and modified Giemsa staining, IHC allows for the accurate H. pylori detection in cases with minimal or atypical infection. Moreover, IHC can be an alternative diagnostic method to qRT-PCR for detection of H. pylori in such cases.