Carmine Staining Research Articles

Molecular characterization of Bertiella studeri infecting a primate in South India.

Bertiella spp. is a mite-borne cestode parasite that inhabits the small intestine of wide range of mammals, including non-human primates. In the present study, the morphological and molecular analysis of Bertiella studeri recovered from the small intestine of a bonnet macaque (Macaca radiata) from Wayanad, Kerala (South India) was performed. Acetic alum carmine staining identified the cestode morphologically based on the characters like broader proglottids, which contain irregularly alternating genital pores, single set of reproductive organs, 280 testes and a tubular transverse uterus. Molecular characterization was done using 18SrRNA, ITS1-5.8S and COX1 genes. Phylogenetic trees were constructed using MEGA X based on the Maximum Likelihood (ML) method (Hasegawa-Kishino-Yano (HKY) model). Cytochrome oxidase I gene could detect the existence of genetic variation in the parasite from two different hosts viz., monkey (Kerala, Argentina, and Kenya) and human (Sri Lanka). A minimum spanning network of haplotypes was generated by the haplotype networking with the above sequences using the popARTv1.7. Haplotype analysis based on COX1 revealed that the parasite haplotype was different in each country with highest population frequency in Sri Lanka.

Novel carbamodithioate regulates cellular hypoxia through chemical activation of prolyl hydroxylase-2 for breast cancer chemoprevention.

Inhibition of prolylhydroxylase-2 (PHD-2) in both normoxic and hypoxic cells is a critical component of solid tumours. The present study aimed to identify small molecules with PHD-2 activation potential. Virtually screening 4342 chemical compounds for structural similarity to R59949 and docking with PHD-2. To find the best drug candidate, hits were assessed for drug likeliness, antihypoxic and antineoplastic potential. The selected drug candidate's PHD-2 activation, cytotoxic and apoptotic potentials were assessed using 2-oxoglutarate, MTT, AO/EtBr and JC-1 staining. The drug candidate was also tested for its in-vivo chemopreventive efficacy against DMBA-induced mammary gland cancer alone and in combination with Tirapazamine (TPZ). Virtual screening and 2-oxoglutarate assay showed BBAP-6 as lead compound. BBAP-6 exhibited cytotoxic and apoptotic activity against ER+ MCF-7. In carmine staining and histology, BBAP-6 alone or in combination with TPZ restored normal surface morphology of the mammary gland after DMBA produced malignant alterations. Immunoblotting revealed that BBAP-6 reduced NF-κB expression, activated PHD-2 and induced intrinsic apoptotic pathway. Serum metabolomics conducted with 1H NMR confirmed that BBAP-6 prevented HIF-1α and NF-κB-induced metabolic changes in DMBA mammary gland cancer model. In a nutshell, it can be concluded that BBAP-6 activates PHD-2 and exhibits anticancer potential.

Novel furan chalcone modulates PHD-2 induction to impart antineoplastic effect in mammary gland carcinoma.

Normoxic inactivation of prolyl hydroxylase-2 (PHD-2) in tumour microenvironment paves the way for cancer cells to thrive under the influence of HIF-1α and NF-κB. Henceforth, the present study is aimed to identify small molecule activators of PHD-2. A virtual screening was conducted on a library consisting of 265,242 chemical compounds, with the objective of identifying molecules that exhibit structural similarities to the furan chalcone scaffold. Further, PHD-2 activation potential of screened compound was determined using in vitro 2-oxoglutarate assay. The cytotoxic activity and apoptotic potential of screened compound was determined using various staining techniques, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 4',6-diamidino-2-phenylindole (DAPI), 1,1',3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1), and acridine orange/ethidium bromide (AO/EB), against MCF-7 cells. 7,12-Dimethylbenz[a]anthracene (DMBA) model of mammary gland cancer was used to study the in vivo antineoplastic efficacy of screened compound. [(E)-1-(4-fluorophenyl)-3-(furan-2-yl) prop-2-en-1-one] (BBAP-7) was screened and validated as a PHD-2 activator by an in vitro 2-oxo-glutarate assay. The IC50 of BBAP-7 on MCF-7 cells is 18.84 µM. AO/EB and DAPI staining showed nuclear fragmentation, blebbing and condensation in MCF-7 cells following BBAP-7 treatment. The red-to-green intensity ratio of JC-1 stained MCF-7 cells decreased after BBAP-7 treatment, indicating mitochondrial-mediated apoptosis. DMBA caused mammary gland dysplasia, duct hyperplasia and ductal carcinoma in situ. Carmine staining, histopathology, and scanning electron microscopy demonstrated that BBAP-7, alone or with tirapazamine, restored mammary gland surface morphology and structural integrity. Additionally, BBAP-7 therapy significantly reduced oxidative stress and glycolysis. The findings reveal that BBAP-7 activates PHD-2, making it a promising anticancer drug.

IDENTIFICATION OF EURYTREMA SPP. IN BRAHMAN CROSS CATTLE SLAUGHTERED IN BANDA ACEH SLAUGHTERHOUSE

Eurytrema spp. is a trematode worm that generally attacks the pancreas of livestock and humans which can cause chronic interstitial pancreatitis, weight loss that leads to decreased livestock productivity and ends in death. The purpose of this study is to identify the parasitic worm Eurytrema spp. and also to find out the prevalence rate in Brahman Cross cattle slaughtered in Banda Aceh slaughterhouse. This study used a sample of 20 pieces of Brahman Cross cattle pancreas from the Banda Aceh slaughterhouse. The worms were collected by taking the pancreas of Brahman Cross cattle that had been cut and stained with the Semicon Carmine staining method. The parameters of this study were the morphological differences in size of the body, oral sucker, ventral sucker and reproductive organs in each species of Eurytrema spp. The results showed that out of the 10 worms found, there was only 1 species, namely E. pancreaticum, which has the characteristics of a wide body with a length of 8-16 mm, the oral sucker is larger than the ventral sucker and lobed testes and ovaries. The prevalence rate of infection with E. pancreaticum is 15%. It was concluded that the worm that infects the Brahman Cross cattle slaughtered in Banda Aceh slaughterhouse is E. pancreaticum.

First report of pentastomiasis and the lung histopathological changes in Oriental Rat Snake (Ptyas mucosa) in Sidoarjo, East Java, Indonesia

Abstract. Plumeriastuti H, Pranashinta GT, Suwanti LT, Proboningrat A, Kusnoto. 2023. First report of pentastomiasis and the lung histopathological changes in Oriental Rat Snake (Ptyas mucosa) in Sidoarjo, East Java, Indonesia. Biodiversitas 24: 2045-2051. Currently, reptiles, especially snakes, are increasingly popular as pets worldwide and are often consumed by several communities in Indonesia. This trend needs attention from a public health perspective because it can potentially cause zoonotic diseases, including parasitic disease such as pentastomiasis. This study aimed to determine: the prevalence of pentastomiasis, identify the parasite and observe the pulmonary histopathology in Oriental Rat Snakes (Ptyas mucosa Linnaeus 1758) collected in Tulangan, Sidoarjo, Indonesia. The methods used were calculating the prevalence of pentastomiasis, semichen-acetic carmine staining, and identifying parasites and eggs based on references as a comparison. Furthermore, histopathological preparation and hematoxylin-eosin staining were performed on the lungs infected with the parasite to observe the microscopic lesions. The results showed that 65% of the snakes had pentastomiasis, and the parasites were called Kiricephalus pattoni Stephens 1908. Furthermore, a microscopical examination revealed mild fibrosis in the pulmonary area due to pentastomid infestation. This study is the first reported pentastomida in P. mucosain Sidoarjo, Indonesia, showing that snakes caught and commonly consumed were infected with zoonotic pentastomic.

Molecular Characterization of Avitellina lahorea Woodland, 1927 (Class Cestoda; Family Anoplocephalidae) Infecting Sheep and Goats Raised in India.

Tapeworms of Avitellina spp. are among those gastrointestinal parasitic helminths which infect wild and domestic ruminants worldwide leading to various clinical manifestations in the ruminant hosts, thereby causing considerable economic losses in livestock production. While these worms are among the major constraints in ruminant livestock raising, there is very meagre molecular information available making their identity error-prone. This study aimed to provide insights into the genetic characterization of these economically important tapeworms. In the present study, we examined 480 guts of slaughtered goats (n = 413) and sheep (n = 67) of which 74 guts were found infected with anoplocephalid cestodes (sheep gut:18; goat gut:56). A total of 27 Avitellina lahorea worms (19 from goat and 8 from sheep) were isolated, fixed, relaxed and stained using Gower's carmine stain. For molecular analyses, the genomic DNA was extracted and fragments of cytochrome c oxidase subunit 1 (cox1) gene, internal transcribed spacer1-5.8S ribosomal RNA (ITS1-5.8S rRNA) gene, and small subunit ribosomal RNA (18S rRNA) gene were amplified and sequenced. Based on snail-shaped paruterine organs and other morphological and morphometric features, the worms were identified as Avitellina lahorea. The phylogenetic analyses, based on our original cox1 gene sequence and those available from NCBI GenBank, showed Avitellina tapeworms as a sister lineage of Thysaniezia with 14 to 17% genetic divergence. Molecular analyses of 18S rRNA gene sequences depicted the present isolate as one of the species of the genus Avitellina clustering with A. centripunctata as a separate species in the phylogenetic tree with 92% homogeneity in sequences. In conjunction with existing data of internal transcribed spacer1-5.8S rRNA (ITS1-5.8S rRNA) gene, the phylogenetic analysis placed the present isolate among the anoplocephalids as one of the species. The present study is the first molecular report on A. lahorea isolated from sheep and goats with the simultaneous use of a morphological approach, and certainly contributes to bridging the existing gaps in the understanding of these economically important parasites.

First report of acanthocephalan parasite in wild-caught Asian vine snake (Ahaetulla prasina) in Indonesia.

Exotic pet snakes are more susceptible to infection, especially parasitic helminths than wild-caught. There is no comprehensive report on the prevalence of acanthocephalan parasite infection in Indonesian snakes. Therefore, this study aimed to estimate the prevalence rate and to identify the acanthocephalan infection in wild-caught Asian vine snake (Ahaetulla prasina) from the Mojokerto District, East Java, Indonesia. A total of 60 snakes were collected from the local sellers in the Mojokerto District, East Java, Indonesia. Then, snakes were euthanized and necropsied to observe various predilections of acanthocephalan larval stage (cystacanth). Morphological identification of the cystacanth was conducted using the carmine staining method and microscopic examination. Acanthocephalan infection was recorded with a high prevalence rate of 80.06%. A total of 696 cystacanths were examined from the muscle, subcutaneous tissues, and visceral with 32.90, 16.37, and 50.71% intensity rates, respectively. Acanthocephalan prevalence rate was recorded at 80.06% in this study. Constant disease monitoring is necessary, considering wild-caught Asian vine snakes were susceptible host and lack of data regarding parasitological surveys. Therefore, further studies are needed in new areas and various species of wild-caught snakes in Indonesia, because of the potential of parasitic helminth transmission between snake and other reptiles.

Meiosis and male fertility in F1 interspecific hybrids (Passiflora vitifolia vs. Passiflora hatschbachii)

Interspecific hybrids can be studied using methodologies in which the male gamete with high reproduction potential, viability, and fertility is prioritized. Passiflora species, with lush, showy, and exotic colors, have great potential for the ornamental plant market. In addition, artificial Passiflora hybrids were developed without many difficulties because of weak reproductive barriers. Thus, meiotic and post-meiotic behaviors were analyzed with 2% acetic carmine staining. Confirmation of interspecific hybridization was performed using SSR markers and GISH technique was used to detect genomic differentiation. The pollen viability of the parental and hybrids genotypes was tested using Alexander`s solution, fluorescein diacetate and in vitro germination tests were performed using culture medium. The meiotic behavior was regular and displayed haploid number n = 9 with nine bivalent (II) chromosomal, and pairing in 90% of the cells in diakinesis. There was a significant difference (p < 0.05) in terminal and interstitial chiasma frequencies. Meiotic irregularities observed were as follows: early and/or delayed chromosomes, disorientation of spindle fibers, transverse spindles, tripolar spindles, and asynchrony; and consequently irregular post-meiotic products were observed: monads, dyads, triads, and polyads. GISH was used in the interspecific hybrids and pairing between homeologous chromosomes, and bivalent and tetravalent formation were observed. From this study, we could conclude that hybrid genotypes are fertile and pollen grains are viable and can be used in breeding programs.

First Report on Molecular Identification of Moniezia expansa in Sheep from Mannavanur, Palani Hills, Tamil Nadu, India.

Tape worm infection is common among sheep at SRRC, Mannavanur, Palani hills, Tamil Nadu, India. The aim of the present study is to find out the cestode species infecting the sheep being maintained at SRRC, Mannavanur, by means of molecular method. During the second week of June 2021, the hogget flock of sheep (comprising both Bharat Merino and Avikalin sheep breeds) was drenched on empty stomach with commercial preparation of anthelmintic drug containing Niclosamide plus Albendazole, as per the standard dose specified by the manufacturer (Niclozole™: each 5ml contains 500mg of Niclosamide and 150mg of Albendazole: dose for sheep-10ml/15kg body weight). The tapeworms expelled in dung by the drug-treated sheep were collected, washed in PBS (pH 7.2), and fixed in between two glass slides using 10% formalin. Furthermore, cytochrome c oxidase subunit I (Cox-I) gene-based PCR was carried out. Only partial sequence (1593bp) of Cox-I gene of Moniezia expansa from Sheep at SRRC, Mannavanur, Tamil Nadu, India was obtained by PCR. The PCR amplified fragment was cloned into pGEM-T vector and the recombinant plasmid was sequenced. The obtained nucleotide sequences of Cox-I gene of the M. expansa from Indian sheep were analysed with that of 27 more cestode species from different mammalian species (available in GenBank) using bioinformatics tools. The species of the tapeworm was identified as Moniezia species by the Department of Veterinary Parasitology, VC& RI, Orathanadu, TANUVAS by the standard Acidic alum carmine staining method. Due to the ambiguity in the conventional method, Cox-I gene-based PCR and subsequent gene sequencing protocols were used for the identification of the species of cestode infecting sheep at SRRC, Mannavanur, and it was confirmed as M. expansa upon BLAST analysis. Moniezia expansa from SRRC, Mannavanur is having 100% sequence identity at nucleotide level with that of M. expansa from Sengal/Ethiopia. M. benedeni shared 87-88% nucleotide identity with Indian M. expansa. With taenids, the share of percent nucleotide identity of Indian M. expansa ranged from 79 to 81%. M. expansa from Indian sheep was clustering with other anaplocephalids from various mammalian species in the analysis of phylogenetic tree based on Cox-I nucleotide sequences. From the present study, it is concluded that M. expansa is the anoplocephalid cestode infecting the sheep at Mannavanur, Tamil Nadu, India. To our knowledge, this is the first report on partial nucleotide sequences of Cox-I gene of M. expansa from Sheep of Indian peninsula. An investigation on the involvement of oribatid mites as the vector in the transmission of M. expansa among sheep at SRRC, Mannavanur needs to be carried out.

Morphology and molecular identification of Eurytrema spp. worm in Aceh cattle, Indonesia

Abstract. Hanafiah M, Helmi TZ, Sutriana A, Bahi M. 2021. Morphology and molecular identification of Eurytrema spp. worm in Aceh cattle, Indonesia. Biodiversitas 22: 5654-5661. The objective of this research was to determine the species of Eurytrema spp. in Aceh cattle which were slaughtered in slaughterhouses located in Banda Aceh, Indonesia. Identification of Eurytrema spp worm species was performed by Semichon’s Carmine staining, SEM and molecular method. The result fromSemichon’s Carmine staining and SEM method showed that worms coded E2 and E3 were Eurytrema cladorchis indicated by clearly visible protruding cirrus while worms coded E1, E4 and E5 were E. pancreaticum indicated by the absence of protruding cirrus. Molecular characterization of Eurytrema spp.produced fragments of approximately 700 bp in length. The phylogenic tree showed that Eurytrema spp.from Aceh cattle consisted of 2 clades: clade 1 was E. pancreaticum, while clade 2 was E. cladorchis. DNA sequencing result showed that the similarity of worm samples coded 1, 4, and 5 were 98.90% similar to E. pancreaticum (KJ767631.1 and KC535543.1), and 96. 33% similar to E. cladorchis (MN566134.5 and MN566140.1), whereas the worm samples coded 2 and 3 were 97.53% similar to E. pancreaticum (KY490000.1 until KY490004.1), and 97.55 % similar to E. cladorchis (MN566135.1 until MN566140.1). This research was able to identify E. pancreaticumand E. cladorchis in Aceh cattle slaughtered in Banda Aceh slaughter houses.

Sparganosis (Spirometra spp.) in Asian Water Monitor (Varanus salvator): A medical implications for veterinarians, breeders, and consumers.

Background and Aim:The high prevalence of sparganosis has been reported globally, especially in Asian countries where the majority of individuals consume raw meat from wild-caught reptiles. In Indonesia, similar cases regarding the high prevalence of sparganosis were recorded from wild reptiles such as snakes that utilized for culinary purposes, whereas, there are no data regarding other species such as water monitor lizard (Varanus salvator), which also provided as daily culinary with a high number of trades. Therefore, this study aims to investigate the prevalence of sparganosis in water monitor lizard (V. salvator), which is frequently utilized as culinary products in East Java Province, Indonesia.Materials and Methods:A total of 313 living wild-caught and captive-bred Asian water monitor lizards were collected from the reptile markets and breeders. All samples were euthanized and observed for the presence of plerocercoid. Identification of the plerocercoid as larval infective stage was made using carmine staining method.Results:The total prevalence of sparganosis was recorded at 69.64%. A total of 393 plerocercoids were collected in which divided 280 (71.24%) infecting muscles and 113 (28.75%) located in subcutaneous tissues.Conclusion:To the best of our knowledge, this study not only recorded as the first evidence but also confirms the role of monitor lizards as sparganosis transmitters in Asia and reveals additional routes of sparganosis transmission in Indonesian reptiles. Local conservation laws should be strengthened to effectively control or ban wildlife trade in traditional markets. Moreover, public awareness regarding sparganosis as a neglected zoonotic disease should be applied to prevent disease transmission in Indonesia.

Effect of Voacamine upon inhibition of hypoxia induced fatty acid synthesis in a rat model of methyln-nitrosourea induced mammary gland carcinoma

BackgroundIn the present study, fatty acid synthesis is targeted to combat mammary gland carcinoma by activating prolyl hydroxylase-2 with Voacamine alone and in combination with Tamoxifen. It was hypothesized that the activation of prolyl hydroxylase-2 would inhibit the hypoxia-induced fatty acid synthesis and mammary gland carcinoma. Mammary gland carcinoma was induced with a single dose administration of N-methyl-N-nitrosourea (50 mg/kg,i.p.) and treatment with Voacamine and Tamoxifen 15 days after carcinogen administration.ResultsAt the end of the study, hemodynamic profiling of animals was recorded to assess the cardiotoxic potential of the drug. Blood serum was separated and subjected to nuclear magnetic resonance spectroscopy. Carmine staining and histopathology of mammary gland tissue were performed to evaluate the anti-angiogenic potential of the drug. The antioxidant potential of the drug was measured with antioxidant markers. Western blotting was performed to study the effect of the drug at the molecular level.ConclusionResults of the study have shown that Voacamine treatment stopped further decrease in body weight of experimental animals. The hemodynamic study evidenced that Voacamine at a low dose is safe in cardiac patients. Microscopic evaluation of mammary gland tissue documented the anti-angiogenic potential of Voacamine and Tamoxifen therapy. Perturbed serum metabolites were also restored to normal along with antioxidant markers. Immunoblotting of mammary gland tissue also depicted restoration of proteins of the hypoxic and fatty acid pathway. Conclusively, Voacamine and its combination with Tamoxifen activated prolyl hydroxylase-2 to combat mammary gland carcinoma.

Early Nutrition with Different Diets Composition versus Fasting on Immunity-Related Gene Expression and Histomorphology of Digestive and Lymphoid Organs of Layer-Type Chicks.

Simple SummaryWith the continuous improvement in the progress of poultry industry, a better understanding of the avian immune system is necessary. A prolonged holding period (36–72 h), along with a delay in access to feed and/or water post-hatching, has a negative influence on performance, intestinal histomorphology, and the immune system development of chicks. Therefore, the present study aimed to investigate the effect of early feeding with different diet composition or delayed feeding during the first 72 h of chick’s life on the expression of immunity-related genes and histomorphology of digestive and lymphoid organs of layer-type chicks. Early nutrition post-hatching had no negative effect on the development of the lymphoid and digestive organs in chicks. Histomorphological examination revealed an increase in cortex and cortex:medulla of thymus and bursa in the early fed groups compared to the fasted ones, with resultant impacts on the primary lymphoid organs. Higher germinal center areas and white pulp of the spleen were recorded in the early fed chicks, implying augmented proliferation and maturation of B cells in the secondary lymphoid organs. In the liver, a strong positive reaction to Best’s carmine stain in the early fed groups, indicating that the liver of these chicks had numerous glycogen granules in the cytoplasm of hepatocytes. The expression levels of splenic-immunity related genes were up-regulated in most of the early fed chicks at 14 day of age. Our findings suggested that early feeding post-hatch can modify the splenic-immunity related genes and modulate the histomorphology of the digestive (liver and proventriculus) and lymphoid organ in layer-type chicks during the early life post-hatching.Early feeding post-hatching (EFPH) can impact the immune response and modify the immunity-related gene expression. Therefore, we aimed to examine the effects of EFPH with different diets composition versus fasting during the first 72 h of chick’s life on the histomorphological structures of the liver, proventriculus, central and peripheral lymphoid organs, and immunity-related genes in layer-type chicks during the brooding period. A total of 400 chicks were randomly allotted into 4 groups with 4 replicates each. The experimental groups during the first 72 h of life were: feed and water deprivation (control, T1), feeding a starter layer diet (20% CP and 11.84 MJ/kg ME, T2), feeding a starter layer diet contained 3% molasses in its composition (20% CP and 11.81 MJ/kg ME; T3), and feeding a starter broiler diet (23% CP and 12.68 MJ/kg ME, T4). After the first 72 h of chick’s life, all chicks were fed ad libitum the T2 diet. EFPH had no negative effect on the development of the lymphoid or digestive organs in chicks. Greater relative weights of the spleen and bursa of Fabricius (p < 0.05) were observed in the early fed chicks compared to control at day 14 of age. Histomorphological examination revealed an increase (p < 0.05) in thymus cortex and cortex:medulla in the T3 and T4 groups compared to the fasted ones at day 28 of age. Pelicae height, follicular width, cortex, and cortex:medulla of bursa were improved (p < 0.01) in the fed groups compared to fasted chicks, with resultant influences on the primary lymphoid organs. Compared to control, higher germinal center areas and white pulp of the spleen (p < 0.05) were recorded in the early fed chicks, implying augmented proliferation and maturation of B cells in the secondary lymphoid organs. In the liver, a strong positive reaction to Best’s carmine stain in the early fed groups, indicating that the liver of these chicks had numerous glycogen granules or greater glycogen density in the cytoplasm of hepatocytes. There was a significant enhancement in the proventriculus mucosal and gland thickness, as well as fold height (p < 0.05) in the early fed chicks. The expression levels of splenic Toll-like receptor 2, interleukin 4, tumor necrosis factor α, and interferon gamma were up-regulated (p < 0.01) in most of the early fed chicks (T2, T3, and T4) compared to fasted ones at 14 day of age. In conclusion, EFPH could modify the splenic-immunity related genes and modulate the histomorphology of the digestive (liver and proventriculus) and lymphoid organs in layer-type chicks during the brooding period.

A Method to Pre-Screen Rat Mammary Gland Whole-Mounts Prior To RNAscope.

RNAscope is a quantitative in situ gene expression measurement technique that preserves the spatial aspect of intact tissue; thus, allowing for comparison of specific cell populations and morphologies. Reliable and accurate measurement of gene expression in tissue is dependent on preserving RNA integrity and the quantitative nature of RNAscope. The purpose of this study was to determine if the quantitative nature of RNAscope was retained following processing and carmine staining of mammary gland whole-mounts, which are commonly used to identify lesions, such as hyperplasia and ductal carcinoma in situ (DCIS). We were concerned that handling and procedures required to visualize microscopic disease lesions might compromise RNA integrity and the robustness of RNAscope. No effect on the quantitative abilities of RNAscope was detected when mammary gland whole-mounts were pre-screened for lesions of interest prior to RNAscope. This was determined in comparison to tissue that had been formalin-fixed and paraffin embedded (FFPE) immediately after collection. The ability to pre-screen whole-mounts allowed unpalpable diseased lesions to be identified without labor-intensive serial sectioning of tissue samples to find diseased tissue. This method is applicable to evaluate mammary gland whole-mounts during normal mammary gland development, function, and disease progression.

Acanthocephaliasis And Sparganosis Occurrence in An Asian Vine Snake (Ahaetulla prasina): A Perspective of Neglected Zoonotic Disease

Ahaetulla prasina commonly known as asian vine snake frequently kept as exotic pet in Indonesia. Snake also susceptible to various parasitic disease and one of them have zoonotic potential such as sparganosis and acanthocephaliasis. Sparganosis is an infection of humans and animals especially reptiles caused by tapeworms which belonging to the genus Spirometra. Sparganosis and acanthocephaliasis has been reported sporadically in reptiles around the world, with higher cases occurs in several Asian countries including Indonesia even though this disease categorized as neglected zoonotic disease. To our knowledge, sparganosis and acanthocephaliasis infection case has not been reported in this snake species. Therefore, this study aims to report sparganosis and acanthocephaliasis infection in asian vine snake which kept as an exotic pet and moreover provide scientific foundation for preventing sparganosis which include in zoonotic disease among animals and humans. A sudden death snake collected as sample from pet owner which suspected with parasites infection. Necropsy method was performed to investigate the distribution of parasites inside the snake body cavity. Identification of parasite infective stages using Carmine staining and examination under light microscope with a magnification of 40x and 100x. The results of this study found that Ahaetulla prasina was infected with Spirometra and Acanthocephalan parasites. The parasites located within subcutaneous tissues, muscular tissues, and coelom cavity of snake. The muscular tissues had highest intensity of parasites infection compared with subcutaneous tissues and coelom cavity sites. Present report of this finding can potentially contributes not only diagnosing parasitic disease in exotic animals but also proposing prevention program for zoonotic disease in humans worldwide. Moreover, education about neglected parasitic disease with zoonotic potential should take into consideration above this fndings and reports.

The spectrum of pathogens in 187 cases of pulmonary fungal disease diagnosed by histopathology-a retrospective analysis

Objective: To investigate the spectrum of pathogens causing lung fungal disease diagnosed by histopathology through histochemical special staining, compared to the fungal culture results, and to further evaluate the diagnostic value of histochemical special staining in pulmonary fungal disease. Methods: We performed a retrospective analysis of 187 cases of pulmonary fungal disease diagnosed by histopathology in Peking Union Medical College Hospital from 2001 to 2015 (including 92 cases with pulmonary resection or open lung biopsy, 33 with percutaneous lung biopsy and 62 ones with fiberoptic bronchoscopic lung biopsy). All cases were treated with hexamine silver, PAS, mucus carmine and acid-fast staining in addition to conventional HE staining. The clinical records and the fungal culture results were reviewed. Results: There were 103 male and 84 female patients, aged from 12 to 70 years [average (48±14) years]. There were 85 cases(45.5%) of pulmonary aspergillosis(including 60 cases of invasive infection and 25 cases of aspergilloma), 51 cases(27.3%) of pulmonary cryptococosis, 6 cases (3.2%)of pulmonary mucormycosis, 3 cases(1.6%) of pulmonary histoplasmosis, 3 cases (1.6%)of pulmonary candidiasis, and 2 cases (1.1%) of pneumocystosis, while in the remaining 37 cases (19.8%) the pathogens could not be clearly classified by microscopy due to limited tissue or degeneration. Among the 88 patients with pulmonary fungal disease diagnosed by histopathology from 2011 to 2015, 35 ones (39.9%) were detected by fungal culture (including lung biopsy, intraoperative swab, blood, bronchoalveolar lavage fluid and sputum, etc.). The diagnostic results of 18 cases were completely consistent between histopathological examination and fungal culture (18/35, 51.4%), while 13 cases (13/35, 37.1%) were diagnosed by histopathology but no fungi were cultured, and in 3 cases (3/35,8.6%) the culture was positive for fungi which could not be classified clearly by histopathology. In another case the pathogen was found to be Cryptococcus histopathologically but the lavage culture grew"candida", but the patient's blood cryptococcal antigen was positive. Conclusions: Among patients with histopathological diagnosis of pulmonary fungal disease, pulmonary aspergillosis was the most common, followed by pulmonary cryptococcosis, pulmonary mucormycosis, pulmonary histoplasmosis, pulmonary candidiasis and pneumocystosis. A small number of cases could not be classified by histopathology through histochemical special staining. There was a high consistency in discovering fungal pathogens between pathological histochemical special staining and culture method, but 37% pulmonary fungal disease diagnosed by histopathology were culture negative. In practice, the role of histochemical special staining in diagnosing pulmonary fungal disease should be paid more attention.

Sparganosis in wild-caught Javanese keelback water snakes (Fowlea melanzostus) from Indonesia

Sparganosis is a globally important water and food-borne parasitic zoonosis, caused by the plerocercoid (spargana) stage of Spirometra spp. However, in Indonesia sparganosis is still categorized as neglected zoonotic disease due to paucity of reports on the prevalence of spargana in naturally infected hosts. Therefore, this study aimed to investigate the prevalence of spargana infection in wild-caught Javanese keelback water snakes (Fowlea melanzostus) from Banyuwangi District, East Java Province, Indonesia. Microscopic examination using semichen-acetic carmine staining method was used to confirm plerocercoid stage of Spirometra spp., in which the anterior end had specific structure (bothria) and was broader compared to the posterior end. The study revealed a very high prevalence rate of 92.72% (51/55) of spargana in these snakes. Spargana were mostly distributed in muscle tissues (57.97%), followed by subcutaneous tissues (25.08%) and visceral organs (13.62%). To our knowledge, this study not only records the first report but also reveals the role of aquatic snakes as transmitters of sparganosis in Asia. Regarding the high prevalence, further studies are recommended to explain the role of various species of snakes which can serve as intermediate hosts in the spread of sparganosis in the natural environment.

Oral indigo carmine for the detection of colon adenoma

Objectives Colonoscopy is currently considered the optimal method to detect colorectal neoplasia; however, some adenomas remain undetected. While indigo carmine staining with a dye-spray catheter has demonstrated promising results for reducing the miss rate, we investigated the oral indigo carmine method. The aim of this study was to determine whether oral indigo carmine intake before standard colonoscopy increases the adenoma (and adenocarcinoma) detection rate (ADR) or the mean number of adenomas per patient (MAP). Materials and methods The oral indigo carmine method was performed from April 2018 to July 2020 in two hospitals. Data were collected in a prospective manner and compared to the conventional group whose data were collected retrospectively and consecutively from January 2016 to March 2018. All data were anonymized. Results Among the 704 patients included, colonoscopies were completely performed in 693 patients (347 in the indigo group). The ADR did not significantly differ between the groups: 42.3% vs. 40.3% (indigo vs. conventional group; odds ratio: 1.13; 95% confidence interval: 0.9–1.33, p = .33). The MAP was significantly greater in the indigo group (1.15) than that in the conventional group (0.82; p = .009). The cecal intubation rate and time to cecal intubation did not differ between the indigo and conventional group (98.6% vs. 98.3%, p = .83, and 6.2 min vs. 5.9 min, p = .39, respectively). Conclusion The routine use of oral indigo carmine does not lead to a higher ADR despite the higher MAP.

Detection of Pentastomida on Crab-Eating Frog (Fejervarya cancrivora) in Surabaya and Sidoarjo Areas

This research was conducted to detect the presence of Pentastomida on Crab-eating Frog (Fejervarya cancrivora) in Surabaya and Sidoarjo areas. A total of 50 samples were taken in three different time and the identification was conducted in Laboratory of Parasitology, Universitas Airlangga by Semichen-Acetic Carmine staining method. Pentastomida that found in the body of Crab-eating Frog (Fejervarya cancrivora) was belong to Maxillopoda class which is included in Crustacea subphylum were matched by macroscopic and microscopic taxonomy and observation. The result of this research showed that there found Pentastomida on Crab-eating Frog collected Surabaya and Sidoarjo areas, East Java.

Comparative Evaluation of Colon Cancer Specific Antigen-2 Test and Chromocolonoscopy for Early Detection of Egyptian Patients with Colorectal Cancer.

Background: Effective screening of colorectal cancer (CRC) in early stage could reduce the advancement of CRC and therefore mortality. Effective screening is based on either stool dependent tests or colon dependent examination.Aims: The aim of the study was a comparative evaluation of chromocolonoscopy and Colon Cancer-Specific Antigen-2 test for early detection of colorectal cancer in Egyptian patients.Methods: This case control study was carried out on 55 patients classified into 3 groups: Group I consisted of twenty patients with precancerous lesions detected by colonoscopy, Group II consisted of twenty patients diagnosed with colorectal cancer and Group III consisted of fifteen individuals (who underwent colonoscopy for other indications) as a control group. All the subjects were subjected to measure occult blood in the stool, measurement of Colon Cancer-Specific Antigen-2 level in serum and tissue and chromo colonoscopy using Indigo Carmine stain.Results: In group II, there was a statistically significant increase in CCSA2 in serum as compared to the other 2 groups. Cutoff >11.3 CCSA2 in serum showed 65% sensitivity, 85% specificity, 81.2% PPV, 70.8% NPV and 70.3% accuracy in the differentiation of group II with cancer colon from group I with premalignant colonic lesions. A cutoff > 9.1 CCSA2 in serum showed 95% sensitivity, 46.67% specificity, 70.4% PPV, 87.5% NPV and 73.5% accuracy in differentiating group II with cancer colon from normal controls (group III).Conclusion: CCSA-2 level in serum was significantly higher in cancer colon. Chromoendoscopy has a role in the detection of polyps, both neoplastic and non-neoplastic.