rRNA-ITS2 Sequences Research Articles

Pouchitis Is Associated with Paneth Cell Dysfunction and Ameliorated by Exogenous Lysosome in a Rat Model Undergoing Ileal Pouch Anal Anastomosis.

Pouchitis is a common complication of restorative proctocolectomy and ileal pouch anal anastomosis (IPAA) for ulcerative colitis (UC), significantly affecting the postoperative quality of life. Paneth cells play an important role in the maintenance of gut homeostasis. This study aimed to investigate the role of Paneth cells in the pathogenesis of pouchitis. Endoscopic biopsies from the pouch body and terminal ileum of UC patients undergoing IPAA with or without pouchitis were obtained to analyze Paneth cell function. Acute pouchitis was induced with 5% dextran sulfate sodium (DSS) for seven consecutive days in a rat model of IPAA. The Paneth cell morphology was examined by immunofluorescence and electron microscopy. The effect of exogenous lysozyme supplementation on pouchitis was also investigated. The fecal microbiota profile after DSS and lysozyme treatment was determined by 16s rRNA ITS2 sequence analysis. Abnormal mucosal lysozyme expression was observed in patients with pouchitis. The rat model of pouchitis showed increased pouch inflammation, increased CD3+ and CD45+ T cell infiltration, and decreased tight junction proteins, including ZO-1 and Occludin. There is a significant deficiency of Paneth cell-derived lysozyme granules in the rat model of pouchitis. Supplementation with exogenous lysozyme significantly ameliorated pouchitis, lowering the levels of inflammatory cytokines such as TNF-α and IL-6 in the pouch tissue. 16s rRNA analysis revealed a higher Lachnospiraceae level after lysosome treatment. Paneth cell dysfunction is prominent in patients and rat models of pouchitis and may be one of its causes. The decrease in Lachnospiraceae, a characteristic of dysbiosis in pouchitis, could be reserved by lysosome treatment. Lysozyme supplementation shows promise as a novel treatment strategy for pouchitis.

Prevalence of Tri­cho­monas gallinae with molecular characterisation and phylogenetic analysis in domestic pigeons using ITS1-5.8s rRNA-ITS2 gene in Alborz province, Iran

Avian trichomonosis is an important parasitic disease throughout the world caused by the protozoan Trichomonas gallinae, commonly seen in pigeons and wild birds. Lesions in the upper gastrointestinal tract (beak and crop) are complications of this disease. Currently, the diagnosis of this organism is made using laboratory methods including direct smear, culture medium and molecular methods. The aim of the present study was to survey the prevalence of T. gallinae in pigeons of Alborz province, Iran through molecular and phylogenetic identification using ITS1-5.8s rRNA-ITS2 gene. A total of 87 samples were collected from domestic pigeons from May to September 2019. The samples were taken directly from the mouth and larynx using an oral swab. Out of 87 collected samples, 28 (32.18 %) were positive using direct smear, culture and polymerase chain reaction (PCR) methods. Based on the results, the size of the amplification product of this gene was 372 base pairs. The results of this study were analysed using a phylogenetic tree and Neighbour-Joining (NJ) method. The present study showed high prevalence of T. gallinae in pigeons. Two types of T. gallinae genotypes, A and B, were found in pigeons. Also, the phylogenetic analysis of ITS1-5.8s rRNA-ITS2 sequences from positive samples, showed high coverage with sequences present in the GenBank.

Inocybe stellatospora и I. teraturgus (Inocybaceae, Agaricales) — первые находки плодовых тел на территории России

Here we present information about the first findings of basidiomes of two species of the genus Inocybe (I. stellatospora, I. teraturgus) in Russia. The species were collected in the foothills and mountains of the western macroslope of the Subpolar and Northern Urals, on the territory of two protected areas: Yugyd Va National Park and Pechoro-Ilychsky Reserve (Komi Republic). Analysis of the ITS1-5.8s rRNA-ITS2 sequences of I. stellatospora and I. teraturgus samples confirmed their identification by morphological features. The article provides morphological descriptions of the species, photographs of microscopic features as well as discusses differences with morphologically similar species.

Parasitological and molecular survey of scattered parasitism by trichomonads in some avian species in Iran

ABSTRACT Outbreaks of avian trichomonosis are being reported worldwide; meanwhile, the genetic and virulence variations are under investigation. In this study, the occurrence and genetic variability of oral or faecal trichomonads among various avian species were investigated. Samples obtained from either the oropharyngeal cavity, crop/oesophagus, droppings/cloaca, or conjunctival swabs of avian species were inspected for flagellates. Phylogenetic analysis of partial ITS1-5.8s rRNA-ITS2 sequences from selected samples was performed to investigate the genetic diversity of the isolates. Investigation of 737 birds revealed an infection rate of 15.7% in the upper gastrointestinal tract, 7.3% in the faecal samples, and 0.7% involvement of the conjunctiva. Phylogenetic analysis of partial ITS1-5.8s rRNA-ITS2 sequences from selected samples, identified genotypes A and B of Trichomonas gallinae and genogroups A-C and E of Tetratrichomonas gallinarum. A novel ITS genotype of intestinal trichomonads was also detected in hooded crow (Corvus cornix) and common mynah (Acridotheres tristis). In the present study, in addition to Columbiformes and Falconiformes, trichomonads were detected in Passeriformes and Galliformes with the involvement of organs other than the gastrointestinal tract. Genotype A T. gallinae was detected in domestic pigeons (Columba livia domestica), a laughing dove (Spilopelia senegalensis), a common kestrel (Falco tinnunculus), a budgerigar (Melopsittacus undulates), and a canary (Serinus canaria). Distinct genotype B was detected in a common mynah and a budgerigar. Genogroups A-C of T. gallinarum were also demonstrated in Galliformes and Anseriformes. Furthermore, two novel trichomonad ITS genotypes were detected in hooded crows and a common mynah warranting detailed multi-locus molecular analysis. RESEARCH HIGHLIGHTS ITS diversity of trichomonads was shown in various avian species. Diversity of the parasites’ target organ and clinical manifestations was demonstrated. Two novel ITS genotype trichomonads from common mynah and hooded crow were identified.

Allobodo chlorophagus n. gen. n. sp., a Kinetoplastid that Infiltrates and Feeds on the Invasive Alga Codium fragile

A novel biflagellate protist that consumed chloroplasts inside material of the invasive marine green alga Codium fragile was reported from the U.S. east coast in 2003. We observed a similar association in C. fragile from five sites in Nova Scotia, Canada during 2013 and 2014. After incubating Codium fragments for 2-3 days, some utricles and filaments contained numerous chloroplast-consuming cells. Transmission electron microscopy (TEM) confirmed that these were kinetoplastids with a pankinetoplast, large electron-dense droplets in the cytoplasm and a connective between the paraxonemal rod bases, but no conspicuous para-cytopharyngeal rod, all consistent with U.S. material observed in 2003. The ITS1-5.8S rRNA-ITS2 sequences from 13 Nova Scotia isolates were identical. SSU rRNA gene phylogenies placed the Codium-associated kinetoplastid in neobodonid clade '1E'. Clade 1E likely contains no previously described species, and branches outside all other major neobodonid groups, either as their sister or as a separate lineage, depending on rooting. These results indicate that the kinetoplastid represents a single species that merits a new genus (and family), and we describe it as Allobodo chlorophagus n. gen., n. sp. The lack of evidence for food sources other than Codium is consistent with a parasitic association, but other possibilities exist (e.g. necrotrophy).

Ascaridia galli isolates with ITS1-5.8rRNA-ITS2 fragment homologous to Ascaridia columbae.

Ascaridia (A.) galli is one of the most commonly occurring nematodes in poultry worldwide, often in hens and broiler chickens. The infection with Ascaridia galli in free-range chickens was even 70%. There is not much information about A. galli genetic features. The present study was conducted to assess the genetic diversity of A. galli isolated from hens in Poland by analyzing the nucleotide sequence of the region ITS1-5.8rRNA-ITS2 and to define its homology within the family Ascaridiidae. Adult A. galli were collected from the intestines of naturally infected hens from two flocks of free-run laying hens from the Wielkopolska region in Poland. From all parasites an identical ITS1-5.8rRNA-ITS2 sequence was obtained, which was homologous in 99% with A. columbae (JQ995321.1) sequence. The high homology sequences of A. galli (KX683286) from Poland and A. columbae (JQ995321.1) isolate from the USA, support the observations of other authors suggesting that A. galli and A. columbae might be closely related. It is the first whole ITS1-5.8rRNA-ITS2 of A. galli in the GenBank database, so there is not enough data for detailed phylogenetic analysis of A. galli. Detailed genetic analysis is necessary to get better insight into the birds' Ascaridia species.

Isolation, enrichment of a halophilic yeast Candida sp. YH and its treatability study of saline wastewater

A strain of halophilic yeast was isolated, enriched and adapted to an increasing salinity started from 5, 10 to 15 g/l NaCl to get it adapted gradually to high saline environment. Then the yeast biomass was developped in batch experiment, with each batch lasted 32 hours each, at further increased salinity to 20, 25 and 30 g/l NaCl with influent COD 5000 mg/l at pH 3.5, temperature 30oC. At salinity 20 g/l NaCl, COD removal efficiency reached over 90% from beginning and reached over 96% after 6 batches with effluent COD 203 mg/l. When the salinity increased to 25 g/l, COD removal efficiency varied within 89% to 90% in first 4 batches and reached 95% after 8 batches with effluent COD 250 mg/l. At salinity 30 g/l NaCl, COD removal efficiency decreased notably, reached less than 90% during first 5 batches and then inceased gradually, reached 93.6% only after 9 batches with effluent COD 320 mg/l. Salinity was the main factor influencing the ion equilibrium inside the yeast cells leading to decrease in substrate degradation and rate of cell. Inceased salt concentration also lead to longer adaptation time of the cells. The cell morphology of YH strain was observed by Scanning Electron Microscopy (SEM). The cells of this yeast strain has lemon-like shape, with 1.3–1.78 in width; and 2.00–2.69 µm in length. Based on the morphological characteristics and the phylogenetic analysis by alignment of ITS1-5.8S rRNA-ITS2 sequences, high homology up to 92% with Candida sp. YS W113A (AM410670)) indicated this strain as, namely, Candida sp. YH.

Phylogenetic analysis and assessment ability of Fusarium isolates for the biodegradation of the pesticide carbofuran

Carbofuran is a pesticide used against insects and nematodes. It is a common environmental pollutant associated with agricultural activities. Carbofuran has harmful effects on target and non-target organisms through the accumulation of acetylcholine at the junction of the nerve cell and the receptor sites system by inhibiting acetylcholine esterase. Since the Kingdom of Saudi Arabia is one of the countries that use the pesticide carbofuran in agricultural applications, it inevitably suffers from this worsening environmental problem. Therefore, removal of this compound from the environment is a necessity to ensure human health. In this study, 20 fungal isolates were isolated from pesticide-contaminated agricultural soils. Three fungal isolates (KKU-F5, KKU-F11, KKU-F17) were selected that have the ability to tolerate and utilise carbofuran. The exact identification of these isolates was determined using ITS1-5.8S rRNA-ITS2 region amplification and sequence determination. Alignment results and the comparison of ITS1-5.8S rRNA-ITS2 region sequences of the isolates to ITS1-5.8S rRNA-ITS2 sequences available in the GenBank database, as well as the phylogenetic analysis, confirmed the accurate identification of the isolates KKU-F5, KKU-F11 and KKU-F17 as Fusarium polyphialidicum, Fusarium oxysporum and Fusarium verticillioides, respectively. The three selected fungi demonstrated a high growth rate on 3 gm/L carbofuran, which is twice the recommended dose of carbofuran, after an incubation period of seven days. The percentage of carbofuran removal determined by HPLC analysis for the isolates KKU-F 5, KKU-F11 and KKU-F17 was 88%, 77% and 90.10% respectively, within this period. RAPD-PCR fingerprinting was performed to determine the differentiation at the molecular level within the three Genetics and Molecular Research 17 (1): gmr16039900 carbofuran-degrading Fusarium isolates (KKU-F5, KKU-F11 and KKU-F17). The RAPD-PCR results exhibited different RAPD banding profiles indicating that the three Fusarium isolates were different from each other and could be clearly distinguished by their amplification patterns.

Induction of Secondary Carotenogenesis in New Halophile Microalgae from the Genus Dunaliella (Chlorophyceae).

We report on the effects of high light irradiance (480 µmol quanta/(m(2)·s)) and salinity (160 and 200 g/liter NaCl) on culture growth as well as on cell lipid pigment and fatty acid (FA) composition in three novel strains of halophile microalga from the genus Dunaliella. Based on the ITS1-5.8S rRNA-ITS2 sequence and on the capability of accumulation of secondary (uncoupled from the photosynthetic apparatus) β-carotene, the strains Dunaliella sp. BS1 and BS2 were identified as D. salina and Dunaliella sp. R5 as D. viridis. Under conditions optimal for growth, chlorophylls and primary carotenoids (mainly lutein) dominated the pigment profile of all investigated strains. The main FA were represented by unsaturated C18 FA typical of thylakoid membrane structural lipids. In all studied cells, stressors caused a decline in chlorophylls and an increase in unsaturated C16 and C18 FA associated with reserve lipids. The carotenogenic species D. salina demonstrated 10-fold increase in carotenoids accompanied by a decline in lutein and a drastic increase in β-carotene (up to 75% of total carotenoids). In D. viridis, only 1.5-fold increase in carotenoid content took place, the ratio of major carotenoids remaining essentially unchanged. The role of the carotenogenic response in mechanisms of protection against photooxidative damage is discussed in view of halophile microalgae stress tolerance and application of the new Dunaliella strains for biotechnological production of β-carotene.

Discrimination and simultaneous detection of two myxozoan parasites belonging to genus Thelohanellus by multiplex polymerase chain reaction

Thelohanellus kitauei and Thelohanellus hovorkai are myxozoan parasites pathogenic in cyprinid fish especially adult Israel carp and common carp. In the present study, the complete 18S rRNA-ITS1–5.8S rRNA-ITS2 sequences of these two Thelohanellus species were cloned with primers designed from information from Genbank and previous studies. The results revealed that ITS1 and ITS2 sequences of T. kitauei and T. hovorkai were clearly distinguished although the 18S rRNA regions of the two parasites were highly conserved. Based on these sequences, new primer sets were designed for specific identification of these two parasites by multiplex PCR. Both single and multiplex PCR methods using these primers could identify these two myxozoan parasites from mixed DNA samples successfully. Our findings provide a powerful tool for the differentiation of the highly similar pathogenic Thelohanellus species for specific detection for the early diagnosis of diseases.

Simultaneous identification of three highly pathogenic Eimeria species in rabbits using a multiplex PCR diagnostic assay based on ITS1-5.8S rRNA-ITS2 fragments

Eimeria stiedai, E. intestinalis, and E. flavescens are highly pathogenic in rabbits, especially rabbits younger than 3 months. In this study, the complete ITS1-5.8S rRNA-ITS2 sequences of six rabbit Eimeria species, E. stiedai, E. intestinalis, E. flavescens, E. media, E. magna, and E. irresidua, were cloned with universal primers for the genus Eimeria and genomic DNA of LY and KF isolates as templates. These results revealed that both ITS1 and ITS2 sequences were specific to each Eimeria species in rabbits. A specific and sensitive multiplex PCR diagnostic assay based on polymorphic sites of ITS1 and ITS2 was developed and used to identify the three highly pathogenic species from rabbits, E. stiedai, E. intestinalis, and E. flavescens. Our findings provide a powerful tool for the clinical differentiation of highly pathogenic Eimeria species in rabbits and the study of the population genetics of rabbit coccidia.

Identification and Authentication of Burdock ( Arctium lappa Linn) Using PCR Sequencing

The traditional visual identification of Chinese herbs is not objective. Molecular biological techniques can be used to accurately identify the origin of each herbal species. This can enable the further purification and control of important herbal species. Molecular techniques involving polymerase chain reaction and sequencing were used to provide a relatively simple and objective means of identifying burdock at the species level. This study proved that the length of the ITS1–5.8S rRNA-ITS2 sequence was 358 base-pairs (bp) for six types of Arctium lappa Linn (the following breeds: Pingtung Gueilai, General Snow, Japanese, Fengshan, Wholesaler, and Tainan). Automatic sequencing analysis found that ITS DNA sequences for Pingtung Gueilai and Japanese breeds were the same, and the General Snow breed differed from them at its 277 bp. This study used DNA sequencing to analyze the high specificity regions of A. lappa Linn ITS, originated in different parts of Taiwan, and discovered the breed identification single-nucleotide polymorphism at the 277 bp position for local differentiation.

Tetratrichomonads from the oral cavity and respiratory tract of humans

To clarify the taxonomy of trichomonads associated with human respiratory diseases, we examined a collection of axenic trichomonad strains isolated from the oral cavity and bronchi of patients from pulmonary diseases clinics in Tallin, Estonia. The oral and bronchial strains were compared mutually as well as with a reference strain of Trichomonas tenax, a common inhabitant of the human oral cavity, and other trichomonad species from humans and animals. Unexpectedly, the morphological studies, as well as DNA sequencing of ITS1-5.8S rRNA-ITS2 regions revealed that the Estonian strains belong to the genus Tetratrichomonas, with a high similarity to the avian species Tetratrichomonas gallinarum. None of the strains belonged to Trichomonas tenax. DNA fingerprinting using the RAPD method separated Estonian strains into 2 distinct groups: 'bronchial' consisting of 5 and 2 strains isolated from bronchi and 'oral' cavity, respectively, and oral consisting of 3 oral strains. Consistent differences between 'bronchial' and 'oral' groups were confirmed by analysis of ITS1-5.8S rRNA-ITS2 sequences. Our results have revealed novel trichomonad species of the human oral cavity and bronchi.