ITS Sequence Analysis of Ophidascaris Baylisi from Burmese Pythons

Members of genus Ophidascaris are common parasitic roundworms in snakes that cause gastric granulomas, adenocarcinomas, intestinal obstruction, death, and serious economic losses in snakes and their products. To identify molecular marker of Ophidascaris baylisi from the Burmese python (Python molurus bivittatus), we amplified internal transcribed spacer (ITS) sequence from this roundworm and analyzed their homology and phylogeny. The amplified ITS sequence length was 1140 bp, comprising the complete ITS1, 5.8S, and ITS2 sequences and a partial 28S sequence. The ITS1+ sequence was homologous (85.8% homology) to the related species Ophidascaris robertsi. The phylogenetic tree revealed a close genetic distance between O. baylisi and O. robertsi, which formed a separate branch within family Ascaridae clade. The results indicated that the identified ITS sequence could be a good molecular marker for further study on the molecular classification and genetic variation of Ophidascaris species.

Molecular phylogenetic study of rare weed-field species of the genus Avena L.

A molecular phylogenetic study of weed-field species of the genus Avena L. using marker sequences ITS1–5.8S rRNA gene–ITS2 was undertaken. In addition, next-generation sequencing (NGS) was performed on the Illuminaplatform for the ITS1 sequence and the beginning of the gene 5.8S rRNA. Sanger sequencing results revealed the separateclade of microspecies with a good level of support and small level of difference between themselves. According to NGSsequencing data, the two most abundant subgenomes in terms of the number of sequences were identified. Among thecommon sequences of hexaploids, those associated with the C-genome were not found. The presence of unique ribotypeswas shown for A. persica and A. georgica.

Molecular Identification of Sarcocystis Halieti in Birds of Prey From Spain

Background: Members of the genus Sarcocystis are protozoan parasites characterized by a prey-predator two-host life cycle. Sarcocysts are formed in muscles or CNS of the intermediate host (IH), while sporocysts develop in the small intestine of the definitive host (DH). Various birds of prey were confirmed to be DH for Sarcocystis spp. By contrast, only two species, S. wobeseri and S. falcatula were identified in the muscles of birds of prey. The latter species is pathogenic and can cause encephalitis in various birds. The aim of the present study was to identify Sarcocystis species in the muscles of birds of prey from Spain. Methods: In the period between 2019 and 2020, muscle tissues of 59 birds collected from Spain were examined for the presence of Sarcocystis spp. Sarcocysts in fresh squashed samples were morphologically characterised under a light microscope (LM). Sarcocystis species were identified by means of 28S rRNA and ITS1 sequence analysis. Results: With the help of methylene blue-staining microscopic sarcocysts were detected in 3/59 (5.1%) birds of prey from Spain. Under LM, one type of sarcocysts was observed. Sarcocysts were thread-like (1050–2160 × 130–158 μm), had a thin (0.7–1.4 μm) and smooth cyst wall. Septa divided the cysts into compartments filled with banana-shaped (5.9 × 1.7 μm) bradyzoites. On the basis of DNA sequence results, S. halieti was identified in the western marsh harrier (Circus aeruginosus) and the black kite (Milvus migrans) for the first time. Sarcocysts of S. halieti detected in the black kite and the western marsh harrier were shorter and wider as compared to those observed in the great cormorant (Phalacrocorax carbo) and the herring gull (Larus argentatus). Hence, S. halieti might infect birds belonging to three different orders, Suliformes, Charadriiformes and Accipitriformes. Conclusions: This is the first report of S. halieti in birds of prey as IH. Due to the inconsistency of research on Sarcocystis spp. from birds of prey, further complex morphological, histopathological, and molecular studies are required.

Taxonomic Re-Examination of Nine Rosellinia Types (Ascomycota, Xylariales) Stored in the Saccardo Mycological Collection

In a recent monograph on the genus Rosellinia, type specimens worldwide were revised and re-classified using a morphological approach. Among them, some came from Pier Andrea Saccardo’s fungarium stored in the Herbarium of the Padova Botanical Garden. In this work, we taxonomically re-examine via a morphological and molecular approach nine different Roselliniasensu Saccardo types. ITS1 and/or ITS2 sequences were successfully obtained applying Illumina MiSeq technology and phylogenetic analyses were carried out in order to elucidate their current taxonomic position. Only the ITS1 sequence was recovered for Rosellinia areolata, while for R. geophila, only the ITS2 sequence was recovered. We proposed here new combinations for Rosellinia chordicola, R. geophila and R. horridula, while for R. ambigua, R. areolata, R. australis, R. romana and R. somala, we did not suggest taxonomic changes compared to the current ones. The name Rosellinia subsimilis Sacc. is invalid, as it is a later homonym of R. subsimilis P. Karst. & Starbäck. Therefore, we introduced Coniochaeta dakotensis as a nomen novum for R. subsimilis Sacc. This is the first time that these types have been subjected to a molecular study. Our results demonstrate that old types are an important source of DNA sequence data for taxonomic re-examinations.

Illuminating type collections of nectriaceous fungi in Saccardo's fungarium

Specimens of Nectria spp. and Nectriella rufofusca were obtained from the fungarium of Pier Andrea Saccardo, and investigated via a morphological and molecular approach based on MiSeq technology. ITS1 and ITS2 sequences were successfully obtained from 24 specimens identified as ' Nectria ' sensu Saccardo (including 20 types) and from the type specimen of Nectriella rufofusca. For Nectria ambigua, N. radians and N. tjibodensis only the ITS1 sequence was recovered. On the basis of morphological and molecular analyses new nomenclatural combinations for Nectria albofimbriata, N. ambigua, N. ambigua var. pallens, N. granuligera, N. peziza subsp. reyesiana, N. radians, N. squamuligera, N. tjibodensis and new synonymies for N. congesta, N. flageoletiana, N. phyllostachydis, N. sordescens and N. tjibodensis var. crebrior are proposed. Furthermore, the current classification is confirmed for Nectria coronata, N. cyanostoma, N. dolichospora, N. illudens, N. leucotricha, N. mantuana, N. raripila and Nectriella rufofusca. This is the first time that these more than 100-yr-old specimens are subjected to molecular analysis, thereby providing important new DNA sequence data authentic for these names.

Using the rDNA Internal Transcribed Spacer 1 to Identify the Invasive Pest Rhagoletis cerasi (Diptera: Tephritidae) in North America

The cherry-infesting fruit fly Rhagoletis cerasi Loew is a significant commercial pest in Europe that has recently invaded North America. To date, it has been trapped only in Canada and northwestern counties of New York. It has the potential to spread further and threaten production and movement of cherry commodities. Timely diagnosis of the pest will facilitate surveys and quick response to new detections. Adult morphology of the pest is distinct from other flies in North America. However, when flies are significantly damaged on traps or the immature life stages are found in fruits, molecular methods of identification are important to confirm presence and host-use records. Other than DNA sequencing of genes from flies which takes over a day to complete, there are no timely methods of molecular identification for this pest. In this study, we report the first sequence record of the internal transcribed spacer 1 (ITS1) from R. cerasi and develop two diagnostic tests for the pest based on ITS1 differences among species in North America. The tests use loop-mediated isothermal amplification (LAMP) and multiplex, conventional polymerase chain reaction (mcPCR) technologies that target the same region of the R. cerasi ITS1 sequence. Both tests performed well when tested against collections of R. cerasi from North America and Europe, generating Diagnostic Sensitivity estimates of 98.4–99.5%. Likewise, the tests had relatively high estimates of Diagnostic Specificity (97.8–100%) when tested against Rhagoletis Loew species present in North America that also use cherry as a developmental host.

Leishmania ITS1 Is Genetically Divergent in Asymptomatic and Symptomatic Visceral Leishmaniasis: Results of a Study in Southern Iran

It has been documented that the genotypic traits in symptomatic and asymptomatic cases of visceral leishmaniasis (VL) may be different. The current study aimed to find out and compare the genotype and intraspecies diversity of Leishmania Internal Transcribed Spacer 1 (ITS1) from asymptomatic and symptomatic VL cases in southern Iran. Methods. Buffy coat samples from seven VL patients, with clinical signs and symptoms, and seven asymptomatic VL cases, were evaluated in this study. Samples of asymptomatic individuals were obtained from children living in a VL endemic area in southern Iran, while the samples of symptomatic subjects were obtained from patients admitted to hospitals with a diagnosis of VL. DNA was extracted from the buffy coats of the samples and PCR-amplified, targeting the ITS1of Leishmania. The PCR products were sequenced, and the consensus sequences were assembled and multiple-aligned with a set of Leishmania strains retrieved from the GenBank, using Clustal W. The phylogenetic tree was rooted, using MEGAX software, and the diversities based on haplotype and nucleotides, as well as the number of polymorphic sites, were measured using DnaSP v5.0 software. The results of ITS1 sequencing in 5 out of 7 asymptomatic VL cases showed 99.25% to 100% similarity with the Leishmania infantum ITS1 sequence (accessed number: MN648746), and one isolate was considered as just Leishmania sp. In one sample, 99.75% similarity was seen with the ITS1 sequence of Crithidia fasciculata. Of the symptomatic VL patients, the PCR product revealed a 340 bp band corresponding to L. infantum in all of the samples. By analyzing the ITS1 sequences, all seven sequences formed a clade somewhat different from other Leishmania species and considered as Leishmania sp. Haplotype and nucleotide diversity were much more prevalent in symptomatic cases where six haplotypes were seen in the ITS1 of Leishmania from symptomatic patients and only two haplotypes were observed in the samples from asymptomatic cases. The findings of the current study showed that the Leishmania ITS1 from symptomatic VL and asymptomatic cases has significant genetic differences. Besides, infection with Crithidia fasciculata was reported, for the first time, in an asymptomatic case, which deserves further study.

The origin of Alopecurus × brachystylus Peterm. according to the results of next-generation sequencing (NGS)

A molecular phylogenetic study of the hybrid species Alopecurus × brachystylus Peterm. and somesupposed ancestral taxa was carried out. Next-generation sequencing (NGS) of the ITS1 sequence and the start of the5.8S rRNA gene was used on the Illumina platform. According to NGS sequencing, genome of the A. × brachystylusforms common subgenomes with representatives of the section Alopecurium: A. geniculatus and A. aequalis, as well asrepresentatives of the type section: A. pratensis, A. arundinaceus, and alpine A. vlassowii. In addition, it was found thatA. vlassowii (section Alopecurus) contains sequences identical to the species of another section, Alopecurium.

Molecular identification of four Sarcocystis species in the herring gull, Larus argentatus, from Lithuania

Background Birds of the family Laridae have not been intensively examined for infections with Sarcocystis spp. To date, sarcocysts of two species, S. lari and S. wobeseri, have been identified in the muscles of gulls. The aim of the present study was to evaluate the species richness of Sarcocystis in the herring gull, Larus argentatus, from Lithuania. Methods In the period between 2013 and 2019, leg muscles of 35 herring gulls were examined for sarcocysts of Sarcocystis spp. Sarcocystis spp. were characterised morphologically based on a light microscopy study. Four sarcocysts isolated from the muscles of each infected bird were subjected to further molecular examination. Sarcocystis species were identified by means of ITS1 sequence analysis. Results Sarcocysts were detected in 9/35 herring gulls (25.7%). Using light microscopy, one morphological type of sarcocysts was observed. Sarcocysts were microscopic, thread-like, had a smooth and thin (about 1 µm) cyst wall and were filled with banana-shaped bradyzoites. On the basis of ITS1 sequences, four Sarcocystis species, S. columbae, S. halieti, S. lari and S. wobeseri, were identified. Furthermore, it was demonstrated that a single infected herring gull could host two Sarcocystis species indistinguishable under light microscopy. Conclusions Larus argentatus is the first bird species found to act as intermediate host of four Sarcocystis spp. According to current knowledge, five species, S. falcatula, S. calchasi, S. wobeseri, S. columbae and S. halieti can use birds belonging to different orders as intermediate hosts.

Prevalence of Eimeria parasites and sulfachloropyrazine sodium resistance in chicken farms in the Hubei and Henan provinces

Background：Coccidiosis is an intestinal parasitic disease that causes huge economic losses to the poultry industry globally. At present, the primary control strategy is administration of anticoccidial drugs with feed. However, overuse of anticoccidials, such as sulfachloropyrazine sodium (SC), has resulted in an increase in the emergence of drug resistance.Methods: We aimed to evaluate coccidiosis prevalence and SC resistance in field isolates to provide reasonable guidance on the use of anticoccidial drugs in the Hubei and Henan provinces. We collected 318 fresh fecal samples from 137 chicken farms. We used internal transcribed spacer 1 (ITS1) sequence of ribosomal DNA to identify the species from 94 samples that were collected from different farms and to assess drug resistance.Results: As shown by genus-specific PCR results, the positivity rate of Eimeria was 97.17 % (309/318), and the most common species were E. mitis (66.67%), E. tenella (46.86%), and E. necatrix (41.51%). Animal experiment demonstrated that 25 strains were completely resistant to SC, among which 16 were from Henan and nine were from Hubei. Twenty-four strains were partially resistant, among which 8 and 16 strains were identified from Hubei and Henan, respectively.Conclusions: In summary, these data indicated that chicken coccidia is ubiquitous and SC resistance is widespread, in the Hubei and Henan provinces. The results provide important insights into the control of chicken coccidiosis in this region.