Clustered Regularly Interspaced Short Palindromic Repeat Analysis of Clonal Complex 17 Serotype III Group B Streptococcus Strains Causing Neonatal Invasive Diseases

Group B Streptococcus (GBS) is an important pathogen of neonatal infections, and the clonal complex (CC)-17/serotype III GBS strain has emerged as the dominant strain. The clinical manifestations of CC17/III GBS sepsis may vary greatly but have not been well-investigated. A total of 103 CC17/III GBS isolates that caused neonatal invasive diseases were studied using a new approach based on clustered regularly interspaced short palindromic repeats (CRISPR) loci and restriction fragment length polymorphism (RFLP) analyses. All spacers of CRISPR loci were sequenced and analyzed with the clinical presentations. After CRISPR-RFLP analyses, a total of 11 different patterns were observed among the 103 CRISPR-positive GBS isolates. GBS isolates with the same RFLP patterns were found to have highly comparable spacer contents. Comparative sequence analysis of the CRISPR1 spacer content revealed that it is highly diverse and consistent with the dynamics of this system. A total of 29 of 43 (67.4%) spacers displayed homology to reported phage and plasmid DNA sequences. In addition, all CC17/III GBS isolates could be categorized into three subgroups based on the CRISPR-RFLP patterns and eBURST analysis. The CC17/III GBS isolates with a specific CRISPR-RFLP pattern were more significantly associated with occurrences of severe sepsis (57.1% vs. 29.3%, p = 0.012) and meningitis (50.0% vs. 20.8%, p = 0.009) than GBS isolates with RFLP lengths between 1000 and 1300 bp. Whole-genome sequencing was also performed to verify the differences between CC17/III GBS isolates with different CRISPR-RFLP patterns. We concluded that the CRISPR-RFLP analysis is potentially applicable to categorizing CC17/III GBS isolates, and a specific CRISPR-RFLP pattern could be used as a new biomarker to predict meningitis and illness severity after further verification.

Genotyping of Malassezia pachydermatis disclosed genetic variation in isolates from dogs in Colombia

ABSTRACT: Malassezia pachydermatis is a lipophilic and lipid-dependent yeast mostly isolated from animals’ skin; hence, it is regarded as a zoophilic species causing otitis externa in dogs. Aspects associated with its epidemiology and pathogenicity is a matter of interest. This study aimed to conduct a molecular characterization of 43 isolates of M. pachydermatis obtained from dogs with otitis externa. For this purpose, the 5.8S internal transcribed spacer 2 (ITS2) and D1/D2 26S rRNA regions were amplified, sequenced and analyzed using restriction fragment length polymorphism (RFLP) with AluI, CfoI, and BstF5I endonucleases. Phylogenetic analyses revealed that these isolates grouped with the sequence types I, IV and V, previously proposed for M. pachydermatis. Interestingly, we found a new polymorphic RFLP pattern using BstF5I, these isolates were associated with the sequence types IV and V, nevertheless an association between polymorphic RFLP patterns, and fosfolipase activity or canine population data was not observed. These findings underline the genetic diversity of M. pachydermatis and provide new insights about the epidemiology of this species in the analyzed population.

Identification of Fasciola Species Isolates from Nghe An Province, Vietnam, Based on ITS1 Sequence of Ribosomal DNA Using a Simple PCR-RFLP Method

Fascioliasis—a disease caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea)—is considered as the most important helminthic infection of bovine, sheep, and buffalo in Vietnam. The aim of this study is to detect the genotype of Fasciola spp. isolated from bovine and buffalo in the Nghe An province, central Vietnam, using PCR-RFLP and sequence analysis of the first nuclear ribosomal internal transcribed spacer (ITS1). Adult Fasciola spp. were isolated from bile ducts of bovine and buffalo in Nghe An province, Vietnam. Overall, 96 adult flukes from livers of slaughtered animals were collected from abattoirs of different areas. They included 7 samples from infected bovine and 89 samples from infected buffalo. 96/96 samples were identified as Fasciola species by ITS1 of rDNA. In this study, a PCR-RFLP method was used to distinguish between F. hepatica and F. gigantica in ITS1 of rDNA (680 bp) with RsaI restriction enzyme. RFLP pattern with RsaI produced a consistent pattern of 360, 100, and 60 bp fragments in F. hepatica, whereas F. gigantica worms had a profile of 360, 170, and 60 bp in size, respectively. The results showed that using PCR-RFLP based on the first internal transcribed spacers (ITS1) of the ribosomal RNA revealed that 93 out of 96 isolates were of Fasciola gigantica type, whereas three isolates presented an intermediate Fasciola. In the present study, F. gigantica and intermediate form were coexisting in bovine and buffalo in the Nghe An province of central Vietnam, whereas F. hepatica was not detected.

Bursaphelenchus gillanii sp. n. (Nematoda: Aphelenchoididae) – a new species of the xylophilus group in packaging wood imported from China

A new Bursaphelenchus species of the xylophilus group was detected in coniferous packaging wood imported with goods from China in 2011. The new species is described herein and compared with other species of the xylophilus group. Bursaphelenchus gillanii sp. n. has a slim body (a = 31 (28-34) and 33 (29-36) in females and males, respectively), c′ = 3.8 (3.2-4.5) and 2.1 (1.7-2.5) in females and males, respectively, a large vulval flap, a 5-7 μm long digitate mucro as a continuation of the female tail, excretory pore at or closely posterior to the median bulb, strongly arcuate spicules, 34 (31-37) μm long as measured along the median line, with prominent pointed rostrum and small cucullus. The ITS-RFLP pattern of the new species obtained by digestion of the PCR product with RsaI, HaeIII, MspI, HinfI and AluI is different from other known Bursaphelenchus species. Results of sequencing the ITS1/2 region demonstrate the close relationship of the new species to B. mucronatus and B. xylophilus.

Grapevine yellows affecting the Croatian indigenous grapevine cultivar Grk

- The grapevine cultivar Grk, a close relative of Crljenak kaštelanski/Zinfandel, is grown exclusively in southern Croatia. Grapevine yellows-like symptoms were observed on vines in the vineyards in Lumbarda (southern Croatia) and in propagated grapevines near Zadar and Zagreb. The majority of the detected phytoplasma isolates belonged to the 16SrI group. However, RFLP pattern and R16F2n/R2 fragment sequence assigned one isolate to the 16SrIII group. Thus far, on cv. Grk, phytoplasmas belonging to three different groups have been detected: 16SrI, 16SrIII, and 16SrXII, which was confirmed previously. Aside from the 16SrI, 16SrV and 16SrXII phytoplasma groups previously found on grapevines in Croatia, the finding of 16SrIII group, which is not common on grapevines in Europe, adds to the diversity of phytoplasmas in a very small geographic region.

Description of Ruehmaphelenchus digitulus sp. n. (Nematoda: Aphelenchoididae) found in packaging wood from Taiwan

Ruehmaphelenchus digitulus sp. n., isolated from coniferous packaging wood from Taiwan, is described and figured. The new species is characterised by a relatively stout body with a = 28.0 (23.7-31.3) and 27.0 (22.9-32.7) for male and female, respectively, three lines in the lateral field, spicules relatively small (chord = 17.0-19.2 μm; arc = 13.8-16.6 μm) with weakly developed condylus and rostrum, bursa absent, vulva positioned at ca 80% of body length, vulva flap absent, vulval lips slightly protruding, post-uterine branch extending slightly over half of vulva to anus distance, and tail conoid, ca 2-3 anal body diam. long, with terminal process 7-10 μm long. The new species is the fourth known for the genus. It can be distinguished from R. martinii, R. asiaticus and R. thailandae mainly by the different shape and size of the spicules and the different shape and length of the female tail terminus. The separate species status is also supported by ITS-RFLP pattern and molecular phylogenetic analysis based on partial SSU ITS1/2 and partial LSU sequences.

Description of Ruehmaphelenchus thailandae n. sp. (Nematoda: Aphelenchoididae) found in dunnage from Thailand

Ruehmaphelenchus thailandae n. sp., isolated from deciduous dunnage from Thailand, is described and figured. The new species is characterised by the relatively stout body (a = 28.6 and 27.6 for males and females, respectively), three lines in the lateral field, spicules relatively small (12.0-13.3 μm) with weakly developed condylus and rostrum, bursa absent, vulva located at 79.5% of body length, vulval lips slightly swollen, length of post-uterine branch more than half of vulva to anus distance, female tail conoid, ca two anal body diam. long and with a broad terminal process up to 12 μm long. The new species is the third known for the genus. It can be distinguished from R. martinii and R. asiaticus mainly by the different shape and size of its spicules and different shape of the female tail terminus. The separate species status is also supported by ITS-RFLP pattern. Additional measurements of paratype R. asiaticus are appended.