Partial Sequence Analysis Research Articles

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There is still unexplored reservoir of microorganisms from sediments and water within Lakes Olbolosat and Oloiden using culture dependent technique. The current study compares bacterial diversity within Lake Olbolosat a freshwater lake and Lake Oloiden a saline alkaline lake. Out of 60 isolates obtained from sediments and water samples, 35 were from Lake Olbolosat and 25 from Lake Oloiden. Microbial count ranged between 0-1.75× 105 cfu/ml from both lakes. There was a significant difference between bacterial density and sampling points (p<0.001, F=6.667), 58 were Gram-positive and 2 Gram-negative. Fifty-five isolates that were rod-shaped, 3 were cocci and 2 filamentous. There was excellent growth of isolates at an optimum growth pH range of 6-10, a temperature range of 25-30°C and a salinity range of 0-5%. There was significant difference at p<0.001 for bacterial growth within physiological parameters. The isolates utilized skimmed milk, starch, olive oil, cellulose powder and xylan, hence the production of extracellular enzymes. There was antimicrobial activity against Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Candida albicans by bacterial isolates. BLAST analysis of partial sequences showed there were 4 different phyla.  Firmicutes scored 77% closely affiliated with 20 strains, Actinobacteria scored 15% closely affiliated with 4 strains, Proteobacteria and Bacteroidetes scored 4% affiliated with 1 strain each. Novel bacteria from this study could provide insights into their diversity and biotechnological applications.   Keywords: Lakes, bacteria, sediment, water, culture-dependent.

Possible nosocomial transmission of virus-associated hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation.

Adenovirus (ADV)- or BK virus (BKV)-associated hemorrhagic cystitis (HC) is a common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Several risk factors have been previously reported; however, it is unclear whether virus-associated HC can be transmitted. To clarify this point, we performed a retrospective cohort study on 207 consecutive patients who underwent allo-HSCT at Kyoto University Hospital between 2012 and 2018. We evaluated the incidence and risk factors of virus-associated HC and performed a phylogenetic analysis of the ADV partial sequence. The median age at transplantation was 50 (range, 17-68) years. Fifty-eight patients (28%) developed HC. ADVs were detected in 18 cases, BKVs were detected in 51, both were detected in 12, and only John Cunningham virus (JCV) was detected in 1 case. No factor was significantly associated with HC. However, both ADV- and BKV-HC occurred intensively between April 2016 and September 2017, which suggested possible nosocomial transmission of ADV and BKV. Genome sequencing of the hexon, E3, and penton regions of detected ADVs identified 7 cases of ADV type 11, 2 cases of type 35, and 3 cases of a type 79-related strain. A sequence analysis revealed that these strains in each type were almost identical, except for one case of a type 79-related strain. In conclusion, ADV-HCs with possible nosocomial transmission were described based on genotyping of the virus and partial sequencing of the viral genome. Although viral HC after allo-HSCT is thought to mainly be due to reactivation of a latent virus, nosocomial transmission of ADV or BKV should also be considered.

Molecular Identification of Parasites Causing Cutaneous Leishmaniasis in Panama.

Isolates from 475 cutaneous leishmaniasis (CL) patients from three endemic regions were studied by three typing techniques. The molecular analysis from lesion scrapings based on hsp70 PCR-restriction fragment length polymorphism (RFLP) showed that 78.1% (371/475) restriction patterns corresponded to Leishmania (Viannia) panamensis, 19% (90/475) to Leishmania (Viannia) guyanensis, and 3.0% (14/475) to Leishmania (Viannia) braziliensis. Promastigotes isolated by culture from lesions of 228 patients (48.0%, 228/475) were identified by multi-locus enzyme electrophoresis. Of them, 95.2% (217/228) were typified as L. (V.) panamensis, 1.3% (3/228) as L. (V.) guyanensis, 2.2% (5/228) as L. (V.) braziliensis, and 1.3% (3/228) as hybrids (L. [V.] braziliensis/L. [V.] panamensis). However, a partial sequencing analysis of the hsp70 gene from 77 selected samples showed 16.9% (13/77) typified as L. (V.) panamensis, 68.8% (53/77) as Leishmania (V.) sp., 1, 3.9% (3/77) as L. (V.) guyanensis, 1.3% (1/77) as L. (V.) braziliensis outlier, 2.6% (2/77) as Leishmania (Viannia) naiffi, 2.6% as (2/77) Leishmania (V.) sp., and 2 and 3.9% (3/77) hybrid isolates of L. (V.) braziliensis/L. (V.) guyanensis. These results confirm L. (V.) panamensis as the predominant species and cause of CL lesions in Panama and that L. (V.) guyanensis, L. (V.) braziliensis, and L. (V.) naiffi are circulating to a lower degree. Furthermore, the determination of parasite isolates belonging to atypical clusters and hybrid isolates suggests the circulation of genetic variants with important implications for the epidemiology and clinical follow-up of CL in Panama. No evidence of the existence of parasites of the Leishmania (Leishmania) mexicana complex in Panamanian territory was found in this study.

Genetic and Antigenetic Characterization of the Novel Kotalahti Bat Lyssavirus (KBLV).

There is a growing diversity of bat-associated lyssaviruses in the Old World. In August 2017, a dead Brandt’s bat (Myotis brandtii) tested positive for rabies and based on partial sequence analysis, the novel Kotalahti bat lyssavirus (KBLV) was identified. Because the bat was in an autolyzed state, isolation of KBLV was neither successful after three consecutive cell passages on cells nor in mice. Next generation sequencing (NGS) was applied using Ion Torrent ™ S5 technology coupled with target enrichment via hybridization-based capture (myBaits®) was used to sequence 99% of the genome, comprising of 11,878 nucleotides (nt). KBLV is most closely related to EBLV-2 (78.7% identity), followed by KHUV (79.0%) and BBLV (77.6%), supporting the assignment as phylogroup I lyssavirus. Interestingly, all of these lyssaviruses were also isolated from bat species of the genus Myotis, thus supporting that M. brandtii is likely the reservoir host. All information on antigenic and genetic divergence fulfil the species demarcation criteria by ICTV, so that we recommend KBLV as a novel species within the Lyssavirus genus. Next to sequence analyses, assignment to phylogroup I was functionally corroborated by cross-neutralization of G-deleted RABV, pseudotyped with KBLV-G by sera from RABV vaccinated humans. This suggests that conventional RABV vaccines also confer protection against the novel KBLV.

Control of damping-off in tomato seedlings exerted by Serratia spp. strains and identification of inhibitory bacterial volatiles in vitro

Serratia marcescens can be a plant growth promoting bacteria (PGPB) and an opportunistic human and plant pathogen. We have identified and characterized strains of related species of Serratia and evaluated their biological control of damping-off of tomato seeds caused by Pythium cryptoirregulare. Serratia ureilytica, S. bockelmannii and S. nevei were identified by phylogenetic analysis of partial gyrB gene sequence and average nucleotide identity (ANI). Tomato seeds inoculated with S. ureilytica ILBB 145 showed higher germination percentage and reduced damping-off in greenhouse experiment resembling a commercial operation, and volatiles produced by this strain caused the nearly complete inhibition in vitro of P. cryptoirregulare. Analysis of volatile organic compounds (VOCs) showed that ILBB 145 produced dimethyl disulfide (DMDS), which can partially account for this inhibition. Serratia bockelmannii ILBB 162 performance against damping-off was intermediate and the inhibition of P. cryptoirregulare in vitro was lower and explained by volatile and diffusible metabolites. Both strains augmented DMDS production in the presence of P. cryptoirregulare, suggesting this compound may play a role in the context of interspecific competition. Serratia nevei ILBB 219 showed the lowest inhibition of P. cryptoirregulare in vitro, no DMDS production, and no biocontrol in planta. Draft genomes of the three strains were annotated and individual genes and biosynthesis gene clusters were identified in relation with the observed phenotypes. We report S. ureilytica – a low risk species- with activity as a biological control agent and DMDS produced by this bacterial species putatively involved in seed and seedling protection against P. cryptoirregulare.

Morphological and molecular characterization of Paratylenchus beltsvillensis n. sp. (Tylenchida: Paratylenchidae) from the rhizosphere of pine tree (Pinus virginiana Mill) in Maryland, USA.

The pin nematode, Paratylechus beltsvillensis n. sp. collected from rhizosphere soil of a Virginia pine tree (Pinus virginiana Mill) growing in Little Paint Branch Park, Beltsville, Prince George’s County, Maryland, USA, is described and illustrated along with light and scanning electron photomicrographs. Females, males, and juveniles of this new species were recovered from soil samples using the sugar centrifugal flotation and Baermann funnel extraction methods. Morphologically, females are short, body length ranging from 245 to 267 μm, stylet from 70 to 75 μm long with anchor shaped knobs, vulva located at 70–73% and small vulval flap, spermatheca large, and ovoid filled with sperms. Lateral field with three incisures, of which the outer two are prominent. Tail slender, having a rounded tail terminus. Males without stylet and have a degenerated pharynx, spicules = 17–20 µm and gubernaculum = 5.0–5.5 µm. Both morphological observations and molecular analysis of ITS and partial 28S ribosomal RNA gene sequences indicated that the specimens collected from the soil at Beltsville Park from rhizosphere soil samples from Virginia pine represents a new pin nematode species.

Circulation of classic and recombinant human astroviruses detected in South Africa: 2009 to 2014.

Astroviruses (AstVs) are associated with diarrhoeal and extra-intestinal infections in human, animal and avian species. A prevalence of 7% was reported in selected regions in SA while AstVs detected from clinical stool specimens were almost identical phylogenetically to strains identified in environmental and water samples. This study investigated the molecular diversity of astroviruses circulating between 2009 and 2014 in South Africa (SA). Astroviruses detected in stool specimens collected from hospitalised children were investigated retrospectively. Astroviruses were characterised using type-specific RT-PCR, partial nucleotide sequence analyses in ORF1 and ORF2 and whole genome sequencing. Different genotypes were compared with clinical features to investigate genotype-related associations. The Vesikari severity scale (VSS) was evaluated for scoring astrovirus diarrhoeal infections. Of 405 astroviruses detected, 49.9 % (202/405) were characterised into 32 genotypes comprising 66.3 % (134/202) putative-recombinants and 33.7 % (68/202) classic strains. No trends by year of collection, age or site were observed. Whole genome analysis in eight strains revealed that genotypes assigned by partial nucleotide sequence analyses to five astroviruses were incorrect. Bivariate analyses showed there were no significant associations between genotypes and clinical symptoms or severity of infection. A comparison of Vesikari parameters with astrovirus-positive proxy values demonstrated that Vesikari scores for duration of diarrhoea and admission temperatures would result in a milder infection rating in astrovirus-positive cases. Diverse genotypes co-circulated with putative-recombinants predominating. Astrovirus classification was complicated by the lack of a consistent characterisation system and reliable reference database. The VSS should be used cautiously to rate astrovirus diarrhoea. While surveillance in communities and out-patient clinics must be continued, screening for human astroviruses in alternate hosts is needed to determine the reservoir species.

Biosorption of heavy metals by dry biomass of metal tolerant bacterial biosorbents: an efficient metal clean-up strategy.

Heavy metals discharge at an unrestrained rate from various industries into the environment pose serious human health problems. Considering this, the present study aimed at exploring the metal biosorbing potentials of bacterial strains recovered from polluted soils. The bacterial strains (CPSB1, BM2 and CAZ3) belonging to genera Pseudomonas, Bacillus and Azotobacter expressing multi-metal tolerance ability were identified to species level as P. aeruginosa, B. subtilis and A. chroococcum, respectively, by 16S rRNA partial gene sequence analysis. The biosorption of cadmium, chromium, copper, nickel, lead and zinc by three dead bacterial genera were studied as a function of metal concentration, variable pH of the medium and reaction (contact) time. The three bacterial strains exhibited a tremendous metal removal ability which continued even at the highest tested concentration of some metals. Later, a decline in the percentage of biosorbed metals was recorded as the metal concentration was increased with the simultaneous generation of a driving force to overcome mass transfer resistance for movement of metal ions between the solution and the surface of adsorbent. Among test bacteria, B. subtilis biosorbed a maximum of 96% chromium at 25μgmL-1 while the maximum percentage (91%) of biosorbed metals recorded at 400μg CdmL-1 was observed for P. aeruginosa. The sorption of metal ions by dead biomass of three bacterial genera at optimum conditions followed the order-(i) B. subtilis BM2: Pb > Cu > Ni > Cd > Cr, (ii) A. chroococcum CAZ3: Cr > Cd > Cu > Ni > Pb and (iii) P. aeruginosa CPSB1: Cd > Cr > Ni > Cu > Pb > Zn. It was found that the optimum pH for metal adsorption ranged between pH8 and 9 which, however, declined substantially at pH5.0 for all three bacterial strains. In general, the biosorption of Cd, Cr, Cu, Ni and Pb by B. subtilis and A. chroococcum and such metals along with Zn by P. aeruginosa occurred maximally up to 60min of bacterial growth. The adsorption data with regard to five metals provide an outstanding fit to the Langmuir and Freundlich isotherms. The biosorptive ability of three bacterial genera correlated strongly (r2 > 0.9) with each metal. The bacteria belonging to two Gram-negative genera Pseudomonas (P. aeruginosa) and Azotobacter (A. chroococcum) and one Gram-positive genus Bacillus (B. subtilis) demonstrated exceptional metal removal efficiency and, hence, provides a comprehensive understanding of metal-bacteria sorption process which in effect paves the way for detoxifying/removing metals from contaminated environment.

Morphological and molecular data of Hepatozoon ursi in two brown bears (Ursus arctos) in Turkey.

Species of Hepatozoon Miller, 1908 are vector-borne parasites that infect domestic and wild animals worldwide. Hepatozoon ursi Kubo, Uni, Agatsuma, Nagataki, Panciera et al., 2008 was reported from bears (Ursidae) in Japan and India. The present study represents the first report of infection with H. ursi in Turkish brown bears (Ursus arctos Linnaeus) by microscopic and molecular analysis. Two dead brown bears were found in Uzundere and Pasinler districts of Erzurum. Blood and visceral organ (spleen and liver) samples were delivered to laboratory by the Nature Conservation and National Parks officers. Detected gamonts were evaluated based on morphological features and confirmed as gamonts of H. ursi. The size of gamonts and parasitemia were 8.2 × 3.5 μm (6.9-8.7 × 3.0-3.9 μm; n = 12) and 0.6% (6/1000 leukocytes), respectively. The blood and visceral organ samples were positive for species of Hepatozoon by PCR targeting partial sequence of 18S rDNA. Sequence analysis of newly obtained sequences of H. ursi showed 98.8-100% identity with previously sequenced isolates of H. ursi. Sequences of H. ursi from Erzurum were identical to each other and showed 100% identity with isolates of H. ursi from ticks Ixodes ricinus (Linnaeus), Rhipicephalus turanicus Pomerantzev and Hyalomma marginatum Koch collected from two brown bears in Turkey (GenBank accession numbers MN463021, MN463022, MN905023). Analysis of partial sequences of the 18S rRNA gene of H. ursi showed that Turkish isolates differ in NT substitutions found at three different positions [72 (A→G), 537 (A→G) and 570 (A→T)]. This study provides morphological and molecular data of H. ursi infection in brown bears from two districts of Erzurum, Turkey. Further studies are needed to elucidate whether brown bears have any eco-epidemiologic importance in the life cycle of H. ursi in wildlife.

Phylogeny and Toxin Profile of Freshwater Pufferfish (Genus Pao) Collected from 2 Different Regions in Cambodia

The species classification of Cambodian freshwater pufferfish is incomplete and confusing, and scientific information on their toxicity and toxin profile is limited. In the present study, to accumulate information on the phylogeny and toxin profile of freshwater pufferfish, and to contribute to food safety in Cambodia, we conducted simultaneous genetic-based phylogenetic and toxin analyses using freshwater pufferfish individuals collected from Phnom Penh and Kratie (designated PNH and KTI, respectively). Phylogenetic analysis of partial sequences of three mitochondrial genes (cytochrome b, 16S rRNA, and cytochrome c oxidase subunit I) determined for each fish revealed that PNH and KTI are different species in the genus Pao (designated Pao sp. A and Pao sp. B, respectively). A partial sequence of the nuclear tributyltin-binding protein type 2 (TBT-bp2) gene differentiated the species at the amino acid level. Instrumental analysis of the toxin profile revealed that both Pao sp. A and Pao sp. B possess saxitoxins (STXs), comprising STX as the main component. In Pao sp. A, the toxin concentration in each tissue was extremely high, far exceeding the regulatory limit for STXs set by the Codex Committee, whereas in Pao sp. B, only the skin contained high toxin concentrations. The difference in the STX accumulation ability between the two species with different TBT-bp2 sequences suggests that TBT-bp2 is involved in STX accumulation in freshwater pufferfish.

Identification, HPG2 Sequence Analysis, and Antimicrobial Susceptibility of Avibacterium paragallinarum Isolates Obtained from Outbreaks of Infectious Coryza in Commercial Layers in Sonora State, Mexico.

This is the first extensive report on the identification and characterization of Avibacterium paragallinarum (AVP) isolates obtained from outbreaks of infectious coryza (IC) in IC-vaccinated layer flocks from Sonora State in Mexico. Isolates obtained from IC outbreaks during the years 2007, 2014, 2015, 2017, and 2019 were identified by conventional PCR test and 16S rRNA gene analysis, serotyped by Page serotyping and genotyped by the recently described partial sequence analysis of the HPG2 region. Furthermore, antimicrobial susceptibility profiles were determined by a recently improved minimal inhibitory concentration (MIC) test. The conventional PCR test and the 16S rRNA analyses confirmed the isolates as AVP. Serotyping results showed the involvement of isolates belonging to serotypes A, B, and C in the IC outbreaks. Genotyping of the HPG2 region revealed the presence of sequence type (ST)1, ST4, and ST11, of which the latter has also been identified in Europe. The MIC susceptibility test showed that all tested isolates were susceptible for the majority of tested antimicrobials, including erythromycin and tetracycline, which are important antibiotics for the treatment of IC. The IC situation in Sonora State, Mexico, is complex because of the presence of serotypes A, B, and C. This finding emphasizes the importance of biosecurity in combination with the application of the most optimal vaccination programs in the control of IC in Sonora State, Mexico.

Molecular characterization of human adenoviruses associated with respiratory infection in Turkey.

Human adenoviruses (HAdVs) are responsible for various clinical diseases. Molecular epidemiological studies of respiratory HAdVs are limited in Turkey. To determine the main genotypes and epidemiological characteristics of HAdVs in patients with respiratory symptoms. HAdV PCR-positive extracts of nasal/nasopharyngeal specimens sent to the Turkish Public Health Institution from various cities of Turkey in 2015-2016 were investigated by seminested PCR. Partial sequence analysis of the hexon gene of HAdVs was performed. SPSSv.24.0 was used. A total of 23/68 (33.82%) HAdV-positive samples were amplified. Mastadenovirus B, C, D, and F were detected and mastadenovirus B (10/23; 43.5%) and C (10/23; 43.5%) were predominant strains. Interestingly, HAdV-F known to have gastrointestinal system tropism was detected in two patients with respiratory symptoms. HAdV-B3 was the most prevalent genotype (9/23; 39.1%). Also, HAdV-B7 is defined as a reemerging pathogen. It is noteworthy that there is a cluster of four HAdV-C strains showing a close paraphyletic relationship with HAdV-2/6 intertypic recombination. To our knowledge, this is the first study showing that HAdV-B7 reemerging pathogen circulating in patients with respiratory infections in our country. It is also necessary to emphasize that HAdV-2/6 recombinant strains were detected in this study for the first time in Turkey.

Ciprofloxacin-Resistant Kerstersia gyiorum Isolated From a Chronic Wound in Brazil: A Case Report

This study evaluated the antimicrobial susceptibility profile of K gyiorum isolated from a chronic wound. An 85-year-old woman with chronic venous insufficiency presented to an intermediate care unit in Niteroi City, Rio de Janeiro, Brazil, with an instep chronic wound of 14 cm² with wound duration of 6 months. K gyiorum was identified by matrix-assisted laser desorption ionization-time of flight, confirmed by 16S rRNA partial sequence analysis, and classified as resistant for ciprofloxacin by reagent strips(minimum inhibitory concentration [MIC] = 32 µg/mL) and the broth macrodilution method (MIC = 8 µg/mL). Intermediate resistance for ciprofloxacin was verified by microscan (MIC = 2 µg/mL). The authors identified the first, to their knowledge, lower leg wound with K gyiorum in Brazil and verified that it was ciprofloxacin resistant.

<strong>A new crenobiontic water mite species of the genus <em>Atractides</em> Koch, 1837 from Montenegro and Bulgaria, based on morphological and molecular data (Acariformes, Hydrachnidia, Hygrobatidae)</strong>

A new species of water mites from Montenegro and Bulgaria, Atractides anae Pešić sp. nov., is described, combining traditional morphological techniques with the analysis of partial mtDNA COI sequences (DNA-barcodes). Based on our molecular and morphological analysis, the new species is most closely related to the crenobiontic A. pennatus (K. Viets 1920) from which it differs by 6.4 (SD = 1.12)% K2P distance. A dataset including the novel COI sequences of 10 Atractides species from Montenegro and Iran is assembled in order to facilitate further studies on the genus by using molecular tools.

Rare/cryptic Aspergillus species infections and importance of antifungal susceptibility testing.

The number of patients infected with Aspergillus rose dramatically in recent years. However, studies on the clinical spectrum and antifungal susceptibilities of non-classical (non-fumigatus, non-flavus, non-niger and non-terreus) pathogenic Aspergillus species are very limited. We examined the clinical spectrum and antifungal susceptibilities of 34 non-duplicated, non-classical Aspergillus isolates collected from Hong Kong, Shenzhen and Shanghai. The Aspergillus isolates were identified by internal transcribed spacer, partial BenA and partial CaM sequencing and phylogenetic analyses. Susceptibility testing against eight antifungals was performed following the European Committee for Antimicrobial Susceptibility Testing's methodology. The 34 Aspergillus isolates were identified as 14 different rare/cryptic species of four sections (Flavi [n=8], Nidulantes [n=8], Nigri [n=17] and Restricti [n=1]). Except for one patient whose clinical history could not be retrieved, 72.7% of the remaining patients had underlying conditions predisposing them to Aspergillus infections. The most common diseases were pulmonary infections (n=15), followed by skin/nail infections (n=6), chronic otitis externa and/or media (n=5), wound infections (n=2) and mastoiditis/radionecrosis (n=1), while three were colonisations. Five patients succumbed due to the infections during the admission, and another two died 5years later because of chronic pulmonary aspergillosis. Antifungal susceptibility testing showed that they possessed different susceptibility profiles compared to the classical Aspergillus species. The majority of isolates characterised were sensitive or wild-type to amphotericin B. The minimum effective concentrations for all the three echinocandins were also low. Susceptibility testing should be performed for infections due to these rare/cryptic Aspergillus species to guide proper patient management.

Phylogeny of dracunculoid nematodes (Chromadorea: Rhabditida: Spirurina: Dracunculoidea) from some Eurasian freshwater fishes.

Sets of small ribosomal DNA (SSU rDNA) and large ribosomal DNA (LSU rDNA) sequences were obtained for Philometroides moraveci Vismanis Yunchis, 1994, Philometra kotlani (Molnár, 1969), Philometra rischta Skrjabin, 1923, Philometra cf. obturans (Prenant, 1886) (Philometridae), Sinoichthyonema amuri (Garkavi, 1972), Agrachanus scardinii (Molnár, 1966), Kalmanmolnaria intestinalis (Dogiel Bychowsky, 1934) and Skrjabillanus tincae Shigin Shigina, 1958 (Skrjabillanidae). Phylogenetic analysis of SSU rDNA data shows that dracunculoid nematodes are divided into two well-supported clades designated as Clade I and Clade II, respectively. Clade I includes the type species of the genus Philonema Kuitunen-Ekbaum, 1933, some species from the family Daniconematidae Moravec Køie, 1987 and two subfamilies of skrjabillanids, Skrjabillaninae Shigin Shigina, 1958 and Esocineminae Moravec, 2006. Clade II unites species from the families Dracunculidae Stiles, 1907, Micropleuridae Baylis Daubney, 1926 and Philometridae Baylis Daubney, 1926. Within the Philometridae, there are several well-supported groups of species, one of which unites freshwater Philometra spp. from the Palearctic cyprinids, identified as P. kotlani, P rischta, P. ovata (Zeder, 1803) and P. cyprinirutili (Creplin, 1825). However, the phylogenetic relationships of most philometrids are unresolved. An analysis of partial SSU and LSU rDNA sequences indicates that there is no direct phylogenetic relationship between Agrachanus Tikhomirova, 1971 (type species Skrjabillanus scardinii Molnár, 1966) and Skrjabillanus Shigin Shigina, 1958 (type species Sk. tincae), which means that the genus Agrachanus can be resurrected. Our study confirms that Philonematinae Ivashkin, Sobolev Khromova, 1971 should be elevated to the family rank. We formally establish the family Philonematidae Ivashkin, Sobolev Khromova, 1971 stat. nov. We also suggest combining the superfamilies Dracunculoidea Stiles, 1907 and Camallanoidea Railliet Henry, 1915 into the infraorder Camallanomorpha Roberts, Janovy Nadler, 2013.

Utilizing attached hard ticks as pointers to the risk of infection by Babesia and Theileria species in sika deer (Cervus nippon yesoensis), in Japan.

Ticks are hematophagous ectoparasites that have a significant impact on their animal hosts. Along with mosquitoes, they are the main arthropod vectors of disease agents in domestic animals, wildlife and humans. To investigate the occurrence and prevalence of piroplasmids in ticks, DNA was extracted from 519 hard ticks collected from 116 hunted Hokkaido sika deer (Cervus nippon yesoensis). The success of the DNA extraction was confirmed by touchdown PCR targeting the mitochondrial 16S rDNA gene of ticks. Touchdown PCR and reverse line blot (RLB) hybridization targeting the 18S rRNA gene were used to detect 14 piroplasm species. All hard ticks parasitizing Hokkaido sika deer were identified as belonging to the genera Ixodes and Haemaphysalis. In total 163 samples (31.4%) were positive for Babesia and Theileria spp. among tick species according to RLB hybridization. Tick DNA hybridized to the oligonucleotide probes of Theileria sp. Thrivae (27.0% of ticks; 140/519), Theileria capreoli (10.6%; 55/519), Babesia divergens-like (1.7%; 9/519), Babesia sp. (Bab-SD) (0.6%; 3/519), Babesia microti U.S. (0.4%; 2/519), and B. microti Hobetsu (0.4%; 2/519). The partial sequencing and phylogenetic analyses of the 18S rRNA gene confirmed the RLB hybridization results. Further investigations are needed to reveal the epidemiology and respective vectors of these pathogens.

Isolation and identification of herbivorous ciliates from contaminated microalgal cultures

Ciliates are a common but understudied group of grazers that can invade microalgal cultures. To estimate the potential impact of ciliates on microalgal culture productivity, the identification of species that can invade these cultures is essential. Furthermore, isolation of these herbivorous ciliates allows to use them in experiments that investigate the impact of ciliate grazing on the productivity of microalgal cultures. The main aims of this study were to isolate and identify ciliates that invade cultures of the freshwater microalgae Chlorella and Chlamydomonas, and to establish a live collection of these ciliates for usage in future experiments. To this end, we optimized a method for isolating ciliates from contaminated microalgal cultures and we developed a new PCR primer set for amplifying the partial 18S rDNA of ciliates belonging to the classes Spirotrichea, Oligohymenophorea and Colpodea. As a result, we isolated 11 ciliates from microalgal enrichment cultures inoculated with non-sterile dust and various freshwater sources. Of these 11 species, 7 were found to be feeding on Chlamydomonas. Ciliate species that fed on Chlorella could not be isolated in this study. Ciliate species feeding on Chlamydomonas were identified based on a combination of morphological observations and molecular analyses of partial 18S rDNA sequences.

Fungicide-Tolerant Plant Growth-Promoting Rhizobacteria Mitigate Physiological Disruption of White Radish Caused by Fungicides Used in the Field Cultivation.

Excessive use of fungicides in agriculture may result in substantial accumulation of active residues in soil, which affect crop health and yield. We investigated the response of Raphanus sativus (white radish) to fungicides in soil and potential beneficial interactions of radish plants with fungicide-tolerant plant growth-promoting rhizobacteria (PGPR). The PGPR were isolated from cabbage and mustard rhizospheres. Morphological and biochemical characteristics measured using standard methods, together with analysis of partial 16S rRNA gene sequences, revealed that fungicide-tolerant PGPR, isolates PS3 and AZ2, were closely related to Pseudomonas spp. These PGPR survived in the presence of high fungicide concentrations i.e., up to 2400 μg mL−1 carbendazim (CBZM) and 3200 μg mL−1 hexaconazole (HEXA). Bacterial isolates produced plant growth stimulants even under fungicide stress, though fungicides induced surface morphological distortion and alteration in membrane permeability of these bacteria, which was proved by a set of microscopic observations. Fungicides considerably affected the germination efficiency, growth, and physiological development of R. sativus, but these effects were relieved when inoculated with PGPR isolates. For instance, CBZM at 1500 mg kg−1 decreased whole dry biomass by 71%, whole plant length by 54%, total chlorophyll by 50%, protein content by 61%, and carotenoid production by 29%. After applying isolate AZ2 for white radish grown in CBZM (10 mg kg−1)-amended soil, it could improve plant growth and development with increased whole plant dry weight (10%), entire plant length (13%) and total chlorophyll content (18%). Similarly, isolate PS3 enhanced plant survival by relieving plant stress with declined biomarkers, i.e., proline (12%), malondialdehyde (3%), ascorbate peroxidase (6.5%), catalase (18%), and glutathione reductase (4%). Application of isolates AZ2 and PS3 could be effective for remediation of fungicide-contaminated soil and for improving the cultivation of radish plants while minimizing inputs of fungicides.

Screening of polyhydroxybutyrate producing indigenous bacteria from polluted lake soil

The prime aim of this study was to enumerate predominant bacteria from polluted lake soil samples, which possess polyhydroxybutyrate (PHB) fabricating potential and identify the suitable growth conditions and nutritional factors for PHB fabrication. From several numbers of bacterial cultures, one culture has the competence to yield PHB, and it was endorsed through Sudan Black B stain, Nile red staining, SEM analysis, and growth in PHB selective media. Under the microscopic observation, the fluorescent cells and polymeric granules were observed in the fluorescent microscope and SEM, respectively. This PHB fabricating isolate was recognized as Bacillus cereus NDRMN001 through 16S rRNA partial sequence analysis. The structural characteristics of PHB produced by B. cereus NDRMN001 were studied through FT-IR, 1H NMR, and 13C NMR analysis. The peak observed at 1759.27 cm−1 on FT-IR analysis is corresponding to the signal band of PHB. In 1H NMR peaks were noticed at 1.67, 2.37 to 2.71, and 3.38 to 7.68 which corresponding to –CH3, –CH2, and –CH protons of PHB. About 4 notable peaks were noticed in 13C NMR analysis at 19.62, 68.27, 40.68, and 169.11 ppm which appeared close to the carboxyl group of PHB. About 10% of inoculum, pH 7.5, 2 g L of yeast extract, 20 g L of rice bran, 35 °C, and 2 days of incubation were recognized as optimal growth conditions for B. cereus NDRMN001 to produce PHB. The identified B. cereus NDRMN001 has the potential to yield 91.48% of PHB as 33.19 g L of PHB from 36.26 g L of culture biomass. The complete results conclude that the B. cereus NDRMN001 screened from polluted lake soil has the competence to produce fine quality and quantity of PHB in a short duration of fabrication process under favorable conditions with the utilization of cheap nutritional factors.