Phylogenetic Analysis of Cryptosporidium Parasites Based on the Small-Subunit rRNA Gene Locus

Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentis belonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known as Cryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed.     

Zygotorulaspora cornina sp. nov. and Zygotorulaspora smilacis sp. nov., Two Novel Ascomycetous Yeast Species Isolated from Plant Flowers and Fruits

Three isolates belonging to the ascomycetous genus Zygotorulaspora were obtained from the fruits of Cornus officinalis and Smilax china, and flowers of Dendranthema zawadskii var. latilobum in Gongju-si, Korea. Phylogenetic Analyses of the LSU D1/D2 domain and ITS region sequences supported the recognition of two new species: Zygotorulaspora cornina sp. nov. (type strain NIBRFGC000500475 = KACC93346P^PP) and Zygotorulaspora smilacis sp. nov. (type strain NIBRFGC000500476 = KACC93347P^PP). The two novel species revealed no growth on D-Galactose, unlike the other six species in the genus Zygotorulaspora. They are distinguished from each other by their phylogenetic differences and phenotypic characteristics such as assimilation of xylitol, 5-keto-D-gluconate, and ethanol. All species in the genus Zygotorulaspora including the two novel species have phenotypic traits of genus Zygotorulaspora: asci are persistent, sucrose and raffinose are assimilated, and m-inositol is not required for growth, and they are mainly associated with plants.     

A new species of Lapparentophis from the mid-Cretaceous Kem Kem beds,Morocco, with remarks on the distribution of lapparentophiid snakes

Two isolated trunk vertebrae from the ?uppermost Albian–lower Cenomanian Kem Kem beds of Morocco are described and assigned to Lapparentophis, an early snake genus known from coeval deposits in Algeria. The Moroccan specimens represent a new species, Lapparentophis ragei, which can be distinguished from the type and only known species, Lapparentophis defrennei, by its smaller size, its more elongate vertebrae, the presence of parazygosphenal foramina, and paradiapophyses extending anteroventrally closer to the cotyle. The discovery of Lapparentophis in the Kem Kem beds adds to the relatively diverse snake assemblage previously reported from this formation and extends the geographical range of the genus. The distribution of Lapparentophis and lapparentophiid-grade (?lapparentophiid) snakes is discussed. This poorly known family of terrestrial snakes seems to be restricted to the latest Albian–early Cenomanian of North Africa, with the exception of Pouitella from the early–middle Cenomanian of France. As for many other vertebrate taxa of this period, this distribution is consistent with a dispersal event from Africa to the western part of the European archipelago.     

A new tapeworm from Compsophis infralineatus (Pseudoxyrhophiidae), an endemic snake of Madagascar: Scratching the surface of undiscovered reptilian parasite diversity

A new species of proteocephalid cestodes, provisionally assigned to the polyphyletic genus Ophiotaenia La Rue, 1911 (Cestoda: Proteocephalidae), is described from Compsophis infralineatus (Günther) (Serpentes: Pseudoxyrhophiidae) endemic to Madagascar. Ophiotaenia oreae n. sp. differs from all African and Asian species of Ophiotaenia by possessing more uterine diverticula (68–82 on one side). It is also characterised by the absence of an apical organ, the relative sizes of the cirrus-sac and ovary, the almost equatorial position of the gonopore, the diameter of the embryophore, and other biometric characteristics. Phylogenetic relationships of the new species indicate its relatedness to Indomalayan and Australasian proteocephalids from reptiles. Ophiotaenia oreae n. sp. formed a well-supported clade composed of species of Australophiotaenia de Chambrier, Beveridge and Scholz, 2018 from Australian snakes, Macrobothriotaenia ficta (Meggitt, 1931) from Xenopeltis unicolor Reinwardt in Boie from Vietnam, Ophiotaenia sp. from Trimeresurus flavomaculatus (Gray) from the Philippines, and Ophiotaenia bungari de Chambrier, Binh and Scholz, 2012 from Bungarus fasciatus (Schneider) from Vietnam. The only proteocephalid from Madagascan snakes sequenced so far, Ophiotaenia lapata Rambeloson, Ranaivoson and de Chambrier, 2012 from Madagascarophis colubrinus (Schlegel), does not form a monophyletic group with the new species. The actual species diversity of reptilian cestodes in Madagascar is undoubtedly underestimated. Because of the assumed strict (oioxenous) host specificity of reptilian proteocephalids and rich fauna of snakes occurring in Madagascar, it is plausible to expect the existence of dozens new species of proteocephalids on this island.     

Meiothermus rufus sp. nov., a new slightly thermophilic red-pigmented species and emended description of the genus Meiothermus

Four red-pigmented isolates, with optimum growth temperatures of approximately 55–60 °C and an optimum pH for growth between 7.5 and 8.5, were recovered from hot springs in Central France. Phylogenetic analysis of the 16S rRNA gene sequences showed that these organisms represented a new species of the genus Meiothermus. The new isolates could be distinguished from other strains of the species of the genus Meiothermus primarily by the glycolipid profile and fatty acid composition because these organisms lacked the hydroxy fatty acids and the glycolipid variant GL-1a found in all other isolates of the species of Meiothermus examined. On the basis of the results presented here we propose the name Meiothermus rufus for the new species, which is represented by strains CAL-4^T (=DSM 22234^T=LMG 24878^T) and CAL-12 (=DSM 22235=LMG 24879). We also propose emending the genus Meiothermus to include strains that have only one glycolipid instead of two glycolipid variants.     

Occurrence,Diversity, and Host Association of Intestinal Campylobacter,Arcobacter, and Helicobacter in Reptiles

Campylobacter, Arcobacter, and Helicobacter species have been isolated from many vertebrate hosts, including birds, mammals, and reptiles. Multiple studies have focused on the prevalence of these Epsilonproteobacteria genera in avian and mammalian species. However, little focus has been given to the presence within reptiles, and their potential zoonotic and pathogenic roles. In this study, occurrence, diversity, and host association of intestinal Epsilonproteobacteria were determined for a large variety of reptiles. From 2011 to 2013, 444 cloacal swabs and fecal samples originating from 417 predominantly captive-held reptiles were screened for Epsilonproteobacteria. Campylobacter, Arcobacter, and Helicobacter genus specific PCRs were performed directly on all samples. All samples were also cultured on selective media and screened for the presence of Epsilonproteobacteria. Using a tiered approach of AFLP, atpA, and 16S rRNA sequencing, 432 Epsilonproteobacteria isolates were characterized at the species level. Based on PCR, Campylobacter, Arcobacter, and Helicobacter were detected in 69.3% of the reptiles; 82.5% of the chelonians, 63.8% of the lizards, and 58.0% of the snakes were positive for one or more of these genera. Epsilonproteobacteria were isolated from 22.1% of the reptiles and were isolated most frequently from chelonians (37.0%), followed by lizards (19.6%) and snakes (3.0%). The most commonly isolated taxa were Arcobacter butzleri, Arcobacter skirrowii, reptile-associated Campylobacter fetus subsp. testudinum, and a putative novel Campylobacter taxon. Furthermore, a clade of seven related putative novel Helicobacter taxa was isolated from lizards and chelonians. This study shows that reptiles carry various intestinal Epsilonproteobacteria taxa, including several putative novel taxa.     

Aliarcobacter vitoriensis sp. nov., isolated from carrot and urban wastewater

Two isolates, one recovered from a carrot and another one from urban wastewater, were characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both isolates clustered together, and were most closely related to Aliarcobacter lanthieri. Multilocus phylogenetic analysis (MLPA) using the concatenated sequences of five housekeeping genes (atpA, gyrA, gyrB, hsp60 and rpoB) suggested that these isolates formed a distinct phylogenetic lineage among the genera derived from the former genus Arcobacter. Whole-genome sequence, in silico DNA-DNA hybridization (isDDH) and the average nucleotide identity (ANI) value between the genome of strain F199^T and those of related species confirmed that these isolates represent a novel species. These strains can be differentiated from its phylogenetically closest species A. lanthieri by its inability to growth on 1% glycine and by their enzyme activity of esterase lipase (C8) and acid phosphatase. Our results, by the application of a polyphasic analysis, confirmed that these two isolates represent a novel species of the genus Aliarcobacter, for which the name Aliarcobacter vitoriensis sp. nov. is proposed. The type strain is F199^T (=CECT 9230^T=LMG 30050^T).     

Meiothermus granaticius sp. nov., a new slightly thermophilic red-pigmented species from the Azores

Two red-pigmented isolates, with optimum growth temperatures between 45 and 50 °C, were recovered from a hot spring in the Furnas, Área da Fonte 1825 on the Island of São Miguel in the Azores. Phylogenetic analysis of the 16S rRNA gene sequences showed that these organisms represented a new species of the genus Meiothermus. These new isolates could be distinguished from other strains of the species of the genus Meiothermus primarily by the fatty acid composition and polar lipid pattern, since they did not possess 2-OH fatty acids or glycolipid variant GL-1a. Moreover, the two new isolates had the lowest growth temperature range of any of the known species of the genus Meiothermus. On the basis of the results presented here we propose the name Meiothermus granaticius for the new species represented by strains AF-68^T (=DSM 23260^T = LMG 25524^T) and AF-49 (=DSM 23259 = LMG 25525).     

Pantoea beijingensis sp. nov., isolated from the fruiting body of Pleurotus eryngii

Four Gram-negative-staining, facultatively anaerobic bacterial isolates were obtained from a fruiting body of the edible mushroom Pleurotus eryngii showing symptoms of soft rot disease in Beijing, China. Partial 16S rRNA gene sequencing, together with partial rpoB sequencing, placed these isolates in the genus Pantoea. Multilocus sequence analysis based on the partial sequences of gyrB, rpoB, infB and atpD revealed Pantoea dispersa and Pantoea gaviniae as their closest phylogenetic relatives and indicated that these isolates constituted a possible novel species. DNA–DNA hybridization studies confirmed the classification of the new isolates as a novel species and phenotypic tests allowed for differentiation from the closest phylogenetic neighbours. The name Pantoea beijingensis sp. nov. [type strain LMG 27579^T = KCTC 32406^T = JZB2120001^T (deposited at Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences)] is proposed.     

Thiobacillus sajanensis sp. nov., a new obligately autotrophic sulfur-oxidizing bacterium isolated from Khito-Gol hydrogen-sulfide springs, Buryatia

Four strains of rod-shaped gram-negative sulfur-oxidizing bacteria were isolated from Khoito-Gol hydrogen-sulfide springs in the eastern Sayan Mountains (Buryatia). The cells of the new isolates were motile by means of a single polar flagellum. The strains were obligately chemolithoautotrophic aerobes that oxidized thiosulfate (with the production of sulfur and sulfates) and hydrogen sulfide. They grew in a pH range of 6.8–9.5, with an optimum at pH 9.3 and in a temperature range of 5–39°C, with an optimum at 28–32°C. The cells contained ubiquinone Q-8. The DNA G+C content of the new strains was 62.3–64.2 mol %. According to the results of analysis of their 16S rRNA genes, the isolates belong to the genus Thiobacillus within the subclass Betaproteobacteria. However, the similarity level of nucleotide sequences of the 16S rRNA genes was insufficient to assign the isolates to known species of this genus. The affiliation to the genus Thiobacillus was confirmed by DNA-DNA hybridization of the isolates with the type strain of the type species of the genus Thiobacillus, T. thioparus DSM 505^T (= ATCC 8158^T). Despite the phenotypic similarity, the hybridization level was as low as 21–29%. In addition, considerable differences were revealed in the structure of the genes encoding RuBPC, the key enzyme of autotrophic CO₂ assimilation, between the known Thiobacillus species and the new isolates. Based on molecular-biological features and certain phenotypic distinctions, the new isolates were assigned to a new Thiobacillus species, T. sajanensis sp. nov., with the type strain 4HG^T (= VKM B-2365^T).     

A new species of Leptobacillim,L. symbioticum,isolated from mites and sori of soybean rust

A verticillium-like fungus forming a whitish colony and mainly solitary phialides that produced fusiform to cylindrical conidia in chains was isolated from uredinia of Phakopsora pachyrhizi, a causal agent of soybean rust. In addition, two similar looking isolates were obtained from Prostigmata mites. Our taxonomic study based on morphology and phylogenetic analysis using ITS rDNA and LSU rDNA D1/D2 region sequences revealed that the three isolates were the same species and assigned to the genus Leptobacillium. These isolates represent a new species, morphologically and phylogenetically distinguished from the type species, L. leptobactrum, and we propose L. symbioticum sp. nov. In addition, Simplicillium chinense and S. coffeanum are assigned to the genus Leptobacillium.     

Finding of Bov-B LINE retroelement in parthenogenetic and bisexual lizard species of the genus Darevskia (Lacertidae)

The Bov-B LINE retrotransposon was first discovered in Ruminantia and was long considered to be specific for this order. Later, this mobile element was described in snakes and some lizard species. Analysis of phylogenetic relationships of Bov-B LINE elements from different ruminants, snakes, and lizard species led to the suggestion on horizontal transfer of this retrotransposon from Squamata to Ruminantia. In the Squamata group, Bov-B LINE element was found in all snakes and some lizard species examined. The element was not detected in the genomes of some species of the genera Lacerta and Podarcis. In the present study, using PCR amplification and sequencing of PCR products, Bov-B LINE element was identified in the genomes of parthenogenetic and bisexual species of the genus Darevskia (Lacertidae), as well as in such species as Lacerta agilis and Zootoca vivipara, where this retrotransposon had not been not detected before.     

Molecular cloning and sequence analysis of cDNAs coding for a serine proteinase and a Kunitz-type inhibitor in the venom gland of the Vipera nikolskii viper

Serine proteinases and Kunitz-type inhibitors are widely represented in the venoms of snakes belonging to different genera. During the studies of the venoms of snakes inhabiting Russia, we have cloned cDNAs coding for novel proteins of these families. A novel serine proteinase that we named nikobin was identified in the venom gland of the Nikolsky viper. The amino acid sequence of nikobin deduced from the cDNA sequence slightly differs from those of the serine proteinases found in other snakes, displaying 15 unique amino acid substitutions. This is the first serine proteinase from a viper of the Vipera genus for which the complete amino acid sequence has been determined. A cDNA coding for a Kunitz-type inhibitor has also been cloned. The deduced amino acid sequence of the inhibitor displays overall homology to the already known sequences of analogous proteins from vipers of the Vipera genus. However, several unusual amino acid substitutions that can cause a change of the inhibitor activity have been detected.     

A new species of Serpentirhabdias Tkach,Kuzmin & Snyder, 2014 (Nematoda: Rhabdiasidae) parasitic in the brown ground snake Atractus major Boulenger (Reptilia: Serpentes: Dipsadidae) in Brazil

Serpentirhabdias atracti n. sp. is described based on specimens discovered in the lung of Atractus major Boulenger from Caxiuanã National Forest, Pará, Brazil. The new species is assigned to Serpentirhabdias Tkach, Kuzmin & Snyder, 2014 based on morphological characters (comparatively thin body cuticle without prominent inflations, arrangement of circumoral papillae in two lateral groups, pre-equatorial position of vulva, eggs in uteri at early cleavage stages), as well as because of its parasitism in snakes. The new species is most similar to S. vellardi (Pereira, 1928) due to the absence of lips and buccal capsule, similar body dimensions, and the specificity to dipsadid snakes in Brazil. The two species differ in the shape of the tail (bulbous dilatation in the posterior part followed by a thread-like tail tip present in S. atracti n. sp.), the width of the oesophagus, and the size of the excretory glands. Serpentirhabdias atracti n. sp. is the sixth species of this genus found in the Neotropical Region.     

First description of two moderately halophilic and psychrotolerant Mycoplasma species isolated from cephalopods and proposal of Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov

Two moderately halophilic and psychrotolerant new Mycoplasma species were isolated from common cephalopods. Three strains were isolated in pure culture from two individual European flying squid (Todarodes sagittatus), and two individual octopuses (Octopus vulgaris). The strains showed optimal growth at 25 °C and a salinity of 3% (w/v) NaCl. Molecular analyses revealed that the isolates belonged to two new, but phylogenetically related species, divergent from all previously described Mollicutes, representing the first marine isolates of the class, and also the first Mycoplasma strains for which NaCl requirement has been demonstrated. A genome search against all available marine metagenomes and 16S rRNA gene databases indicated that these two species represent a novel non-free-living marine lineage of Mollicutes, specifically associated with marine animals. Morphology and physiology were compatible with other members of this group, and genomic and phenotypic analyses demonstrated that these organisms represent two novel species of the genus Mycoplasma, for which the names Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov. are proposed; the type strains are PE^T (DSM 105487^T, CIP 111404^T) and 5H^T (DSM 105,488^T, CIP 111405^T), respectively.     

<Emphasis Type="Italic">Calcarisporium xylariicola</Emphasis> sp. nov. and introduction of <Emphasis Type="Italic">Calcarisporiaceae</Emphasis> fam. nov. in Hypocreales

During a survey of fungicolous fungi, a novel taxon from the surface of stroma of an unidentified Xylaria species was collected. Phylogenetic analyses showed that this taxon clustered with Calcarisporium sp. and C. arbuscula isolates, but was resolved as a distinct species. A detailed morphological examination coupled with phylogenetic analysis indicated that the taxon represented a new species. Calcarisporium xylariicola sp. nov. is thus introduced. The new taxon is characterized by short conidiophores with swollen bases and less length/width ratio of conidia that distinguish it from other Calcarisporium species. Calcarisporium is presently placed in Hypocreales genera, incertae sedis genus. Species in the genus are largely fungicolous, or occasionally caulicolous or foliicolous, and have hyaline, erect, verticillate conidiophores and sympodial, polyblastic conidiation. A phylogenetic analysis of combined SSU, ITS, LSU, TEF and RPB2 sequence data from Calcarisporium species and other taxa in Hypocreales indicate that Calcarisporium is a distinct lineage from other families. Therefore, a new family, Calcarisporiaceae, in Hypocreales is introduced.     

Diversity of haemogregarine parasites infecting Brazilian snakes from the Midwest and Southeast regions with a description of two new species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae)

Although Brazil is a hotspot for snake species, there is a lack of information on the biodiversity of haemoparasites infecting these hosts. Thus, the present study aimed to bring new insights on the diversity of species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) infecting Brazilian snakes from the Midwest and Southeast regions. The snakes were captured from 2018 to 2020 from the states of Mato Grosso, Mato Grosso do Sul, Goiás, and São Paulo. Three to five blood smears were made and the remaining blood sample was stored for further molecular analysis. Moreover, histopathological slides of the organs were stained with haematoxylin-eosin. Regarding molecular diagnosis, PCR was performed targeting different regions of the 18S rRNA gene of apicomplexan parasites. From the 13 free-living snakes screened, ten (76.92%) were found infected with Hepatozoon spp. Based on morphological and morphometric tools, five different morphotypes of species of Hepatozoon gamonts were detected. Molecular data and phylogenetic analysis support the morphological data, identifying five species of Hepatozoon from snakes, of which three species belong to previously described species, Hepatozoon cevapii, Hepatozoon cuestensis, and Hepatozoon quagliattus, with a genetic similarity of 100% (based on the 18S rRNA genetic marker). The present study identifies and describes two new species of Hepatozoon, Hepatozoon annulatum sp. nov. infecting the snake Leptodeira annulata and Hepatozoon trigeminum sp. nov. infecting the snake Oxyrhopus trigeminus. Thus, based on morphological and molecular data the present study provides new insights on haemogregarine diversity infecting Brazilian snakes from the Midwest and Southeast regions.