Preliminary findings on the gastrointestinal parasites of the brown bear (Ursus arctos) in the Cantabrian mountains, Spain.

No study is currently available on the parasitofauna of the population of brown bears (Ursus arctos) inhabiting the Cantabrian Mountains in Spain. The aim of the present study was to obtain novel information on diversity and prevalence of gastrointestinal parasites in these individuals. During August 2016 and from May to July 2017, 14 fecal samples were collected from the western Cantabrian bear subpopulation, in the Somiedo Natural Park, in the Spanish province of Asturias. The prevalence of parasites detected was 71% and two genera were identified: Dicrocoelium sp. and Trichuris sp. Since the impact that pathogens such as endoparasites can have on the health of bears, together with other stressors, is still poorly understood, research efforts that include disease surveillance are critical to the successful protection of this emblematic species. Our preliminary findings require further investigations, with a wider sampling effort, and bring awareness for the need of carrying further studies on this area as a part of a proactive species management plan.

Genetic variability, cryptic species and phylogenetic relationship of six cyathostomin species based on mitochondrial and nuclear sequences.

Cyathostomins are important intestinal nematode parasites of equines and include 50 accepted species. Their taxonomy has been frequently revised and the presence of cryptic species suggested. Furthermore, usually molecular- and morphology-based phylogenetic analyses give divergent results. In this study, the nucleotide sequences of the nuclear second internal transcribed spacer (ITS-2) and the mitochondrial partial cytochrome c oxidase subunit I (COI) were determined for adults of six cyathostomin species (Coronocyclus coronatus, Coronocyclus labiatus, Cylicocyclus nassatus, Cylicostephanus calicatus, Cylicostephanus longibursatus, Cylicostephanus minutus) collected from different equine species within two geographic regions. Maximum likelihood trees were calculated for ITS-2, COI, and concatenated data. No obvious differentiation was observed between geographic regions or equine host species. As previously reported, Coronocyclus coronatus and Cylicostephanus calicatus revealed a close relationship. Cryptic species were detected in Cylicostephanus minutus and Cylicostephanus calicatus. Cylicocyclus nassatus and Coronocyclus labiatus showed diverse mitochondrial and nuclear haplotypes occurring in different combinations, while Cylicostephanus longibursatus was comparatively homogenous. In conclusion, a combined analysis of nuclear and mitochondrial haplotypes improved resolution of the phylogeny and should be applied to the remaining cyathostomin species and across additional equine host species and geographic regions.

Nuclear and mitochondrial marker sequences reveal close relationship between Coronocyclus coronatus and a potential Cylicostephanus calicatus cryptic species complex.

The Cyathostominae (Nematoda, Strongyloidea) parasitising equines represent a diverse group currently including 50 species. However, their taxonomy has been repeatedly revised and occasionally the presence of cryptic genospecies was suggested. Moreover, molecular- and morphology-based phylogenetic analyses give divergent results. For instance, molecular data have suggested close relationship between Coronocyclus coronatus and Cylicostephanus calicatus, although morphology-based taxonomy places them in different genera. Here, nuclear (internal transcribed spacer 2, ITS-2) and mitochondrial (cytochrome oxidase I, COI) sequences were obtained from the same individual, morphologically identified worms. In both morphospecies, two ITS-2 sequences types were observed: In Cor. coronatus, a small PCR product of 278 bp (nuclear haplotype group nHGBco) was always present but often in combination with a larger 369-370 bp fragment (nHGAco). In Cys. calicatus, either a large 370 bp product (nHGAca) or a short 281 bp amplicon (nHGBca) were found, but never both. Sequence identity between morphospecies was up to 100%. The smaller differed from the larger fragments by deletion of the region 110-198 in Cor. coronatus and 112-203 in Cys. calicatus. In COI, three and five mitochondrial haplotype groups (HGs), mtHG1co-mtHG3co and mtHG1ca-mtHG5ca were identified for Cor. coronatus and Cys. calicatus, respectively. In Cor. coronatus, there was no particular association of mtHG with nuclear genotypes (only nHGBco vs. both nHGBco plus nHGAco). In Cys. calicatus the nHGAca was always associated with the mtHG1ca, mtHG2ca or mtHG5ca whereas nHGBca was exclusively associated with mtHG3ca or mtHG4ca. Despite up to 100% identity in the nHGs, no mixing of mtHGs was observed between both species. Clear separation of certain nHGs with particular mtHGs in Cys. calicatus, despite the fact that the same host individuals were infected with both groups simultaneously, suggests presence of two non-interbreeding genospecies within Cys. calicatus, which needs further confirmation using additional samples from diverse geographical origins.

Lower within-community variance of negative density dependence increases forest diversity.

Local abundance of adult trees impedes growth of conspecific seedlings through host-specific enemies, a mechanism first proposed by Janzen and Connell to explain plant diversity in forests. While several studies suggest the importance of this mechanism, there is still little information of how the variance of negative density dependence (NDD) affects diversity of forest communities. With computer simulations, we analyzed the impact of strength and variance of NDD within tree communities on species diversity. We show that stronger NDD leads to higher species diversity. Furthermore, lower range of strengths of NDD within a community increases species richness and decreases variance of species abundances. Our results show that, beyond the average strength of NDD, the variance of NDD is also crucially important to explain species diversity. This can explain the dissimilarity of biodiversity between tropical and temperate forest: highly diverse forests could have lower NDD variance. This report suggests that natural enemies and the variety of the magnitude of their effects can contribute to the maintenance of biodiversity.

Screening for anthelmintic resistance in equid strongyles (Nematoda) in Romania.

Resistance to anthelmintic medication of equid strongyles is a worldwide phenomenon and for this reason systematic investigations of resistant parasite populations are necessary. The purpose of the present study was to investigate the presence and distribution of equid strongyles resistant to the anthelmintics used in Romania, as well as the pre-treatment and post-treatment prevalence of species of strongylid nematodes. The Faecal Egg Count Reduction Test was performed between 2010 and 2013 on a total number of 588 horses and 23 donkeys from 26 locations (subgroups). Animals of the first group (I) consisting of subgroups no. 1-11 were treated with Albendazole (ABZ), those of the second group (II) consisting of subgroups no. 12-23 with Fenbendazole (FBZ), while Ivermectin (IVM) was used on animals of the third group (III) consisting of subgroups no. 24-26. Resistant strongyles have been found in 82% (average lower limit of the 95% confidence interval, LCL95%, was 65) of the total equids from the group treated with ABZ. In the group of horses treated with FBZ, resistant strongyles were identified in 75% of the subgroups (LCL95% = 44). No resistant strongyles have been identified in IVM-treated horse groups (LCL95% = 98). The pre-treatment prevalence of the species of the Strongylinae Müller, 1780 was 22%, whereas that with nematodes of the subfamily Cyathostominae Molin, 1861 78%. Post-treatment reduction of strongyline nematodes was observed (5%), which demonstrates the sensitivity of large strongyles to common anthelmintics. The post-treatment prevalence of cyathostomes was of 95%, which proves their resistance, especially to ABZ- and FBZ-based anthelmintics.

Mutation scanning-based analysis of Theileria orientalis populations in cattle following an outbreak.

Bovine theileriosis is a tick-borne disease caused by one or more hemoprotozoan parasites of the genus Theileria. In the past, Theileria infection in cattle in Australia was largely asymptomatic and recognized to be associated with Theileria buffeli. However, outbreaks of theileriosis have occurred in beef and dairy cattle in subtropical climatic regions (New South Wales) of Australia. There is also one published report of a recent theileriosis outbreak in a beef farm near Seymour in the southeastern state of Victoria. In order to gain an improved insight into the genetic composition of Theileria populations following this outbreak, we undertook herein an integrated PCR-coupled mutation scanning-sequencing-phylogenetic analysis of sequence variation in part of the major piroplasm surface protein (MPSP) gene within and among samples from cattle involved in the outbreak. Theileria DNA was detected in 89.4% of 94 cattle in the Seymour farm; the genetic analysis showed that the ikeda and chitose genotypes representing the Theileria orientalis complex were detected in 75 and 4.8% of 84 infected cattle, respectively, and that mixed populations of these two genotypes were found in 20.2% of infected cattle. Given unpublished reports of a significant increase in the number of outbreaks in Victoria, future investigations should focus sharply on elucidating the epidemiology of Theileria to subvert the economic impact on the cattle industry in this state. Although used here to explore genetic variation within the T. orientalis complex in Australia, a mutation scanning-based approach has broad applicability to other species of Theileria in other countries.

Pervasive sign epistasis between conjugative plasmids and drug-resistance chromosomal mutations.

Multidrug-resistant bacteria arise mostly by the accumulation of plasmids and chromosomal mutations. Typically, these resistant determinants are costly to the bacterial cell. Yet, recently, it has been found that, in Escherichia coli bacterial cells, a mutation conferring resistance to an antibiotic can be advantageous to the bacterial cell if another antibiotic-resistance mutation is already present, a phenomenon called sign epistasis. Here we study the interaction between antibiotic-resistance chromosomal mutations and conjugative (i.e., self-transmissible) plasmids and find many cases of sign epistasis (40%)--including one of reciprocal sign epistasis where the strain carrying both resistance determinants is fitter than the two strains carrying only one of the determinants. This implies that the acquisition of an additional resistance plasmid or of a resistance mutation often increases the fitness of a bacterial strain already resistant to antibiotics. We further show that there is an overall antagonistic interaction between mutations and plasmids (52%). These results further complicate expectations of resistance reversal by interdiction of antibiotic use.

Survey of infectious and parasitic diseases in stray cats at the Lisbon Metropolitan Area, Portugal.

A survey of infectious and parasitic diseases of stray cats was carried out using biological samples collected from animals captured during a catch-neuter-release programme in four counties of the Lisbon Metropolitan Area. The main objective was to investigate the potential threat of stray cats for animal and public health. Samples of blood, stool, hair and auricular swabs were collected from 231 cats in 27 colonies. Anti-Toxoplasma gondii antibodies were detected in 47/194 samples (24.2%); anti-Leishmania infantum antibodies in 1/180 cats (0.6%); intestinal parasites in 23/74 samples (Toxocara cati, Isospora felis, Ancylostoma tubaeforme, Dipylidium caninum, Uncinaria stenocephala, Toxascaris leonina) and Otodectes cynotis in 4/182 cats (2.2%); dermatophyte fungi were isolated in 40/136 samples (29.4%); feline immunodeficiency virus antibodies were detected in 23/226 samples (10.2%); feline leukaemia virus antigen in 14/198 samples (7.1%); and feline coronavirus RNA in 9/127 samples (7.1%). Our results revealed that zoonotic agents, namely dermatophyte fungi and Toxocara cati were present in stray cat colonies in the investigated counties. Overall the low frequency of major pathogens suggests a balanced relationship between host and agents.

Analysis of genetic variation in Globocephaloides populations from macropodid marsupials using a mutation scanning-based approach.

Three species of Globocephaloides, parasitic nematodes occurring in macropodid marsupials in different areas of Australia, were characterized by the sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA. Samples were subjected to PCR-coupled SSCP analysis and targeted sequencing, in order to assess genetic variation within and among individuals from different host species. Both SSCP and sequence data supported the current classification of morphospecies. Contrary to a previous hypothesis that cryptic species exist within the Globocephaloides trifidospicularis "complex", no or minor (0.2%) variation was detected among individuals from different hosts or geographical origins. Within G. macropodis populations, there was a consistent difference in both the ITS-1 (5.2%) and ITS-2 (7.1%) sequences between individuals derived from Macropus agilis and those from Macropus dorsalis. Although the results suggested that G. macropodis from each host species represented sibling species, future morphological study of individuals representing each G. macropodis genotype is warranted to provide further support for this hypothesis. (Nucleotide sequence data have been deposited in the GenBank database under accession nos. GQ131400-GQ131409.).

1H and 13C NMR signal assignments of some new spiro[7H-benzo[de]anthracene-naphthopyrans].

The complete 1H and 13C NMR signal assignment of four new photochromic spiro[7H-benzo[de]anthracene-naphthopyrans] was achieved using one- and two-dimensional methods (DEPT, COSY, HSQC, HMBC, NOESY).

Arbuscular mycorrhizal fungal propagules in a salt marsh.

The tolerance of indigenous arbuscular mycorrhizal fungi (AMF) to stressful soil conditions and the relative contribution of spores of these fungi to plant colonization were examined in a Portuguese salt marsh. Glomus geosporum is dominant in this salt marsh. Using tetrazolium as a vital stain, a high proportion of field-collected spores were found to be metabolically active at all sampling dates. Spore germination tests showed that salt marsh spores were not affected by increasing levels of salinity, in contrast to two non-marsh spore isolates, and had a significantly higher ability to germinate under increased levels of salinity (20 per thousand) than in the absence of or at low salinity (10 per thousand). Germination of salt marsh spores was not affected by soil water levels above field capacity, in contrast to one of the two non-marsh spore isolates. For the evaluation of infectivity, a bioassay was established with undisturbed soil cores (containing all types of AM fungal propagules) and soil cores containing only spores as AM fungal propagules. Different types of propagules were able to initiate and to expand the root colonization of a native plant species, but spores were slower than mycelium and/or root fragments in colonizing host roots. The AM fungal adaptation shown by this study may explain the maintenance of AMF in salt marshes.

A new xanthone from Hedychium gardnerianum.

A new xanthone (2), 3-(2-hydroxyethoxy)xanthone, and three known compounds, 1-hydroxyxanthone, salicylic acid and the sesquiterpene oplopanone, were isolated for the first time from the rhizomes of Hedychium gardnerianum Rosc. The structures of these compounds were elucidated on the basis of detailed spectral analysis, including 2D NMR experiments.