Sarcocystis neurona and Neospora caninum in Brazilian opossums (Didelphis spp.): Molecular investigation and in vitro isolation of Sarcocystis spp.

Sarcocystis neurona and Neospora spp. are protozoan parasites that induce neurological diseases in horses and other animal species. Opossums (Didelphis albiventris and Didelphis virginiana) are definitive hosts of S. neurona, which is the major cause of equine protozoal myeloencephalitis (EPM). Neospora caninum causes abortion in cattle and infects a wide range of animal species, while N. hughesi is known to induce neurologic disease in equids. The aims of this study were to investigate S. neurona and N. caninum in tissues from opossums in the northeastern Brazil, and to isolate Brazilian strains of Sarcocystis spp. from wild opossums for comparison with previously isolated strains. Carcasses of 39 opossums from Bahia state were available for molecular identification of Sarcocystis spp. and N. caninum in their tissues, and for sporocyst detection by intestinal scraping. In addition, Sarcocystis-like sporocysts from nine additional opossums, obtained in São Paulo state, were tested. Sarcocystis DNA was found in 16 (41%) of the 39 opossums' carcasses; N. caninum DNA was detected in tissues from three opossums. The sporocysts from the nine additional opossums from São Paulo state were tested by bioassay and induced infection in nine budgerigars, but in none of the gamma-interferon knockout mice. In vitro isolation was successful using tissues from all nine budgerigars. The isolated strains were maintained in CV-1 and Vero cells. Three of nine isolates presented contamination in cell culture and were discarded. Analysis of six isolates based on five loci showed that these parasites were genetically different from each other and also distinct from S. neurona, S. falcatula, S. lindsayi, and S. speeri. In conclusion, opossums in the studied regions were infected with N. caninum and Sarcocystis spp. and represent a potential source of infection to other animals. This is the first report of N. caninum infection in tissues from black-eared opossum (D. aurita or D. marsupialis) and white-eared opossum (D. albiventris). Brazilian opossums are probably infected by different Sarcocystis spp. distinct from S. neurona and S. falcatula, or present a high level of genetic recombination.

Anthropogenic impact on environmental filamentous fungi communities along the Mediterranean littoral.

We hypothesised that anthropogenic influences impact the filamentous fungi community structure and that particular species or species patterns might serve as markers to characterise ecosystems. This study aimed to describe the filamentous fungi community structure in various biotopes along the Mediterranean shore that were exposed to various levels of anthropogenic influence. We sampled filamentous fungi from yellow-legged gull faecal samples at five study sites along the Mediterranean littoral in southern France. The sites were characterised by variable anthropogenic influence, ranging from building rooftops in two cities to a natural reserve. The sites also included two suburban ecoclines, one of which was exposed to sewer pollution. Filamentous fungal colonies were quantified and identified via MALDI-TOF mass spectrometry. Interestingly, we found that both fungal diversity and abundance were low in urban areas compared with suburban ecocline or environments little affected by anthropogenic influence. Furthermore, some fungal species were clearly associated with particular environments. In particular, Mucor circinelloides was associated with a natural environment with little anthropogenic impact and distant from human settlements. Whereas, Scedosporium apiospermum was associated with an ecocline polluted by sewage. Our findings indicate that particular fungal species or species combination might be used as surrogate markers of ecosystems exposed to anthropogenic pollution.

Gut yeast communities in Larus michahellis from various breeding colonies.

Yellow-legged gulls have been reported to carry antibiotic-resistant Enterobacteriaceae; however, the gut mycobiota of these birds has not yet been described. In this study, we analyzed the gut yeast communities in five yellow-legged gull breeding colonies along the Mediterranean littoral in southern France. Gull fecal samples were inoculated onto four types of culture media, including one supplemented with itraconazole. Yeast species richness, abundance, and diversity were estimated, and factorial analysis was used to highlight correspondences between breeding colonies. Yeast grew in 113 of 177 cultures, and 17 distinct yeast species were identified. The most frequent species were Candida krusei (53.5%), Galactomyces geotrichum (44.1%), C. glabrata (40.9%), C. albicans (20.5%), and Saccharomyces cerevisiae (18.1%). Gut yeast community structure in the gulls at both Pierre-Blanche Lagoon (PB) and Frioul Archipelago (F) were characterized by greater species richness and diversity than in those at the two cities of La Grande-Motte (GM) and Palavas-les-Flots (PF) as well as Riou Archipelago (R). Gulls in these latter three sites probably share a similar type of anthropogenic diet. Notably, the proportion of anthropic yeast species, including C. albicans and C. glabrata, in the gull mycobiota increased with gull colony synanthropy. Antifungal resistance was found in each of the five most frequent yeast species. We found that the gut yeast communities of these yellow-legged gulls include antifungal-resistant human pathogens. Further studies should assess the public health impact of these common synanthropic seabirds, which represent a reservoir and disseminator of drug-resistant human pathogenic yeast into the environment.

Opportunistic fungal pathogen Candida glabrata circulates between humans and yellow-legged gulls.

The opportunistic pathogenic yeast Candida glabrata is a component of the mycobiota of both humans and yellow-legged gulls that is prone to develop fluconazole resistance. Whether gulls are a reservoir of the yeast and facilitate the dissemination of human C. glabrata strains remains an open question. In this study, MLVA genotyping highlighted the lack of genetic structure of 190 C. glabrata strains isolated from either patients in three hospitals or fecal samples collected from gull breeding colonies located in five distinct areas along the French Mediterranean littoral. Fluconazole-resistant isolates were evenly distributed between both gull and human populations. These findings demonstrate that gulls are a reservoir of this species and facilitate the diffusion of C. glabrata and indirect transmission to human or animal hosts via environmental contamination. This eco-epidemiological view, which can be applied to other vertebrate host species, broadens our perspective regarding the reservoirs and dissemination patterns of antifungal-resistant human pathogenic yeast.

Ecological implications on the aggregation of Amblyomma fuscum (Acari: Ixodidae) on Thrichomys laurentius (Rodentia: Echimyidae), in northeastern Brazil.

We investigated the Amblyomma fuscum load on a pullulating wild rodent population and the environmental and biological factors influencing the tick load on the hosts. One hundred and three individuals of Thrichomys laurentius were caught in an Atlantic forest fragment in northeastern Brazil, as part of a longitudinal survey on ticks infesting non-volant small mammals. Ticks (n = 342) were found on 45 individuals and the overall mean intensity of infestation was 7.6 ticks per infested rodent. Ticks were highly aggregated in the host population and the negative binomial distribution model provides a statistically satisfactory fit. The aggregated distribution was influenced by sex and age of the host. The microhabitat preference by T. laurentius probably increases contact opportunities between hosts and aggregated infesting stages of the ticks and represents important clues about the habitat suitability for A. fuscum.

Exposure of small mammals to ticks and rickettsiae in Atlantic Forest patches in the metropolitan area of Recife, North-eastern Brazil.

Between December 2007 and March 2009, small mammals were captured in 6 Atlantic Forest patches in Brazil. We assessed tick-host associations and whether they differ among forest strata, sites, seasons, and host age classes or between sexes. Moreover, we assessed the exposure of animals to Rickettsia spp. In total, 432 animals were captured and 808 ticks were found on 32·9% of them. Significant differences were found among host species, collection sites, and forest strata; microhabitat preference was a strong risk factor for tick infestation. The highest tick density rates were recorded in forest fragments settled in rural areas; 91·3% of the ticks were collected from animals trapped in these forest fragments. A high prevalence (68·8%) of antibodies to Rickettsia spp. was detected among animals. This study suggests that disturbed Atlantic Forest fragments provide an environment for ticks and small mammals, which are highly exposed to rickettsiae. It also indicates that forest patches settled in rural areas are usually associated with higher small mammal diversity as well as with higher tick density rates.

Relative importance of habitat and landscape scales on butterfly communities of urbanizing areas.

Agricultural decline and urbanization entail rapid alterations of the patterns of organization of rural landscapes in Europe. The spread of the urban footprint to the adjacent countryside contributes to the development of new anthropogenic ecosystems in formerly rural hinterlands. In this study, butterflies are considered as biological indicators of these rapid environmental changes. Our purpose is to better understand changes in biodiversity related to the evolution of available habitats in a mutating landscape. In this study, we investigate butterfly communities of four land-use types (fallow lands, gardens, vineyards, woodlands) within different landscape contexts. Our results reveal that variations in structure and functional composition of these communities are related to different levels of human disturbance at both landscape scale and habitat scale.

Staphylococcus aureus mastitis in a white-eared opossum (Didelphis albiventris) in the Atlantic Forest of northeast Brazil.

This is the first brief communication of clinical mastitis caused by Staphylococcus aureus occurring in a white-eared opossum (Didelphis albiventris) captured in a remnant of the Atlantic Forest in northeast Brazil. The bacterium was cultured from a milk sample by using sheep blood and Levine agars. Phenotyping of S aureus was performed with coagulase biochemical tests, DNase agar, mannitol agar, and anaerobic glucose and mannitol fermentation.

Dengue infection in neotropical forest mammals.

In South America, dengue is the arbovirus-transmitted disease with the highest incidence. Unlike other arboviruses, wild mammals have no confirmed role in the cycle of dengue in the neotropics, although serological studies have suggested a possible secondary amplification cycle involving mammals other than nonhuman primates. In French Guiana, where all four serotypes (DENV-1, DENV-2, DENV-3, DENV-4) are present, the disease is endemic with outbreak events. To determine whether wild mammals can be infected by DENV, rodents, marsupials, and bats were captured over several periods, from 2001 to 2007, at two sites. The first location is a secondary forest surrounded by an urban area where dengue is endemic. The second location is a forest edge site where the disease has not yet emerged. A total of 10,000 trap-nights were performed and 616 mammals were captured. RNAs representing the four DENV serotypes were detected at both sites by reverse-transcriptase polymerase chain reaction in the livers and/or sera of 92 mammals belonging to 14 out of 32 species distributed among all the orders investigated: Rodentia (33 positive/146 tested), Marsupialia (40/318), and Chiroptera (19/152). Sequence analyses of a portion of the capsid and premembrane junction revealed that mammal strains of DENV-1, DENV-2, DENV-3, and DENV-4 had only 92.6%, 89%, 95%, and 95.8% identity, respectively, with strains circulating in the human population during the same periods. Regarding DENV-2, strains related (99% identity) to those responsible for an epidemic event in humans in French Guiana concurrent to the capture sessions were also evidenced, suggesting that wild mammals in edge habitats can be infected by circulating human strains. Our results demonstrate, for the first time, that neotropical wild mammals can be infected with dengue virus. The question of whether mammals maintain DENV in enzootic cycles and can play a role in its reemergence in human populations remains to be answered.

Gardens in urbanizing rural areas reveal an unexpected floral diversity related to housing density.

The increasing urbanization of rural areas leads to a strong development of horticultural flora, which is the main source of alien and invasive plants. In order to assess the pool of cultivated species under different urbanization pressures, the diversity and distribution of horticultural flora were studied in 120 Mediterranean gardens belonging to three housing density types. The results showed a great richness and heterogeneity of this flora, and similarities in species composition between gardens of the same housing density types. Twenty-four percent of the cultivated species are well adapted to the Mediterranean climate, and 21 species known to be invasive on the French territory have emanated from gardens. Inventorying areas adjoining gardens would be useful in identifying escaped garden plants and to assess the associated risks for biological diversity. The results also suggested a detailed analysis of the influence of social, economic and regional factors on planting practices, in order to identify the drivers of these original floral patterns.

Determination of mitochondrial genetic diversity in mammals.

Mitochondrial DNA (mtDNA) is one of the most popular population genetic markers. Its relevance as an indicator of population size and history has recently been questioned by several large-scale studies in animals reporting evidence for recurrent adaptive evolution, at least in invertebrates. Here we focus on mammals, a more restricted taxonomic group for which the issue of mtDNA near neutrality is crucial. By analyzing the distribution of mtDNA diversity across species and relating it to allozyme diversity, life-history traits, and taxonomy, we show that (i) mtDNA in mammals does not reject the nearly neutral model; (ii) mtDNA diversity, however, is unrelated to any of the 14 life-history and ecological variables that we analyzed, including body mass, geographic range, and The World Conservation Union (IUCN) categorization; (iii) mtDNA diversity is highly variable between mammalian orders and families; (iv) this taxonomic effect is most likely explained by variations of mutation rate between lineages. These results are indicative of a strong stochasticity of effective population size in mammalian species. They suggest that, even in the absence of selection, mtDNA genetic diversity is essentially unpredictable, knowing species biology, and probably uncorrelated to species abundance.

Ecomorphological diversification among South American spiny rats (Rodentia; Echimyidae): a phylogenetic and chronological approach.

The phylogeny of South American spiny rats (Rodentia; Echimyidae) was studied using the exon 28 of the von Willebrand Factor nuclear gene (vWF). Sequences were analysed separately and in combination with a mitochondrial dataset (cyt b, 12S and 16S rRNAs) used in previous publications. The basal polytomy of echimyids was partially resolved and unexpected intergeneric clades were recovered. Thus, the intimate nested position of Myocastor within echimyids is evidenced. A well-supported clade is identified, including all the arboreal genera, and a group formed by Myocastor, Thrichomys, and Proechimys+Hoplomys. The clustering of Euryzygomatomys+Clyomys with Trinomys is also suggested. On the opposite, the phylogenetic position of Capromys as well as the relationships among arboreal genera remain unclear. Molecular divergence times were estimated using a Bayesian relaxed molecular clock and suggest a Middle Miocene origin for most of modern genera. The ecomorphological diversification of echimyids is discussed in the light of these new results and past environmental modifications in South America.

Napoleon Bonaparte and the fate of an Amazonian rat: new data on the taxonomy of Mesomys hispidus (Rodentia: Echimyidae).

The spiny rat Mesomys hispidus is one of many South American rodents that lack adequate taxonomic definition. The few sampled populations of this broadly distributed trans-Amazonian arboreal rat have come from widely separated regions and are typically highly divergent. The holotype was described in 1817 by A.-G. Desmarest, after Napoleon's army brought it to Paris following the plunder of Lisbon in 1808; however, the locality of origin has remained unknown. Here we examine the taxonomic status of this species by direct comparison of 50 extant individuals with the holotype at the morphometric and genetic levels, the latter based on 331 bp of the mitochondrial cytochrome b gene retrieved from a small skin fragment of the holotype with ancient DNA technology. Extensive sequence divergence is present among samples of M. hispidus collected from throughout its range, from French Guiana across Amazonia to Bolivia and Peru, with at least seven mitochondrial clades recognized (average divergence of 7.7% Kimura 2-parameter distance). Sequence from the holotype is, however, only weakly divergent from those of recent samples from French Guiana. Moreover, the holotype clusters with greater that 99% posterior probability with samples from this part of Amazonia in a discriminant analysis based on 22 cranial and dental measurements. Thus, we suggest that the holotype was originally obtained in eastern Amazonia north of the Amazon River, most likely in the Brazilian state of Amapá. Despite the high level of sequence diversity and marked morphological differences in size across the range of M. hispidus, we continue to regard this assemblage as a single species until additional samples and analyses suggest otherwise.