Integrated taxonomy: traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda)

<p>Abstract</p> <p>Background</p> <p>We compared here the suitability and efficacy of traditional morphological approach and DNA barcoding to distinguish filarioid nematodes species (Nematoda, Spirurida). A reliable and rapid taxonomic identification of these parasites is the basis for a correct diagnosis of important and widespread parasitic diseases. The performance of DNA barcoding with different parameters was compared measuring the strength of correlation between morphological and molecular identification approaches. Molecular distance estimation was performed with two different mitochondrial markers (<it>coxI </it>and 12S rDNA) and different combinations of data handling were compared in order to provide a stronger tool for easy identification of filarioid worms.</p> <p>Results</p> <p>DNA barcoding and morphology based identification of filarioid nematodes revealed high coherence. Despite both <it>coxI </it>and 12S rDNA allow to reach high-quality performances, only <it>coxI </it>revealed to be manageable. Both alignment algorithm, gaps treatment, and the criteria used to define the threshold value were found to affect the performance of DNA barcoding with 12S rDNA marker. Using <it>coxI </it>and a defined level of nucleotide divergence to delimit species boundaries, DNA barcoding can also be used to infer potential new species.</p> <p>Conclusion</p> <p>An integrated approach allows to reach a higher discrimination power. The results clearly show where DNA-based and morphological identifications are consistent, and where they are not. The coherence between DNA-based and morphological identification for almost all the species examined in our work is very strong. We propose DNA barcoding as a reliable, consistent, and democratic tool for species discrimination in routine identification of parasitic nematodes.</p

Les phlébotomes (Diptera-Psychodidae) de l'île de Chypre.II - Presence de Leishmania (Leishmania) infantum Nicolle, 1908 (zymodeme MON 1) chez Phlebotomus (Larroussius) tobbi Adler et Theodor, 1930

Dans le foyer leishmanien cypriote, les auteurs ont disseque 2910 femelles de phlebotomes appartenant a 11 especes : Phlebotomus papatasi, P. sergenti, P. jacusieli, P. alexandri, P. tobbi, P. galilaeus, P. mascittii, P. economidesi, Sergentomyia fallax, S. minuta et S. azizi . Les deux larroussius (P. galilaeus et P. tobbi ) sont les especes les plus abondantes. Elles representent plus de 60 % des captures realisees avec des pieges CDC. Des promastigotes ont ete observees chez un seul specimen appartenant a l'espece P. tobbi . Elles ont ete mises en culture puis identifiees selon la methode isoenzymatique. La souche de Leishmania isolee appartient a l'espece Leishmania infantum , zymodeme MON 1. Le meme zymodeme a ete aussi isole et identifie chez quatre chiens de l'ile. En l'absence des vecteurs habituels de L. infantum dans l'est mediterraneen (P. neglectus ef P. syriacus) et en raison de sa repartition a Chypre, P. tobbi constitue vraisemblablement un bon vecteur local. Sa faible anthropophilie expliquerait peut-etre le tres faible nombre de cas humains. Le role de P. galilaeus dans la transmission de la leishmaniose a Chypre reste a preciser

Immunosenescence patterns differ between populations but not between sexes in a long-lived mammal

International audienceIn animals, physiological mechanisms underlying reproductive and actuarial senescence remain poorly understood. Immunosenescence, the decline in the ability to display an efficient immune response with increasing age, is likely to influence both reproductive and actuarial senescence through increased risk of disease. Evidence for such a link has been reported from laboratory animal models but has been poorly investigated in the wild, where variation in resource acquisitions usually drives life-history tradeoffs. We investigated immunosenescence patterns over 7 years in both sexes of two contrasting roe deer populations (Capreolus capreolus). We first measured twelve immune markers to obtain a thorough identification of innate and adaptive components of immunity and assessed, from the same individuals, the age-dependent variation observed in parasitic infections. Although the level of innate traits was maintained at old age, the functional innate immune traits declined with increasing age in one of two populations. In both populations, the production of inflammatory markers increased with advancing age. Finally, the adaptive response declined in late adulthood. The increasing parasite burden with age we reported suggests the effective existence of immunosenescence. Age-specific patterns differed between populations but not between sexes, which indicate that habitat quality could shape agedependent immune phenotype in the wild

Spiculopteragia mathevossiani Ruchliadev, 1948 is the minor morph of Spiculopteragia spiculoptera (Gushanskaya, 1931): molecular evidence

Although Spiculopteragia spiculoptera is primarily a parasite of cervids, it can also but less often contaminate domestic livestock. Little is known about its epidemiology and its pathogenicity in domestic ruminants and other unusual cervid species. Its taxonomic status remains unclear since the hypothesis of morphological polymorphism among males has been proposed. However, accurate taxonomy is fundamental in the identification and survey of potentially pathogenic species of parasites. The second internal transcribed spacer of rDNA (ITS-2) and the mitochondrial (mt) DNA-derived ND4 gene were used to study the polymorphism hypothesis for S. spiculoptera. ND4 evolves more quickly than ITS-2 and is considered to be more discriminant in the characterization of closely related species. DNA sequences of ITS-2 and ND4 were studied in 18 individual males of morphological type spiculoptera and in 3 of morphological type of mathevossiani from Red deer (Cervus elaphus), Roe deer (Capreolus capreolus) and Chamois (Rupicapra rupicapra). Intraindividual ITS-2 variations were detected within and between each morphotype of Spiculopteragia but these differences did not separate the two morphs mathevossiani and spiculoptera. Similarly, although ND4 showed a high level of nucleotide substitution, the morphotypes S. mathevossiani and S. spiculoptera were clustered together. Our genetic data support the dimorphic male hypothesis for the species S. spiculoptera

Mosquitoes of North-Western Europe as potential vectors of Arboviruses: a review

Background: The intensification of trade and travel is linked to the growing number of imported cases of dengue, chikungunya or Zika viruses into continental Europe and to the expansion of invasive mosquito species such as Aedes albopictus and Aedes japonicus. Local outbreaks have already occurred in several European countries. Very little information exists on the vector competence of native mosquitoes for arboviruses. As such, the vectorial status of the nine mosquito species largely established in North-Western Europe (Aedes cinereus and Aedes geminus, Aedes cantans, Aedes punctor, Aedes rusticus, Anopheles claviger s.s., Anopheles plumbeus, Coquillettidia richiardii, Culex pipiens s.l., and Culiseta annulata) remains mostly unknown. Objectives: To review the vector competence of both invasive and native mosquito populations found in North-Western Europe (i.e., France, Belgium, Germany, United Kingdom, Ireland, The Netherlands, Luxembourg and Switzerland) for dengue, chikungunya, Zika, West Nile and Usutu viruses. Methods: A bibliographical search with research strings addressing mosquito vector competence for considered countries was performed. Results: Out of 6357 results, 119 references were related to the vector competence of mosquitoes in Western Europe. Eight species appear to be competent for at least one virus. Conclusions: Aedes albopictus is responsible for the current outbreaks. The spread of Aedes albopictus and Aedes japonicus increases the risk of the autochthonous transmission of these viruses. Although native species could contribute to their transmission, more studies are still needed to assess that risk

MALDI-TOF mass spectrometry: a new tool for rapid identification of cercariae (Trematoda, Digenea)

Identification of cercariae was long based on morphological and morphometric features, but these approaches remain difficult to implement and require skills that have now become rare. Molecular tools have become the reference even though they remain relatively time-consuming and expensive. We propose a new approach for the identification of cercariae using MALDI-TOF mass spectrometry. Snails of different genera (Radix, Lymnaea, Stagnicola, Planorbis, and Anisus) were collected in the field to perform emitting tests in the laboratory. The cercariae they emitted (Trichobilharzia anseri, Diplostomum pseudospathaceum, Alaria alata, Echinostoma revolutum, Petasiger phalacrocoracis, Tylodelphys sp., Australapatemon sp., Cotylurus sp., Posthodiplostomum sp., Parastrigea sp., Echinoparyphium sp. and Plagiorchis sp.) were characterized by sequencing the D2, ITS2 and ITS1 domains of rDNA, and by amplification using specific Alaria alata primers. A sample of each specimen, either fresh or stored in ethanol, was subjected to a simple preparation protocol for MALDI-TOF analysis. The main spectral profiles were analyzed by Hierarchical Clustering Analysis. Likewise, the haplotypes were analyzed using the maximum likelihood method. Analytical performance and the log-score value (LSV) cut-off for species identification were then assessed by blind testing. The clusters obtained by both techniques were congruent, allowing identification at a species level. MALDI-TOF enables identification at an LSV cut-off of 1.7 without false-positives; however, it requires more data on closely related species. The development of a “high throughput” identification system for all types of cercariae would be of considerable interest in epidemiological surveys of trematode infections

MALDI-TOF: A new tool for the identification of Schistosoma cercariae and detection of hybrids

International audienceSchistosomiasis is a neglected water-born parasitic disease caused by Schistosoma affecting more than 200 million people. Introgressive hybridization is common among these parasites and raises issues concerning their zoonotic transmission. Morphological identification of Schistosoma cercariae is difficult and does not permit hybrids detection. Our objective was to assess the performance of MALDI-TOF (Matrix Assistated Laser Desorption-Ionization-Time Of Flight) mass spectrometry for the specific identification of cercariae in human and non-human Schistosoma and for the detection of hybridization between S. bovis and S. haematobium. Spectra were collected from laboratory reared molluscs infested with strains of S. haematobium, S. mansoni, S. bovis, S. rodhaini and S. bovis x S. haematobium natural (Corsican hybrid) and artificial hybrids. Cluster analysis showed a clear separation between S. haematobium, S. bovis, S. mansoni and S. rodhaini. Corsican hybrids are classified with those of the parental strain of S. haematobium whereas other hybrids formed a distinct cluster. In blind test analysis the developed MALDI-TOF spectral database permits identification of Schistosoma cercariae with high accuracy (94%) and good specificity (S. bovis: 99.59%, S. haematobium 99.56%, S. mansoni and S. rodhaini: 100%). Most misidentifications were between S. haematobium and the Corsican hybrids. The use of machine learning permits to improve the discrimination between these last two taxa, with accuracy, F1 score and Sensitivity/Specificity > 97%. In multivariate analysis the factors associated with obtaining a valid identification score (> 1.7) were absence of ethanol preservation (p < 0.001) and a number of 2-3 cercariae deposited per well (p < 0.001). Also, spectra acquired from S. mansoni cercariae are more likely to obtain a valid identification score than those acquired from S. haematobium (p<0.001). MALDI-TOF is a reliable technique for high-throughput identification of Schistosoma cercariae of medical and veterinary importance and could be useful for field survey in endemic areas.Author summary:Schistosomiases are neglected tropical diseases, affecting approximately 200 million people worldwide. They are transmitted during contact with water contaminated with the infesting stage of the parasite (the cercaria stage). Species-level recognition of cercariae present in water has important implications for field campaigns aimed at eradicating schistosomiasis. In addition, Schistosomes are able to hybridize between different species. Identification of Schistosomes cercariae on microscopy is difficult because of their similarity, and it does not allow hybrids to be distinguished. Molecular biology techniques allow a reliable diagnosis but are expensive. MALDI-TOF is a recent technique that permits an inexpensive identification of micro-organisms in a few minutes. In this paper, we evaluate MALDI-TOF identification of Schistosomes cercariae. We have implemented a database of MALDI-TOF cercariae spectra obtained from parental strains and hybrids of species of medical or veterinary interest, allowing reliable identification with an accuracy of 94%. The identification errors mainly come from confusion between the natural Corsican hybrid (S. haematobium x S. bovis) and S. haematobium. The use of machine learning algorithms permits to obtain an accuracy of more than 97% in the recognition of these two parasites. In conclusion, MALDI-TOF is a promising tool for the identification of Schistosome cercariae

Anthelmintic Treatment and the Stability of Parasite Distribution in Ruminants

International audienceParasites are generally overdispersed among their hosts, with far-reaching implications for their population dynamics and control. The factors determining parasite overdispersion have long been debated. In particular, stochastic parasite acquisition and individual host variation in density-dependent regulation through acquired host immunity have been identified as key factors, but their relative roles and possible interactions have seen little empirical exploration in parasite populations. Here, Taylor's power law is applied to test the hypothesis that periodic parasite removal destabilises the host-parasite relationship and increases variance in parasite burden around the mean. The slope of the power relationship was compared by analysis of covariance among 325 nematode populations in wild and domestic ruminants, exploiting that domestic ruminants are often routinely treated against parasite infections. In Haemonchus spp. and Trichostrongylus axei in domestic livestock, the slope increased with the frequency of anthelmintic treatment, supporting this hypothesis. In Nematodirus spp., against which acquired immunity is known to be strong, the slope was significantly greater in post-mortem worm burden data than in faecal egg counts, while this relationship did not hold for the less immunogenic genus Marshallagia. Considered together, these findings suggest that immunity acting through an exposure-dependent reduction in parasite fecundity stabilises variance in faecal egg counts, reducing overdispersion, and that periodic anthelmintic treatment interferes with this process and increases overdispersion. The results have implications for the diagnosis and control of parasitic infections in domestic animals, which are complicated by overdispersion, and for our understanding of parasite distribution in free-living wildlife. Parasite-host systems, in which treatment and immunity effectively mimic metapopulation processes of patch extinction and density dependence, could also yield general insights into the spatio-temporal stability of animal distributions