Schistosomiasis 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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Phlebotomine (Diptera, Psychodidae, Phlebotominae) taxonomy has been studied extensively, primarily due to the role of these flies as vectors of various parasites, including species of Leishmania, ...Bartonella and arboviruses that cause diseases in humans and other vertebrates. We present some topics discussed at a round-table on phlebotomine taxonomy held at the Ninth International Symposium on Phlebotomine Sandflies (ISOPS IX) in Reims, France, in June 2016. To date, approximately one thousand phlebotomine species have been described worldwide, although in varying languages and mostly without standardization of characters and terminology. In the interest of standardization, we list the characters that should minimally be considered in the description of new phlebotomine taxa as well as annotated illustrations of several characters. For these characters, multiple illustrations are provided to show some of the variations. The preferred terms for all pertinent characters are listed as well as their synonyms in English, Portuguese, and French. Finally, we offer an updated list of abbreviations to be used for generic and subgeneric names.
Morphological characteristics of eggshells are important in sand fly ootaxonomy. In this study, eggshells from Phlebotomus stantoni Newstead, Sergentomyia khawi (Raynal), and Grassomyia indica ...(Theodor) sand flies collected in Chiang Mai province, Thailand were examined and characterized using light microscopy (LM) and scanning electron microscopy (SEM). Then, eggshell morphology of these three species was described for the first time. Each gravid female was forced to lay eggs by decapitation and the eggs were collected for SEM analysis. Egg laying females were identified by morphological examination and molecular typing using cytochrome b (Cytb) as a molecular marker. The chorionic sculpturing of Ph. stantoni eggs combines two patterns on the same egg: unconnected parallel ridges and reticular patterns. Sergentomyia khawi and Gr. indica have similar chorionic polygonal patterns, but their exochorionic morphology and aeropylar area are different. Results indicate that eggshell morphological characteristics such as chorionic pattern, exochorionic morphology, inter-ridge/boundary area, aeropylar area (including the number of aeropyles) and basal layer, can be useful to develop morphological identification keys of eggs. These can serve as an additional tool to distinguish species of sand flies. In addition, the chorionic sculpturing of the eggs of the three species of sand flies observed by LM is useful for species identification in gravid females with spermathecae obscured by eggs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Phlebotomine sand flies are the main natural vectors of
Leishmania
, which cause visceral and tegumentary tropical diseases worldwide. However, their taxonomy and evolutionary history remain poorly ...studied. Indeed, as for many human disease vectors, their small size is a challenge for morphological and molecular works. Here, we successfully amplified unbiased copies of whole genome to sequence thousands of restriction-site associated DNA (RAD) markers from single specimens of phlebotomines. RAD markers were used to infer a fully resolved phylogeny of the subgenus
Paraphlebotomus
(11 species + 5 outgroups, 32 specimens). The subgenus was not recovered as monophyletic and we describe a new subgenus
Artemievus
subg. nov. Depaquit for
Phlebotomus alexandri
. We also confirm the validity of
Ph
.
riouxi
which is reinstated as valid species. Our analyses suggest that
Paraphlebotomus sensu nov
. originated
ca
12.9–8.5 Ma and was possibly largely distributed from peri-Mediterranean to Irano-Turanian regions. Its biogeographical history can be summarized into three phases: i) a first split between
Ph
.
riouxi
+
Ph
.
chabaudi
and other species that may have resulted from the rise of the Saharan belt ca 8.5 Ma; ii) a Messinian vicariant event (7.3–5.3 Ma) during which the prolonged drought could have resulted in the divergence of main lineages; iii) a recent radiation event (3–2 Ma) that correspond to cycles of wet and dry periods in the Middle East and the East African subregions during the Pleistocene. Interestingly these cycles are also hypothetical drivers of the diversification of rodents, in the burrows of which
Paraphlebotomus
larvae develop. By meeting the challenge of sequencing pangenomics markers from single, minute phlebotomines, this work opens new avenues for improving our understanding of the epidemiology of leishmaniases and possibly other human diseases transmitted by arthropod vectors.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
West Nile virus (WNV) and Usutu virus (USUV) are two arthropod-borne viruses that circulate in mainland France. Assessing vector competence has only been conducted so far with mosquitoes from ...southern France while an increasingly active circulation of WNV and USUV has been reported in the last years. The main vectors are mosquitoes of the Culex genus and the common mosquito Culex pipiens. Here, we measure the vector competence of five mosquito species (Aedes rusticus, Aedes albopictus, Anopheles plumbeus, Culex pipiens and Culiseta longiareolata) present in northeastern France. Field-collected populations were exposed to artificial infectious blood meal containing WNV or USUV and examined at different days post-infection. We show that (i) Cx. pipiens transmitted WNV and USUV, (ii) Ae. rusticus only WNV, and (iii) unexpectedly, Ae. albopictus transmitted both WNV and USUV. Less surprising, An. plumbeus was not competent for both viruses. Combined with data on distribution and population dynamics, these assessments of vector competence will help in developing a risk map and implementing appropriate prevention and control measures.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background
In South-East Asia, Thailand is the country with the highest number of human autochthonous cases of leishmaniases mostly due to
Leishmania martiniquensis
. Their transmission remains ...unresolved to date even though sand flies are known vectors of leishmaniases. As such, we focused a study on the sand fly fauna of a cave in Thailand to explore the biodiversity of potential
Leishmania
vectors.
Main results
We carried out an inventory in Pha Tong cave. We caught and identified 570 Phlebotomine sand flies (452 females and 118 males) and identified 14 species belonging to the genera
Phlebotomus
,
Idiophlebotomus
,
Chinius
,
Sergentomyia
and
Grassomyia
. Among these 14 species, two could not be related to known sand fly species. Herein, we propose the description of two new sand fly species, previously unknown to science.
The first new species,
Phlebotomus shadenae
n. sp. is a sand fly of the subgenus
Anaphlebotomus
. It is morphologically close to
Ph
.
stantoni
, a species widely distributed throughout Southeast Asia. However, it differs by the length of the genital filaments in males or by the length of the ducts of the spermathecae in females as well as the high divergence of cytochrome b sequences. Additionally, we revised the systematics of the subgenus
Anaphlebotomus
and reinstated, by examination of its holotype, the validity of
Ph
.
maynei
, an Indian wrongly considered as a synonym of
Ph
.
stantoni
in the past.
The second new species,
Sergentomyia maiae
n. sp., differs from a species in the same group,
Se
.
barraudi
, by an original cibarial double row of vertical teeth as well as by molecular data.
Conclusions
We propose the description of two new sand fly species for Science with morphological and molecular evidence.
Ph
.
shadenae
n. sp. was also found to be distributed in the south of Thailand and in Laos. Future studies need to determine whether these two species can play a role as vectors of
Leishmania
parasites, Trypanosomatids or Phlebovirus. Most of the species caught in the present study are strictly cavernicolous except
Grassomyia
sp. and a few
Sergentomyia
.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background
The Greek island of Crete is endemic for both visceral leishmaniasis (VL) and recently increasing cutaneous leishmaniasis (CL). This study summarizes published data on the sand ...fly fauna of Crete, the results of new sand fly samplings and the description of a new sand fly species.
Methods
All published and recent samplings were carried out using CDC light traps, sticky traps or mouth aspirators. The specific status of
Phlebotomus
(
Adlerius
)
creticus
n. sp., was assessed by morphological analysis,
cytochrome b
(
cytb
) sequencing and MALDI-TOF protein profiling.
Results
Published data revealed the presence of 10
Phlebotomus
spp. and 2
Sergentomyia
spp. During presented field work, 608 specimens of 8 species of
Phlebotomus
and one species of
Sergentomyia
were collected. Both published data and present samplings revealed that the two most common and abundant species were
Phlebotomus neglectus
, a proven vector of
Leishmania infantum
causing VL, and
Ph. similis
, a suspected vector of
L. tropica
causing CL. In addition, the field surveys revealed the presence of a new species,
Ph.
(
Adlerius
)
creticus
n. sp.
Conclusions
The identification of the newly described species is based on both molecular and morphological criteria, showing distinct characters of the male genitalia that differentiate it from related species of the subgenus
Adlerius
as well as species-specific sequence of
cytb
and protein spectra generated by MALDI-TOF mass spectrometry.
to BLAST analyses (https://blast.ncbi.nlm.nih.gov/Blast. cgi). The YFV Uganda 2016 strain envelope sequence was aligned with reference YFV genomes by using MAFFT through the EMBL-EBI server ...(www.ebi.ac.uk), and phylogenies were generated with BEAST 1.8.4 (9), as previously described (7). BLAST analyses determined that the highest percentage identity (95%) is shared between the Uganda 2016 strain and strains from South Sudan 2003 in the envelope region (the only region for which data from the Sudan strain are available) versus 83% with Angola 2016 strains from the same region. Furthermore, the Uganda 2016 sequences corresponding to the NS genes NS3 and NS5 have the highest percentage identities (94% and 95%, respectively) with a Uganda 1948 strain relative to 85% and 84% with the Angola 2016 strains in the same regions. Together these BLAST analyses indicate that the Uganda 2016 YFV is most similar to strains in the East African genotype. Phylogenetic analyses confirm the BLAST analyses and place the Uganda 2016 YFV in a well-supported clade along with these East African genotype strains, whereas the Angola 2016 strains group with an Angola 1971 YFV (Figure), indicating that the Uganda outbreak in 2016 was not seeded by the Angola outbreak. These findings reiterate the endemicity of YFV throughout the tropical regions of Africa because at least 2 concurrent yellow fever outbreaks of independent origins were identified in 2016. Our findings also highlight the importance of assessing the molecular epidemiology of the virus in outbreak investigations. These data improve our understanding of YFV epidemiology in Africa and support the previous studies of Mutebi and colleagues (2). In addition, removal of contaminating ribosomal RNA proved to be an effective method for unbiased enrichment of viral RNA in degraded samples to enhance sequencing sensitivity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Phlebotomine sand flies are the vectors of the leishmaniases, parasitic diseases caused by Leishmania spp. Little is known about the prevalence and diversity of sand fly microflora colonizing the ...midgut or the cuticle. Particularly, there is little information on the fungal diversity. This information is important for development of vector control strategies.
FIVE SAND FLY SPECIES: Phlebotomus papatasi, P. sergenti, P. kandelakii, P. perfiliewi and P. halepensis were caught in Bileh Savar and Kaleybar in North-Western Iran that are located in endemic foci of visceral leishmaniasis. A total of 35 specimens were processed. Bacterial and fungal strains were identified by routine microbiological methods. We characterized 39 fungal isolates from the cuticle and/or the midgut. They belong to six different genera including Penicillium (17 isolates), Aspergillus (14), Acremonium (5), Fusarium (1), Geotrichum (1) and Candida (1). We identified 33 Gram-negative bacteria: Serratia marcescens (9 isolates), Enterobacter cloacae (6), Pseudomonas fluorescens (6), Klebsiella ozaenae (4), Acinetobacter sp. (3), Escherichia coli (3), Asaia sp. (1) and Pantoea sp. (1) as well as Gram-positive bacteria Bacillus subtilis (5) and Micrococcus luteus (5) in 10 isolates.
Our study provides new data on the microbiotic diversity of field-collected sand flies and for the first time, evidence of the presence of Asaia sp. in sand flies. We have also found a link between physiological stages (unfed, fresh fed, semi gravid and gravid) of sand flies and number of bacteria that they carry. Interestingly Pantoea sp. and Klebsiella ozaenae have been isolated in Old World sand fly species. The presence of latter species on sand fly cuticle and in the female midgut suggests a role for this arthropod in dissemination of these pathogenic bacteria in endemic areas. Further experiments are required to clearly delineate the vectorial role (passive or active) of sand flies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rapid, accurate and high-throughput identification of vector arthropods is of paramount importance in surveillance programmes that are becoming more common due to the changing geographic occurrence ...and extent of many arthropod-borne diseases. Protein profiling by MALDI-TOF mass spectrometry fulfils these requirements for identification, and reference databases have recently been established for several vector taxa, mostly with specimens from laboratory colonies.
We established and validated a reference database containing 20 phlebotomine sand fly (Diptera: Psychodidae, Phlebotominae) species by using specimens from colonies or field-collections that had been stored for various periods of time.
Identical biomarker mass patterns ('superspectra') were obtained with colony- or field-derived specimens of the same species. In the validation study, high quality spectra (i.e. more than 30 evaluable masses) were obtained with all fresh insects from colonies, and with 55/59 insects deep-frozen (liquid nitrogen/-80 °C) for up to 25 years. In contrast, only 36/52 specimens stored in ethanol could be identified. This resulted in an overall sensitivity of 87 % (140/161); specificity was 100 %. Duration of storage impaired data counts in the high mass range, and thus cluster analyses of closely related specimens might reflect their storage conditions rather than phenotypic distinctness. A major drawback of MALDI-TOF MS is the restricted availability of in-house databases and the fact that mass spectrometers from 2 companies (Bruker, Shimadzu) are widely being used. We have analysed fingerprints of phlebotomine sand flies obtained by automatic routine procedure on a Bruker instrument by using our database and the software established on a Shimadzu system. The sensitivity with 312 specimens from 8 sand fly species from laboratory colonies when evaluating only high quality spectra was 98.3 %; the specificity was 100 %. The corresponding diagnostic values with 55 field-collected specimens from 4 species were 94.7 % and 97.4 %, respectively.
A centralized high-quality database (created by expert taxonomists and experienced users of mass spectrometers) that is easily amenable to customer-oriented identification services is a highly desirable resource. As shown in the present work, spectra obtained from different specimens with different instruments can be analysed using a centralized database, which should be available in the near future via an online platform in a cost-efficient manner.
PDF
