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1.
Parasit Vectors ; 13(1): 580, 2020 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-33203446

RESUMO

BACKGROUND: Ethiopia is affected by human leishmaniasis caused by several Leishmania species and transmitted by a variety of sand fly vectors of the genus Phlebotomus. The sand fly fauna in Ethiopia is highly diverse and some species are closely related and similar in morphology, resulting in difficulties with species identification that requires deployment of molecular techniques. DNA barcoding entails high costs, requires time and lacks reference sequences for many Ethiopian species. Yet, proper species identification is pivotal for epidemiological surveillance as species differ in their actual involvement in transmission cycles. Recently, protein profiling using MALDI-TOF mass spectrometry has been introduced as a promising technique for sand fly identification. METHODS: In our study, we used an integrative taxonomic approach to identify most of the important sand fly vectors of leishmaniasis in Ethiopia, applying three complementary methods: morphological assessment, sequencing analysis of two genetic markers, and MALDI-TOF MS protein profiling. RESULTS: Although morphological assessment resulted in some inconclusive identifications, both DNA- and protein-based techniques performed well, providing a similar hierarchical clustering pattern for the analyzed species. Both methods generated species-specific sequences or protein patterns for all species except for Phlebotomus pedifer and P. longipes, the two presumed vectors of Leishmania aethiopica, suggesting that they may represent a single species, P. longipes Parrot & Martin. All three approaches also revealed that the collected specimens of Adlerius sp. differ from P. (Adlerius) arabicus, the only species of Adlerius currently reported in Ethiopia, and molecular comparisons indicate that it may represent a yet undescribed new species. CONCLUSIONS: Our study uses three complementary taxonomical methods for species identification of taxonomically challenging and yet medically import Ethiopian sand flies. The generated MALDI-TOF MS protein profiles resulted in unambiguous identifications, hence showing suitability of this technique for sand fly species identification. Furthermore, our results contribute to the still inadequate knowledge of the sand fly fauna of Ethiopia, a country severely burdened with human leishmaniasis.

2.
Parasit Vectors ; 13(1): 276, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32487217

RESUMO

BACKGROUND: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts. METHODS: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve. RESULTS: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10-3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents. CONCLUSIONS: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

3.
PLoS Negl Trop Dis ; 13(8): e0007667, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31425506

RESUMO

BACKGROUND: Ochollo is a village in southern Ethiopia burdened with cutaneous leishmaniasis (CL), where Phlebotomus pedifer is the only vector for Leishmania aethiopica and hyraxes are confirmed reservoir hosts. A detailed description of the different players of transmission, and the ecology and seasonality of the vector needs to be established in order to accomplish efficient control programs. METHODS AND FINDINGS: Between March 2017 and February 2018, a monthly sandfly collection was carried out in different habitats and records of temperature and humidity were taken. Rodents and hyraxes were trapped in the dry and wet season. All samples were screened for Leishmania kinetoplast DNA (kDNA). Positive samples were further processed for determination of the Leishmania species and the species of the sandfly/small mammal that was found infected. Additionally, the species of 400 sandfly specimens from different habitats and seasons was identified. 17,190 Sergentomyia and Phlebotomus sandflies were caught and showed an overall kDNA prevalence of 2.6%, all were L. aethiopica infections only found in P. pedifer. The overall sandfly and P. pedifer abundance peaked in the dry season and was negatively correlated with the %RH. The kDNA prevalence varied over the months and was negatively correlated with the temperature. Total sandfly abundance did not differ between the sampled habitats, but P. pedifer was the distinct predominant species only in caves. Moreover, significantly more infected sandflies were found in caves. Only 1/192 rodents were kDNA positive, while 20.0% (5/25) of Heterohyrax brucei were found infected. CONCLUSIONS: This study suggests that caves may be a source of multiplication of the infection. If an outdoor control program would be considered, it would be useful to focus on caves in the wet season, when the sandfly abundance is lowest. The captured rodent species appear not important for transmission and the contribution of hyraxes in transmission should be further investigated.


Assuntos
DNA de Protozoário/análise , Reservatórios de Doenças , Vetores de Doenças , Procaviídeos/parasitologia , Leishmania/genética , Leishmaniose Cutânea/epidemiologia , Psychodidae/parasitologia , Animais , DNA de Protozoário/genética , Transmissão de Doença Infecciosa , Etiópia/epidemiologia , Feminino , Humanos , Umidade , Leishmaniose Cutânea/transmissão , Masculino , Carga Parasitária , Prevalência , Psychodidae/crescimento & desenvolvimento , Estações do Ano , Temperatura
4.
C R Biol ; 342(3-4): 108-117, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31056422

RESUMO

The taxonomy of African shrew species is still unresolved due to their conserved morphology. This also affects knowledge concerning their geographic distribution. In Nigeria, using mitochondrial Cytochrome b gene sequences, we carried out a survey for shrews from the genus Crocidura across various ecological zones to determine taxa that are present and also to assess their phylogeographic structure. Our analyses include 183 specimens collected with Sherman traps from 19 localities around the country. We detected six taxa: Crocidura olivieri lineages II, III and IV, C. hildegardeae, C. jouvenetae, and C. foxi. Among these, C. hildegardeae and C. jouvenetae are reported in Nigeria for the first time. Phylogenetic comparison of our genetic sequences to those generated from other parts of Africa demonstrate that all species in our study, as currently defined, are in need of taxonomic revision. Geographically, Nigeria seems to represent the easternmost boundary for C. olivieri lineage II and C. jouvenetae, and the western distribution limit of C. olivieri lineage IV and C. hildegardeae. The Niger River appears to be the most significant topographical barrier restricting these taxa. This information is vital to preserving the diversity but also managing the epidemiological potential of these small mammals.


Assuntos
Citocromos b/genética , Genes Mitocondriais , Filogenia , Musaranhos/classificação , Animais , Ecologia , Nigéria , Filogeografia
5.
Sci Rep ; 7: 39596, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-28054584

RESUMO

The spirochete bacterium Borrelia afzelii is the most common cause of Lyme borreliosis in Europe. This tick-borne pathogen can establish systemic infections in rodents but not in birds. However, several field studies have recovered larval Ixodes ricinus ticks infected with B. afzelii from songbirds suggesting successful transmission of B. afzelii. We reviewed the literature to determine which songbird species were the most frequent carriers of B. afzelii-infected I. ricinus larvae and nymphs. We tested experimentally whether B. afzelii is capable of co-feeding transmission on two common European bird species, the blackbird (Turdus merula) and the great tit (Parus major). For each bird species, four naïve individuals were infested with B. afzelii-infected I. ricinus nymphal ticks and pathogen-free larval ticks. None of the co-feeding larvae tested positive for B. afzelii in blackbirds, but a low percentage of infected larvae (3.33%) was observed in great tits. Transstadial transmission of B. afzelii DNA from the engorged nymphs to the adult ticks was observed in both bird species. However, BSK culture found that these spirochetes were not viable. Our study suggests that co-feeding transmission of B. afzelii is not efficient in these two songbird species.


Assuntos
Grupo Borrelia Burgdorferi/isolamento & purificação , Ixodes/patogenicidade , Aves Canoras/parasitologia , Animais , Grupo Borrelia Burgdorferi/genética , Grupo Borrelia Burgdorferi/patogenicidade , Europa (Continente) , Ixodes/genética
6.
Sci Rep ; 5: 10445, 2015 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-26022445

RESUMO

Arenaviruses can cause mild to severe hemorrhagic fevers. Humans mainly get infected through contact with infected rodents or their excretions, yet little is known about transmission dynamics within rodent populations. Morogoro virus (MORV) is an Old World arenavirus closely related to Lassa virus with which it shares the same host species Mastomys natalensis. We injected MORV in its host, and sampled blood and excretions at frequent intervals. Infection in adults was acute; viral RNA disappeared from blood after 18 days post infection (dpi) and from excretions after 39 dpi. Antibodies were present from 7 dpi and never disappeared. Neonatally infected animals acquired a chronic infection with RNA and antibodies in blood for at least 3 months. The quantified excretion and antibody patterns can be used to inform mathematical transmission models, and are essential for understanding and controlling transmission in the natural rodent host populations.


Assuntos
Infecções por Arenaviridae/transmissão , Arenavirus/patogenicidade , Vírus Lassa/patogenicidade , Animais , Anticorpos Antivirais/sangue , Infecções por Arenaviridae/patologia , Infecções por Arenaviridae/virologia , Arenavirus/genética , Reservatórios de Doenças/virologia , Humanos , Vírus Lassa/genética , Murinae/virologia , RNA Viral/sangue , Tanzânia
7.
Am J Trop Med Hyg ; 90(3): 463-8, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24445202

RESUMO

The prevalence and identity of Rickettsia and Bartonella in urban rat and flea populations were evaluated in Kisangani, Democratic Republic of the Congo (DRC) by molecular tools. An overall prevalence of 17% Bartonella species and 13% Rickettsia typhi, the agent of murine typhus, was found in the cosmopolitan rat species, Rattus rattus and Rattus norvegicus that were infested by a majority of Xenopsylla cheopis fleas. Bartonella queenslandensis, Bartonella elizabethae, and three Bartonella genotypes were identified by sequencing in rat specimens, mostly in R. rattus. Rickettsia typhi was detected in 72% of X. cheopis pools, the main vector and reservoir of this zoonotic pathogen. Co-infections were observed in rodents, suggesting a common mammalian host shared by R. typhi and Bartonella spp. Thus, both infections are endemic in DRC and the medical staffs need to be aware knowing the high prevalence of impoverished populations or immunocompromised inhabitants in this area.


Assuntos
Bartonella/genética , Insetos Vetores/microbiologia , Ratos/microbiologia , Rickettsia typhi/genética , Sifonápteros/microbiologia , Animais , Infecções por Bartonella/epidemiologia , Infecções por Bartonella/microbiologia , DNA Bacteriano/análise , República Democrática do Congo/epidemiologia , Vetores de Doenças , Infestações por Pulgas/microbiologia , Humanos , Reação em Cadeia da Polimerase , Prevalência , Infecções por Rickettsia/epidemiologia , Infecções por Rickettsia/microbiologia , Tifo Endêmico Transmitido por Pulgas/epidemiologia , Tifo Endêmico Transmitido por Pulgas/microbiologia , População Urbana
8.
Hereditas ; 150(4): 45-52, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24164457

RESUMO

Theory suggests that hermaphroditic plants and animals should be either entirely outcrossing or entirely selfing. As such, very few hermaphroditic plants and basommatophoran snails have a mixed breeding system. However, reliable estimates of selfing rates are lacking for most hermaphroditic animals. This partly prevents to delineate the relative contributions of the selective factors that determine selfing and outcrossing rates in hermaphroditic animal taxa. Here, we studied the population genetic structure of, and breeding system in, 11 populations of the hermaphroditic land slug Arion intermedius using five polymorphic microsatellite loci. Moreover, genotype frequencies deviated significantly from Hardy-Weinberg equilibrium expectations for most of the loci in all populations suggesting some level of selfing. Estimates of the selfing level s, suggest moderate levels of outcrossing (mean s based on FIS = 0.84; mean s based on the two-locus heterozygosity disequilibrium = 0.20, or with a ML approach = 0.22). Our study therefore suggests that A. intermedius has a mixed breeding system. A re-analysis of allozyme data from another arionid slug ( subgenus Carinarion) indicates that mixed breeding may be more common in arionid slugs than hitherto was assumed. These results seem therefore at variance with current theoretical and empirical predictions and opens perspectives for the study on the evolutionary factors driving mixed breeding systems in animals.


Assuntos
Gastrópodes/genética , Hibridização Genética , Alelos , Animais , Evolução Molecular , Frequência do Gene , Variação Genética , Genética Populacional , Repetições de Microssatélites
9.
Genetica ; 141(7-9): 281-92, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23887892

RESUMO

The hermaphroditic terrestrial snail Rumina decollata has a mixed breeding system with a high prevalence of self-fertilization. In the Montpellier area (France), the species is represented by a dark and a light color morph. Based on allozyme data, both morphs have been reported as single, homozygous multilocus genotypes (MLG), differing at 13 out of 26 loci, but still showing occasional hybridization. Recent DNA sequence data suggest that each morph is a different phylogenetic species. In order to further evaluate this new taxonomic interpretation, the present contribution explores to what extent populations or color morphs indeed consist of single or few MLG. As such it is shown that both morphs are not single, homozygous MLG, but instead reveal a considerable amount of allelic variation and substantial numbers of heterozygous microsatellite genotypes. This suggests that outcrossing may be more prevalent than previously reported. Nevertheless, both morphs maintain a diagnostic multimarker differentiation in the presence of outcrossing in sympatric conditions, implying that they may be interpreted as species under the biological species concept. Finally, our data challenge the idea that simultaneous hermaphrodites should be either strict selfers or strict outcrossers.


Assuntos
Frequência do Gene , Pigmentação/genética , Caramujos/genética , Alelos , Animais , Loci Gênicos , Homozigoto , Endogamia , Isoenzimas/genética , Repetições de Microssatélites , Polimorfismo Genético , População/genética , Caramujos/anatomia & histologia , Caramujos/classificação
10.
Genetica ; 128(1-3): 227-39, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17028953

RESUMO

Allozyme variation was determined in two land snail species (Cepaea nemoralis and Succinea putris) from four localities in northern Belgium. In each locality we selected a polluted and a nearby, less-polluted, reference plot. We examined whether (i) genetic variability differed between the polluted and reference plots, (ii) populations from polluted plots experienced recent bottlenecks, and (iii) certain allele or genotype frequencies were associated with the pollution. Our results suggest that (i) about 13% of the genetic differentiation in C. nemoralis and 5% in S. putris was due to differences among polluted and reference plots, (ii) polluted and reference plots had comparable levels of genetic variation, but in C. nemoralis observed heterozygosities were higher in polluted plots, (iii) most plots showed significant evidence for recent bottlenecks, irrespective of the degree of pollution, so that bottlenecks seem poor indicators of pollution-induced stress in land snails, and (iv) mutagenic or pollution-induced modifications did not seem to account for new allozyme variants in polluted sites. The observed patterns of genetic variation may be explained by the action of genetic drift, pollution-mediated selection, restricted gene flow, or a combination of these processes.


Assuntos
Caramujos/genética , Alelos , Animais , Bélgica , Poluentes Ambientais/toxicidade , Evolução Molecular , Fluxo Gênico , Deriva Genética , Variação Genética , Genética Populacional , Isoenzimas/genética , Metais Pesados/toxicidade , Seleção Genética , Caramujos/efeitos dos fármacos , Caramujos/enzimologia , Especificidade da Espécie
11.
Genetica ; 125(2-3): 155-71, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16247689

RESUMO

Several European species of the terrestrial slug genus Arion have been introduced into North America. A case in point is the species complex A. subfuscus s.l. which has become one of the most abundant slug taxa in North America. In Europe this complex consists of at least two cryptic species, viz. A. fuscus and A. subfuscus s.s., the latter of which is further subdivided in five strongly divergent mtDNA lineages (A. subfuscus S1-S5). In order to determine which of these A. subfsucus s.l. taxa are present in the NE USA and in order to assess their population genetic structure, we compared mtDNA, nDNA and allozyme variation between populations from the NE USA and Europe. Our results show that (1) at least A. subfuscus S1 has become successfully established in the NE USA, (2) founder effects are the most likely explanation for the loss of a large amount of molecular genetic variation in populations from the NE USA (i.e. a loss of 96% of the 16S rDNA haplotypes, 67% of the ITS1 alleles and 46% of the alleles at polymorphic allozyme loci), and (3) part of the remaining genetic variation in NE USA populations was probably due to multiple introductions from the British Isles and the European mainland, and the hybrid structure of most of these source populations. Apparently, the extreme loss of molecular genetic variation in this introduced species has not prevented it from successfully establishing and spreading in novel environments.


Assuntos
Gastrópodes/genética , Alelos , Animais , Sequência de Bases , DNA Mitocondrial/genética , DNA Ribossômico/genética , Europa (Continente) , Efeito Fundador , Gastrópodes/classificação , Variação Genética , Genética Populacional , Haplótipos , New England , Filogenia , Especificidade da Espécie
12.
Mol Phylogenet Evol ; 37(1): 132-43, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16085432

RESUMO

The Azorean representatives of the Leptaxini (Pulmonata) are single island endemics, where a high-spired shell distinguishes the monotypic genus Helixena from two slightly different low-spired forms within Leptaxis (azorica and caldeirarum type). We studied the evolutionary history of putative taxa and the three shell-types using 12 allozyme loci and sequences of nuclear (ITS-1 and ITS-2) and mitochondrial DNA (COI and 16S rRNA). While little variation was found in both ITS genes, allozyme and mtDNA divergence was among the highest reported for pulmonate land snails. Generally, phylogeographic patterns are indicative of allopatric differentiation via the successive colonization of (younger) islands, while a major role for adaptive evolution is not supported. The azorica shell-type is monophyletic and has no common history with other sympatric shell-types on the same islands. The (ambiguous) position of Helixena sanctaemariae makes Leptaxis paraphyletic on the Azores and possibly also the caldeirarum shell-type. Helixena can therefore not be distinguished as a separate genus on the Azores. Following a lineage-based concept, representatives on all (ancient) islands should be considered distinct species.


Assuntos
Evolução Biológica , Caramujos/classificação , Caramujos/genética , Animais , Açores , Sequência de Bases , DNA Mitocondrial , Frequência do Gene , Variação Genética , Isoenzimas , Filogenia , RNA Ribossômico , Alinhamento de Sequência , Análise de Sequência de DNA
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