Axenic amastigote cultivation and in vitro development of Leishmania orientalis

Leishmania (Mundinia) orientalis is a recently described new species that causes leishmaniasis in Thailand. To facilitate characterization of this new species, an in vitro culture system to generate L. orientalis axenic amastigotes was developed. In vitro culture conditions of the axenic culture-derived amastigotes were optimized by manipulation of temperature and pH. Four criteria were used to evaluate the resulting L. orientalis axenic amastigotes, i.e., morphology, zymographic analysis of nucleases, cyclic transformation, and infectivity to the human monocytic cell line (THP-1) cells. Results revealed that the best culture condition for L. orientalis axenic amastigotes was Grace's insect medium supplemented with FCS 20%, 2% human urine, 1% BME vitamins, and 25 μg/ml gentamicin sulfate, pH 5.5 at 35 °C. For promastigotes, the condition was M199 medium, 10% FCS supplemented with 2% human urine, 1% BME vitamins, and 25 μg/ml gentamicin sulfate, pH 6.8 at 26 °C. Morphological characterization revealed six main stages of the parasites including amastigotes, procyclic promastigotes, nectomonad promastigotes, leptomonad promastigotes, metacyclic promastigotes, and paramastigotes. Also, changes in morphology during the cycle were accompanied by changes in zymographic profiles of nucleases. The developmental cycle of L. orientalis in vitro was complete in 12 days using both culture systems. The infectivity to THP-1 macrophages and intracellular growth of the axenic amastigotes was similar to that of THP-1 derived intracellular amastigotes. These results confirmed the successful axenic cultivation of L. orientalis amastigotes. The axenic amastigotes and promastigotes can be used for further study on infection in permissive vectors and animals

Morfometria e morfologia comparadas de ovos de Anopheles aconitus formas B e C à microscopia eletrônica de varredura

Comparative morphometric and morphological studies of eggs under scanning electron microscope (SEM) were undertaken in the three strains of two karyotypic forms of Anopheles aconitus, i.e., Form B (Chiang Mai and Phet Buri strains) and Form C (Chiang Mai and Mae Hong Son strains). Morphometric examination revealed the intraspecific variation with respect to the float width [36.77 ± 2.30 µm (Form C: Chiang Mai strain) = 38.49 ± 2.78 µm (Form B: Chiang Mai strain) = 39.06 ± 2.37 µm (Form B: Phet Buri strain) >; 32.40 ± 3.52 µm (Form C: Mae Hong Son strain)] and number of posterior tubercles on deck [2.40 ± 0.52 (Form B: Phet Buri strain) = 2.70 ± 0.82 (Form B: Chiang Mai strain) ; 32,40 ± 3,52 µm (Forma C: linhagem Mae Hong Son)] e número de tubérculos posteriores sobre a superfície livre [2,40 ± 0,52 (Forma B: linhagem Phet Buri) = 2,70 ± 0,82 (Forma B: linhagem Chiang Mai) < 3,10 ± 0,32 (Forma C: linhagem Chiang Mai) = 3,20 ± 0,42 (Forma C: linhagem Mae Hong Son)] embora a topografia de superfície dos ovos entre as três linhagens de duas formas cariotípicas tenham sido morfologicamente semelhantes

Mitochondrial pseudogenes in the nuclear genome of Aedes aegypti mosquitoes: implications for past and future population genetic studies

<p>Abstract</p> <p>Background</p> <p>Mitochondrial DNA (mtDNA) is widely used in population genetic and phylogenetic studies in animals. However, such studies can generate misleading results if the species concerned contain nuclear copies of mtDNA (Numts) as these may amplify in addition to, or even instead of, the authentic target mtDNA. The aim of this study was to determine if Numts are present in <it>Aedes aegypti </it>mosquitoes, to characterise any Numts detected, and to assess the utility of using mtDNA for population genetics studies in this species.</p> <p>Results</p> <p>BLAST searches revealed large numbers of Numts in the <it>Ae. aegypti </it>nuclear genome on 146 supercontigs. Although the majority are short (80% < 300 bp), some Numts are almost full length mtDNA copies. These long Numts are not due to misassembly of the nuclear genome sequence as the Numt-nuclear genome junctions could be recovered by amplification and sequencing. Numt evolution appears to be a complex process in <it>Ae. aegypti </it>with ongoing genomic integration, fragmentation and mutation and the secondary movement of Numts within the nuclear genome.</p> <p>The PCR amplification of the putative mtDNA nicotinamide adenine dinucleotide dehydrogenase subunit 4 (<it>ND4</it>) gene from 166 Southeast Asian <it>Ae. aegypti </it>mosquitoes generated a network with two highly divergent lineages (clade 1 and clade 2). Approximately 15% of the <it>ND4 </it>sequences were a composite of those from each clade indicating Numt amplification in addition to, or instead of, mtDNA. Clade 1 was shown to be composed at least partially of Numts by the removal of clade 1-specific bases from composite sequences following enrichment of the mtDNA. It is possible that all the clade 1 sequences in the network were Numts since the clade 2 sequences correspond to the known mitochondrial genome sequence and since all the individuals that produced clade 1 sequences were also found to contain clade 2 mtDNA-like sequences using clade 2-specific primers. However, either or both sets of clade sequences could have Numts since the BLAST searches revealed two long Numts that match clade 2 and one long Numt that matches clade 1. The substantial numbers of mutations in cloned <it>ND4 </it>PCR products also suggest there are both recently-derived clade 1 and clade 2 Numt sequences.</p> <p>Conclusion</p> <p>We conclude that Numts are prevalent in <it>Ae. aegypti </it>and that it is difficult to distinguish mtDNA sequences due to the presence of recently formed Numts. Given this, future population genetic or phylogenetic studies in <it>Ae. aegypti </it>should use nuclear, rather than mtDNA, markers.</p

Development of Leishmania orientalis in the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) and the biting midge Culicoides soronensis (Diptera: Ceratopogonidae)

Leishmania (Mundinia) orientalis is a newly described species causing human leishmaniasis in Thailand whose natural vector is unknown. L. orientalis infections in sand flies and/or biting midges under laboratory conditions have not been previously investigated. In this study, the development of L. orientalis in two experimental vectors, Lutzomyia longipalpis sand flies and Culicoides sonorensis biting midges was investigated for the first time using light microscopy, scanning electron microscopy, and histological examination. The results showed that L. orientalis was unable to establish infection in Lu. longipalpis. No parasites were found in the sand fly gut 4 days post-infected blood meal (PIBM). In contrast, the parasite successfully established infection in C. sonorensis. The parasites differentiated from amastigotes to procyclic promastigotes in the abdominal midgut (AMG) on day 1 PIBM. On day 2 PIBM, nectomonad promastigotes were observed in the AMG and migrated to the thoracic midgut (TMG). Leptomonad promastigotes appeared at the TMG on day 3 PIBM. Clusters of leptomonad promastigotes and metacyclic promastigotes colonized around the stomodeal valve with the accumulation of a promastigote secretory gel-like material from day 3 PIBM onwards. Haptomonad-like promastigotes were observed from day 5 PIBM, and the proportion of metacyclic promastigotes reached 23% on day 7 PIBM. The results suggest that biting midges or other sand fly genera or species might be vectors of L. orientalis

Suscetibilidade de duas formas cariotípicas de Anopheles aconitus (Diptera: Culicidae) a Plasmodium falciparum e P. vivax

Four laboratory-raised colonies of two karyotypic forms of Anopheles aconitus, i.e., Form B (Chiang Mai and Phet Buri strains) and C (Chiang Mai and Mae Hong Son strains), were experimentally infected with Plasmodium falciparum and P. vivax using an artificial membrane feeding technique and dissected eight and 12 days after feeding for oocyst and sporozoite rates, respectively. The results revealed that An. aconitus Form B and C were susceptible to P. falciparum and P. vivax, i.e., Form B (Chiang Mai and Phet Buri strains/P. falciparum and P. vivax) and Form C (Chiang Mai and Mae Hong Son strains/P. vivax). Comparative statistical analyses of the oocyst rates, average number of oocysts per infected midgut and sporozoite rates among all strains of An. aconitus Form B and C to the ingroup control vectors, An. minimus A and C, exhibited mostly no significant differences, confirming the high potential vector of the two Plasmodium species. The sporozoite-like crystals found in the median lobe of the salivary glands, which could be a misleading factor in the identification of true sporozoites in salivary glands were found in both An. aconitus Form B and C.Quatro colônias desenvolvidas em laboratório, de duas formas cariotípicas de Anopheles aconitus i.e. forma B (cepa Chiang Mai e Phet Buri) e C (Cepa Chiang Mai e Mae Hong Son), foram infectadas experimentalmente com Plasmodium falciparum e P. vivax usando técnica de alimentação com membrana artificial e dissecados oito e 12 dias após alimentação da média de oocistos e esporozoitos, respectivamente. Os resultados revelaram que An. aconitus formas B e C foram suscetíveis ao P. falciparum e P. vivax isto é, forma B (cepa Chiang Mai e Phet Buri/P. falciparum e P. vivax) e forma C (cepa Chiang Mai e Mae Hong Son/P. vivax). Análises estatísticas comparativas das taxas de oocistos, número médio de oocistos por intestino médio infectado e taxas de esporozoitos entre todas as cepas de An. aconitus formas B e C ao grupo interno de vetores controles, An. minimus A e C, não exibiram nenhuma diferença significante, confirmando o alto potencial vetor das duas espécies de Plamodium. Os cristais semelhantes a esporozoitos encontrados no lobo médio das glândulas salivares que poderiam ser um fator enganoso na identificação de esporozoitos verdadeiros nas glândulas salivares foram encontrados em ambos An. aconitus formas B e C

Multi-level analyses of spatial and temporal determinants for dengue infection

BACKGROUND: Dengue is a mosquito-borne viral infection that is now endemic in most tropical countries. In Thailand, dengue fever/dengue hemorrhagic fever is a leading cause of hospitalization and death among children. A longitudinal study among 1750 people in two rural and one urban sites in northern Thailand from 2001 to 2003 studied spatial and temporal determinants for recent dengue infection at three levels (time, individual and household). METHODS: Determinants for dengue infection were measured by questionnaire, land-cover maps and GIS. IgM antibodies against dengue were detected by ELISA. Three-level multi-level analysis was used to study the risk determinants of recent dengue infection. RESULTS: Rates of recent dengue infection varied substantially in time from 4 to 30%, peaking in 2002. Determinants for recent dengue infection differed per site. Spatial clustering was observed, demonstrating variation in local infection patterns. Most of the variation in recent dengue infection was explained at the time-period level. Location of a person and the environment around the house (including irrigated fields and orchards) were important determinants for recent dengue infection. CONCLUSION: We showed the focal nature of asymptomatic dengue infections. The great variation of determinants for recent dengue infection in space and time should be taken into account when designing local dengue control programs

Molecular markers for analyses of intraspecific genetic diversity in the Asian Tiger mosquito, Aedes albopictus

BACKGROUND: The dramatic worldwide expansion of Aedes albopictus (the Asian tiger mosquito) and its vector competence for numerous arboviruses represent a growing threat to public health security. Molecular markers are crucially needed for tracking the rapid spread of this mosquito and to obtain a deeper knowledge of population structure. This is a fundamental requirement for the development of strict monitoring protocols and for the improvement of sustainable control measures. METHODS: Wild population samples from putative source areas and from newly colonised regions were analysed for variability at the ribosomal DNA internal transcribed spacer 2 (ITS2). Moreover, a new set of 23 microsatellite markers (SSR) was developed. Sixteen of these SSRs were tested in an ancestral (Thailand) and two adventive Italian populations. RESULTS: Seventy-six ITS2 sequences representing 52 unique haplotypes were identified, and AMOVA indicated that most of their variation occurred within individuals (74.36%), while only about 8% was detected among populations. Spatial analyses of molecular variance revealed that haplotype genetic similarity was not related to the geographic proximity of populations and the haplotype phylogeny clearly indicated that highly related sequences were distributed across populations from different geographical regions. The SSR markers displayed a high level of polymorphism both in the ancestral and in adventive populations, and F(ST) estimates suggested the absence of great differentiation. The ancestral nature of the Thai population was corroborated by its higher level of variability. CONCLUSIONS: The two types of genetic markers here implemented revealed the distribution of genetic diversity within and between populations and provide clues on the dispersion dynamics of this species. It appears that the diffusion of this mosquito does not conform to a progressive expansion from the native Asian source area, but to a relatively recent and chaotic propagule distribution mediated by human activities. Under this scenario, multiple introductions and admixture events probably play an important role in maintaining the genetic diversity and in avoiding bottleneck effects. The polymorphic SSR markers here implemented will provide an important tool for reconstructing the routes of invasion followed by this mosquito

Antennal morphology and sensilla ultrastructure of the malaria vectors, Anopheles maculatus and An. sawadwongporni (Diptera: Culicidae).

Mosquitoes rely mainly on the olfactory system to track hosts. Sensilla contain olfactory neuron receptors that perceive different kinds of odorants and transfer crucial information regarding the surrounding environment. Anopheles maculatus and An. sawadwongporni, members of the Maculatus Group, are regarded as vectors of malaria in Thailand. The fine structure of their sensilla has yet to be identified. Herein, scanning electron microscopy is used to examine the sensilla located on the antennae of adults An. maculatus and An. sawadwongporni, collected from the Thai-Myanmar border. Four major types of antennal sensilla are discovered in both species: chaetica, coeloconica, basiconica (grooved pegs) and trichodea. The antennae of female An. maculatus have longer lengths (μm, mean&nbsp;±&nbsp;SE) in the long sharp-tipped trichodea (40.62&nbsp;±&nbsp;0.35&nbsp;&gt;&nbsp;38.20&nbsp;±&nbsp;0.36), blunt-tipped trichodea (20.39&nbsp;±&nbsp;0.62&nbsp;&gt;&nbsp;18.62&nbsp;±&nbsp;0.35), and basiconica (7.84&nbsp;±&nbsp;0.15&nbsp;&gt;&nbsp;7.41&nbsp;±&nbsp;0.12) than those of An. sawadwongporni. Using light microscopy, it is found that the mean numbers of large sensilla coeloconica (lco) on both flagella in An. maculatus (left: 32.97&nbsp;±&nbsp;0.48; right: 33.27&nbsp;±&nbsp;0.65) are also greater when compared to An. sawadwongporni (left: 30.40&nbsp;±&nbsp;0.62; right: 29.97&nbsp;±&nbsp;0.49). The mean counts of lco located on flagellomeres 1-3, 6, and 9 in An. maculatus are significantly higher than those of An. sawadwongporni. The data in this study indicate that two closely related Anopheles species exhibit similar morphology of sensilla types, but show variations in length, and likewise in the number of large sensilla coeloconica between them, suggesting they might be causative factors that affect their behaviors driven by the sense of smell

Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0% oocyst rates were obtained, in comparison to the 86.67-100% oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0% sporozoite rates were obtained, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09%, 6.67% and 11.76% sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67% and 64.29% sporozoite rates were obtained, respectively, in comparison to 90% sporozoite rates recovered from An. cracens