Species composition and population dynamics of phlebotomine sand flies in a Leishmania infected area of Chiang Mai, Thailand.

Phlebotomine sand flies are established vectors of leishmaniasis in humans. In Thailand, Leishmania martiniquensis and "Leishmania siamensis" have been described as causative agents of leishmaniasis. In this study, a survey of sand flies in the Leishmania infected area of Hang Dong district, Chiang Mai, Thailand was performed using CDC light traps for eight consecutive months, from January to August 2016. A total of 661 sand flies were collected, and of 280 female sand flies, four species of the genus Sergentomyia including Sergentomyia gemmea, S. barraudi, S. indica, and S. hivernus and one species of the genus Phlebotomus, Phlebotomus stantoni, were identified. S. gemmea and S. hivernus were found in Chiang Mai for the first time. The density of captured female sand flies was high in warm and humid periods from June to August, with temperatures of around 26°C and relative humidity about 74%. In addition, S. gemmea was the most predominant species in the area. Further studies as to whether or not these sand fly species could be a vector of Leishmaniasis in Thailand are required.

Didilia sp. infecting Phlebotomus stantoni in Thailand.

Nematode infection in wild caught Phlebotomine sand flies was investigated in Thailand. Light microscopy (LM) and scanning electron microscopy (SEM) were used to detect and morphologically characterize entomopathogenic nematodes that presented in the sand flies. Didilia sp. nematodes were found for the first time in the body cavity of wild caught male Phlebotomus stantoni sand flies. The Didilia sp. was identified based on the morphology of the adult nematodes, from their stylet and teeth at the anterior tip, body length, and egg shell sculpture. It was noted that every infected male sand fly had unrotated genitalia, which would not allow them to mate, thus leading to the loss of their offspring. This finding provided information that might lead to study on whether or not the Didilia sp. has the potential to control sand fly population.

Chronic generalized fibrotic skin lesions from disseminated leishmaniasis caused by Leishmania martiniquensis in two patients from northern Thailand infected with HIV.

BACKGROUND: Leishmaniasis is a newly emerging infection in Thailand. Most of the previous human cases have presented with the clinical features of visceral leishmaniasis and were mainly found in southern Thailand. Here we report the first two patients from northern Thailand presenting with disseminated cutaneous leishmaniasis. OBJECTIVES: To determine the nature of the infection of leishmaniasis and to identify the species of parasite responsible. METHODS: Clinical investigations included the taking of biopsy samples and histology. Parasitological diagnosis was performed by establishment of Leishmania promastigote cultures, and identification was performed by DNA sequencing of four independent gene loci (ribosomal RNA internal transcribed spacer 1; large subunit of RNA polymerase II; heat shock protein 70; RPL23a intergenic sequence). RESULTS: Both patients were infected with HIV, and had multiple cutaneous lesions and accompanying visceral leishmaniasis. They had similar cutaneous manifestations characterized by chronic generalized fibrotic lesions, which were more prominent on traumatic areas. In both patients the parasite was identified as Leishmania martiniquensis. This is a recently described species that is distinct and only distantly related to the classical agents of cutaneous leishmaniasis in Asia (Leishmania major and Leishmania tropica) or of visceral leishmaniasis (Leishmania donovani and Leishmania infantum). Each patient responded well to therapy with intravenous amphotericin B followed by oral itraconazole. CONCLUSIONS: Leishmania martiniquensis is a cause of cutaneous leishmaniasis in Thailand.

Detection of Leishmania martiniquensis DNA in various clinical samples by quantitative PCR.

Leishmaniasis is a neglected tropical parasitic disease affecting a large number of countries in the world. Early diagnosis of Leishmania infections is essential for therapeutic reasons, as it can decrease morbidity and mortality. L. siamensis and L. martiniquensis are novel Leishmania species recently described in Thailand and Myanmar. The disease is usually found in immunocompromised patients, especially those who have AIDS. Currently, the diagnosis of Leishmania infection in Thailand relies on microscopy, microbial culture, and polymerase chain reaction (PCR). In this study, we established a quantitative PCR (qPCR) method for detection of L. martiniquensis DNA in various types of clinical specimens, including whole blood, buffy coat, saliva, and urine of L. martiniquensis infected patients. The results of the qPCR assay were positive in all saliva samples. The assay is therefore effective to detect L. martiniquensis DNA even in noninvasive specimens, and it could be used for the diagnosis, follow up, and survey of L. martiniquensis infections.

Exsheathment and midgut invasion of nocturnally subperiodic Brugia malayi microfilariae in a refractory vector, Aedes aegypti (Thailand strain).

Exsheathment and midgut invasion of nocturnally subperiodic Brugia malayi microfilariae were analyzed using light and scanning electron microscopy in a refractory vector, Aedes aegypti (Thailand strain). Results showed that exsheathed microfilariae represented only approximately 1% of the total microfilaria midguts dissected at 5-min post-infected blood meal (PIBM). The percentage of exsheathed microfilariae found in midguts progressively increased to about 20, 60, 80, 90, and 100% at 1-, 2-5-, 6-12-, 18-36-, and 48-h PIBM, respectively. Importantly, all the microfilariae penetrating the mosquito midguts were exsheathed. Midgut invasion by the exsheathed microfilariae was observed between 2- and 48-h PIBM. SEM analysis revealed sheathed microfilariae surrounded by small particles and maceration of the microfilarial sheath in the midguts, suggesting that the midguts of the refractory mosquitoes might have protein(s) and/or enzyme(s) and/or factor(s) that induce and/or accelerate exsheathment. The microfilariae penetrated the internal face of the peritrophic matrix (PM) by their anterior part and then the midgut epithelium, before entering the hemocoel suggesting that PM was not a barrier against the microfilariae migrating towards the midgut. Melanized microfilariae were discovered in the hemocoel examined at 96-h PIBM suggesting that the refractory mosquitoes used melanization reactions against this parasite. This study provided evidence that A. aegypti (Thailand strain) has refractory mechanisms against B. malayi in both midgut and hemocoel.

Morphological and protein analyses of adult female salivary glands of Anopheles barbirostris species A1 (Diptera: Culicidae).

Morphology and protein profiles of female salivary glands of Anopheles barbirostris species A1 were analyzed. Female glands consisted of a distinctive tri-lobed structure connected to a main salivary canal, a single medial and two lateral lobes with proximal and distal portions. Cellular architecture was similar among the lobes, with secretory material appearing as large masses. Cells of the proximal-lateral lobes contained secretory masses with a finely filamentous aspect. In the distal-lateral lobes, cells had a dense secretory product with mottled pattern. Cells of the medial lobe had secretory masses which were uniformly stained and highly electron dense. Following emergence, the glands accumulated secretory material rapidly and developed completely within three days. Degenerative changes including loss of stored secretion and increase of cytoplasmic vacuolation and concentric lamellar structures were observed from day 16 post emergence that correlated with total amount of the salivary gland proteins determined during development. SDS-PAGE, nanoLC-MS, and glycoprotein analysis revealed at least eleven major protein bands, of which each morphological region contained different major proteins. Two glycoproteins, apyrase/5'-nucleotidase and D7, were identified. These results form a basis for further studies on details of cytopathological changes of malarial infected glands and roles of the proteins in disease transmission.

Metaphase karyotypes of Anopheles paraliae (Diptera:Culicidae) in Thailand and evidence to support five cytological races.

Sixteen isoline colonies of Anopheles paraliae were established from wild-caught females collected from cow-baited traps at 4 locations in Thailand. They showed 3 types of X (X1, X2, X3) and 5 types of Y (Y1, Y2, Y3, Y4, Y5) chromosomes based on the number and amount of major block(s) of heterochromatin present in the heterochromatic arm, and were designated as Forms A (X3, Y1), B (X1, X2, X3, Y2), C (X3, Y3), D (X1, X2, X3, Y4) and E (X3, Y5). Form A was found in Songkhla Province, Form B was obtained in Ratchaburi, Nakhon Si Thammarat and Songkhla Provinces, Form C was acquired in Chanthaburi Province, Form D was recovered in Ratchaburi and Songkhla Provinces, and Form E was encountered in Ratchaburi Province. Hybridization experiments among the 7 isoline colonies, which represented the 5 karyotypic forms of An. paraliae, revealed genetic compatibility in providing viable progenies and synaptic salivary gland polytene chromosomes through F2-generations, and thus suggest the conspecific nature of these karyotypic forms. These results were supported by the very low intraspecific sequence divergence (mean genetic distance = 0.000-0.002) of the nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII) of the 5 forms.

Polygamy: the possibly significant behavior of Aedes aegypti and Aedes albopictus in relation to the efficient transmission of dengue virus.

The polygamous behavior of male Aedes aegypti (L.) and Ae. albopictus (Skuse) was investigated by co-habiting a newly-emerged male and females in a 30 cm3 cage (1 male: 20 females) for up to 5 consecutive days. As determined by insemination rates, the results indicated that one Ae. aegypti and Ae. albopictus male could successfully mate with 1.10 (0-4), 4.10 (1-8), 5.40 (4-8), 5.10 (2-8), 5.15 (3-9) and 0.20 (0-3), 1.70 (0-3), 2.35 (1-4), 2.30 (0-4), 2.35 (1-4) Ae. aegypti and Ae. albopictus females, respectively on day 1,2,3,4 and 5 consecutively. The possibly significant role of their polygamy in relation to dengue virus transmission is discussed.