Detection of amastigote-like forms in the valve of Phlebotomus papatasi infected with Leishmania major

A massive and homogeneous amount of amastigote-like forms was detected in the stomodeal valve (SV) and the thoracic mid-gut (TMG) of Leishmania major-infected Phlebotomus papatasi, which received a second blood meal 13 to 21 days post-infection on healthy anaesthetized hamsters. After re-feeding, the infected sand flies were dissected out to examine the morphology of the parasite in SV, TMG and the abdominal mid-gut (AMG). Different promastigote forms were seen in the infected flies. Among these included typical promastigotes (nectomonads and haptomonads), paramastigotes, metacyclic promastigotes and, in some samples, the here-reported amastigote-like forms. The Leishmania amastigote-like forms were detected in the SV of sand flies with 14, 18 and 21 days of infection as well as in the TMG at 13 and 18 days post-infection. However, the amastigote-like forms were not detected in the AMG. Factors such as the acidic pH predominating the TMG and the SV, as well as the temperature of the ingested blood, among others, are suggested as contributing to the transformation of the typical promastigotes into the amastigote-like forms. The significance of this finding is discussed and the possible biological advantage for transmission of Leishmania is considered.

Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

Effect of mouse antisera targeting the Phlebotomus papatasi midgut chitinase PpChit1 on sandfly physiology and fitness

In sandflies, the absence of the peritrophic matrix (PM) affects the rate of blood digestion. Also, the kinetics of PM secretion varies according to species. We previously characterised PpChit1, a midgut-specific chitinase secreted in Phlebotomus papatasi (PPIS) that is involved in the maturation of the PM and showed that antibodies against PpChit1 reduce the chitinolytic activity in the midgut of several sandfly species. Here, sandflies were fed on red blood cells reconstituted with naïve or anti-PpChit1 sera and assessed for fitness parameters that included blood digestion, oviposition onset, number of eggs laid, egg bouts, average number of eggs per bout and survival. In PPIS, anti-PpChit1 led to a one-day delay in the onset of egg laying, with flies surviving three days longer compared to the control group. Anti-PpChit1 also had a negative effect on overall ability of flies to lay eggs, as several gravid females from all three species were unable to lay any eggs despite having lived longer than control flies. Whereas the longer survival might be associated with improved haeme scavenging ability by the PM, the inability of females to lay eggs is possibly linked to changes in PM permeability affecting nutrient absorption.

Leishmaniasis transmission: distribution and coarse-resolution ecology of two vectors and two parasites in Egypt

Introduction: In past decades, leishmaniasis burden has been low across Egypt; however, changing environment and land use has placed several parts of the country at risk. As a consequence, leishmaniasis has become a particularly difficult health problem, both for local inhabitants and for multinational military personnel. Methods: To evaluate coarse-resolution aspects of the ecology of leishmaniasis transmission, collection records for sandflies and Leishmania species were obtained from diverse sources. To characterize environmental variation across the country, we used multitemporal Land Surface Temperature (LST) and Normalized Difference Vegetation Index (NDVI) data from the Moderate Resolution Imaging Spectroradiometer (MODIS) for 2005-2011. Ecological niche models were generated using MaxEnt, and results were analyzed using background similarity tests to assess whether associations among vectors and parasites (i.e., niche similarity) can be detected across broad geographic regions. Results: We found niche similarity only between one vector species and its corresponding parasite species (i.e., Phlebotomus papatasi with Leishmania major), suggesting that geographic ranges of zoonotic cutaneous leishmaniasis and its potential vector may overlap, but under distinct environmental associations. Other associations (e.g., P. sergenti with L. major) were not supported. Mapping suitable areas for each species suggested that northeastern Egypt is particularly at risk because both parasites have potential to circulate. Conclusions: Ecological niche modeling approaches can be used as a first-pass assessment of vector-parasite interactions, offering useful insights into constraints on the geography of transmission patterns of leishmaniasis.

Bionomics of phlebotomine sand flies (Diptera: Psychodidae) in the province of Al-Baha, Saudi Arabia

The bionomics of phlebotomine sand flies (Diptera: Psychodidae) were studied for two successive years (January 1996-December 1997) at 12 collecting stations representing six sectors of the province of Al-Baha, Saudi Arabia. The predominant species was Phlebotomus bergeroti (41.7%), followed by lesser numbers of Phlebotomus sergenti (11%), Phlebotomus arabicus (10.6%), Sergentomyia tiberiadis (10.5%), Phlebotomus papatasi (10.2%), Sergentomyia antennata (9.6%), Phlebotomus alexandri (3%), Phlebotomus orientalis (2.3%) and Sergentomyia clydei (1.1%). The distribution of the collected species including species that are elsewhere known to act as vectors of human cutaneous leishmaniasis were distributed across different altitudes in Al-Baha. P. bergeroti, P. papatasi and P. arabicus were more abundant indoors; however, P. sergenti was more abundant outdoors. Sand fly populations exhibited three patterns of seasonal abundance in terms of their monthly activity. P. bergeroti, P. sergenti and P. arabicus were found to be naturally infected with Leishmania-like flagellates at an infection rate of 0.2%.

Sandflies (Diptera: Psychodidae) in a focus of visceral leishmaniasis in White Nile, Sudan

Visceral leishmaniasis (VL) has been known to occur since the 1980s on the western bank of the White Nile River (Central Sudan), 150 km south of Khartoum, and has resulted in high mortality. The most recent outbreak of the disease in this area began in 2006. Entomological surveys were carried out during May 2008, June 2010 and May and July 2011 in the White Nile area. Sandflies were collected using Centers for Disease Control light traps and sticky oil traps in the village of Kadaba and the nearby woodland. Phlebotomus females were dissected for the presence of Leishmania promastigotes. A total of 17,387 sandflies, including six species of Phlebotomus and 10 species of Sergentomyia, were identified. The Phlebotomus species recorded were Phlebotomus orientalis, Phlebotomus papatasi, Phlebotomus bergeroti, Phlebotomus duboscqi, Phlebotomus rodhaini and Phlebotomus saevus. P. orientalis was collected in both habitats. The relative abundance of P. orientalis in the woodland habitat was higher than that recorded in the village habitat. In the woodland habitat, there was a notable increase in the relative abundance of P. orientalis during the surveys conducted in 2008 and 2010 compared to 2011. None of the 311 P. orientalis females dissected were infected with Leishmania promastigotes, although relatively high parous rates were recorded in both habitats. Based on the distribution of P. orientalis recorded in this study, this species is the most likely vector of VL in the endemic focus in the White Nile area. Further investigation is required to elucidate the seasonal abundance and distribution of the vector, as well as the transmission season of VL in both habitats so that appropriate control strategies for the vector can be designed.

Detection of Wolbachia pipientis, including a new strain containing the wsp gene, in two sister species of Paraphlebotomus sandflies, potential vectors of zoonotic cutaneous leishmaniasis

Individual, naturally occurring Phlebotomus mongolensis and Phlebotomus caucasicus from Iran were screened for infections with the maternally inherited intracellular Rickettsia-like bacterium Wolbachia pipientis via targeting a major surface protein gene (wsp). The main objective of this study was to determine if W. pipientis could be detected in these species. The sandflies were screened using polymerase chain reaction to amplify a fragment of the Wolbachia surface protein gene. The obtained sequences were edited and aligned with database sequences to identify W. pipientis haplotypes. Two strains of Wolbachia were found. Strain Turk 54 (accession EU780683) is widespread and has previously been reported in Phlebotomus papatasi and other insects. Strain Turk 07 (accession KC576916) is a novel strain, found for first time in the two sister species. A-group strains of W. pipientis occur throughout much of the habitat of these sandflies. It is possible that Wolbachia is transferred via horizontal transmission. Horizontal transfer could shed light on sandfly control because Wolbachia is believed to drive a deleterious gene into sandflies that reduces their natural population density. With regard to our findings in this study, we can conclude that one species of sandfly can be infected with different Wolbachia strains and that different species of sandflies can be infected with a common strain.

Ecology of cutaneous leishmaniasis in Sinai: linking parasites, vectors and hosts

Cutaneous leishmaniasis (CL) is a neglected clinical form of public health importance that is quite prevalent in the northern and eastern parts of Egypt. A comprehensive study over seven years (January 2005-December 2011) was conducted to track CL transmission with respect to both sandfly vectors and animal reservoirs. The study identified six sandfly species collected from different districts in North Sinai: Phlebotomus papatasi, Phlebotomus kazeruni, Phlebotomus sergenti, Phlebotomus alexandri, Sergentomyia antennata and Sergentomyia clydei. Leishmania (-)-like flagellates were identified in 15 P. papatasi individuals (0.5% of 3,008 dissected females). Rodent populations were sampled in the same districts where sandflies were collected and eight species were identified: Rattus norvegicus (n = 39), Rattus rattus frugivorous (n = 13), Rattus rattus alexandrinus (n = 4), Gerbillus pyramidum floweri (n = 38), Gerbillus andersoni (n = 28), Mus musculus (n = 5), Meriones sacramenti (n = 22) and Meriones crassus (n = 10). Thirty-two rodents were found to be positive for Leishmania infection (20.12% of 159 examined rodents). Only Leishmania major was isolated and identified in 100% of the parasite samples. The diversity of both the vector and rodent populations was examined using diversity indices and clustering approaches.

The taxonomic status of Phlebotomus oliverioi (Diptera, Psychodidae) and a lectotype designation for Psathyromyia brasiliensis

Phlebotomus oliverioi Barretto & Coutinho, 1941 was originally described based only on the male holotype and has since been considered junior-synonym of Psathyromyia brasiliensis (Costa Lima, 1932). The study of the holotype of Ph. oliverioi allowed us to conclude that the head of this specimen belongs to the genus Psychodopygus and the wings, thorax and abdomen belong to a different species of Psathyromyia. Thus, Ph. oliverioi is a not valid species, and must be removed from the synonym and excluded from the provisions of the International Code of Zoological Nomenclature (Articles 17.2 and 23.8). The specimen was measured, drawn, photographed and the morphological characters are discussed. Lectotype and paralectotypes to Pa. brasiliensis are designated.

Sôbre o aparelho genital interno do macho de Phlebotomus longipalpis (Lutz et Neiva, 1912): (Diptera, Psychodidae)

O aparelho genital interno do macho de Phlebotomus longipalpis compõe-se de dois testículos simples e dos vasos deferentes que se unem em um hilo e continuam, depois, como canal ímpar, penetrando na pompeta que representa uma bomba de sucção e pressão para o transporte do esperma. Os vasos deferentes, antes de formar um hilo, possuem regiões glandulares. Também uma parede do hilo é glandular. As duas secreções misturam-se na cavidade do hilo onde se realiza a suspensão dos espérmios neste líquido. A pompeta consta de um corpo de compressão e de um pistilo comprido que penetra neste por ação muscular. Os músculos que comprimem o esperma para dentro dos canais ejaculatórios, localizam-se entre oo corpo de compressão e a extremidade do pistilo, de onde partem alguns fascículos de músculos para a antecosta do 5º segmento abdominal onde se inserem: êste músculo representa o retrator do pistilo. A construção da pompeta e dos canais ejaculatórios é explicada nos esquemas das figs. 1, 4 e 5. Nos tergitos dos 7º e 8º segmentos abdominais do macho existe uma grande glândula odorífera de origem hipodermal. Esta foi encontrada em Phlebotomus longipalpis e P. quinquefer, sendo êste órgão ausente em P. renei e nas fêmeas de tôdas as três espécies.

Observações histológicas na musculatura de Phlebotomus na Renei e P. Longipalpis

Descrevem-se 4 tipos histológicos diferentes de músculos em Phlebotomus renei, sendo êstes comparados com os correspondentes de Phlebotomus longipalpis. Obtivemos os seguintes resultados: 1. De uma forma primitiva embriogenética, cujos feixes de fibrilas se agrupam em fitas radiais e cuja massa sarcoplasmática é muito elevada em comparação com os elementos contráteis, desenvolveu-se um músculo, pobre em sarcoplasma, que funciona como músculo direto do vôo, condicionado para contrações fortes, modificando a posição das partes da articulação das asas para a orientação do vôo. 2. O tipo que se assemelha mais à forma primitiva, é o músculo do tipo lamelar que se encontra em tôdas as partes do corpo e que efetua os movimentos gerais. 3. Por meio de desintegração das fitas de feixes fibrilares resulta um músculo, rico em sarcoplasma, com número reduzido de elementos contráteis. 4. O último tipo é considerado como sendo um músculo que exerce contraturas "tônicas" de ação lenta, porém persistentes. Encontra-se no basipodito do aparelho de cópula do macho e em outras regiões do corpo onde são necessárias contraturas prolongadas. 5. É de supor que o músculo indireto do vôo se formou, por meio de desintegração progressiva dos seus elementos contráteis, sendo o mesmo também rico em sarcoplasma. 6. A estriação transversal do músculo indireto do vôo é de interêsse especial, porque a linha K (de contração) desenvolve-se, aparentemente, na altura da zona h. 7. Observa-se que diferentes regiões do músculo indireto do vôo mostram ondas de contração intermitentes; dêste fato conclui-se que, por meio de entradas e saídas aceleradas, de ondas de contração, é favorecida a alta freqüência das asas.

Sôbre o Phlebotomus Brasiliensis Costa Lima, 1932 (Diptera, Psychodidae)

In 1939, Mangabeira obtained, under laboratory conditions, the development of eggs of Phlebotomus brasiliensis Costa Lima, 1932, collected at Lassance (typical locality), Minas Gerais, Brasil. He then studied the female and immature stages of this Phlebotomus. The results of these observations plus some more recent data on the male, geographical distribution and bionomics are presented. Morphologically it is closest to Phlebotomus runoides. However, the male Phlebotomus brasiliensis differs from all other Phlebotomus because of its very long spicules, similar to those of Brumptomyia. The female differs by its longer ducts, and by possessing only four horizontal teeth in the buccal cavity, whereas P. runoides has approximately 12 teeth. The pupae of P. brasiliensis is characterized by its two pre-alar setae, which are very simple and small and by the abdominal setae, which are not planted on a protruding tubercle. The fourth stage larvae main characteristics are very thin antennae, inserted on a protruding tuberculum, and slightly brush-like hind frontal setae. P. brasiliensis is here reported, for the first time, for the State of Bahia (Cachoeira, Pojuca and Salvador). The species has almost always been found in armadillo burrows. In the State of Bahia it is more frequent during the dry season. Under laboratory conditions, the female lays about 53 eggs.

Sôbre alguns Phlebotomus e Brumptomyia da Colômbia (Diptera, Psychodidae)

The first case of Kala-azar in Colombia was discovered in Soledad, S. Vicente do Chucuri, Dept. Santander, by Gast-Galvis who viscerotomized a three year old girl deceased in December, 1943. In 1944, fifty-three Phlebotominae were collected in the chicken pen of the girl's house, two new species included. Mangabeira helped by A. Gast Galvis, Juan Antonio Montoya and E. Osorno Mesa, collected some Phlebotomus in that country. The geographical distribution of the species of Phlebotomus collected in Colombia (P. abonnenci, P. camposi, P. columbianus, P. dubitans, P. gasti, P. montoyai, P. saulensis, P. serranus, P. triramulus) and two species of Brumptomyia (B. beaupertuyi and b mesari), are included. our description of the male P. columbianus is based on some specimens found in association with females. However, doubts exist about such association of sexes. There is no correspondence between the length of the spicules and the ducts of spermathecae. Besides, the specimens were not obtained by raising. The following new species are described and compared with previously known ones: a) Phlebotomus gasti sp. n. differs from the other species by a protruding tubercle in the gubernaculum. It has also fewer setae in the tuft of the basistyle, a different length of the inferior gonapophyses, and a differently shaped clasper. b) Phlebotomus dubitans sp. n. differs from P. walkeri and P. deanei (according to personal information from O. Theodor, who examined the types, they are identical to P. williamsi and P. sericeus respectively), mainly because these species have the inferior gonapophyses larger than the basistyle and fewer setae in the basistyle. P. evandroi is separated by the shape of the claspers and by the tuft of setae of the basistyle. P. marajoensis is the closest relative to P. dubitans. There is a possibility of their being synonymous. On the other hand, they can be differentiated by the existence of three extra distal spines in P. marajoensis. There is also a difference in their palpal indexes: for marajoensis I - II - IV - III - V, and for dubitans I - IV (III - II) - V. We notice, too, that the inferior gonapophyses in P. marajoensis is a little shorter. P. marajoensis has a long seta in the basistyle (clearly shown in the original drawing), not found in the new species. c) Phlebotomus montoyai sp. n.: The closest relatives are P. noguchii, P. peruensis, P. pescei, P. quinquifer and P. rickardi. They differ from the new species by the number and length of the setae of the basistyle tuft which are more numerous and longer in the new species. The shapes of their claspers are also different. Other differences are: the basal portion of the basistyle in P. noguchii is very wide (in montoyai it is narrower); the intermediate spine of the dististyle is located on a protruding tubercle ( in the new species there is hardly a tubercle); the spicules are long, and the inferior gonapophyses is longer than the basistyle. P. quinquifer and P. rickardi have a shorter dististyle and narrower wings, with different venation. The main difference, however lies, in the M4, which ends almost at the level of the junction of M1 with M2 (in P. montoyai the M4 ends far behind). In P. peruensis and P. pescei the intermediary spine of the dististyle is closer to the distal spine than to the basal one, whereas in the new species it is situated between the two pairs. Their inferior gonapophyses is longer than the basistyle. d) Brumptomyia mesai sp. n. - Closest relatives are: B. hamatus, B. pentacanthus, B. beaupertuyi which are easily separated from the new species because the tufts of their basistyle have thin and differently shaped hairs. Also their claspers are shaped differently. B. avellari is also easily recognized on account of the twisted aspect of its clasper and because the basal tuft of the basistyle has few setae, B. brumpti tuft of setae arise directly from the basistyle; these setae are stronger than those of the new species. It has 8 blade-like setae located on the inner surface of the distal half, whereas the new species has only six setae. In B. brumpti, there are three median and two terminal spines in the dististyle; in the new species, there are two median and two terminal spines and one between them, which is closer to the two median spines. The comparison with B. galindoi is based in a specimen determined by Fairchild and deposited in the entomological collection of the "Faculdade de Higiene e Saúde Pública da Universidade de S. Paulo". The genitalia of the new species is much shorter, in galindoi the inferior gonapophyses is 0,8 mm long whereas in B. mesai it hardly reaches 0,6 mm. The shape of the clasper and the distribution of its setae are different. The sub-median lamellae, besides being longer in B. galindoi are also longer in comparison with the other parts of the genitalia. The gubernaculum of the new species is longer, thinner, and more pointed; in B. galindoi it is shorter and triangular. In the drawing published by Fairchild and Hertig 91947), the basistyle shows 8 blade-like setae on the distal half, whereas in the new species only six are found.