The race to discover the insect vector of kala-azar: a great saga of tropical medicine 1903-1942.

In the 19(th) century, a devastating epidemic of visceral leishmaniasis (kala-azar) swept through northeast India. After identification of the pathogenic agent, Leishmania donovani, in 1903, the question of its transmission remained to be resolved. In 1904, thanks to work by L. Rogers on cultures of this parasite it became probable that a haematophagous arthropod was responsible for transmission. J.A. Sinton suggested, in 1925, the distribution of the sand fly Phlebotomus argentipes was similar to that of the disease and, thereafter, two independent teams led by H.E. Shortt in Assam and R. Knowles and L. Napier in Calcutta concentrated on this potential vector. Parallel work was in progress in China, directed by E. Hindle and W. S. Patton for the Royal Society Kala-azar Commission, on another species of sand fly. In 1942 the Assam workers transmitted L. donovani to five human volunteers by the bites of colonised P. argentipes and the race was over.

Mark-release-recapture of sand flies fed on leishmanial dogs: the natural life-cycle of Leishmania infantum in Phlebotomus ariasi.

Wild-caught Phlebotomus ariasi Tonnoir permitted to feed on dogs infected with Leishmania infantum Nicolle were marked with fluorescent powder and released into their natural habitat in an uninhabited area of the Cévennes in southern France. Over a period of 29 days after release, 253 females were recaptured with CDC miniature light traps or by active search at night with portable UV lamps. The ovaries and infections in the alimentary tract were then examined. The females oviposited 6 nights after in infecting blood meal. Second blood meals were never taken during the maturation of eggs. During the first ovarian cycle, midgut infections with promastigotes were only moderately heavy. The intensity of infection increased markedly during the second ovarian cycle and, in the third ovarian cycle, the first pharynx infected with paramastigotes was seen (on day 19). From day 19 to day 29, 76% of the flies had pharyngeal infections. Three out of 19 sand flies with pharyngeal infections recaptured during this period had metacyclic promastigotes in their mouthparts. The long time required for parasites to reach the proboscis in completely natural conditions suggests that their presence in the mouthparts is not a prerequisite for transmission by bite. It is more likely that transmission is most commonly by the regurgitation of metacyclic promastigotes from the thoracic midgut following damage to the stomodaeal valve by chitinase produced by the parasite during its development in the gut of the fly. Nevertheless, it is reasonable to assume that the bite of a fly with metacyclic promastigotes in the proboscis (or salivary glands) would also be infective.

Deltamethrin-impregnated dog collars have a potent anti-feeding and insecticidal effect on Lutzomyia longipalpis and Lutzomyia migonei.

Deltamethrin-impregnated PVC dog collars were tested to assess if they were effective in protecting dogs from sand fly bites of Lutzomyia longipalpis and Lu. migonei. A protective effect against Old World species Phlebotomus perniciosus was demonstrated before. Four dogs wearing deltamethrin collars and three dogs wearing untreated collars (not impregnated with deltamethrin) were kept in separate kennels for over eight months in a village on the outskirts of Fortaleza in Ceará, Brazil. Periodically, a dog from each group was sedated, placed in a net cage for 2 h in which 150 female sand flies had been released 10-15 min before. Lu. longipalpis were used 4, 8, 12, 16, 22, 27, and 35 weeks after the attachment of the collars. Lu. migonei were used 3, 7, 11, 15, 22, 26, and 36 weeks after attachment. During 35 weeks, only 4.1% (81 of 2,022) Lu. longipalpis recovered from the nets with the deltamethrin collared dogs were engorged, an anti-feeding effect of 96%. Mortality initially was over 90% and at 35 weeks was 35% with half of the sand flies dying in the first 2 h. In contrast, 83% of the 2,094 Lu. longipalpis recovered from the nets containing the untreated collared dogs were engorged and the mortality ranged from zero to 18.8% on one occasion with 1.1% dying in the first 2 h. Similar findings were found with Lu. migonei: of 2,034 sand flies recovered over this period, only 70 were engorged, an anti-feeding effect of 96.5%, and mortality ranged from 91% initially to 46% at 36 weeks. In contrast, engorgement of controls ranged from 91 to71% and a mortality ranged from 3.5 to 29.8%. These studies show that deltamethrin impregnated collars can protect dogs against Brazilian sand flies for up to eight months. Thus, they should be useful in a program to control human and canine visceral leishmaniasis.

Molecular systematics of the phlebotomine sandflies of the subgenus Paraphlebotomus (diptera, psychodidae, phlebotomus) based on ITS2 rDNA sequences. Hypotheses Of dispersion and speciation.

Phylogenetic Paraphlebotomus relationships are inferred by a study based on the sequences of ITS2, which has been sequenced in nine Paraphlebotomus species: P. alexandri, P. andrejevi, P. jacusieli, P. kazeruni, P. mireillae, P. mongolensis, P. saevus, P. sergenti and P. similis and in two out-groups species of the subgenus Phlebotomus: P. papatasi and P. duboscqi. Paraphlebotomus alexandri appears as the sister group of all other Paraphlebotomus sandflies. Among the other species, three groupings are clearly highlighted: andrejevi and mongolensis; mireillae and saevus; jacusieli, kazeruni, sergenti and similis. These groupings are related to speculations about the migration of Paraphlebotomus from a centre of dispersion located in the Middle East sometime from the early Eocene to the late Miocene.

Courtship and mating by the sandfly Phlebotomus duboscqi, a vector of zoonotic cutaneous leishmaniasis in the Afrotropical region.

Courtship behaviour of males of the Afrotropical sandfly Phlebotomus duboscqi Neveu-Lemaire (Diptera: Psychodidae) involved mounting the female and clasping her 'waist' with the male coxites placed between the female's thorax and abdomen. This behaviour, which we call 'piggy-backing', was preceded by male wing beating, perhaps involving mate recognition and contact pheromones. It did not seem to be pre- or postcopulatory mate guarding. Piggy-backing was attempted by P. duboscqi males on females of other species (P. papatasi and P. perniciosus) and even on other male P. duboscqi. The majority of female P. duboscqi piggy-backed by males were already inseminated, and most of the courting did not lead to copulation. This, coupled with the presence of a mating plug (semen) in each spermatheca of inseminated females, suggests that female P. duboscqi are monogamous for at least the first gonotrophic cycle. Male courtship with piggy-backing was more intense when females could feed on a hamster than when a hamster was present but the females were denied access to the host. It is suggested that, when a hamster was available to the females, the conditions in the laboratory are similar to those in rodent holes, the natural habitat of P. duboscqi.

The genital atrium as a good taxonomic character to distinguish between species of phlebotomine sandflies (Diptera: Psychodidae) from Venezuela.

The shape and size of the genital atria of 17 phlebotomine sandflies from Venezuela were examined. The atria were found to express constant characters among individuals of the same species and to be sufficiently different between species to allow taxonomic separation. The spines on the genital armature are described and the characters that can be used to classify individual specimens to species level identified. It is suggested that these characters can be of use on specimens where the spermathecae have been lost or where cryptic species are concerned. Dissection procedures to display the atrium are given.

[Description of Phlebotomus (Paraphlebotomus) riouxi n. sp. (Diptera:Psychodidae) of northern Africa]. / Description de Phlebotomus (Paraphlebotomus) riouxi n. sp. (Diptera-Psychodidae) d'Afrique du Nord.

Description of a new palearctic species from Morocco, Tunisia and Spain: Phlebotomus (Paraphlebotomus) riouxi closely related to P. chabaudi. Differential identification with the neighbouring species. The aedeagus of the male is unrecurved, a character which distinguishes P. riouxi from other species with recurved aedeagus and the basal process of the coxite is bigger and more tufted than in P. chabaudi. For female, which has the same spermatheca with a typical terminal ring, the only available character to differentiate from P. chabaudi is the aspect of the armature in the genital atrium.

Mitochondrial haplotypes and phylogeography of Phlebotomus vectors of Leishmania major.

Haplotypes of eight phlebotomine species were characterized by cycle sequencing a mitochondrial (mt) DNA fragment (cytochrome b to NADH1) amplified from single sandflies by PCR. Phlebotomus (Phlebotomus) papatasi displayed little variation throughout its large geographical range. We conclude that this vector of Leishmania major suffered a population bottleneck late in the Pleistocene and then radiated out from the eastern Mediterranean subregion. There was no support for a recent domestic lineage of P. papatasi. The mtDNA molecular clock in phlebotomines (subgenera Phlebotomus and Larroussius) was calibrated by reference to palaeogeographical events in Africa and the Mediterranean subregion. It fitted a pairwise nucleotide sequence divergence rate of 1.0-2.5% per million years. Co-evolution of L. major, its Phlebotomus vectors and mammalian reservoirs is discussed.

Phlebotomine sandflies of Kenya (Diptera: Psychodidae). V. Phlebotomus (Paraphlebotomus) mireillae n.sp.

A new species of the sandfly subgenus Paraphlebotomus Theodor from Kenya is described and named Phlebotomus mireillae. The type locality is Utut Reserve, near Gilgil, Nakuru District. The description is based on 10 males and 10 females reared from individual egg batches of wild-caught females. Males of the new species differ from nine other species of the subgenus by the shape of the aedeagus, which is straight, not curved or hooked. They are separated from the remaining three species by other differences in the aedeagus, and in the style and coxite process. Female P. mireillae have backwardly pointing spines inside the distal quarter of the spermathecal ducts, a character not previously described for phlebotomine sandflies. Additional characters which distinguish females of the new species from others of the subgenus are differences in the pharyngeal armature and spermathecae and the lengths of the third antennal segment and labium.

Protection of dogs from bites of phlebotomine sandflies by deltamethrin collars for control of canine leishmaniasis.

Dog collars made of PVC plastic impregnated with the pyrethroid insecticide deltamethrin at 40 mg/g were investigated for their protective efficacy against phlebotomine sandflies. Collared dogs were kept separately (two untreated control dogs lived together) in outdoor enclosures, each with a kennel, in the Cévennes, southern France. To measure sandfly mortality and anti-feeding effects due to the deltamethrin-impregnated collars worn continuously by the dogs for up to 8 months, each dog was periodically sedated and exposed for 2h to 150-200 laboratory-reared Phlebotomus perniciosus females (plus c. 25 males) inside a net (1.2 m square, 1.8 m high) indoors. After dogs were removed from the nets, allowed to recover and returned to their kennels, any dead sandflies were collected from inside the net and counted. Surviving flies were kept overnight, then scored according to whether they were still alive or dead, unfed or blood-fed. From tests 2, 3, 4, 13, 20, 26 and 34 weeks after the dogs began wearing collars, the overall numbers of blood-fed female sandflies recaptured were 75 from two dogs with collars, compared with 1911 from two collarless dogs. Thus, for every 100 flies which fed on collarless dogs, only 4 fed on collared dogs, i.e. the collars protected dogs from 96% of the bites and this activity was maintained for up to 34 weeks. During the same period, the percentage of recaptured female sandflies that had fed on collared dogs was 0-12% compared to 55-95% on collarless dogs. Immediately after dogs were taken out of the nets, 21-60% of flies confined with the collared dogs were found dead, compared to 0-12% with the controls. It is concluded that, at least in the Mediterranean subregion, this insecticidal collar would protect a dog from the majority of sandfly bites and retain a killing effect for a complete sandfly season. Moreover, it seems likely that the use of collars on all dogs in a focus of Leishmania infantum would reduce contact between sandfly vectors and canine reservoir hosts sufficiently to diminish the risk of infection for humans as well as dogs.

Post-engorgement dynamics of haemagglutination activity in the midgut of phlebotomine sandflies.

Haemagglutination activity (HA) was studied in gut extracts of both sexes of adults of six sandfly species. HA was sex dependent, with the activity in males more than 50 times lower than that of unfed females. In females, high HA was demonstrable in both the thoracic and abdominal midgut but not in the hindgut. In unfed flies the activity was similar in the midgut wall and the gut contents whereas, in fed females, a high increase was seen in the midgut contents. After blood-feeding, HA was elevated, reaching peak titres 2 days later and then falling to the base level or less immediately after defaecation. The magnitude of the HA response differed according to species, ranging from 2-fold in Lutzomyia carmelinoi up to 16-fold in Phlebotomus duboscqi. Quantitative differences between sandflies in their HA response may influence their ability to support the development of Leishmania spp.

Agglutination of Leishmania promastigotes by midgut lectins from various species of phlebotomine sandflies.

Lectins which agglutinate Leishmania promastigotes were demonstrated in gut lysates from laboratory colonies of five Phlebotomus and two Lutzomyia species. In general, the highest agglutination titres were found in P. halepensis and Lu. longipalpis (Jacobina). Marked differences were found in the agglutination of promastigotes of various Leishmania species and strains and high agglutination titres were observed in some natural vector-parasite combinations, such as Phlebotomus argentipes and Le. donovani. Intraspecific variability, in agglutination of Le. major strains, could be related to the varying infectivity of the strains to laboratory animals. Similar carbohydrates, of which the most effective were D-mannosamine and N-acetyl-D-glucosamine, inhibited the agglutination of Le. major and Le. donovani promastigotes by midgut extracts from P. papatasi and Lu. longipalpis. D-Mannosamine and N-acetyl-D-glucosamine inhibited agglutination of promastigotes in all vector-parasite combinations. The results of the carbohydrate-inhibition tests indicate that the lectin specificities in Phlebotomus are similar to those in Lutzomyia.

A laboratory model of canine leishmaniasis: the inoculation of dogs with Leishmania infantum promastigotes from midguts of experimentally infected phlebotomine sandflies.

Twenty-five dogs (beagles) were infected with Leishmania infantum by the intradermal inoculation of an estimated 5-8,000 metacyclic promastigotes harvested from the midguts of 320 experimentally infected P. perniciosus. Details are given of the methods of infecting the flies and harvesting the parasites. All dogs developed small, self-healing chancres at the sites of inoculation. Parasites were isolated from lymph nodes, bone marrow or spleen of 21 dogs, 12 of which developed signs of disease and raised IFAT litres to Leishmania. Nine of the 21 remained healthy over a five-year observation period. Six of the nine were shown to have a cell mediated immune response to Leishmania. No parasites were isolated from four of the 25 dogs, two of which had a demonstrable cell mediated immunity and another had low transitory IFAT titres. The fourth had chancres at the sites of inoculation. The results show that dogs can be readily infected with promastigotes from the midguts of sandflies. However, a high proportion develop a cell mediated immunity and show on signs of disease. It is suggested that serological surveys of dogs for canine leishmaniasis reveal neither the true prevalence of infection nor the intensity of transmission. The efficacy of controlling human visceral leishmaniasis caused by L. infantum by destroying seropositive dogs is questioned.

The armature in the genital atrium as a new taxonomic character distinguishing females of Phlebotomus papatasi and P. duboscqi (Diptera: Psychodidae).

Descriptions are given of armatures in the genital atria of females of the two morphologically similar sandfly species, Phlebotomus papatasi and P. duboscqi. The species are distinguishable by the size and shape of the armature, the grouping of the spines in the armature and the length and shape of the spines. These characters have been shown to separate females of other closely related species of phlebotomine sandflies.

Phlebotomine sandflies of Kenya (Diptera: Psychodidae). IV. The armature in the genital atrium of female Larroussius as a means of identification.

Descriptions are given of armatures in the genital atria of the six known Kenyan species of phlebotomine sandflies of the subgenus, Larroussius, namely Phlebotomus aculeatus, P. elgonensis, P. guggisbergi, P. longipes, P. orientalis and P. pedifer. Phlebotomus aculeatus, P. longipes and P. pedifer can be recognized by the shapes of their armatures. Differences in the length and arrangement of the spines in the armature of P. elgonensis and P. longipes are diagnostic features. The distinguishing feature of P. guggisbergi is a wide variation in spine thickness. The most notable feature of P. orientalis is the angle at which the spines lie. The appearance of the base of the spermathecal duct remains the method of choice for the identification of all six but, if this feature is not well displayed in dissected females, they can be distinguished by the armature. It is suggested that descriptions of new species should include an illustration of the armature in the genital atrium.