Phlebotomus guggisbergi (Diptera: Psychodidae), a vector of Leishmania tropica in Kenya.

Sand flies were collected in light traps and on oiled papers at four active case sites of human cutaneous leishmaniasis due to Leishmania tropica at Muruku Sublocation, Laikipia District, Kenya. Nearly 5,200 females of five species, including Phlebotomus guggisbergi, were dissected and examined for flagellates. Of 3,867 P. guggisbergi females collected at a multiple case site, 168 (4.3%) harbored mature infections (to include metacyclic promastigotes) of flagellates morphologically identical to Leishmania, while all other flies were negative. Of the infected flies, 164 were collected in a cave near the patients' home, three from crevices on an escarpment immediately behind the house, and one from the bedroom of one of the patients. One hundred sixty-four of the isolates were successfully grown in Schneider's Drosophila medium and harvested for typing by cellulose-acetate electrophoresis. Isoenzyme profiles of the first 22 of these were compared with those of WHO reference strains and well characterized local strains using 12 enzyme loci. The isolates yielded isoenzyme migration patterns that were indistinguishable from those of two L. tropica reference strains and of six L. tropica patient isolates from the same locality. This is the first reported isolation of L. tropica from a sand fly in Kenya, the first reported isolation of Leishmania parasites from P. guggisbergi, and the first confirmed isolation of this Leishmania from a sand fly other than P. sergenti. The finding of such a large number of P. guggisbergi naturally harboring mature infections of L. tropica at an active case site of cutaneous leishmaniasis due to this agent strongly implicates this fly as a vector.

Development of Leishmania major in Phlebotomus duboscqi and Sergentomyia schwetzi (Diptera: Psychodidae).

The extrinsic development of Leishmania major was observed in 2 man-biting sand flies, Phlebotomus duboscqi, a known vector, and Sergentomyia schwetzi, an assumed non-vector. Flies fed on a leishmanial lesion on the nose of a hamster were examined for infection at 0, 6, 12, 18, 24, 36, 48, and 60 hr and at approximately 24 hr intervals from day 3 to day 14 post-feeding. Infection rates, determined by light microscopy, were 47% (n = 258) in P. duboscqi and 5% (n = 162) in S. schwetzi. Transformation from amastigotes to "procyclic" promastigotes occurred in both species at 6-18 hr post-feeding. In P. duboscqi, the parasites multiplied rapidly and developed through as many as 10 forms, including at least 3 dividing-promastigote forms. Metacyclic promastigotes, the "infective" form, appeared at 6 days post-feeding, first in the region of the stomodeal valve, then in the pharynx, cibarium, and proboscis. In a single attempt 14 days post-feeding, a P. duboscqi transmitted L. major to a mouse by bite. In contrast, the parasites multiplied slowly in S. schwetzi, and did not develop beyond "procyclic" promastigotes. The parasites did not migrate anteriorly nor survive beyond 90 hr post-feeding, indicating that S. schwetzi is not a vector of L. major. Classical strategies for vector incrimination may be confounded by the isolation of non-infective early developmental forms of Leishmania from wild-caught non-vectors.

Phlebotomine sand fly (Diptera: Psychodidae) seasonal distribution and infection rates in a defined focus of Leishmania tropica.

A two-year study was conducted of phlebotomine sand fly fauna in a defined focus of Leishmania tropica. A total of 17,947 sand flies representing 10 species were collected from the location. Phlebotomus guggisbergi, a vector of L. tropica in Kenya, was the most prevalent species through the entire period, representing about 80% of the total catch. There was marked seasonal fluctuation in the populations of the three most common species, with highest population levels reached in December and lowest levels reached in July and August. Leishmania-like infections were encountered in 489 P. guggisbergi. No flagellate infections were observed in any other species of sand fly. Although infected P. guggisbergi were collected during each month of the year, the percent parous infected flies was highest (27.5%) during the November through January time period. These data show that the greatest risk of transmission to humans at this focus occurs during December, when the vector is prevalent and infections are common.

A new focus of kala-azar due to Leishmania donovani sensu lato in Kenya.

Three Masai children from Kekonyokie South Location, Kajiado District were diagnosed with visceral leishmaniasis (kala-azar). Leishmanial isolates from the patients were characterized as Leishmania donovani sensu lato, by cellulose acetate electrophoresis. Case histories indicated that the disease was acquired locally. A survey of 409 children at 7 local primary schools and one nursery school revealed no additional case. Sandfly surveys using light traps and sticky paper traps recovered 10 species of sandfly, including 2 Phlebotomus species. P. martini was prevalent throughout the area. P. orientalis was only rarely encountered, but it was the first collection record of this species in the southern portion of the Rift Valley in Kenya. Although no Leishmania-infected sandfly was found, P. martini is probably the vector of kala-azar in the location, as it is elsewhere in Kenya.

A new rural focus of cutaneous leishmaniasis caused by Leishmania tropica in Kenya.

We have identified a new rural focus of cutaneous leishmaniasis caused by Leishmania tropica in Muruku sublocation, Salama location, Laikipia district, Rift Valley province, Kenya. Based on a few available case histories, previous reports of L. tropica in Kenya indicated a tentative geographical distribution. Recently 6 indigenous Kenyans from the new focus, who had never travelled outside Kenya, developed cutaneous lesions on the face and/or extremities found to contain Leishmania by culture and smear. Most of the patients manifested the typical 'urban' dry sore which grew slowly into a nodule measuring 2 x 1 cm to 9.5 x 3 cm, and after some months formed a central crust surrounded by small satellite papules. After treatment with Pentostam (sodium stibogluconate), about 40% of the sores failed to heal completely, either scarring centrally with fulminating papules at the edges and spreading peripherally, or healing but then recrudescing at the edge of the scar. Stationary-phase promastigotes from culture isolates were analysed by cellulose acetate electrophoresis. Isoenzyme profiles of 6 isolates were compared with those of World Health Organization reference strains using 12 enzyme loci; they were indistinguishable from those of 2 L. tropica reference strains. All 6 case sites lay within a radius of 4 km. Several other suspected cases from the same area are being investigated.

Animal reservoirs of leishmaniasis in Marigat, Baringo District, Kenya.

A total of 1128 rodents belonging to seven genera were examined for leishmanial parasites over a period of sixteen months. Parasites were isolated from 36 (12.5%) Tatera robusta, 3 (0.5%) Arvicanthis niloticus, and 2 (0.8%) Mastomys natalensis. All isolates were characterised by isoenzyme analysis using nine enzymes. The enzymes examined were: malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucose phosphate isomerase (GPI), isocitrate dehydrogenase (ICD), nucleoside hydrolase (NH), glucose 6-phosphate dehydrogenase (G6PD), mannose phosphate isomerase (MPI), malic enzyme (ME) and 6-phosphogluconate dehydrogenase (6PGD). The enzyme profiles from these isolates were compared with those from Leishmania reference strains and also with isolates of Leishmania major from man and sandfly, P. duboscqci from the same area. All the isolates except one from a Mastomys were identified as L. major. The isolate from Mastomys was trypanosome-like and remains unidentified. The results in this study show that Tatera robusta is the main reservoir of cutaneous leishmaniasis in Baringo District. None of the animals trapped were found infected with Leishmania donovani suggesting that rodents do not play a role in the transmission of visceral leishmaniasis in this area.

Leishmania major-Phlebotomus duboscqi interactions: inhibition of anti-LPG antibodies and characterisation of two proteins with shared epitopes.

OBJECTIVES: To assess the effect of monoclonal antibodies (MABS) raised against L. major derived LPG on L. major development in vitro and in its natural vector P. duboscqi. Also determine whether LPG molecule and the sand fly the gut lysates have shared epitopes. DESIGN: A laboratory based study. SETTING: Colony bred P. duboscqi sand flies and all other experiments were done under laboratory conditions. METHODS: Laboratory reared sand flies were allowed to feed beneath a blood filled membrane feeder containing 1 x 10(6) amastigotes in 20 microl mixed with 0.5 ml of defibrinated rabbit blood with a 1:100 dilution of anti-LPG MABS. Control blood contained a similar number of amastigotes but no MABS. At least five female previously fed sand flies were later dissected on days two, four, and six post-feeding and examined for promastigote forms and parasite loads in the sand fly mid gut. In vitro, the same number of amastigotes in 100 microl complete Schneider's Drosophila medium was mixed in a 96 well plate with either 100 microl of 1:100 anti-LPG MABS, 1:1000 anti LPG MABS or undiluted sera from L. major infected mice. The control well contained a similar number of amastigotes but no antibodies added. Following an overnight incubation in a CO2 incubator at 37 degrees C and growth at 26 degrees C, parasites were assessed at 3, 6 and 24 hour intervals for changes in their developmental forms. RESULTS: 1:100 dilution of anti-LPG MABS when mixed with amastigotes were effective in reducing L. major development at the early log phase or procyclic stage both in vitro and within the sand fly (p<0.05). The control cultures or sand flies that fed on amastigotes alone and no MABS supported full parasite development up to the metacyclic stage. Results also showed that flies, which had fed on MABS, showed low parasitemia levels of 2+, compared to a high density of 4+ for their controls (p<0.5). CONCLUSIONS: These findings showed that anti-LPG MABS were effective in reducing sand fly infections. This study also showed that P. duboscqi gut lysates and proteins present in L. major-derived LPG share two common proteins of molecular weights 105 kDa and 106 kDa. Further analysis of these individual proteins from the gut should be studied with a view of determining their vaccine potential.

Host feeding preference of Phlebotomus guggisbergi, a vector of Leishmania tropica in Kenya.

Recently the sandfly Phlebotomus guggisbergi was found to be a vector of Leishmania tropica causing cutaneous leishmaniasis in the Laikipia focus, Kenya, but extensive searches have shed no light on the identity of the rural reservoir host(s). In order to discover more about the biology of the vector, a host feeding preference study was conducted on wild sandflies in their natural cave environment over a 6-month period. Solid state Army miniature (SSAM) traps, without light bulb, were suspended over cages with potential hosts or an empty cage control. The animals tested included sheep, goat, dog, cat, hamster, rabbit, giant rat (Cricetomys gambianus), crested rat (Lophiomys imhausi) and rock hyrax (Procavia capensis), all of which (except hamsters) are normally found in the vicinity of the study site. Sandfly collections from traps baited with goat, sheep, cat, dog, rabbit, or wild rodent species were significantly higher than the control, whereas trap collections with hamster and rock hyrax were not significantly different from the control. Numbers of sandflies collected from the goat, sheep and cat were significantly greater than from the rabbit and rodents. The sex ratio also varied between collections: larger animals attracted a higher proportion of female P. guggisbergi than did the smaller animals (P > 0.05). Therefore greater emphasis should be placed on surveying larger animals to assess their status as reservoir hosts for L. tropica in Kenya.

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.

Identification of phlebotomine sandfly bloodmeals from Baringo District, Kenya, by direct enzyme-linked immunosorbent assay (ELISA).

Direct enzyme-linked immunosorbent assay (ELISA) was used to identify the sources of bloodmeals in phlebotomine sandflies from Baringo District, Rift Valley Province, Kenya. Some bloodmeals had been stored for over 4 years before being analysed. Among 356 sandflies identified, 62.9% were Phlebotomus martini, 14.8% Sergentomyia antennatus, 10% S.schwetzi, 6% S.clydei, 1.9% S.adleri, 1.6% P.duboscqi, 1.4% S.africanus and 0.8% S.bedfordi. Out of 224 P.martini bloodmeals, host source was identified for 69. The order of host preference for P.martini was: goat 28.5%, rabbit 22.7%, human 8.9% and others 8.9%. Evidence of mixed feeding was shown by four species comprising sixteen specimens, twelve of which were P.martini. The most effective methods for trapping bloodfed P. martini were sticky paper traps in termite hills, followed by light-traps. Of the 224 P.martini trapped, 58.9% were collected with traps in termite hills, and 22.7% with light traps. Roles of the three most popular hosts for P.martini should be investigated to ascertain whether they act as reservoirs in the transmission of Leishmania donovani causing visceral leishmaniasis in Kenya.

Phlebotomine sandflies of Kenya (Diptera: Psychodidae). I. The validity of Phlebotomus (Larroussius) elgonensis Ngoka, Madel and Mutinga.

Sandflies collected in Kitum cave on the Kenyan side of Mount Elgon were identified as Phlebotomus (Larroussius) elgonensis Ngoka, Madel and Mutinga, a species synonymized with P. (L.) aculeatus Lewis, Minter and Ashford, by D. J. Lewis. A comparison of the flies from Kitum cave with P. aculeatus from Lake Elmentaita, Kenya, shows marked differences in the lengths of: (a) the spermathecal ducts of the female; (b) the style, coxite, genital pump and aedeagus of the male; and (c) the palpal segments of both sexes. Other notable differences are in the shape of the base of the spermathecal ducts; the position of spines on, and the shape of, the tip of the aedeagus; and the appearance of the pharyngeal armatures of both sexes. Phlebotomus elgonensis is redescribed and it is concluded that it is sufficiently different from P. aculeatus to deserve recognition as a valid species.

Abundances and nocturnal activities of phlebotomine sandflies (Diptera: Psychodidae) in termite hills and animal burrows in Baringo District, Kenya.

The abundance of phlebotomine sandflies was studied in a leishmaniasis endemic area in Baringo District, Rift Valley Province, Kenya. This was based on sandfly behaviors at night in their natural habitats of termite hills and animal burrows. Sandflies were collected with an entry-exit trap which has a sieve fixed at the middle to control sandfly movements. Flies were trapped from 1800-0600 hours. Phlebotomus martini (Parrot) were significantly more abundant in termite hills than in animal burrows (P <0.05). Phlebotomus duboscqi (Neveu-Lemaire) were found to favour animal burrows more than termite hills (P <0.05). Nocturnal movements and activities showed that the majority of the sandflies were exiting from termite hills and animal burrows from 1800-2400 hours while from 2400-0600 hours, the majority of the sandflies were entering termite hills and animal burrows (P<0.05). Sergentomyia (Theodor) species showed mixed preferences for these two habitats. Sergentomyia schwetzi (Aldler, Theodor & Pam favoured termite hills more than animal burrows but S. clydei (Sinton) showed great preference for animal burrows. The nocturnal activities or preferences of habitats by phlebotomine sandflies have epidemiological significance in that control measures can be directed to these habitats with a certain degree of accuracy for the vectors of visceral and cutaneous Leishmaniasis in Kenya.

Phlebotomine sandflies of Kenya (Diptera: Psychodidae). III. The identification and distribution of species of the subgenus Larroussius.

The six species of phlebotomine sandflies of the subgenus Larroussius recorded in Kenya are Phlebotomus aculeatus, P. elgonensis, P. guggisbergi, P. longipes, P. orientalis and P. pedifer. Five are proven vectors of leishmaniasis in that country or elsewhere. Males of all six can be identified by the morphology of the aedeagus or the number and position of the hairs on the inner surface of the coxite. Additional features separating some of the species are the sizes of the palpal and antennal segments. The females have usually been considered difficult or impossible to distinguish. A comparison of the base of the spermathecal ducts is made and it is shown that all six can be identified by this character alone. A map of Kenya is given, showing the presently known distribution of the six Larroussius species. Further surveys are necessary in parts of the country that have not been systematically surveyed.