A simple micro-assay method for estimating blood meal size of the sand fly, Phlebotomus langeroni (Diptera: Psychodidae).

The accurate measurement of blood meal size in Phlebotomus langeroni, the potential vector of infantile visceral leishmaniasis in Egypt, is important to determine the number of parasites taken in fully engorged insects. A simple protein content micro-assay is introduced for that purpose. The accuracy of this method was confirmed by hemoglobin estimation method. Laboratory bred P. langeroni were fed artificially on defibrinated human blood and the fully engorged flies were carefully dissected on ice, within 1-10 min after feeding, since the time of dissection is critical. Serial concentrations of the defibrinated human blood were required as standards. Results show that the full blood meal taken by P. langeroni ranged from 0.76-0.94 mm3 of blood with a mean volume of 0.85 +/- 0.02 mm3 and from 0.71- 0.99 mm3 of blood with a mean volume of 0.83 +/- 0.02 mm3 as measured by protein content and hemoglobin estimation methods respectively. The data showed that there is no significant difference (P=0.27) between the two methods in estimating the blood meal size of P. langeroni. In addition, protein content micro-assay had the advantages of being accurate, rapid, sensitive and reliable.

Evaluation of avermectins as sandfly control agents.

The potential of avermectins as environmentally safe agents for the control of the sandfly vectors of Leishmania spp. was investigated in the laboratory. Female Phlebotomus papatasi and P. langeroni were fed either bloodmeals containing laboratory-grade ivermectin or sugarmeals containing a commercial-product based on abamectin. Low concentrations of either avermectin killed the sandflies, with median lethal concentrations (LC(50)) of just 13 ng ivermectin or 0.5 ng abamectin/ml for P. papatasi and 44 ng ivermectin or 35 ng abamectin/ml for P. langeroni. The feeding of female sandflies of both species with generally sublethal doses (LC(30)) of ivermectin in blood led to markedly reduced survival and fecundity (i.e. number of eggs laid/ovipositing female). However, addition of ivermectin to the bloodmeal (or of abamectin to the sugarmeal) of the females had no statistically significant effect on the proportion of their eggs that hatched. The results indicate that very small amounts of avermectin in their blood- or sugar-meals could control P. papatasi and P. langeroni, by killing many flies and, in the case of ivermectin, by reducing the fecundity of the survivors.

The effect of Leishmania major on some biological parameters of Phlebotomus papatasi (Diptera: Psychodidae) from endemic and non-endemic areas in Egypt.

Biological activities of two populations of Phlebotomus papatasi collected from two different areas in Egypt (Sinai and Aswan) and the effect of Leishmania major on such activities were investigated under insectary conditions. A significant reduction (P < 0.05) was observed in the number of eggs laid by the infected females when compared to the non-infected females of the tested populations. The immatures of the Aswan population required a longer period (P < 0.05) to complete larval development. Life expectancy at emergence for infected and non-infected females was almost 13 days for the two populations. No significant difference was observed for the effect of L. major on the survivorship of tested populations. The mean generation time for the non-infected and infected females of Sinai and Aswan were 47.5, 49.8, 50.8, 49.5 days, respectively. Mean productivity (the number of females produced by one female) of the non-infected females of the Sinai population (18.1 female female/female) was significantly higher (P < 0.05) than the Aswan population (12.3 female female/female) while the productivity for infected females for both Sinai and Aswan populations (10.8 and 7.3 female female/female) was significantly reduced (P < 0.05). These results show Leishmania infection affects the fertility and productivity of both populations and may be a factor in controlling the insect population size in nature.

Susceptibility to Leishmania major of different populations of Phlebotomus papatasi (Diptera: Psychodidae) from endemic and non-endemic regions of Egypt.

The ability of three populations of Phlebotomus papatasi collected from different areas of Egypt (Sinai, Aswan and Delta regions) to acquire successfully and transmit Leishmania major (Sinai sandfly isolate IPAP/EG/89/SI-177) was evaluated in the laboratory. Flies from each population were fed artificially on infected blood suspensions, using a chick-skin-membrane feeding apparatus, and naturally on infected mice. Samples of fed flies were dissected and examined microscopically to determine the infection rate and intensity of parasite infection. The Aswan population had a significantly lower feeding rate (16.2%) than the Sinai (51.2%) and Delta (69.7%) populations (P < 0.0001). The infection rate for the Sinai population was significantly higher (65.9%) than the rate for the Delta (52.3%; P < 0.05) and slightly higher than that for the Aswan (62.5%). No differences were observed in the intensity of L. major infection in the midguts of the sandflies examined from any of the three populations. When flies from each population were fed naturally on BALB/c mice infected with L. major, the feeding rates of the three populations showed a similar pattern to that seen in the membrane feeds, with the Aswan population having the lowest rate. In each of two separate trials for each population, a group of artificially infected flies was refed on uninfected BALB/c mice. Thirty-six days following exposure to the infected sandflies in the Sinai population, a leishmanial lesion was observed on the corner of one animal's mouth. These observations provide the final piece of evidence that P. papatasi is a vector of L. major in Egypt.

Vector-host-parasite inter-relationships in leishmaniasis. I. The effect of Leishmania parasites on rate of digestion of blood proteins from various vertebrate hosts by the sand fly Phlebotomus langeroni (Diptera: Psychodidae).

Protein digestion in the gut of Phlebotomus langeroni (Nitzulescu) was studied at four subsequent 24 hour intervals post feeding on human, dog (Canis familiaris), rat (Rattus rattus) and turkey (Melagris gallopava) bloods with and without Leishmania infantum or L. major promastigotes. Most of the proteins of the studied blood meals were digested within 96 hours. The percent of blood proteins digested in the first 48 hours was higher than in the second 48 hours in all cases of the studied blood meals except the normal blood of the turkey in which the ratio of the digested blood proteins in the two periods was 1:1. During the first 48 hours, the percent of the digested blood proteins was lower than normal in the presence of L. infantum in case of human and dog blood meals. The reverse was true in case of the rat and turkey blood meals in the presence of L. infantum and in the blood meals from each of the four vertebrate hosts in the presence of L. major. The significance of these findings in considering L. infantum as a natural parasite of P. langeroni in El Agamy focus was discussed.

Vector-host-parasite inter-relationships in leishmaniasis. II. Influence of blood meal from natural vertebrate hosts with and without Leishmania infantum and L. major on the proteolytic activity in the gut of Phlebotomus langeroni (Diptera: Psychodidae).

Proteolytic activity in the gut of Phlebotomus langeroni (Nitzulescu) was studied at four subsequent 24 hours intervals post feeding on human, dog (Canis familiaris), rat (Rattus rattus) and turkey (Melagris gallopava) bloods with and without Leishmania infantum or L. major promastigotes. The gut proteolytic activity increased gradually after feeding to reach a maximum at 48 hours post feeding on any of the 12 studied blood meals. In all cases, the activity declined after 48 hours and almost terminated by 96 hours. In case of normal bloods, the proteolytic activity, at 48 hours post feeding, was the highest in case of dog followed by human, rat and turkey respectively. At this time interval the activity was relatively lower in case of human and dog blood mixed with L. infantum promastigotes than in their respective normal blood. The reverse was true in all other blood meal combinations. Promastigotes were alive and active in fresh gut smears of P. langeroni fed on human, dog and rat bloods mixed with either L. infantum or L. major, throughout the digestion period (1-4 days). They were arrested in P. langeroni within the first day post feeding on turkey blood mixed with either Leishmania species. The results of the present study indicate that the kind of blood meal and the Leishmania species affect the proteolytic activity of P. langeroni. The decrease or increase of the proteolytic activity of P. langeroni has no effect on the survival of Leishmania parasites present in the gut and the kind of blood meal is responsible for their development.

Vector-host-parasite inter-relationships in leishmaniasis. III. Impact of blood meal from natural vertebrate hosts on the survival and the development of Leishmania infantum and L. major in Phlebotomus langeroni (Diptera: Psychodidae).

Phlebotomus langeroni collected from a leishmaniasis endemic focus at Et Agamy, Alexandria, Egypt, were found to have fed on blood from man, dogs (Canis familiaris) and rats (Rattus rattus). The effect of the kind of blood meal on the development and the life-cycle of L. infantum and L. major in laboratory reared P. langeroni was therefore investigated. A membrane feeding technique was used to infect sand flies. Gut smears of infected females were examined immediately after feeding and daily for 16 days. Nectomonads and short promastigote forms of L. infantum or L. major were detected in females fed on human, dog and rat bloods at all intervals. Paramastigotes (infective stage) were present only in females fed on dog blood containing L. infantum or L. major and in those fed on rat blood containing L. major. It is concluded that among the factors influencing the Leishmania-phlebotomus relationship is the natural medium in which the parasite is present in vivo. The blood of the natural reservoir host(s) is the key factor for the development of the infective parasite form in the sand fly and P. langeroni could be considered a potential vector for transmitting L. infantum from dogs and L. major from rats and dogs but not from man. This investigation offers a new concept for the study of interactions among vector, host and parasites in Leishmania transmission.

Vector-host-parasite inter-relationships in leishmaniasis. IV. Electrophoretic studies on proteins of four vertebrate bloods with and without Leishmania infantum or L. major.

Fifty five protein bands with relative mobilities of 8,954 to 245,471 kilo Daltons (kD) were electrophoretically separated from 12 feeding media of blood from 4 natural vertebrate hosts of Phlebotomus langeroni. The feeding media included human, dog (Canis familiaris), rat (Rattus rattus) and turkey (Melagris gallopava) bloods without or with Leishmania infantum or L. major promastigotes. Protein bands were identical among the feeding media of one host's blood but varied in number (24-28 bands) and relative mobilities among the various hosts' blood. Some protein fractions were common among the various hosts blood, others were only present in two or three hosts' blood and some were restricted to one host blood and were unique for each host. This study provides data which may help in understanding why blood from different natural hosts may variably influence the life cycle of Leishmania parasite in the sand fly gut.

Experimental infection and transmission of Leishmania major by laboratory-reared Phlebotomus bergeroti parrot.

The ability of colony-reared Phlebotomus bergeroti Parrot to successfully acquire and transmit Leishmania major (strain IPAP/EG 89/SI-177) was demonstrated in the laboratory. Female P. bergeroti were fed naturally on infected mice and artificially on infected blood suspension using a chick-skin membrane apparatus. Groups of sand flies, either infected on mice or by membrane feeding, were dissected and examined using light microscopy at 2-6, 8, 10, and 11 days postfeeding. Heavy promastigote infection of the thoracic and abdominal midgut was observed in 10% (2 of 20) of the naturally infected flies. Promastigote maturation was observed in 87% (81 of 93) of the artificially infected sand flies, with promastigotes observed in the cibarium and mouthparts at five days postinfection, and infective metacyclic stage promastigotes observed at eight days postinfection. Ten days postinfection, 31% (10 of 32) of the remaining artificially infected sand flies refed on an uninfected BALB/c mouse. Twenty-eight days following exposure to the infective sand flies, leishmanial lesions were observed on the pads of the mouse's front feet. The development of lesions on mouse foot pads clearly suggests the potential of P. bergeroti to serve as a vector for L. major.

Reduced longevity and fecundity in Leishmania-infected sand flies.

Phlebotomus papatasi and P. langeroni were infected with Leishmania major and L. infantum by membrane feeding. Each sand fly ingested approximately 200 parasites per blood meal. Higher mortality in both sand fly species was seen with mixed infections than with a single parasite species. There was no significant difference between infections with either L. major or L. infantum in their natural vectors or experimental hosts. Infection significantly depressed the mean number of eggs laid per female.

Enzyme polymorphism and genetic variability of one colonized and several field populations of Phlebotomus papatasi (Diptera: Psychodidae).

The Alexandria laboratory colony and five field populations of Phlebotomus papatasi (Scopoli) from Egypt were analyzed for genetic variation at 17 enzyme loci. The laboratory colony was characterized by a low level of genetic variation as measured by the average number of alleles per locus (A = 1.70 +/- 0.16) and the average expected heterozygosity (He = 0.06 +/- 0.02). Polymorphism was observed at 23.5% of the examined loci, and genotype frequencies at two loci (PGM, AK-2) were found to deviate slightly from the Hardy-Weinberg equilibrium. In contrast, the average number of alleles per locus for field populations ranged from A = 2.35 +/- 0.20 to 2.76 +/- 0.10, and He ranged from 0.15 +/- 0.03 to 0.21 +/- 0.05. All loci of field populations exhibited polymorphism, ranging from 47.0% to 76.5%, and four to seven loci in each population were found to deviate from the Hardy-Weinberg equilibrium. Deviations in both colonized and field populations were caused by heterozygote deficiency. Despite geographic isolation and some individual deviations from the Hardy-Weinberg equilibrium, no evidence of significant genetic difference was obtained for any of the populations sampled. Calculated indices of genetic distance and genetic identity for the five field populations showed minor variation but were collectively representative of a single, genetically uniform population.

The development of Leishmania infantum in Phlebotomus langeroni nitzulescu (Diptera: Psychodidae).

The extrinsic development of Leishmania infantum was observed in Phlebotomus langeroni the potential vector of visceral leishmaniasis in El-Agamy, Egypt. Flies were infected with L. infantum isolated from the same area, using membrane feeding technique. Flies were examined for infection at 1-10 days post-infection. The initial establishment of the parasites was in the posterior midgut region beginning at sixth day post-infection. Parasites were first observed in the esophagus at day 8, and in the posterior armature region of the pharynx at day 9. The pattern of development of L. infantum in P. langeroni was typically suprapylarian.

Adult diet as a factor affecting biology of the sandfly Phlebotomus papatasi (Diptera: Psychodidae).

The effects of adult nutrients on egg retention, immature development and adult survival of P. papatasi, the important vector of leishmaniasis in Egypt were investigated. The tested nutrients were distilled water, overripe fig fruits, guinea pig blood, sucrose solution and alternative meals of blood and sucrose. Egg retention was observed in females irrespective to the type of offered nutrient (r = 0.21) but higher proportion (47%) of blood fed females had retained eggs. Duration of the life cycle was higher for the progeny of fig fed females (P less than 0.05) and mean generation time was longer for sucrose fed females (P less than 0.05). Such nutritional effect on life cycle was observed only for pre-oviposition periods and no extend effect on larval or pupal durations. The survivorship rates for eggs through adults were similar (P greater than 0.05). It is estimated that the population would increase by Ca. 15, 11, 10 and 7 folds if the mother female was fed blood, sucrose, fig or distilled water respectively. The mean life time differed significantly (P less than 0.05) among females fed on different nutrients with the highest co (life expectancy at emergence) value (14.98 +/- 2.75 days) for sucrose fed females. Males fed on distilled water, fig fruits or sucrose solution were with similar longevities P greater than 0.05). In respect to leishmania transmission, the calculated expectancies for female life beyond the infective age indicated that blood-sucrose fed females have higher capability than those fed on sucrose blood or blood alone.

The breeding habitats of Phlebotomus sand flies (Diptera:Psychodidae) in El Agamy, Alexandria Egypt.

A prolonged search for the breeding habitats of Phlebotomus sand flies in El Agamy, resulted in the collection of 105 newly emerged P. papatasi and P. langeroni. These flies were from poultry sheds, rubbish on stone piles and rubbish piles.

Electrophoretic comparison of the Leishmania vectors Phlebotomus papatasi and P. langeroni (Diptera: Psychodidae).

Cellulose acetate electrophoresis was employed to detect 22 different enzyme systems in laboratory-reared populations of the sympatric Leishmania vectors, Phlebotomus papatasi (Scopoli) and P. langeroni Nitzulescu. Electrophoretic conditions sensitive enough to permit as many as eight separate enzyme assays to be performed on individual specimens were developed. Under these conditions, 18 enzymes were detected with high resolution and regularity. Evidence was obtained which suggested that a number of enzymes in both species are under multilocus genetic control. Polymorphism was observed in 14 of 25 (56%) loci detected in P. papatasi and in eight of 24 (33%) loci detected in P. langeroni. Differences in electrophoretic profiles of malic enzyme, 6-phosphogluconate dehydrogenase, and fumarate hydratase were considered to be genetically fixed in both sexes of P. papatasi and P. langeroni. Their detection may permit an accurate and more rapid separation of these vectors in field collections.

Sandflies (Diptera: Psychodidae) in Mersa Matruh and Siwa oasis, A.R. Egypt.

Sandflies were surveyed 3 times during 1989 in Mersa Matruh city and Siwa oasis to investigate their status. Only Phlebotomus papatasi was identified from inside houses and outdoor sites. More flies were collected in Mersa Matruh than in Siwa. Results document for the first time the presence of P. papatasi in Mersa Matruh and verify its presence in Siwa oasis.

Feeding patterns of Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae) in El Agamy, Egypt.

Blood meals from 602 Phlebotomus papatasi (Scopoli) and 49 Phlebotomus langeroni Nitzulescu were collected in El Agamy, Egypt, and were identified using counterimmunoelectrophoresis. Blood meals were tested against specific antisera of eight vertebrate hosts (human, cat, dog, rat, sheep, goat, general avian, and general bovine). Of 597 P. papatasi collected indoors, 594 contained human blood and three had mixed blood meals (human-dog, human-rat, and human-avian). Four of five P. papatasi collected outdoors contained human blood and one contained avian blood. All 39 P. langeroni collected indoors had fed on humans. Six of 10 outdoor-collected P. langeroni had fed on human blood, 2 on dog, 1 on cat, and 1 on rat blood. Both P. papatasi and P. langeroni feed predominantly on humans in El Agamy, Egypt. The documented feeding on humans and dogs by P. langeroni supports the role of this species as the primary vector of visceral leishmaniasis at the El Agamy focus.