Elimination of symbiotic Aeromonas spp. from the intestinal tract of the medicinal leech, Hirudo medicinalis, using ciprofloxacin feeding.

The use of the medicinal leech (Hirudo medicinalis) in promoting venous drainage in tissues whose vitality is threatened by venous congestion and obstruction, especially in plastic and reconstructive surgery, has been complicated by infections caused by Aeromonas spp. These are leech endosymbionts for which patients undergoing hirudotherapy frequently receive systemic chemoprophylaxis. In order to evaluate the possibility of rendering leeches safe for use on patients, H. medicinalis were fed artificially with a 2 g/L arginine solution (used as a phagostimulant) supplemented with ciprofloxacin (100 mg/L). Aeromonads were detected in 57 out of 80 control leeches (71.3%), but in none of the 56 leeches treated with ciprofloxacin (p <0.001). Treated leeches survived for up to 4 months. Tested weekly, 61% of these leeches took human blood for at least 4 weeks after treatment and all remained negative for aeromonads. All water samples in which leeches were kept before treatment were contaminated with Aeromonas spp.; none were detected in any of the NaCl/arginine solutions with which treated animals were fed. Molecular characterization of two phenotypically distinct isolates using gyrB sequencing showed that one clustered tightly with A. veronii and the other was closely related to A. media. Other environmental bacteria and fungi were isolated from 26.5% of treated leeches that had taken a blood meal 1-4 weeks after treatment. Ciprofloxacin reduced the number of leech-associated aeromonads to undetectable levels for extended periods. Most treated leeches were ready to take a blood meal after treatment, suggesting the possibility of using ciprofloxacin-treated leeches instead of chemoprophylaxis in patients undergoing hirudotherapy.

Antibacterial substances of low molecular weight isolated from the blowfly, Lucilia sericata.

Low molecular weight compounds were isolated by high-performance liquid chromatography from the maggot or haemolymph extracts of Lucilia sericata (Meigen) (Diptera: Calliphoridae). Using gas chromatography-mass spectrometry analysis, three compounds were obtained: p-hydroxybenzoic acid (molecular weight 138 Da), p-hydroxyphenylacetic acid (molecular weight 152 Da) and octahydro-dipyrrolo[1,2-a;1',2'-d] pyrazine-5,10-dione (molecular weight 194 Da), also known as the cyclic dimer of proline (or proline diketopiperazine or cyclo[Pro,Pro]). All three molecules revealed antibacterial activity when tested against Micrococcus luteus and/or Pseudomonas aeruginosa, and the effect was even more pronounced when these molecules were tested in combination and caused lysis of these bacteria.

Antibacterial properties of whole body extracts and haemolymph of Lucilia sericata maggots.

OBJECTIVE: To partially characterise maggot-secreted antibacterial substances and determine their range of activity against different bacteria. METHOD: Sterile and non-sterile maggots maintained in the laboratory and taken from chronic wounds of treated patients were used. Whole body extracts and haemolymph were fractionated and their range of activity against bacteria was tested using the zone of inhibition assay. The mode of action of bacterial destruction was examined by viable counts, influx of K+ and changes in the membrane potential by scanning electron microscope (SEM). RESULTS: Extracts of sterile and non-sterile maggots showed an activity of 200 arbitrary units (AU)/ml and 400AU/ml respectively. Maggots removed from chronic wounds had an activity of 1200AU/ml. Injuring sterile maggots with a sterile needle doubled the antibacterial activity within 24 hours, while the antibacterial activity of haemolymph increased fourfold after injury with a sterile needle and sixteenfold with an infected needle. The fractions with a molecular weight of < 1kDa and 3-10kDa showed antibacterial activity against Gram-positive and Gram-negative bacteria including Pseudomonas aeruginosa, Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA) isolated from wounds. The fraction with a molecular weight of < 1kDa lysed over 90% of the bacteria within 15 minutes by causing an influx of K+ and changing the membrane potential of bacteria. CONCLUSION: The nature of the antibacterial materials extracted from maggots not only indicates their ability to ingest the necrotic tissue on the wound, but also their potential significance in wound healing,

Prevalence of vectors of the spotted fever group Rickettsiae and murine typhus in a Bedouin town in Israel.

A survey of the vectors of spotted fever group Rickettsiae and of murine typhus was carried out in Rahat, a Bedouin town in the Negev Desert, where the diseases are endemic. Houses with known cases of spotted fever group Rickettsiae or murine typhus were compared with those without reported clinical cases. A neighboring Jewish community, Lehavim, where no cases of spotted fever group Rickettsiae and murine typhus were reported in recent years, was used as a control. In the houses of patients with spotted fever group Rickettsiae in Rahat, an average of 7.4 times more ticks were found than in control houses. Out of 190 ticks isolated from sheep and goats or caught by flagging in Rahat, 90% were Rhipicephalus sanguineus (Latreille), 7.9% Rhipicephalus turanicus Pomerantzev, and 2.1% were Hyalomma sp. In the houses of patients with murine typhus, three times more rats were caught and, on the average, each rat was infested with 2.2 times more fleas than rats in the control houses. Out of 323 fleas collected from 35 Norwegian rats (Rattus norvegicus Berkenhout), 191 were Xenopsylla cheopis Rothschild and 132 Echidnophaga murina Tiraboschi. Thus, there was a six to seven times higher probability of encountering a tick or flea vector where infections had occurred than in control houses in Rahat. The percentage of rats seropositive to Rickettsia typhi was similar in study and control households (78.3 and 76.2, respectively). In the control settlement, Lehavim, only three Mus musculus L. were caught, which were not infested with ectoparasites and their sera were negative for murine typhus. Out of 10 dogs examined in this settlement, 15 R. sanguineus and eight specimens of the cat flea (Ctenocephalides felis felis Bouché) were isolated. No rats were caught in this settlement. These data indicate that there is a correlation among the density of domestic animals, their ectoparasites, and the incidence of spotted fever group Rickettsiae and murine typhus in Rahat.

Maggot therapy for the treatment of intractable wounds.

BACKGROUND: Fly maggots have been known for centuries to help debride and heal wounds. Maggot therapy was first introduced in the USA in 1931 and was routinely used there until the mid-1940s in over 300 hospitals. With the advent of antimicrobiols, maggot therapy became rare until the early 1990s, when it was re-introduced in the USA, UK, and Israel. The objective of this study was to assess the efficacy of maggot therapy for the treatment of intractable, chronic wounds and ulcers in long-term hospitalized patients in Israel. METHODS: Twenty-five patients, suffering mostly from chronic leg ulcers and pressure sores in the lower sacral area, were treated in an open study using maggots of the green bottle fly, Phaenicia sericata. The wounds had been present for 1-90 months before maggot therapy was applied. Thirty-five wounds were located on the foot or calf of the patients, one on the thumb, while the pressure sores were on the lower back. Sterile maggots (50-1000) were administered to the wound two to five times weekly and replaced every 1-2 days. Hospitalized patients were treated in five departments of the Hadassah Hospital, two geriatric hospitals, and one outpatient clinic in Jerusalem. The underlying diseases or the causes of the development of wounds were venous stasis (12), paraplegia (5), hemiplegia (2), Birger's disease (1), lymphostasis (1), thalassemia (1), polycythemia (1), dementia (1), and basal cell carcinoma (1). Subjects were examined daily or every second day until complete debridement of the wound was noted. RESULTS: Complete debridement was achieved in 38 wounds (88.4%); in three wounds (7%), the debridement was significant, in one (2.3%) partial, and one wound (2.3%) remained unchanged. In five patients who were referred for amputation of the leg, the extremities was salvaged after maggot therapy. CONCLUSIONS: Maggot therapy is a relatively rapid and effective treatment, particularly in large necrotic wounds requiring debridement and resistant to conventional treatment and conservative surgical intervention.

Factors modulating the blood feeding behavior and the electrophysiological responses of labral apical chemoreceptors to adenine nucleotides in the mosquito Aedes aegypti (Culicidae).

The feeding of Aedes aegypti (L.) on blood is induced by the presence of phagostimulants: adenine nucleotides. Three chemoreceptive cells in the labral apical sensilla can distinguish the presence of adenine nucleotides depending on the other stimulus components. This work aims at correlating the sensory information arising from the labral apical sensilla with the feeding behavior in response to the same stimuli. The saline stimulating solution, containing adenine nucleotides, is modulated by changing one of the following components: salt concentration, buffer or pH. Cell 3 that responds to NaCl in a dose dependent manner seems to have another unique modality. The response of this cell is unaffected by ATP when the stimulating solution is NaCl buffered by NaHCO(3). It responds at a higher spike frequency to the presence of ATP in a NaCl solution without NaHCO(3). Thus in the presence of ATP Cell 3 detects whether the NaCl solution is buffered by NaHCO(3). Both the blood feeding response and the sensory information from Cell 2 (which responds at high spike frequencies to the presence of ATP) are modulated by pH in a similar way. Both responses present a bi-modal response, with a major peak at pH 4.0 and a moderate peak at the most alkaline pH value tested.

Sensitivity of the mosquito Aedes aegypti (Culicidae) labral apical chemoreceptors to blood plasma components.

The phagostimulants from the cellular fraction of blood induce gorging of Aedes aegypti (L.), and this process is enhanced by some plasma components. This project examines the responses of the labral apical chemoreceptors to plasma components enhancing phagostimulation. From the electrophysiological responses of the labral apical chemoreceptors four cells were identified by the waveform of their action potentials. Three of the cells (Cell 2, Cell 3 and Cell 4) responded in a dose dependent manner to NaCl. The responses of Cell 2 and Cell 3 to NaCl concentrations from 1 to 500 mmol/l can be described by a logarithmic equation. The response of Cell 2 to 150 mmol/l NaCl is modulated when a buffer is added. The magnitude of the modulation of the response is determined by the nature of the buffer: NaHCO(3) inhibits while Na(2)HPO(4) enhances the response. High osmotic pressure inhibits the response of Cell 4, regardless of how it is achieved. Cell 4 responds with a high frequency to the presence of L-alanine, the C-terminal amino acid of albumin, but shows a reduced response to the same concentration of albumin. From these results it can be concluded that labral apical chemoreceptors of A. aegypti are capable of detecting the plasma components involved in blood recognition.

Sensitivity of the mosquito Aedes aegypti (Culicidae) labral apical chemoreceptors to phagostimulants.

The feeding of Aedes aegypti (L.) on blood and nectar is induced by phagostimulants: adenine nucleotides and sugars respectively. This work examines the responses of the four chemoreceptor cells in the labral apical sensilla to these phagostimulants. The apical chemoreceptors can detect the presence of adenine nucleotides. This part of the response is in good agreement with the gorging behavior. The output of the chemoreceptors cannot distinguish among different adenine nucleotides or among their concentrations (0.01-1 mmol/l), whereas gorging behavior is affected by the identity of adenine nucleotides and by their concentrations. Hence the gorging behavior cannot be driven by the output of these chemoreceptors alone. To the presence of adenine nucleotides Cell 2 was the only cell that responded with high frequencies, while the response of Cell 4 was almost abolished. The response of Cell 2 to ATP depended on the mosquito's physiological state. This dependence accorded well with the gorging behavior; Cell 2 responded with a higher frequency to ATP in the gorging state, than when not in a gorging state. The responses to sucrose and fructose constituted the only case recorded in which all these chemoreceptors failed to respond. This depression of response implies that other chemoreceptors must be present as sugar detectors.

[Maggot therapy for gangrene and osteomyelitis].

5 patients with diabetic-foot were treated by maggot therapy. The most serious case was in a 75-year-old man who had gangrene and osteomyelitis of the right foot. Proteus mirabilis, Enterococcus sp., Providencia stuartii and Staphylococcus spec. (coagulase positive) were isolated from lesions which did not respond to antibiotic therapy. The patient had twice refused amputation but agreed to maggot therapy. Larvae of the sheep blowfly Phoenicia (Lucilia) sericata were used for twice-weekly treatment over a period of 7 months. Sterile larvae were applied to the wound and replaced every 3-4 days. After 4 months of treatment, the necrotic tissue around the toes and on the sole of the foot detached from the healthy tissue. During the last 3 months of treatment the larvae removed the remaining infected tissue. As therapy progressed, new layers of healthy tissue covered the wound. The offensive odor associated with the necrotic tissue and the intense pain in the foot decreased significantly. At the end of therapy, during which there were no complaints of discomfort, he was able to walk. In the 4 other patients who had relatively superficial gangrene, the maggots debrided the wounds within 2-4 weeks. Thereafter treatment was continued with antibiotics. Maggot therapy can be recommended in cases of intractable gangrene and osteomyelitis, when treatment with antibiotics and surgical debridement have failed.

Rhipicephalus sanguineus and R. turanicus (Acari:Ixodidae): closely related species with different biological characteristics.

Life cycle parameters of 2 closely related tick species, Rhipicephalus sanguineus Latreille and R. turanicus Pomerantsev, were studied under laboratory conditions. Both Rhipicephalus, which have small adults, demonstrated the same adaptations as large tick species inhabiting deserts and semideserts: high reproductive rate, decrease in egg size, and an increase in interstage growth to compensate for the smaller size at birth. Pronounced quantitative differences between both species were discerned in relation to these adaptations. Female R. turanicus produced twice as many eggs as R. sanguineus which was facilitated by the greater amount of blood engorged by females and by the smaller egg weight in R. turanicus as compared with R. sanguineus. In all developmental stages, the weight increase from unfed to fed ticks was greater in R. turanicus than in R. sanguineus (23% higher in larvae, 118% in nymphs, and 26% in females). The increase in weight in R. turanicus from the unfed larva (0.013 mg) to the unfed female (3.31 mg) was 254-fold, and in R. sanguineus it was 127-fold (from 0.021 to 2.54 mg). In nymphal R. turanicus, the higher density and the greater height of the dorsal epicuticular folds, as well as procuticular indentations found inside the folds allow this tick to stretch its alloscutum during blood engorgement to a greater extent than R. sanguineus. The rates of blood ingestion (for nymphs and females), egg maturation, and metamorphosis were 1.1-1.7 times greater in R. turanicus than in R. sanguineus. A life cycle strategy with both a higher reproductive rate and faster development in R turanicus may be explained by its greater dependence on environmental factors than that in R. sanguineus.

Immunization of rabbits with faecal extract of Pediculus humanus, the human body louse: effects on louse development and reproduction.

Immunization of rabbits with a faecal extract of the human body louse (Pediculus humanus) induced a high titre of specific IgG. The mean weight of blood taken by females fed on the immunized rabbits was significantly lower (29%) than taken by females fed on the control rabbits. The mean number of eggs per female fed on the immunized rabbits was significantly lower than for females fed on the control rabbits. The hatchability of the eggs laid by lice fed on immunized rabbits (91%) was significantly lower than of those fed on control rabbits (94%). The rate of development of nymphs fed on control rabbits was significantly higher than those fed on the immunized rabbits. There was no difference in survival rates of lice fed on immunized and control rabbits.

Prevalence of spotted fever group rickettsiae in ticks from southern Israel.

The prevalence of spotted fever group rickettsiae was studied in questing ticks collected from southern Israel. Ticks were examined from an agricultural settlement (kibbutz Ze'elim), an endemic site for Mediterranean spotted fever, and from another kibbutz (Re'im) in an adjacent area where the disease has not been reported. Ticks were collected by flagging and CO2 traps from vegetation inside and outside the settlements during 1989/1990 and 1994. In Ze'elim, 98% of the ticks collected were brown dog tick, Rhipicephalus sanguineus (Latreille), whereas in Re'im the predominant species was Rhipicephalus turanicus Pomerantzev (84.6%), with 11.8% of the ticks R. sanguineus and 3.6% Hyalomma excavatum Koch. The presence of rickettsiae in the hemolymph of the ticks was demonstrated by immunofluorescence using polyclonal antibodies made against the human pathogenic Moroccan strain of Rickettsia conorii. Of 549 ticks examined in 1989/1990 from Ze'elim, 7.3% were positive for spotted group rickettsiae, and in Re'im 2.2% of 156 R. turanicus were positive. In 1994, 51 out of 186 (27.4%) ticks in Ze'elim and 3 out of 115 (2.6%) ticks in Re'im were positive. All 20 specimens of H. excavatum were negative for spotted fever group rickettsiae. To our knowledge, this is the 1st report of spotted fever group rickettsiae in R. turanicus from Israel.

Characterization of body louse midgut proteins recognized by resistant hosts.

The human body louse, Pediculus humanus, showed eighteen midgut proteins ranging between 12 and 117 kDa, when analysed by SDS-PAGE electrophoresis. Seven of them (12 kDa, 17 kDa, 29 kDa, 35 kDa, 40 kDa, 55 kDa and 97 kDa) were major bands based on their intensity of staining. The immunization of rabbits with a midgut extract elicited the production of protective polyclonal antibodies. These antibodies reacted strongly with all major midgut proteins as well as with 63 kDa and 117 kDa proteins when tested by the Western blot technique. The analysis of the proteins revealed that the 12 kDa, 25 kDa, 29 kDa, 35 kDa, 45 kDa, 87 kDa and 97 kDa proteins are glycosylated and none of them contained a lipid moiety. By electroelution, the proteins of 35 kDa and 63 kDa were purified. On trypsinization, the proteins of 35 kDa and 63 kDa produced four major fragments (F1, F2, F3, and F4) when resolved on a 18% SDS-PAGE. The F1 fragment of the 35 kDa protein reacted with the polyclonal antibodies by the immunoblot technique.

Localization of immunogenic antigens on midgut of the human body louse Pediculus humanus humanus (Anoplura: Pediculidae).

Immunogenic midgut antigens of the human body louse, Pediculus humanus humanus L., were localized using rabbit antisera against a louse-midgut extract followed by a 2nd antibody conjugated to either fluorescein or colloidal gold. Strong fluorescence was observed on the outer membrane of the epithelial cell of the midgut. The immunogold technique revealed that most of the antigens were localized on the microvilli of the midgut cells. Small numbers of gold particles were also seen in the gut lumen and within the cell cytoplasm. Only a few gold particles were seen in the lumen of the gut sections incubated with control sera.

Permethrin resistance in the head louse Pediculus capitis from Israel.

Head lice, Pediculus capitis, were collected from children aged 3-12 years in Maale Adumin, a town near Jerusalem, after reports of control failure with the pyrethroid insecticide permethrin. A total of 1516 children were examined: living lice and eggs were found on 12.1% of the children; or another 22.8% of the children only nits were found. Twice as many girls as boys (8.1% v 4%) were infested with lice and or nits. Head lice collected from infested children were exposed to permethrin impregnated filter-papers. Log time probit mortality (ltp) regression lines were calculated for mortality data and compared to ltp lines for a similar collection of head lice made in 1989. The regression lines for the two years were significantly different, with a 4-fold decrease in susceptibility at the LT50 level between 1989 and 1994. The slopes of the lines also suggested that the 1994 population was more heterogenous in its response to permethrin than the 1989 population. In contrast, a laboratory population of body lice (Pediculus humanus) tested with the same batch of permethrin-impregnated papers showed a slight but non-significant increase in susceptibility between 1989 and 1994. The results suggest that resistance to pyrethroids has developed rapidly among head lice since permethrin was introduced in 1991 as a pediculicide in Israel.

Immunization of rabbits with a midgut extract of the human body louse Pediculus humanus humanus: the effect of induced resistance on the louse population.

Resistance to human body lice, Pediculus humanus humanus L, induced by feeding on rabbits immunized with an extract of louse gut was studied. The mortality of lice fed on immunized rabbits was 73%, significantly higher than that of lice fed on control rabbits (52%) (P < 0.01). The proportion of dead nymphs and female lice with ruptured guts was significantly higher in lice fed on immunized rabbits (P < 0.01). The size of the bloodmeal was 35% greater in female lice fed on control rabbits than on immunized rabbits. Lice fed on immunized rabbits laid 40% less eggs than those fed on the controls, they also demonstrated a significant decrease in the number of eggs per female over time (P < 0.01). 86% of the eggs laid by lice fed on immunized animals hatched, compared with 92% hatching of eggs laid by the lice fed on control animals (P < 0.01). With the exception of the first bloodmeal the percentage of hatched eggs which were laid between any two bloodmeals was significantly smaller (P < 0.01) in the lice fed on immunized rabbits than in the control group. The first nymphal stage of lice fed on immunized rabbits took an average of 5.2 days to moult to the second stage, compared with 4 days for those fed on control rabbits.

Gorging response of culicine mosquitoes (Diptera: Culicidae) to blood fractions.

Although anopheline mosquitoes will ingest plasma without blood cells, Culex spp. and Aedes spp. require the phagostimulatory effect of blood cells; this effect can be duplicated by the addition of adenine-nucleotides to plasma. Because activation of platelets released ADP and ATP into the plasma, they were suspected as the major source of the phagostimulant. This paper describes quantitatively the role of platelets in ingestion by Aedes aegypti (L.) and Culex univittatus Theobald. We found that about 10(6)/mm3 inactivated platelets are required to induce engorgement by 80-90% of the mosquitoes of both species. Thrombin activation of the platelets reduced the effective dose to < 2 x 10(4)/mm3. Other blood fractions also were tested as possible sources of stimulation. A series of washed red blood cells (RBC) dilutions was tested; 5 x 10(5) RBC/mm3 were required to induce 90% engorgement. Several types of leukocytes derived from blood by standard methods also induced engorgement at their physiological concentrations. Macrophages and cultured lymphocytes that do not contain any platelets induced gorging in Cx. univittatus, but not in Ae. aegypti. Because RBC and leukocytes do not release nucleotides unless broken, we suggest that their phagostimulatory effect is due to platelet contamination, which invariably occurs during standard methods of blood fractionation.

Ecological studies on the brown dog tick Rhipicephalus sanguineus (Acari: Ixodidae) in southern Israel and its relationship to spotted fever group rickettsiae.

An outbreak of spotted fever group rickettsiae (SFGR) was investigated by studying free-living and parasitic stages of ticks in two settlements of equal size and population located 20 km apart in the Negev Desert. Although high morbidity from SFGR was found in one of the settlements (Kibbutz Ze'elim), no clinical cases were observed in the second (Kibbutz Re'im). Using flagging and CO2-trapping, approximately 9 times more ticks were collected in Ze'elim than in Re'im. Rhipicephalus sanguineus (Latreille) was the dominant species in Ze'elim, whereas in Re'im R. turanicus Pomerantzev was the most abundant species. Several physical factors that may account for these differences were investigated. Significantly higher maximum soil temperature as well as ambient temperature above the soil were found in Ze'elim. Differences in soil composition in the two sites were also observed. Tick numbers were especially high during April through October in Ze'elim, whereas in Re'im, ticks were found mainly from April to July. A significant positive correlation was found between temperature and tick population size in Ze'elim. Dogs, sheep, goats, Mus musculus, and Meriones crassus, were more heavily infested with ticks in Ze'elim than in Re'im. The percentage of mice and dogs seropositive to SFGR was the same in both sites. In Ze'elim, 7.1% of dog owners acquired Mediterranean spotted fever during the period 1984-1989 compared with only 1.4% of people without dogs.