SNPs in Entire Mitochondrial Genome Sequences (≈15.4 kb) and cox1 Sequences (≈486 bp) Resolve Body and Head Lice From Doubly Infected People From Ethiopia, China, Nepal, and Iran But Not France.

Some people host lice on the clothing as well as the head. Whether body lice and head lice are distinct species or merely variants of the same species remains contentious. We sought to ascertain the extent to which lice from these different habitats might interbreed on doubly infected people by comparing their entire mitochondrial genome sequences. Toward this end, we analyzed two sets of published genetic data from double-infections of body lice and head lice: 1) entire mitochondrial coding regions (≈15.4 kb) from body lice and head lice from seven doubly infected people from Ethiopia, China, and France; and 2) part of the cox1 gene (≈486 bp) from body lice and head lice from a further nine doubly infected people from China, Nepal, and Iran. These mitochondrial data, from 65 lice, revealed extraordinary variation in the number of single nucleotide polymorphisms between the individual body lice and individual head lice of double-infections: from 1.096 kb of 15.4 kb (7.6%) to 2 bps of 15.4 kb (0.01%). We detected coinfections of lice of Clades A and C on the scalp hair of three of the eight people from Nepal: one person of the two people from Kathmandu and two of the six people from Pokhara. Lice of Clades A and B coinfected the scalp hair of one person from Atherton, Far North Queensland, Australia. These findings argue for additional large-scale studies of the body lice and head lice of double-infected people.

Second-generation sequencing of entire mitochondrial coding-regions (∼15.4 kb) holds promise for study of the phylogeny and taxonomy of human body lice and head lice.

The Illumina Hiseq platform was used to sequence the entire mitochondrial coding-regions of 20 body lice, Pediculus humanus Linnaeus, and head lice, P. capitis De Geer (Phthiraptera: Pediculidae), from eight towns and cities in five countries: Ethiopia, France, China, Australia and the U.S.A. These data (∼310 kb) were used to see how much more informative entire mitochondrial coding-region sequences were than partial mitochondrial coding-region sequences, and thus to guide the design of future studies of the phylogeny, origin, evolution and taxonomy of body lice and head lice. Phylogenies were compared from entire coding-region sequences (∼15.4 kb), entire cox1 (∼1.5 kb), partial cox1 (∼700 bp) and partial cytb (∼600 bp) sequences. On the one hand, phylogenies from entire mitochondrial coding-region sequences (∼15.4 kb) were much more informative than phylogenies from entire cox1 sequences (∼1.5 kb) and partial gene sequences (∼600 to ∼700 bp). For example, 19 branches had > 95% bootstrap support in our maximum likelihood tree from the entire mitochondrial coding-regions (∼15.4 kb) whereas the tree from 700 bp cox1 had only two branches with bootstrap support > 95%. Yet, by contrast, partial cytb (∼600 bp) and partial cox1 (∼486 bp) sequences were sufficient to genotype lice to Clade A, B or C. The sequences of the mitochondrial genomes of the P. humanus, P. capitis and P. schaeffi Fahrenholz studied are in NCBI GenBank under the accession numbers KC660761-800, KC685631-6330, KC241882-97, EU219988-95, HM241895-8 and JX080388-407.

Babesiosis: an underdiagnosed disease of children.

Babesiosis is a malaria-like illness caused by the intraerythrocytic parasite Babesia microti and is transmitted by the same tick that transmits Borrelia burgdorferi, the causative agent of Lyme disease. Babesiosis is well recognized in adult residents of southern New England and New York but has been described in only five children. To determine whether children are infected with B microti less often than are adults, a prospective serosurvey was carried out on Block Island, RI, where babesiosis is endemic. Randomly recruited subjects completed a questionnaire and provided a blood sample. Antibodies against B microti and B burgdorferi were measured using a standard indirect immunofluorescence assay and enzyme-linked immunosorbent assay, respectively. Of 574 subjects, 9% tested positive for B microti, including 12% of the 52 children (7 months through 16 years) and 8% of the 522 adults (not significant, P less than .6). Although babesiosis had not been diagnosed in any of the Babesia-seropositive subjects, 25% of the children and 20% of the adults reported symptoms compatible with this infection during the previous year. Of the 6 children and 45 adults seropositive for B burgdorferi, 17% and 14%, respectively, were also seropositive for B microti. It is concluded that children are infected with B microti no less frequently than are adults and that this infection is underdiagnosed in all age groups. Physicians who practice where Lyme disease is endemic should become familiar with the clinical presentation and diagnosis of babesiosis, both in adults and children.

Overdiagnosis and consequent mismanagement of head louse infestations in North America.

BACKGROUND: Lay personnel and many health care workers in the United States believe that head louse infestations caused by Pediculus capitis are exceedingly transmissible and that infested children readily infest others. Schoolchildren therefore frequently become ostracized and remain so until no signs of their presumed infestations are evident. Repeated applications of pediculicidal product and chronic school absenteeism frequently result. METHODS: To determine how frequently louse-related exclusions from schools and applications of pediculicidal therapeutic regimens might be inappropriate, we invited health care providers as well as nonspecialized personnel to submit specimens to us that were associated with a diagnosis of pediculiasis. Each submission was then characterized microscopically. RESULTS: Health care professionals as well as nonspecialists frequently overdiagnose pediculiasis capitis and generally fail to distinguish active from extinct infestations. Noninfested children thereby become quarantined at least as often as infested children. Traditional anti-louse formulations are overapplied as frequently as are "alternative" formulations. Pediculicidal treatments are more frequently applied to non-infested children than to children who bear active infestations. CONCLUSIONS: Pediculicidal treatments should be applied solely after living nymphal or adult lice or apparently viable eggs have been observed. Because health care providers as well as lay personnel generally misdiagnose pediculiasis, and because few symptoms and no direct infectious processes are known to result, we suggest that the practice of excluding presumably infested children from school may be more burdensome than the infestations themselves.

Differential permethrin susceptibility of head lice sampled in the United States and Borneo.

BACKGROUND: Pediculiasis is treated aggressively in the United States, mainly with permethrin- and pyrethrin-containing pediculicides. Increasingly frequent anecdotal reports of treatment failure suggest the emergence of insecticidal resistance by these lice. OBJECTIVE: To confirm or refute the susceptibility of head lice sampled in the United States to permethrin. DESIGN: Survey. Head lice were removed from children residing where pediculicides are readily available and where such products are essentially unknown. Their survival was compared following exposure to residues of graded doses of permethrin in an in vitro bioassay. SETTING: School children from Massachusetts, Idaho, and Sabah (Malaysian Borneo). SUBJECTS: In the United States, 75 children aged 5 to 8 years. In Sabah, 59 boys aged 6 to 13 years. Virtually all sampled US children had previously been treated with pediculicides containing pyrethrins or permethrin; none of the Sabahan children were so exposed. MAIN OUTCOME MEASURE: Survival of head lice exposed to permethrin. RESULTS: Permethrin did not affect head lice sampled from chronically infested US children who had previously been treated for pediculiasis. The slope of the dose-response regression line for these lice did not differ significantly from zero (P = .66). This pediculicide immobilized lice sampled in Sabah. Mortality correlated closely with permethrin concentration (P = .008). CONCLUSIONS: Head lice in the United States are less susceptible to permethrin than are those in Sabah. The pyrethroid susceptibility of the general population of head lice in the United States, however, remains poorly defined. Accordingly, these relatively safe over-the-counter preparations may remain the pediculicides of choice for newly recognized louse infestations.

Emerging vector-borne infections.

The principles that regulate the transmission of vector-borne infectious agents are briefly described. In particular, the circumstances that may lead to human exposure to various North American zoonoses are analyzed.

Enhanced development in nature of larval Anopheles arabiensis mosquitoes feeding on maize pollen.

To determine whether pollen produced by maize (Zea m. mays) may contribute to the development of larval Anopheles gambiae complex mosquitoes, the main African vectors of malaria, we correlated duration of larval development, pupation success, and size of the resulting adults with degree of access to this potential nutriment. Maize pollen is abundant during the wet season on the surface of water near maize plantings in a malaria-endemic region of Ethiopia, and larval Anopheles arabiensis readily ingest these particles in nature. Larvae develop to the pupal stage more rapidly, more frequently, and produce larger adults where maize pollen is abundant than do those that have little access to this food. The force of transmission of malaria in sub-Saharan Africa might be reduced if maize plantings were excluded from the immediate vicinity of homes or, perhaps, if pollen of such maize were to express entomotoxins.

Further studies on the development of onchocerca spp. (Nematoda: Filarioidea) in Nearctic black flies (Diptera: Simuliidae).

Of three species of Nearctic black flies tested, Simulium pictipes Hagen proved the most efficient laboratory vector of the bovine parasite Onchocerca lienalis. Among flies inoculated intrathoracially with 40 microfilariae, numbers of 3rd-stage larvae per fly were 7.63 for S. pictipes, 7.54 for S. vittatum Zetterstedt, and 0.83 for S. decorum Walker. S. pictipes survived the longest under laboratory conditions, with 83.3% of the females remaining alive 10 days after inoculation with 40 microfilariae of O. lienalis. Using an artificial membrane feeding system, S. pictipes could be routinely infected with O. lienalis by mouth. This black fly was also susceptible to infection with the Guatemalan strain of O. volvulus. Among flies injected with 10 microfilariae the rate of infection with 3rd-stage larvae was 93%, with a mean of three 3rd-stage larvae per fly. Successful techniques for the large-scale recovery and cryopreservation of 3rd-stage larvae of O. lienalis were also developed. A motility rate of 92.7% was observed in larvae cryopreserved within vector black flies.

Issues in public health entomology.

Public health entomology focuses on the population biology of vector-borne infections, seeking to understand how such pathogens perpetuate over time and attempting to devise methods for reducing the burden that they impose on human health. As public health entomology passes its centennial, a series of pervasive research themes and spirited debates characterize the discipline, many reflecting a tension between field and laboratory research. In particular, institutional support for population-based research and training programs has fallen behind that for those using modern lab-based approaches. Discussion of modes of intervention against vector-borne infections (such as deployment of genetically modified vectors, the role of DDT in malaria control, host-targeted acaricides for Lyme disease risk reduction, and truck-mounted aerosol spraying against West Nile virus transmission) illustrates the discipline's need for strengthening population-based research programs. Even with the advent of molecular methods for describing population structure, the basis for anophelism without malaria (or its eastern North American counterpart, ixodism without borreliosis) remains elusive. Such methods have not yet been extensively used to examine the phylogeography and geographical origins of zoonoses such as Lyme disease. Basic ecological questions remain poorly explored: What regulates vector populations? How may mixtures of pathogens be maintained by a single vector? What factors might limit the invasion of Asian mosquitoes into North American sites? Putative effects of "global warming" remain speculative given our relative inability to answer such questions. Finally, policy and administrative issues such as the "no-nits" dictum in American schools, the Roll Back Malaria program, and legal liability for risk due to vector-borne infections serve to demonstrate further the nature of the crossroads that the discipline of public health entomology faces at the start of the 21st Century.

Time limitation and the role of research in the worldwide attempt to eradicate malaria.

The U.S. Congress established an intense, time-limited, worldwide malaria eradication program in 1958 and assigned operational responsibility to the U.S. Agency for International Development (and its predecessors). When the program was terminated on schedule in 1963, approximately $400 million had been consumed and malaria prevalence had greatly been reduced. Transmission began to increase thereafter. The open-ended WHO global eradication effort began in 1955 ended in 1969 and consumed approximately $15 million during the 1958-1963 period of progress, mainly provided by the United States. Intensified anti-malaria interventions continued after Congress discontinued direct support. Although malariological research was discouraged during the period of time limitation, it was embraced as the conceptual basis for the open-ended period of intervention that followed. This effort saved many lives but expended our ability to intervene against future epidemics and reduced human herd immunity. To avoid the "great gamble" inherent in any ambitious intervention against this disease, future programs should be designed to seek incremental, local antimalaria gains.

Rectal infusion and aspiration of material through the guts of ixodid ticks (Acari: Ixodidae).

A technique for inoculating and removing substances via the anus of vector ticks was devised to define features of vector competence precisely. Calibrated inocula (greater than 5 nanoliter) containing aqueous dye and polystyrene beads as well as infectious agents were infused into the rectal sacs of ticks using glass microcapillary pipettes placed within the expanded anal orifice. The guts of preadult and adult ticks, Ixodes dammini Spielman, Clifford, Piesman & Corwin, Dermacentor variabilis (Say), Hyalomma impeltatum Schulze & Schlottke, and Amblyomma americanum (L.), were thereby infused with these inocula. Distribution of inocula was determined by examining hemolymph and sectioned ticks and confirmed that material placed in the rectal sac spread throughout the midgut diverticula. Ticks survived for greater than 6 mo after this procedure and were able to feed, molt to the next stage, or oviposit. In contrast, fewer ticks survived after intracelomic inoculation. The course of infection in ticks receiving anal infusions of Borrelia burgdorferi (the Lyme disease spirochete) was assessed. Such infections appear to differ from those established by feeding on infected hosts. Contents of the tick gut can be sampled nondestructively by anal perfusion to diagnose infection by this spirochete.

Enema infusion technique inappropriate for evaluating viral competence of ticks.

To determine whether anal infusion of virus simulates the natural route of infection rather than intracoelomic injection, we compared the course of Venezuelan equine encephalitis (VEE) virus infection in Amblyomma cajennense (F.) ticks that had been exposed to virus by enema infusion with that in ticks fed on a viremic host or exposed by intracoelomic inoculation. Although virus was detected in virtually all ticks 14 d after exposure, orally exposed ticks contained significantly less virus (10(1.9) plaque-forming units [PFU] per tick) than did ticks infected by enema (10(4.1) PFU per tick) or intracoelomically (10(4.2) PFU per tick). At 42 d after virus exposure, only 1% of 512 orally exposed ticks contained virus, but most enema (77%, n = 43) or intracoelomically (79%, n = 29) exposed ticks were infected. Replication of VEE virus in A. cajennense ticks exposed to virus by enema infusion, therefore, appeared more similar to that of ticks inoculated intracoelomically than to those exposed orally. Thus, because enema infusion may bypass potential midgut infection and escape barriers, this procedure may not be appropriate for determining vector competence in ixodid ticks.

Interrupted blood-feeding by Culiseta melanura (Diptera: Culicidae) on European starlings.

To determine whether Culiseta melanura (Coquillett) mosquitoes tend to take multiple blood meals when birds of certain species serve as hosts, we compared the frequencies with which such mosquitoes fed upon caged starlings and robins and determined whether similar volumes of blood were imbibed from each. The blood of robins (Turdus migratorius) and European starlings (Sturnus vulgaris) was marked contrastingly by injecting birds with rubidium or cesium salts. Caged birds were placed together in a natural wetland setting overnight. Mosquitoes captured nearby on the following morning were analyzed for each of the elemental markers. Where marked robins and starlings were equally abundant, 43% of freshly engorged Cs. melanura fed on more than or equal to two hosts. More Cs. melanura fed on robins than on starlings. Individual mosquitoes tended to contain far more robin- than starling-associated marker, indicating that mosquitoes "feasted" on robins but only "nibbled" on starlings. Mosquitoes marked with both elements apparently fed meagerly on the starlings then abundantly on the robins. Our estimates of bloodmeal volume indicate that 85% of mosquitoes that fed on marked starlings obtained < 0.5 microliter of blood from them. We suggest that defensive behavior by starlings interrupts mosquito blood-feeding and that, in a communal roost of starlings, each mosquito will tend to feed on more than one bird, thereby promoting rapid transmission of such ornithonotic arboviruses as eastern equine encephalomyelitis virus and West Nile virus.

Studies of the growth-regulating effects of ivermectin on larval Onchocerca lienalis in vitro.

At concentrations of 0.1-100 ng/ml ivermectin inhibited L3-L4 molting by Onchocerca lienalis in vitro. The degree of inhibition was dose-dependent with a significant effect apparent at 0.1 ng/ml and complete inhibition occurring at 100 ng/ml. The ED50 for molt inhibition was 0.19 ng/ml. Molt-inhibiting levels of the drug were not acutely toxic to the worms. In the presence of 10 ng/ml, a concentration giving 95% molt inhibition, motility at day 7 postinoculation was 71% of that seen in nontreated controls. A more pronounced effect on motility was apparent in larvae under long-term cultivation in the presence of ivermectin. Kinetic studies indicated that the majority of the larvae respond irreversibly to the drug within the first 2 hr of exposure. Twenty-four hours of exposure were required for a maximal response. The inhibitory effects of ivermectin were less pronounced if larvae were allowed to develop under normal culture conditions for 24 or more hours prior to the initiation of drug treatment.

Analysis of glutathione-enhanced differentiation by microfilariae of Onchocerca lienalis (Filarioidea: Onchocercidae) in vitro.

Reduced glutathione (GSH), but not its oxidized form (GSSG), stimulated development of Onchocerca lienalis microfilariae to the late first-larval stage in vitro. The degree and frequency of development was dose-related with a peak of activity at 15 mM, a concentration that is similar to known intracellular levels of GSH. To determine the mode(s) of action of this multifunctional compound, other reducing agents (L-cysteine, dithiothreitol), cysteine delivery agents (N-acetyl-L-cysteine, L-thiazolidine-4-carboxylic acid, L-2-oxothiazolidine-4-carboxylic acid), cysteine analogues (S-methyl-L-cysteine, D-glucose-L-cysteine, cysteine ethyl ester), free-component amino acids of GSH (glutamic acid, cysteine, and glycine), a specific metabolic inhibitor of gamma-glutamyl synthetase (buthionine sulfoximine), and an inhibitor of gamma-glutamyl transpeptidase (gamma-glutamyl glutamic acid) were also tested at concentrations of 0.01-50 mM in this system. N-acetyl-L-cysteine at 1-5 mM and D-glucose-L-cysteine at 2.5-10 mM significantly enhanced development. In contrast to those worms maintained in GSH-supplemented medium, microfilariae exposed to GSH for only the first 24 hr showed no enhancement by day 7 in culture. Neither buthionine sulfoximine nor gamma-glutamyl glutamic acid at 0.01-35 mM inhibited the effects of 15 mM GSH or 1 mM N-acetyl-L-cysteine. Results indicate that GSH or other cysteine analogues possessing a free sulfhydryl group must be present in the extranematodal environment to support microfilarial differentiation in vitro.

A nestable fiber pot for sampling resting mosquitoes.

To enhance the effectiveness of an arbovirus monitoring program, we evaluated a commercially available device for sampling resting vector mosquitoes. Diverse Anopheles, Culiseta, and Culex mosquitoes were taken in these nestable fiber pots. The pots sample about as many Culiseta melanura mosquitoes per device as do conventional resting boxes, but fewer than do boxes fitted with expanded frames. More Cs. melanura, and more bloodfed mosquitoes, but fewer species of mosquitoes are harvested with fiber pots than with CDC light traps. Fiber pots are more readily used, transported, and stored and are less expensive than conventional resting box devices or CDC light traps. A monitoring program based on the use of fiber pots, therefore, expends fewer resources than one using conventional resting boxes and collects about as many vector mosquitoes.

Standardization of medium for culturing Lyme disease spirochetes.

To standardize the procedure for isolating and culturing Lyme disease spirochetes, we modified the composition of the medium generally used for this purpose (BSK-II) and developed a system for its distribution. This medium contains no gelatin or agarose, and various components are used in proportions that differ from those in BSK-II. Each of the major proteinacious components was screened by substitution in samples of the complete product. The final medium was evaluated for the capacity to grow related spirochetes including Borrelia burgdorferi N40, Guilford, and JD-1 as well as strains of Borrelia hermsii (HS-1) and of Borrelia coriaceae (CO53). Each isolate developed from inocula containing as few as one to five organisms. Doubling time of B. burgdorferi during log-phase growth at 37 degrees C was 10 to 12 h. Lyme disease spirochetes were isolated in this medium from ear punch biopsies and dermal aspirates from naturally infected mice and rabbits, from dermal biopsies from a human patient, and by sampling field-collected deer ticks (Ixodes dammini). Cultured spirochetes remained infective to mice and to ticks. The medium can be stored at -20 degrees C or lower temperatures for at least 8 months without effect on its ability to support growth of small inocula to densities exceeding 10(8) spirochetes per ml. Lyme disease spirochetes remained infective to mice after being stored at -80 degrees C in this medium for at least 8 months. We anticipate that the availability of this standardized medium (Sigma Chemical Co.), supplemented with prescreened rabbit serum, will facilitate comparison of research results between laboratories and may eventually permit definitive clinical diagnosis of Lyme disease based on demonstration of the pathogen. The standardized medium is designated BSK-H.

Rapid dissemination by the agent of Lyme disease in hosts that permit fulminating infection.

We determined whether the agent of Lyme disease (Borrelia burgdorferi) disseminates more rapidly following deposition in hosts that permit fulminating infection than in hosts in which infection is relatively benign. Thus, individual infected nymphal deer ticks (Ixodes dammini) were permitted to engorge on the ears of C3H mice, and the site of attachment was excised at intervals thereafter. Infection in each mouse was determined by serology and by examining previously noninfected ticks that had engorged on these mice. These results were compared with data obtained similarly by using the CD-1 strain of mice in which the agent is relatively nonpathogenic. When the site of inoculation was ablated within 2 days after the infected tick became replete, dissemination was aborted. Spirochetemia could not be demonstrated in any of these mice. We conclude that Lyme disease spirochetes disseminate from the feeding lesion of an infecting tick more rapidly in certain highly spirochete-susceptible mice than in others in which pathogenesis is less severe.

Delayed dissemination of Lyme disease spirochetes from the site of deposition in the skin of mice.

To determine whether the agent of Lyme disease disseminates in vertebrate hosts directly after deposition by an infecting tick, a 6-mm disk of skin was excised from the sites where nymphal Ixodes dammini ticks infected by Lyme disease spirochetes, Borrelia burgdorferi, had fed. Infection in each mouse was tested by examining xenodiagnostic ticks that had engorged on these mice 4 weeks later and by serologic testing. Generalized infection was aborted when the site of inoculation was excised within 2 days after the infecting tick detached but not after 2 weeks. In contrast, all mice became infected when the bite site remained intact. Spirochetes could be cultured from the tissues around the site of attachment solely when the sample was ablated within a week after infecting ticks detached. These observations suggest that infecting ticks deliver the agent of Lyme disease directly into the skin and that such spirochetes multiply locally for some days before disseminating to remote sites.

Development of onchocerca lienalis and O. volvulus from the third to fourth larval stage in vitro.

Third-stage larvae of Onchocerca volvulus and O. lienalis were observed to molt to the fourth stage in various cell-free in vitro systems. The percentage of O. lienalis completing the molt was similar in the three culture media and two gas phases tested ranging from 44.8% (1:1 IMDM:NCTC + 5% CO2: 95% N2) to 56.7% (L-15 + 5% CO2: 95% air). Percent molting in O. volvulus ranged from 0% (F12(K) + 5% CO2: 95% N2) to 33.3% (L-15 + 5% CO2: 95% N2). All media were supplemented with either 20% FCS or 20% horse serum. Molting by O. lienalis occurred on days 2-5 in culture. Molting by O. volvulus was observed as early as day 5 and as late as day 10. Incomplete casting of the third-stage cuticle was frequently observed in O. volvulus. Larvae of both species entered a lethargus 24-48 hours prior to the onset of molting. Maximum survival in culture was 42 days for O. lienalis and 25 days for O. volvulus. Significant growth of larval O. lienalis was noted early in the culture period, but neither species continued development to the fifth stage.