Rabbit antibody responses to experimental infestation with Dermatobia hominis

The three larval stages of Dermatobia hominis (Linnaeus) have been evaluated for their immunogenicity by ELISA and immunodiffusion (ID) using sera from experimentally infested rabbits. During a primary infestation, first instar D. hominis were found to cause most reaction and allowed the earliest diagnosis by ELISA. An inhibition of the antibody response against second and third instars was observed. The inhibition disappeared after departure of the larvae from the host. In experimentally immunized hosts the antibody response, following challenge, was highest against second and third instar antigens. Antibody remained elevated during the infestation but fell immediately after the larvae had left the host.     

Immunologic parameters of monkeys infected by urogenital mycoplasmas

In monkeys contained in captivity conditions in open-air cages or in group cages human mycoplasmas are often detected: antigens of Mycoplasma hominis in blood serum were revealed in 33.3% of cases, and antibodies to it--in 15.6% of cases. IgM to M. hominis were detected more often than IgG. In 8 monkeys both types of immunoglobulins were detected. Rates of detection of Ureaplasma urealyticum antigens and specific antibodies were 43.1% and 31.1% respectively, and IgG were found more frequently than IgM (in 22 cases both types of immunoglobulins were revealed). High rates of M. hominis and U. urealyticum antigens and antibodies detection in blood serum of both healthy monkeys and monkeys with urogenital tract diseases show prevalence of human mycoplasmas carriage among monkeys contained in captivity conditions.     

Eosinophil and mast cell expression in host skin during larval development of the human bot fly Dermatobia hominis

Eosinophils and mast cells in the skin of Wistar rats (Rattus norvegicus) infested with Dermatobia hominis larvae were quantified and analysed. Eosinophils in parasitised skin increased markedly until 10 days post-infestation (dpi) and then decreased up to 28 dpi, close to the point at which third stage larvae (L3) emerged from the host. In ascending order, the highest numbers of eosinophils were seen in rats at 1, 4, 28, 20, 15 and 10 dpi, corresponding to the first, (1 and 4) third (20 and 28) and second (10 and 15) instars. Except for 1 dpi, eosinophil numbers were significantly higher than those seen in control animals. Mast cell numbers were highest in early infestation (4 dpi), followed by those at 20 dpi. In increasing order, numbers of mast cells were greatest at 10, 28, 15, 1, 20 and 4 dpi, although significant differences with control animals were only seen at 10 and 28 dpi. Eosinophils and mast cells showed negative correlation only in animals with second instar larvae (10 and 15 dpi). Comparative analyses were also carried out after considering the skin into four distinct regions. The results suggest that the expression of both cell types, particularly eosinophils, is an important host response to infestation by D. hominis.     

Activated host neutrophils in the larval midgut lumen of the human bot fly Dermatobia hominis

Light microscopy (LM) and transmission electron microscopy (TEM) were used to observe activated polymorphonuclear neutrophils from mammalian hosts as well as invading bacteria in the midgut lumen of larvae of the human bot fly Dermatobia hominis. Other resident or recruited cells associated with dermal myiasis were fed on by larvae and digested more rapidly than neutrophils. The latter were observed moving towards bacteria and particles of food, extending the filopodia and engulfing material to be digested within phagosomes. The larval midgut lumen, thus, appears to be a suitable environment to produce neutrophil activation at least for short periods, as seen in mammalian hosts. Although interactions between phagocytes and bacteria in the midgut lumen may be important in bot fly larval development, further studies are required to confirm this.     

Attempts to culture the parasitic stage of Dermatobia hominis (L. Jr.) in vitro (Diptera: Cuterebridae)

Dermatobia hominis larvae were cultured in a semidefined liquid medium. First-instar larvae (L1) grew well up to 44 days; 29.1% molted in a mean period of 8.62 days. Two larvae reached the third instar but lived only 1 and 18 days, respectively, after the second molt. The increase in size, measured in 4 larvae, was about 10-fold. Second- and third-instar larvae, obtained from the skin of cattle, survived and grew in the medium for up to 2 mo; 39.0% of the L2 molted while 77.3% of the L3 pupated, and some produced flies when transferred to sand after 14.84 +/- 10.08 days in the culture medium. Some maturation factor, obtained from the skin, may be necessary for the larvae to grow satisfactorily and to complete the full parasitic cycle in vitro.     

Inflammatory reaction to the human bot-fly,Dermatobia hominis,in infested and reinfested mice

Two groups of mice were infested with first stage larvae of the human bot-fly, Dermatobia hominis (Linnaeus Jr) (Diptera: Oestridae). In the first group, skin biopsies were carried out 1, 3, 5, 7, 10 and 18 days after infestation. The second group was also infested but had all the larvae removed 5 days after infestation. The mice in the latter group were reinfested 4 weeks later and skin biopsies were carried out 1, 3, 5, 7, 10 and 18 days after reinfestation. In the first group, an inflammatory reaction began slowly, the neutrophils being the main inflammatory cells, eosinophils being scarce. The reaction progressed with time, developing a necrotic halo around the larvae containing inflammatory cells surrounded by fibroblasts. The inflammation invaded the adjacent tissue. In the second group, the inflammatory reaction was intense on the day immediately after reinfestation, the pattern being changed by the presence of a large number of eosinophils. Activated fibroblasts surrounding the necrotic area around the larvae appeared 3 days after reinfestation in the second group and 7 days after infestation in the first group. The results demonstrated that the previous contact with the antigens elicited the early arrival of eosinophils, probably through the chemotactic factors liberated by mast cells in the anaphylactic reaction.     

Topically applied ivermectin: efficacy against torsalo (Diptera: Cuterebridae)

To determine the systemic efficacy and persistence of a 0.5% ivermectin topical formulation against natural infestation of torsalo, Dermatobia hominis (L., Jr.), larvae in cattle, two trials in Colombia and two trials in Brazil were done. Of 56 infested cattle used in these trials, 28 were treated and 28 served as untreated controls. The formulation was poured along the back of each animal at a rate of 0.5 mg (AI)/kg body weight. Significantly fewer larvae were counted in the treated animals than on controls on days 9 and 10; greater than 99% control resulted. The first reinfesting larvae were observed in a few animals on days 30 and 31.     

Scanning electron microscopy studies on the first-instar larva of Dermatobia hominis

Abstract. First-instar larvae of Dermatobia hominis collected 1, 4 and 7 days after having penetrated experimentally infected rats, were studied by scanning electron microscope (SEM) observation. On the pseudocephalon there are basiconic and trichoid sensilla (antennal sensory complex), and basiconic, coeloconic and campaniform sensilla (maxillary sensory complex). The thoracic segments bear several rows of small, backwardly pointed, spines, and trichoid, campaniform, coeloconic and pit sensilla. The anterior spiracle is a minute opening. Both small and large spines directed posteriorly are on the first to fourth abdominal segments, which also bear coeloconic and companiform sensilla. These sensilla are present on the unarmed (fifth and sixth) and armed (seventh) abdominal segments. The seventh and the last (eight) abdominal segments have forwardly directed spines. Each spiracular plate has two spiracular openings and four spatulate-like structures called sun rays. The anus and the coeloconic sensilla are proeminent on the last segment. The results are compared with other parasitic dipteran larvae, and emphasize that the multiple types of sensilla on D. hominis larva may have importance in establishing the parasitic phase of the life cycle of this insect.     

Morphology of the second- and third-instar larvae of Dermatobia hominis by scanning electron microscopy

Larvae of Dermatobia hominis 10–27 days old were collected from experimentally infected rats and their morphology was studied by scanning electron microscopy. The moult from the second to third instar occurs at 18 days, with emergence from the host at 30 days post-infection. The second-instar larvae bear on the pseudocephalon, antennae (coeloconic sensilla), and coeloconic and basicoconic sensilla on the maxillary sensory complex. The thoracic segments bear small backwardly-directed spines anteriorly and ventral trichoid and campaniform sensilla. The first four abdominal segments have small and large backwardly-directed spines that are absent on segments five and six. The seventh and eighth abdominal segments have medium-sized forwardly-directed spines. Abdominal segments are encircled by campaniform sensilla. The terminal end of the eighth abdominal segment bears the anus, prominent anal lobes and two spiracular openings on each spiracular plate. Spiracular plates show a radial sun ray pattern. The rear abdomen also bears an ecdysal aperture, several pores and eight coeloconic sensilla. Although there are slight morphological differences, the spines (predominantly flat and thorn-like) and sensilla (campaniform and coeloconic) of the third-instar larvae show a similar arrangement to that of second-instar larvae. Thoracic trichoid sensilla are not seen in third-instar larvae. A perispiracular gland aperture is situated above each posterior spiracular opening. These morphological features are compared with those of other cuterebrid larvae.     

Genetic Approaches for Studying Myiasis-causing Flies: Molecular Markers and Mitochondrial Genomics

“Myiasis-causing flies” is a generic term that includes species from numerous dipteran families, mainly Calliphoridae and Oestridae, of which blowflies, screwworm flies and botflies are among the most important. This group of flies is characterized by the ability of their larvae to develop in animal flesh. When the host is a live vertebrate, such parasitism by dipterous larvae is known as primary myiasis. Myiasis-causing flies can be classified as saprophagous (free-living species), facultative or obligate parasites. Many of these flies are of great medical and veterinary importance in Brazil because of their role as key livestock insect-pests and vectors of pathogens, in addition to being considered important legal evidence in forensic entomology. The characterization of myiasis-causing flies using molecular markers to study mtDNA (by RFLP) and nuclear DNA (by RAPD and microsatellite) has been used to identify the evolutionary mechanisms responsible for specific patterns of genetic variability. These approaches have been successfully used to analyze the population structures of the New World screwworm fly Cochliomyia hominivorax and the botfly Dermatobia hominis. In this review, various aspects of the organization, evolution and potential applications of the mitochondrial genome of myiasis-causing flies in Brazil, and the analysis of nuclear markers in genetic studies of populations, are discussed.     

Experimental skin lesions from larvae of the bot fly Dermatobia hominis

Skin biopsies from larvae of Rattus norvegicus, experimentally infested with Dermatobia hominis (Linnaeus Jr) (Diptera: Cuterebridae), were processed for histopathological studies. Two days after infestation, the first-stage larvae (L1) were located deep in the dermis, surrounded by an inflamed area infiltrated predominantly by neutrophils. On the fourth day a thin necrotic layer could be seen close to the larvae, surrounded by large numbers of neutrophils, lymphocytes, macrophages with a few eosinophils and mast cells. A small warble was formed after the fourth day, increasing in size until the seventh day, when the L1 moulted to the second-stage larva (L2). The inflammatory process continued with increasing numbers of neutrophils, macrophages, lymphocytes, eosinophils and mast cells invading the area, as well as the proliferation of fibroblasts and endothelial cells and the appearance of a few localized haemorrhages. After 18-20 days, the L2 moulted to the third-stage larva (L3), when a few plasma cells could be seen in the inflamed area. At 25-30 days there was a reduction in the necrotic layer, as well as in the number of neutrophils and lymphocytes, although large amounts of eosinophils, plasma cells, and collagen fibres were seen. The L3 usually left the host after 30 days. Two days later, the larval cavity was reduced, mast cells infiltrated the region and collagen fibre production were increased. After 7 days, an intense infiltration of plasma cells and scattered necrotic areas could be seen. A scar formed after 10 days. This study showed the laboratory rat to be a suitable model for studies of D. hominis infestation.     

Localized reversible frameshift mutation in an adhesin gene confers a phase-variable adherence phenotype in mycoplasma

The variable adherence-associated (Vaa) antigen of Mycoplasma hominis is an abundant surface lipoprotein adhesin that may mediate important interactions of this wall-less prokaryotic pathogen with the human host. Extensive mutational variation of Vaa size, as well as sequence and antigenic divergence, has been described previously. Using a series of clonal isolates representing an isogenic lineage of variants oscillating in Vaa expression, Vaa is further shown in this study to undergo high-frequency phase variation in expression, which correlated precisely with the ability of M . hominis to adhere to cultured human cells. Although no DNA rearrangements or sequence differences in the 5' regions flanking vaa alleles were detected between Vaa⁺ and Vaa⁻ variants, intragenic vaa sequences from this lineage revealed an oscillating mutation involving a single nucleotide deletion/insertion in a short tract of adenine residues near the 5' end of the mature Vaa coding sequence, which created a translational frameshift resulting in either a complete Vaa ORF or an in-frame UAG stop codon immediately downstream of the poly-A tract. Evidence for the occurrence of this high-frequency frameshift mutation in vivo was obtained from analysis of PCR-generated vaa sequences amplified from the joint synovial fluid of a patient with M . hominis -associated arthritis, which indicated that Vaa phase variation occurs during M . hominis infection in the natural host. These results identify a distinctive frameshift mutator element in the vaa gene that governs M . hominis adherence and highlight the importance of mutational alteration of primary gene products on the mycoplasma surface as a means of generating and maintaining functional diversity in the host.     

A case of cutaneous myiasis caused by Dermatobia hominis]

Due to increasing international travelling of European population, tropical parasitic diseases may be imported more frequently in temperate countries. We describe a subject who travelled in Brasil and returned to Italy with a subcutaneous nodule containing a phase-II Dermatobia hominis larva; such larva is reported with photographic documentation.     

First report of human myiasis in GoiáS state, Brazil: frequency of different types of myiasis, their various etiological agents, and associated factors

The objective of this study was to show which species of flies are responsible for human myiasis in the Brazilian state of Goiás and to determine the frequency of cases. Patients at the Clinical Hospital of the Federal University of Goiás (UFG) were examined, and any fly larvae found in their wounds were collected for taxonomic identification. First instar larvae were observed using light microscopy; second and third instars were examined using stereoscopy. The following screwworm flies were observed, in decreasing order of prevalence: Cochliomyia hominivorax, Sarcodexia lambens, Dermatobia hominis, Chrysomya albiceps, Chrysomya megacephala, Lucilia cuprina, and Eristalis tenax. Myiasis was most frequent in the legs in adults, male patients, elderly people, and people of reproductive age. It was lowest in children, females, and patients with neurologic or psychiatric disorders. Frequency was high in patients living in low socioeconomic conditions with poor personal hygiene. Education and sanitation measures are needed to counteract this situation.     

Dermatobia, the neotropical warble fly

The neotropical warble fly, Dermatobia hominis (Fig. 1), has plagued neotropical America since preColombian times, and has become an economically important pest causing substantial losses to the meat, milk and leather industries from northern Mexico down to northern Argentina. Its life cycle (Box 1) is astonishingly complex, requiring another insect as a phoretic carrier of its eggs to the skin of its mammal hosts. Here Eugenio Sancho discusses factors that contribute to the current economic and public health importance of this myiasis-causing fly.     

Electrophoretic analysis of proteins from Mycoplasma hominis strains detected by SDS-PAGE, two-dimensional gel electrophoresis and immunoblotting

The proteins of 14 strains of Mycoplasma hominis were compared by SDS-PAGE in gradient gels, by two-dimensional (2D) gel electrophoresis of extracts of 35S-labelled cells and by immunoblot analysis of cell proteins. The strains examined included the M. hominis type strain PG21 and 13 others isolated variously from genital tract, mouth, blood, upper urinary tract and a wound. These 14 strains shared 76-99% of proteins in SDS-gradient gel analysis and 41-72% in the 2D gels. As expected, the immunoblot analysis likewise revealed the existence of an extensive common protein pattern in M. hominis, in addition to a number of antigens shared only by some strains.     

Cuterebra buccata: immune response in myiasis of domestic rabbits

Naturally infected rabbits (Oryctolagus) were used to define further the nature of the immune response in myiasis due to Cuterebra buccata. Third instar larvae were dissected into four fractions; (1) alimentary tract with attached organs, (2) hemolymph, (3) fat body with tracheae, and (4) cuticle with attached muscles. The antigens provoking immune phenomena in naturally infected rabbits were found to reside in the alimentary tract and hemolymph fractions only. All rabbits which were skin tested were found to exhibit delayed hypersensitivity and to have serum precipitins with specificity against these antigens. Passive cutaneous anaphylaxis activity was demonstrated only in sera from rabbits also exhibiting reaginic and/or Arthus-type skin hypersensitivities. With the use of immunoelectrophoresis four separate antigens were demonstrated in alimentary tract fractions. Larval dissections revealed the alimentary tract to be filled with cellular elements of rabbit whole blood. The immunologic findings are discussed in relation to this newly recognized feeding pattern and it is proposed that sensitization of the host occurs as a consequence of exogenous larval secretions injected at the time of feeding.     

The intensity and duration of primary Heligmosomoides polygyrusinfection in TO mice modify acquired immunity to secondary challenge

The effect of dose and duration of immunizing infections of Heligmosomoides polygyrus on protection against homologous challenge was studied in female TO mice. Primary infections were terminated at various levels with pyrantel embonate (adult infections) or ivermectin (larval infections) and mice were then challenged with 500 infective larvae (L3). The level of protection to secondary challenge positively correlated with the intensity of the primary immunizing infection but truncation of larval infection produced significantly better protection than termination of the adult nematode infection. The duration of the primary larval infection (1-6 days) positively correlated with the level of protection to secondary challenge, antibody responses and the proportion of circulating eosinophils. Histological changes in the gastrointestinal tract, peripheral leucocytic changes and antibody responses of the mice to H. polygyrus adult somatic antigens indicate both a cellular and humoral basis of host immunity to secondary challenge. Although the TO mice are slow responders in that they harbour chronic infections, immunization by intramucosal killing of the larval stage produced strong protection against secondary challenge infection. The presence of dead immunogenic larval stages within the intestinal wall may well be an important factor, since it exposes the host to stage specific antigens at an appropriate location. The implications of the findings for the control of gastrointestinal nematode infections are also discussed.     

Ascaris suum: immunoperoxidase and fluorescent probe analysis of host proteases and parasite proteinase inhibitors in developing eggs and second stage larvae

Live Ascaris suum females were incubated in medium containing chymotrypsin liganded to fluorescein-5-isothiocyanate, and eggs in the parasite's genital tract took up the probe and fluoresced. Eggs passed by these worms into the medium containing fluorescent probe retained their fluorescence in formaldehyde-saline and by 65 days had developed into second stage infective larvae. Eggs passed naturally by untreated worms were incubated in media containing fluorescent probes and all of the eggs exposed to chymotrypsin liganded to fluorescein-5-isothiocyanate were extensively labeled. Control eggs were labeled sporadically and less intensely, indicating specificity in the uptake of environmental proteins. Chymotrypsin from the parasite's environment can bind to A. suum eggs, and this occurs both inside the worm's genital tract and outside of the parasite. Immunoperoxidase studies showed that IgG developed against chymotrypsin or against A. suum chymotrypsin/elastase isoinhibitors A or C, binds to antigens in cross sections of second stage larvae and their egg shell coats. This suggests that host chymotrypsin is retained during development and may be complexed to A. suum isoinhibitors A and C.     

Permeability studies on taenid metacestodes: I. Uptake of proteins by larval stages of Taenia taeniaeformis, T. crassiceps, and Echinococcus granulosus.

Host immunoglobulins of several different classes were detected within the bladder fluids of Taenia taeniaeformis, T. crassiceps, and Echinococcus granulosus. Radioiodinated proteins were taken up in vitro by larvae of both T. taeniaeformis and T. crassiceps and were shown to retain their physicochemical and antigenic characteristics. Rates of uptake were similar in the 2 species and were not related to the molecular weight of the proteins. Immunoglobulins were taken up both in vitro and in vivo by larvae of T. taeniaeformis. Absorbed immunoglobulins were shown to retain both antigen binding capacity and biologic functions associated with the Fc portion of the molecules. Not all cysts of E. granulosus contained detectable host proteins. Uptake of 125I occurred when hydatid cysts were exposed to labeled proteins in vitro, but it appeared that rapid degradation of the labeled carrier led to the appearance of dialysable fragments. This may be attributable to proteolysis within the bladder fluid. We conclude that taeniid metacestodes are capable of absorbing a variety of proteins, and that these macromolecules can retain their structural and functional integrity following transport. This absorptive capacity accounts for the presence of host serum components within bladder fluids.