Marjorie S. Morgan

THE DEVELOPMENT OF PROTECTIVE IMMUNITY IN CANINE SCABIES

Seven of eight dogs that had been previously infested with Sarcoptes scabiei var. canis and then cured, expressed protective immunity when experimentally reinfested with scabies. All seven dogs that expressed resistance were spontaneously cleared of scabies by 64 days after they were experimentally reinfested. Five of the eight dogs were free of scabies by 24 days. The sequential changes in the inflammatory/immune cellular infiltrate in the scabietic lesions of each dog were determined during the sensitizing infestation, cure and the subsequent experimental reinfestation (challenge). During the initial infestation and in the subsequent challenge reinfestation, dogs developed mixed cellular infiltrates in their scabietic lesions that contained mononuclear cells, neutrophils, plasma cells and mast cells. Reinfestation induced more rapid increases in the densities of these cells than had occurred during the sensitizing infestation. Mononuclear and mast cells were the most numerous infiltrating cells during the sensitizing phase. During the challenge phase the most numerous infiltrating cells were mononuclear cells and neutrophils. The sensitizing and challenge infestations induced circulating scabies-specific antibody responses, but the response was more rapid during the reinfestation challenge. Both the cell-mediated response in the skin and the circulating antibody response waned in parallel with clearing of the mites following reinfestation.

Acaricidal Activity of Eugenol Based Compounds against Scabies Mites

Backgound Human scabies is a debilitating skin disease caused by the “itch mite” Sarcoptes scabiei. Ordinary scabies is commonly treated with topical creams such as permethrin, while crusted scabies is treated with topical creams in combination with oral ivermectin. Recent reports of acaricide tolerance in scabies endemic communities in Northern Australia have prompted efforts to better understand resistance mechanisms and to identify potential new acaricides. In this study, we screened three essential oils and four pure compounds based on eugenol for acaricidal properties. Methodology/Principal Findings Contact bioassays were performed using live permethrin-sensitive S. scabiei var suis mites harvested from pigs and permethrin-resistant S. scabiei var canis mites harvested from rabbits. Results of bioassays showed that clove oil was highly toxic against scabies mites. Nutmeg oil had moderate toxicity and ylang ylang oil was the least toxic. Eugenol, a major component of clove oil and its analogues –acetyleugenol and isoeugenol, demonstrated levels of toxicity comparable to benzyl benzoate, the positive control acaricide, killing mites within an hour of contact. Conclusions The acaricidal properties demonstrated by eugenol and its analogues show promise as leads for future development of alternative topical acaricides to treat scabies.

High-resolution melt analysis for the detection of a mutation associated with permethrin resistance in a population of scabies mites

Abstract Permethrin as a topical acaricide cream is widely used to treat scabies. The neuronal voltage‐sensitive sodium channel (Vssc), necessary for the generation of action potentials in excitable cells, is the target of pyrethroid acaricides such as permethrin. Pyrethroid resistance has been linked to specific mutations in the Vssc gene. Following the partial sequencing of the Vssc gene in the scabies mite Sarcoptes scabiei (L.) (Astigmata: Sarcoptidae), we compared Vssc gene sequences from permethrin‐sensitive and ‐tolerant S. scabiei var. canis Gerlach mites, and identified a G to A single nucleotide polymorphism (SNP) in permethrin‐tolerant mites resulting in an amino acid change from glycine to aspartic acid in domain III S6. The mutation is in a region of the gene where mutations have been identified in a range of pyrethroid‐resistant arthropods. Results of in vitro permethrin exposure assays showed that survival rates for mites bearing the mutation were similar to those previously reported for mites from human subjects where clinical tolerance to permethrin had been observed. A real‐time polymerase chain reaction−high‐resolution melt (PCR‐HRM) assay was developed to detect this SNP. This assay provides a useful methodology for screening for this and other mutations associated with permethrin resistance in scabies mite populations and thus facilitates surveillance for acaricide resistance.

The effect of insecticide synergists on the response of scabies mites to pyrethroid acaricides.

Background Permethrin is the active component of topical creams widely used to treat human scabies. Recent evidence has demonstrated that scabies mites are becoming increasingly tolerant to topical permethrin and oral ivermectin. An effective approach to manage pesticide resistance is the addition of synergists to counteract metabolic resistance. Synergists are also useful for laboratory investigation of resistance mechanisms through their ability to inhibit specific metabolic pathways. Methodology/Principal Findings To determine the role of metabolic degradation as a mechanism for acaricide resistance in scabies mites, PBO (piperonyl butoxide), DEF (S,S,S-tributyl phosphorotrithioate) and DEM (diethyl maleate) were first tested for synergistic activity with permethrin in a bioassay of mite killing. Then, to investigate the relative role of specific metabolic pathways inhibited by these synergists, enzyme assays were developed to measure esterase, glutathione S-transferase (GST) and cytochrome P450 monooxygenase (cytochrome P450) activity in mite extracts. A statistically significant difference in median survival time of permethrin-resistant Sarcoptes scabiei variety canis was noted when any of the three synergists were used in combination with permethrin compared to median survival time of mites exposed to permethrin alone (p<0.0001). Incubation of mite homogenates with DEF showed inhibition of esterase activity (37%); inhibition of GST activity (73%) with DEM and inhibition of cytochrome P450 monooxygenase activity (81%) with PBO. A 7-fold increase in esterase activity, a 4-fold increase in GST activity and a 2-fold increase in cytochrome P450 monooxygenase activity were observed in resistant mites compared to sensitive mites. Conclusions These findings indicate the potential utility of synergists in reversing resistance to pyrethroid-based acaricides and suggest a significant role of metabolic mechanisms in mediating pyrethroid resistance in scabies mites.

Serum antibody to Sarcoptes scabiei and house dust mite prior to and during infestation with S. scabiei.

In this study, serum antibodies to Sarcoptes scabiei var. canis (SS), Dermatophagoides farinae (DF), and D. pteronyssinus (DP) were determined in 19 healthy, random-source dogs prior to infestation with scabies then again during a primary infestation, cure and challenge infestation with scabies. Prior to scabies infestation, serum of 11 dogs contained faintly detectable amounts of IgE and/or IgG to proteins in SS extract, probably resulting from sensitization to dust mites that share cross-reactive antigenic epitopes with SS. After becoming infested with scabies, the response to SS antigens became stronger with antibodies appearing to more antigens as the scabies infestation progressed. Three of the newly recognized proteins were 170, 155 and 142/133kD and could be used in a diagnostic test since antibodies to them appeared during the primary infestation. In addition, during the primary infestation, 14 of 15 dogs developed IgE to 1-11 new SS proteins in addition to an increase in IgE binding to those proteins recognized prior to infestation. Overall, the strongest antibody responses (IgE and IgG) were exhibited during cure of the first infestation, when dead mites were still present in the stratum corneum. As expected, the antibody response was strong and rapid during challenge when the infestation self-cured. The immunogenic SS proteins identified by serum antibody binding during challenge, when the hosts self-cured, are candidates for inclusion in a vaccine. These candidate proteins are 200, 185, 170, 155, 142/133, 112, 97, 74, 57, 45/42, 32 and 22kD. Some of the proteins in SS that exhibited new or increased antibody binding during the experiment also had IgE and IgG binding to proteins with similar molecular weights in DF and DP extracts. These results illustrate the difficulties involved in understanding and interpreting serum antibody for developing a serological test for the diagnosis of scabies, isolating relevant SS antigens that could be included in a vaccine for prevention of scabies, and for understanding the immune response mechanism to scabies.

SKEWED TH1/TH2 IMMUNE RESPONSE TO SARCOPTES SCABIEI

Scabies is a contagious skin disease of humans and many other species of mammals. Previous studies suggested that the balance between the Th1 and Th2 immune responses may influence the outcome of a scabies infestation in a sensitized host. Therefore, in this study, we examined the T-helper cell cytokine profiles of splenocytes and lymph node cells in BALB/c mice that were immunized with scabies extract (primary response), infested with scabies mites (primary response), or immunized and then infested (secondary response). Lymphocyte cytokine expression was analyzed by flow cytometry after staining for intracellular cytokines. Immunization with scabies extract induced production of interferon-γ (IFNγ) (Th1 response) by both spleen and lymph node cells. Mice that were infested with scabies increased production of interleukin-4 by lymph node cells and of IFNγ by splenocytes. Mice that were first immunized and then infested with mites increased production of IFNγ by both spleen and lymph node cells. However, this increased level of IFNγ was only about half of that induced by immunization alone. These results suggest that live scabies mites produced something that inhibited IFNγ production in the lymph nodes of scabies-immunized mice. Our data also indicate that lymphocytes in the spleen and lymph nodes can present different cytokine response profiles.

Increased transcription of Glutathione S-transferases in acaricide exposed scabies mites

BackgroundRecent evidence suggests that Sarcoptes scabiei var. hominis mites collected from scabies endemic communities in northern Australia show increasing tolerance to 5% permethrin and oral ivermectin. Previous findings have implicated detoxification pathways in developing resistance to these acaricides. We investigated the contribution of Glutathione S-transferase (GST) enzymes to permethrin and ivermectin tolerance in scabies mites using biochemical and molecular approaches.ResultsIncreased in vitro survival following permethrin exposure was observed in S. scabiei var. hominis compared to acaricide naïve mites (p < 0.0001). The addition of the GST inhibitor diethyl maleate restored in vitro permethrin susceptibility, confirming GST involvement in permethrin detoxification. Assay of GST enzymatic activity in mites demonstrated that S. scabiei var. hominis mites showed a two-fold increase in activity compared to naïve mites (p < 0.0001). Increased transcription of three different GST molecules was observed in permethrin resistant S. scabiei var. canis- mu 1 (p < 0.0001), delta 1 (p < 0.001), and delta 3 (p < 0.0001). mRNA levels of GST mu 1, delta 3 and P-glycoprotein also significantly increased in S. scabiei var. hominis mites collected from a recurrent crusted scabies patient over the course of ivermectin treatment.ConclusionsThese findings provide further support for the hypothesis that increased drug metabolism and efflux mediate permethrin and ivermectin resistance in scabies mites and highlight the threat of emerging acaricide resistance to the treatment of scabies worldwide. This is one of the first attempts to define specific genes involved in GST mediated acaricide resistance at the transcriptional level, and the first application of such studies to S. scabiei, a historically challenging ectoparasite.

Cross-reactivity between storage and dust mites and between mites and shrimp.

Many patients have sensitivities to multiple species of storage and house dust mites. It is not clear if this is because patients have multiple sensitivities to species-specific mite allergens or if these mites share many cross-reacting allergens. Our objective was to further define the cross-allergenicity between several species of storage and house dust mites using crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), immunoblotting, and ELISA. CIE and CRIE reactions revealed that storage mites shared two cross-antigenic molecules and one of these bound IgE in a serum pool from mite allergic patients. Antibody in anti-sera built to each species of mite recognized many SDS–PAGE resolved proteins of other mite species and this suggested the potential for other cross-reactive allergens. Among patient sera, IgE bound to many different proteins but few had IgE that bound to a protein with common molecular weights across the mite species and this suggested mostly species-specific allergens. Antiserum built to each mite species precipitated one protein in shrimp extracts that bound anti-Der p 10 (tropomyosin) and IgE in the serum pool. Anti-Der p 10 showed strong binding to shrimp tropomyosin but very little to any of the mite proteins. ELISA showed the mite extracts contained very little tropomyosin. The storage and dust mites investigated contain mostly species-specific allergens and very small amounts of the pan-allergen tropomyosin compared to shrimp and snail.

Extracts of Scabies Mites (Sarcoptidae: Sarcoptes scabiei) Modulate Cytokine Expression by Human Peripheral Blood Mononuclear Cells and Dendritic Cells

Abstract We performed a series of experiments to determine if human peripheral blood mononuclear cells (PBMCs) from a healthy donor and dendritic cells (NHDCs) derived from these PBMCs reacted to molecules in a scabies extract. PBMCs extravasate from the circulatory system and enter tissues such as scabietic lesions, where monocytes become macrophages. Cells were cultured in medium alone or medium containing 50 μg/ml of Sarcoptes scabiei (SS) extract, 50 ng/ml E. coli lipopolysaccharide (LPS), or SS + LPS together. Supernatants were collected and assayed by enzyme-linked immunosorbent assay (ELISA) for specific cytokines. PBMCs stimulated with SS or LPS exhibited moderately upregulated production of interleukin (IL)-1β and huge increases in secretions of IL-6, IL-8 and TNF-α. Cells co-stimulated with both SS and LPS generally secreted more of these cytokines than cells stimulated with either SS or LPS alone. LPS induced a small amount of IL-1α secretion, whereas SS did not, and neither additive resulted in the production of IL-10. NHDCs did not produce IL-1α, IL-1β, IL-6, IL-8, or IL-10 in response to stimulation with SS. These cells did produce IL-6, IL-8, and tumor necrosis factor (TNF)-α in response to LPS. When cells were co-stimulated with both LPS and SS, the production of IL-6 and IL-8 was significantly reduced compared with the levels secreted after LPS stimulation alone. These studies show that molecules in a whole body extract of S. scabiei modulate the function of PBMCs (probably monocytes) and dendritic cells.

Circulating IgE in Patients with Ordinary and Crusted Scabies

Abstract Serum from seven patients with ordinary scabies and six with crusted scabies were screened by immunoblotting for IgE- and IgG-specific proteins in an extract of the mite, Sarcoptes scabiei variety canis. Sera from atopic individuals without sensitivity to house dust mites were used as controls. Serum from all of the patients with crusted scabies showed strong IgE binding to 11–21 and IgG binding to 1–7 scabies proteins. In contrast, three of the seven patients with ordinary scabies showed IgE binding to one to six scabies proteins, and their antibody binding was much weaker. Patients with crusted scabies had serum antibody that reacted with larger molecular weight proteins compared with patients with ordinary scabies. The results of this study indicate that patients with crusted scabies showed a pronounced IgE response to scabies mites, whereas patients with ordinary scabies did not.