Association between louse abundance and MHC II supertypes in Galápagos mockingbirds.

Major histocompatibility complex class II (MHC II) is an essential molecule triggering the adaptive immune response by the presentation of pathogens to helper T cells. The association between individual MHC II variants and various parasites has become a frequent finding in studies of vertebrate populations. However, although bird ectoparasites have a significant effect on their host's fitness, and the host's immune system can regulate ectoparasitic infections, no study has yet investigated the association between MHC II polymorphism and ectoparasite infection in the populations of free-living birds. Here, we test whether an association exists between the abundance of a chewing louse (Myrsidea nesomimi) and MHC II polymorphism of its hosts, the Galápagos mockingbirds (Mimus). We have found that the presence of two MHC II supertypes (functionally differentiated clusters) was significantly associated with louse abundance. This pattern supports the theory that a co-evolutionary interaction stands behind the maintenance of MHC polymorphism. Moreover, we have found a positive correlation between louse abundance and heterophil/lymphocyte ratio (an indicator of immunological stress) that serves as an additional piece of evidence that ectoparasite burden is affected by immunological state of Galápagos mockingbirds.

Lip b 1 is a novel allergenic protein isolated from the booklouse, Liposcelis bostrychophila.

BACKGROUND: Booklice, belonging to the order Psocoptera, are small household insect pests that are distributed worldwide. Liposcelis bostrychophila, a common home-inhabiting species of booklouse, infests old books, sheets of paper, and stored food. Recent entomological and serological studies demonstrated that L. bostrychophila accounted for the majority of detectable insects in house dust and could be a potent inducer of respiratory allergy. Our recent proteomic analysis identified a potent allergenic protein from L. bostrychophila, designated Lip b 1, and determined its partial amino acid sequences. METHODS: Cloning of cDNAs for Lip b 1 was performed by large-scale transcriptome analysis (RNA-seq) and subsequent reverse transcription polymerase chain reaction. The full-length amino acid sequences deduced from Lip b 1 cDNAs were bioinformatically analyzed. The recombinant proteins of glutathione S-transferase (GST)-fused Lip b 1 were analyzed by Western blot and enzyme-linked immunosorbent assay. RESULTS: Lip b 1 cDNAs encoding two types of 254-amino acid proteins were cloned. The clones shared 87% identity, and the deduced molecular weights and isoelectric points were consistent with those determined in our previous study. The two types of Lip b 1 proteins in the GST-fused form were similarly reactive with sera from allergic patients sensitized with L. bostrychophila. CONCLUSIONS: Lip b 1 is a novel protein possibly causing booklouse allergy.

Paranucleospora theridion (Microsporidia) infection dynamics in farmed Atlantic salmon Salmo salar put to sea in spring and autumn.

The microsporidian Paranucleospora theridion (syn. Desmozoon lepeophtheirii) is a parasite of Atlantic salmon Salmo salar and also a hyperparasite of the salmon louse Lepeophtheirus salmonis. The parasite develops 2 types of spores in salmon, cytoplasmic spores in phagocytes and intranuclear spores in epidermal cells. The former type of development is assumed to be propagative (autoinfection), while the epidermal spores transfer the parasite to lice. Development in lice is extensive, with the formation of xenoma-like hypertrophic cells filled with microsporidian spores. We show that salmon are infected in the absence of lice, likely through waterborne spores that initiate infections in the gills. During summer and autumn the parasite propagates in the kidney, as evidenced by peaking normalised expression of P. theridion rRNA. Lice become infected during autumn, and develop extensive infections during winter. Lice mortality in winter and spring is likely responsible for a reservoir of spores in the water. Salmon transferred to sea in November (low temperature) did not show involvement of the kidney in parasite propagation and lice on such fish did not become infected. Apparently, low temperatures inhibit normal P. theridion development in salmon.

Cutaneous cytokine gene expression and cellular responses in lambs infested with the louse, Bovicola ovis, and following intradermal injection of crude louse antigen.

The present study was undertaken to investigate further the immunological responses in the skin of lambs to natural louse infestation and following intradermal allergen challenge. Bovicola ovis-infested (n=7) and naïve (n=7) Romney lambs received four intradermal injections each of crude louse Ag and diluent control solutions on the dorso-lateral chest. From each lamb, skin samples were obtained from untreated skin and, at 4, 24, 48 and 72 h following injection, from one each of the Ag- and diluent-injected skin sites. Levels of acetylcholinesterase-positive Langerhans and MHC II(+) cells in the epidermis as well as MHC II(+), CD1b(+), T19(+) and IgE(+) cells, eosinophils, and diffuse IgE staining in the dermis were significantly elevated in infested compared to naïve lambs (all p< or =0.01). Additionally, gene expression of interleukin-4 (IL-4), IL-5, IL-13 (all p< or =0.001) and IL-10 (p< or =0.05) was significantly higher in the skin of infested compared to naïve lambs while TNF-alpha and IFN-gamma gene expression were not significantly different between the two groups. Intradermal injection of louse Ag led to immediate and late phase responses in the infested lambs while the naïve lambs showed only minimal responses. Levels of dermal MHC II(+), CD1b(+), T19(+)and IgE(+) cells, eosinophils and diffuse IgE staining in infested lambs following injection of louse Ag were similar to or exceeded those in untreated skin and, with few exceptions, were higher than in naïve lambs. Additionally, cytokine gene expression of IL-4, IL-5, IL-13 and IL-10 increased to peak levels 4 h following Ag injection in the infested lambs (p< or =0.001, < or =0.05, < or =0.05 and < or =0.001 respectively compared to untreated controls) and remained significantly elevated compared to that observed in the naïve controls for the duration of the experiment. Significant elevations of MHC II(+) cells and IL-4, IL-5, IL-13 and IL-10 gene expression were observed in the louse-naïve lambs following injection of louse Ag but were much less pronounced than in the infested lambs. These results indicated that louse infestation in lambs elicited a highly skewed Th2 immuno-inflammatory response with many characteristics similar to those seen with other parasitic infections and also in atopic dermatitis.

Salmon louse rhabdoviruses: Impact on louse development and transcription of selected Atlantic salmon immune genes.

Recently, it has been shown that the salmon louse (Lepeophtheirus salmonis) is commonly infected by one or two vertically transmitted Lepeophtheirus salmonis rhabdoviruses (LsRVs). As shown in the present study, the viruses have limited effect on louse survival, developmental rate and fecundity. Since the LsRVs were confirmed to be present in the louse salivary glands, the salmon cutaneous immune response towards LsRV positive and negative lice was analyzed. In general, L. salmonis increased the expression of IL1ß, IL8 and IL4/13A at the attachment site, in addition to the non-specific cytotoxic cell receptor protein 1 (NCCRP-1). Interestingly, LsRV free lice induced a higher skin expression of IL1ß, IL8, and NCCRP-1 than the LsRV infected lice. The inflammatory response is important for louse clearance, and the present results suggest that the LsRVs can be beneficial for the louse by dampening inflammation. Further research is, however; needed to ascertain whether this is a direct modulatory effect of secreted virions, or if virus replication is altering the level of louse salivary gland proteins.

Changes in morphology and key cytokine gene expression after intradermal injection of louse (Bovicola ovis) antigen in sheep with naturally occurring atopic dermatitis.

Groups of louse-infested and louse-naïve lambs (n=6 or 7) were used in two experiments to determine the sequential tissue response (macroscopical, microscopical and key cytokine mRNA) to intradermal injection of crude louse (Bovicola ovis) antigen over a period of 72 or 96 h. Histamine diphosphate and phosphate-buffered saline/glycerol (antigen vehicle control) solutions were also injected intradermally in each lamb for comparison. In both experiments, louse-infested lambs showed immediate and late-phase responses (LPRs) to louse antigen that differed significantly from the responses in the louse-naïve lambs. In experiment 1, biopsy samples taken at 7, 24, 48 and 96 h after injections showed more extensive dermal inflammation and leucocyte infiltration in response to louse antigen in louse-infested than in louse-naïve lambs. Eosinophils were significantly more numerous in the dermis of louse-infested lambs after all treatments and increased substantially in these lambs after antigen injection. Additionally, the louse-infested lambs differed from the naïve lambs in showing significantly higher mononuclear leucocyte and basophil infiltration and significantly lower neutrophil infiltration after antigen injection. In experiment 2, biopsy samples taken 4, 24, 48 and 72 h after injections showed trends in eosinophil infiltration of the dermis similar to those observed in experiment 1. Peak IL-4 mRNA expression was detected 4 h after antigen injection in the louse-infested lambs and remained significantly elevated at 24 h as compared with the results in the louse-naïve lambs. No significant difference in IFN-gamma mRNA expression between the louse-infested and the louse-naïve lambs was observed. These results indicated that louse-infested lambs show a cutaneous LPR analogous to that observed in atopic human beings and dogs. However, some differences were observed, including the longer duration of the LPR, the profuse eosinophil infiltration, and an absence of increased IFN-gamma mRNA expression.

Ingestion of host antibodies by Bovicola ovis on sheep.

Sheep body lice, Bovicola ovis, collected from moderately heavy infestations on Merino wethers, were assayed by ELISA for their content of host-derived specific immunoglobulin (Ig). Relative concentrations of anti-horse myoglobin antibodies in the lice and in sera from their hosts were used to estimate the total quantities of functional Ig (that which remained capable of binding specifically to its antigen) present, giving a mean of 0.21 +/- 0.20 mg/g of lice. An attempt to demonstrate the presence of antibodies against B. ovis antigens in naturally-infested host sheep using ELISA produced inconclusive results. The implications of the quantities of Ig ingested by feeding B. ovis are discussed in relation to the feasibility of immunological control of this species on sheep.

Proliferative responses of lymphocytes in Bovicola ovis-infested lambs.

Proliferative responses of peripheral blood and prescapular lymph node lymphocytes from 8 B. ovis-infested and 8 naive lambs to concanavalin A (Con A) and soluble antigen of B. ovis were examined in vitro. The numbers of lice and the extent of cockle were also assessed on each lamb. Prescapular lymph node lymphocytes from infested lambs showed significantly higher responses to B. ovis antigen than those from naive lambs. Only a marginal difference was observed between infested and naive lambs in the response of lymphocytes from peripheral blood. Proliferative responses to Con A by prescapular lymph node or blood cells were not significantly different between groups. The results indicated a relationship between the intensity of louse infestation and the proliferative response of prescapular lymph node lymphocytes to B. ovis antigen.

Comparison of local and systemic responsiveness of lymphocytes in vitro to Bovicola ovis antigen and concanavalin A in B. ovis-infested and naive lambs.

The in vitro proliferation assay was used to determine lymphocyte responsiveness to soluble antigen of B. ovis and to Concanavalin A (Con A) in peripheral blood, spleen and various lymph nodes from B. ovis-infested and naive lambs. From March to July, an assay of monthly blood samples showed generally higher proliferative responses to antigen and Con A in B. ovis-infested than naive lambs. The proliferative response of cells from the skin-draining prescapular lymph nodes to B. ovis antigen was significantly higher in B. ovis-infested than naive lambs. Responses of cells from the medial iliac, mediastinal and mesenteric lymph nodes (which do not receive lymph from the skin) and spleen showed no significant differences between groups. Within the B. ovis-infested lambs, the response of cells from the prescapular lymph node was significantly higher than that from any other lymphoid organ examined. Responsiveness of the prescapular, medial iliac and mesenteric lymph node and spleen cells to Con A was not significantly different between groups, while mediastinal lymph node cells showed a significantly higher response in B. ovis-infested lambs. The data indicate that the antigen-specific cellular immune response is operating mainly locally, at the level of the skin and draining lymph nodes. Responses to the T cell mitogen Con A did not support non-specific immunodepression as reported in other ectoparasite/host systems.

Localization of the major immunogen and other glycoproteins of the louse Polyplax spinulosa.

A 31-kDa.immunogenic glycoprotein of Polyplax spinulosa was localized on louse sections using indirect immunofluorescence with specific antibodies of louse-infested rats and using fluorescein-labelled lectins. Antibodies of infested rats reacted specifically with midgut epithelium and semi-digested content of the gut. They did not bind to other louse tissues including salivary glands. The lectins concanavalin A and wheat germ agglutinin bound strongly to the immunogen, the gut and its semidigested contents. Electrophoretic and blotting analysis showed the presence of a 31-kDa glycoprotein component reacting with specific rat antibodies, concanavalin A and wheat germ agglutinin in louse faeces. The result indicates that the 31-kDa immunogen is secreted by the midgut epithelium into the gut lumen and faeces. We suggest that rats become immunized by scratching the lice faeces into the skin.

Do sheep regulate the size of their mallophagan louse populations?

Alternatives to chemicals for controlling parasites are required to minimise problems from resistance, residues in animal products and occupational exposure. Utilisation of host response to parasites through selection of resistant types or vaccination is an appealing option. To date most studies have been with haematophagous or invasive parasites which directly contact elements of the host immune system. Sheep lice (Bovicola ovis) feed superficially on the skin of sheep ingesting lipid, scurf, bacteria and loose stratum corneum squames. Evidence is presented that despite their surface feeding habit Bovicola ovis stimulate an immune response in sheep and that this response may play a part in regulating the size of louse populations.

Pruritus and dermal response to insect antigens in sheep infested with Bovicola ovis.

This study examined the relationships among louse density, pruritus and dermal response to insect antigens in sheep infested with Bovicola ovis. Polypay and Columbia ewes were allocated to two groups, infested and naive, and louse densities and pruritus were monitored for 15 months. Ten months after the initial infestation, all sheep were tested for hypersensitivity on the midside and ears by intradermal injection of soluble extracts of B. ovis, Stomoxys calcitrans and Musca autumnalis. The areas of skin reactions were measured at 20 min, 1, 3 and 24 h after injection and skin thickness was measured at 24 h. Louse densities on Polypays were approximately 10 times greater than on Columbias, and pruritus was correlated with louse numbers at most inspections. Most pruritic behaviour was directed to the sides of infested sheep. Wheal and flare reactions developed rapidly to all extracts in both infested and naive ewes. Reactions to louse extract were larger in infested than naive sheep at all four times after injection. In the infested Polypays, reactions to louse extract were greater than to the fly extracts, but in naive sheep there was little difference among extracts. Reactions in naive Columbias were larger than in naive Polypays at 20 min, 1 and 3 h, but had almost completely abated in both groups at 24 h. Reactions in infested Columbias were greater than in infested Polypays at 20 min, but at 24 h reactions in the Polypays were larger. Louse numbers and pruritus were correlated with wheal areas and skin thickness at 24 h, but there was little relationship with the size of reactions at earlier times. These findings are consistent with the development of a hypersensitive response to B. ovis and suggest that dermal reactions to lice may influence sheep susceptibility.

Detection of homocytotropic antibody in lambs infested with the louse, Bovicola ovis, using a basophil histamine-release assay.

The utility of a basophil histamine-release assay using washed whole blood cells was examined in lambs and was used to determine if homocytotropic antibody with specificity for Bovicola ovis was produced in response to infestation with the louse. Maximal histamine release in the assay in response to Concanavalin A, anti-ovine IgE monoclonal antibody and, in sensitized lambs, to B. ovis antigen ranged from 18 to 48%. Histamine release from blood cells in response to B. ovis antigen was significantly higher in louse-infested lambs than in louse-naive lambs and was significantly correlated with louse and cockle scores. Passive cutaneous anaphylaxis (PCA) tests were negative with sera obtained from the lambs at the same time as blood for the basophil histamine-release assay. Serum histamine levels also were significantly higher in the louse-infested lambs than in louse-naive lambs and were significantly correlated with louse and cockle scores. The present results support a role for B. ovis-specific homocytotropic antibody in the development of cockle and indicate that the basophil histamine-release assay is more sensitive than the PCA test.

Ivermectin: its effect on the immune system of rabbits and rats infested with ectoparasites.

The influence of subcutaneously administered ivermectin on the specific immune response was studied in rabbits infested with mites (Psoroptes cuniculi) and in rats infested with lice (Polyplax spinulosa). A pronounced specific antibody activity and a change in immunoblotting pattern was observed in rabbits after the ivermectin treatment. However, in rats the antibody activity decreased and the profile of specific antibodies, tested by immunoblotting, remained the same as before the treatment. The specific immune response in rabbits artificially immunized with whole-body Psoroptes cuniculi extract was not affected by ivermectin. It was concluded that ivermectin has no direct effect on the immune response of rabbits and rats and that the enhanced immune response in the mite-infested rabbits was caused by the massive release of antigens associated with the synchronous death of the mites.

Isolation and characterization of an immunogen from the louse Polyplax spinulosa.

The reaction of Polyplax spinulosa protein components with antigen-specific rat antisera and with five plant lectins is described. In immunoblotting, the antisera from rats infested with the louse P. spinulosa specifically recognized from 7 to 11 antigenic components of louse whole-body homogenates. Affinoblotting analysis with lectins showed that 10 of these components were glycoproteins with different types of glycan structure. Only one immunogenic antigen was isolated using immunoaffinity chromatography with the gammaglobulin fraction of an antigen-specific antiserum as immunospecific ligand. Further electrophoretic and immunochemical analyses revealed that it was a glycoprotein with relative molecular weight (RMW) 31 kDa and with high mannose type of oligosaccharide.

Variation among Merino sheep in susceptibilty to lice (Bovicola ovis) and association with susceptibility to trichostrongylid gastrointestinal parasites.

Sheep of two bloodlines of Merino were artificially infested with equal numbers of lice (Bovicola ovis) and the resulting louse populations were monitored over the following 20 months. The sheep were shorn 6 and 17 months after infestation and, for analysis, the louse counts considered in 3 years separated by shearings. Nematode faecal egg counts (FECs) were assessed on faecal samples collected on five occasions, three times following natural challenge and twice after artificial challenge with 40,000 trichostrongyloid larvae (84% Trichostrongylus vitrinus). In addition, blood samples were collected and measured for B. ovis-specific immunoglobulins (predominantly IgG), B. ovis-specific IgE and serum total IgE. Bloodlines differed significantly in the size of louse populations at the end of year 2, FEC after both natural and artificial challenge and in serum levels of all three antibodies (p<0.05). There were also large variations in louse counts and FEC among sheep within bloodlines. Louse counts at inspections after louse populations had been allowed to build up were highly repeatable, both between and within years. However, correlations with counts at inspections soon after initial infestation and following shearing were lower. FEC after natural challenge was correlated with louse counts in year 2 (r=0.45, p<0.01) and year 3 (r=0.38, p<0.05), but the correlation with counts in year 1 was not significant (r=0.25, p>0.05). FEC following artificial challenge was significantly correlated with louse counts in year 3 (r=0.36, p<0.05), but not in year 2 (r=0.25, p>0.05) or year 1 (r=0.04, p>0.05). Louse counts in the 3 years were significantly correlated with anti-B. ovis antibody concentration (r=0.60, 0.48, 0.36), but not with levels of either anti-B. ovis or total serum IgE. These results suggest that sheep with greater resistance to gastrointestinal parasites also tend to be less susceptible to lice. Whether this is due to interaction of the effects of the parasites or to correlation in underlying resistance mechanisms requires clarification.

Host-mediated site segregation of ectoparasites: an individual-based simulation study.

Site segregation of coexisting ectoparasite species may result either from a direct interaction such as resource competition between them or from a host-mediated interaction. Here we present an individual-based model for the coevolution of 1 host and 2 parasite species to study this latter hypothesis. Parasite species are generalists at the start of the simulation and develop site specificities under the following assumptions. Parasite populations are not subject to resource limitations but are limited directly by host defense as predation. Hosts have 2 sites that need different defensive abilities to reduce their parasite burden. Parasites need to exhibit different evasive abilities to survive on different sites. Host grooming selects parasites for an increasing capability for evasion, whereas parasites select hosts for an increasing efficiency of grooming. Two trade-offs are incorporated into the model: one between host defensive abilities on the 2 sites, and another between parasite evasive abilities on the 2 sites. We conclude that, under these assumptions, the optimization of host defense and parasite evasion strategies may select ectoparasites for site segregation and this may stabilize the coexistence of parasite species.

Antigenic characterization of rat louse Polyplax spinulosa.

Number and relative molecular weights of proteins in whole body homogenates of the louse, Polyplax spinulosa were determined using SDS-PAGE. 13 protein components from the total of 28 bands were defined as Con A binding glycoproteins. In immunoblotting sera of lice infested laboratory rats of BN strain specifically recognized 3 major protein components with r.m.w. of 77,000, 105,000 and 230,000 Da and from 6 to 8 minor protein components with r.m.w. of 31,000-180,000 Da.

Polyplax spinulosa infestation and antibody response in various strains of laboratory rats.

The degree and duration of Polyplax spinulosa Burm. infestation depend on the sex, age and genetic factors of the host. Various rat strains differ in the severity of lousiness. Host grooming is an important factor in the rat-louse relationship. After longlasting infestations rats become partially resistant. Serum antibody level depends mainly on the severity and the duration of the infestation, and follows the dynamics of louse population. Serum antibodies have no direct negative effect on lice.

Identification of an abundant allergen from the sheep louse, Bovicola ovis.

Infestation of sheep with the louse Bovicola ovis is common worldwide and leads to an allergic dermatitis referred to as 'scatter cockle'. IgE from an infested lamb was used in immunoaffinity chromatography to purify allergens from crude preparations of whole B. ovis and its faeces. SDS-PAGE of the affinity-purified eluates from both preparations showed a dominant band with M(r) of 28.5 kDa. Spleen cells from a mouse immunised with B. ovis faecal antigens were used to produce hybridomas which were screened by ELISA to identify those producing monoclonal antibodies (mAb) to the allergens purified by IgE immunoaffinity chromatography. Western blotting demonstrated that all of the mAbs examined recognised the 28.5 kDa allergen. The allergen, purified using immunoaffinity columns constructed with one of the specific mAbs, was shown to cause immediate and late-phase responses on intradermal skin testing in B. ovis-infested but not in naïve lambs. Levels of serum IgE specific for the purified allergen were significantly higher in infested than in naïve lambs (P < or = 0.0025). N-terminal and internal amino acid (aa) sequences obtained from the purified 28.5 kDa allergen were used to design primers to amplify a partial cDNA probe from B. ovis cDNA by PCR. The amplified probe was radiolabeled and used to screen a B. ovis cDNA library. The complete nucleotide sequence of the allergen was determined from the sequences of the positive clones from the library. The full-length cDNA encodes a 255 aa protein including a secretory leader sequence of 26 aas and a mature protein of 229 aas. The encoded protein showed strong homology to several hypothetical proteins of unknown function from diverse species and weak homology with lipid-binding proteins of Xenopus tropicalis and Galleria mellonella. This is the first allergen to be identified from a louse and it has been designated Bov o 1 in accordance with the criteria of the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Subcommittee.