A bioinformatic study of antimicrobial peptides identified in the Black Soldier Fly (BSF) Hermetia illucens (Diptera: Stratiomyidae).

Antimicrobial peptides (AMPs) play a key role in the innate immunity, the first line of defense against bacteria, fungi, and viruses. AMPs are small molecules, ranging from 10 to 100 amino acid residues produced by all living organisms. Because of their wide biodiversity, insects are among the richest and most innovative sources for AMPs. In particular, the insect Hermetia illucens (Diptera: Stratiomyidae) shows an extraordinary ability to live in hostile environments, as it feeds on decaying substrates, which are rich in microbial colonies, and is one of the most promising sources for AMPs. The larvae and the combined adult male and female H. illucens transcriptomes were examined, and all the sequences, putatively encoding AMPs, were analysed with different machine learning-algorithms, such as the Support Vector Machine, the Discriminant Analysis, the Artificial Neural Network, and the Random Forest available on the CAMP database, in order to predict their antimicrobial activity. Moreover, the iACP tool, the AVPpred, and the Antifp servers were used to predict the anticancer, the antiviral, and the antifungal activities, respectively. The related physicochemical properties were evaluated with the Antimicrobial Peptide Database Calculator and Predictor. These analyses allowed to identify 57 putatively active peptides suitable for subsequent experimental validation studies.

Immunosuppressive mechanism of Hypoderma lineatum secreted serine esterase, a potential modulatory method used to inhibit transplant rejection

Background: Although immunosuppressive therapies have made organ transplantation a common medical procedure worldwide, chronic toxicity has a major issue for long-term treatment. One method to improve therapies and methods is the application of immunomodulatory agents from parasites such as Hypoderma lineatum. Hypodermin A (HA) is a serine esterase secreted by the larvae of Hypoderma lineatum, several studies demonstrated its immunosuppressive mechanism in vitro, and recently we discovered that HA inhibits the expression of interferon (IFN)-γ and interleukin (IL)-2 and activates IL-10 expression. Therefore, we hypothesized that it might be a potential agent used to block allograft rejections. However, most studies of the immunosuppressive mechanisms associated with HA were undertaken at the cellular level. In order to augment these studies, we evaluated the immunosuppressive effects of HA in vivo using an HA transgenic mouse model. Result: Our results revealed similar findings to those reported by in vitro studies, specifically that HA induced prostaglandin E2 expression, downregulated IFN-γ and IL-2 expression, and promoted IL-10 secretion via E-type prostanoid receptor 4. Additionally, we observed that HA overexpression inhibited lipopolysaccharide-induced TLR4 activation. These findings provide insight into a new potential agent capable of blocking graft rejection. Conclusion: Our founding suggested that HA-related treatment could be a promising option to improve the viability of grafts in human.

Hypodermin A, a potential agent for prevention of allogeneic acute rejection.

Immunosuppressive agents play an important role in the success of organ transplantation, however the chronic toxicity of these agents is a major issue over the long-term. Hypodermin A (HA) is an enzyme secreted by the larvae of Hypoderma lineatum (Diptera: Oestridae), and has been implicated in immunosuppression in cattle. Malassagne et al. have demonstrated that HA can degrade the C3 protein, and could be used to prevent hyperacute xenogeneic rejection. We found that overexpression of HA in RAW264.7 cells induced significant secretion of prostaglandin E2 (PGE2), which mediates a variety of innate and adaptive immune responses through four E-type prostanoid (EP) receptor subtypes (EP1-4). PGE2 is useful in the management of allogeneic acute rejection. In addition, we found that induction of PGE2 expression downregulates the expression of interferon (IFN)-γ and interleukin (IL)-2, and promotes the secretion of IL-10 in vitro through the EP4 receptor. It was previously shown that activation of IL-2 and IFN-γ is involved in allograft acute rejection. IL-10 is known to prevent inflammation, and can improve allograft survival rates. We concluded that besides preventing hyperacute xenogeneic rejection, HA might also be a potential therapeutic candidate for ameliorating acute rejection during allotransplantation.

Histological and immunohistochemical characterization of Hypoderma lineatum (Diptera: oestridae) warbles.

Hypoderma larvae are tissue invading parasites which spend several months migrating within the host tissues before completing their development in the sub-dermal tissues of the back. Subcutaneous stages of the parasite produce an inflammatory reaction in the skin called "warbles", as well as holes through which larvae breathe. In order to elucidate the microscopical structure of the warbles, three hides from warbled cows were collected in a slaughterhouse in Lugo (NW, Spain) between March and May 2012. A total of 60 skin samples, including warbles at different phases of development, were chosen for histopathological and immunohistochemical examination. Microscopic lesions were classified into three groups, according to the predominance and distribution of different cell populations. In warbles containing living or recently dead larvae with apparently well preserved cuticle (type 1), plasma cells were observed in high number. However, macrophages and lymphocytes were the predominant cells in granulomas (type 2) formed in relation to remnants of the dead parasite, containing or not remains of the altered cuticle. Scars (type 3) were characterized by granulation tissue. Immunohistochemistry showed that B lymphocytes and IgG(+) cells were predominant in the lesions, as long as the cuticle of the larvae is intact. On the other side, CD3(+) lymphocytes increased once cuticle is destroyed and a granuloma is formed. Macrophages, revealed by CD68(+), MAC387(+) and lysozyme immunolabelling, were detected in all types of lesions, but they were more abundant in type 2 and scarce in scars. These cells appeared isolated around the intact larvae or forming aggregates around its remains in the granuloma. Moreover, a strong immunolabelling against MAC387 antibody was registered in the squamous epithelium covering the breathing pore. This finding may be associated with the expression of calprotectin, a molecule involved on the healing process of the skin after larvae outcome. Our results suggest the predominance of a humoral response inside the warble as long as larvae are intact. Once they are destroyed, cellular response occurred, isolating and destroying the remains of the larvae until healing process completes and scars with low numbers of inflammatory cells appear.

[Cellular immune system of surgical maggots Lucilia sericata (Diptera, Calliphoridae)].

In the hemolymph of surgical maggots Lucilia sericata seven types of hemocytes were revealed. These are prohemocytes, stable and unstable hyaline cells, thrombocytoids, spindle cells, larval plasmatocytes and plasmatocytes I-IV, which represent sequential stages of one cell line differentiation. In contrast to Calliphora hyaline cells, this type of hemocytes in cropemptying larvae of Lucilia is elongated or vermiform in shape. Hyaline cells may be transformed to both prothrombocytoids and unstable prophenoloxydase-producing cells. Appearance and differentiation of each hemocyte type is rigidly linked with a definite stage of development. In cellular defense the main role play juvenile plasmatocytes, plasmatocytes II and III and trombocytoides. Juvenile plasmatocytes are the most active ones. After charcoal particles injection they were instantly surrounded by the thick envelope of adhered alien particles and form uniform morules aggregations or conglomerates together with thrombocytoidal agglutinates. Plasmatocytes II and III during the early stages of differentiation may be involved in adhesion and phagocytosis of alien particles and during the last stages in the engulfing of apoptose desintegrated tissues. Thus the cellular defense reaction is assisted by 4 hemocyte types--prophenoloxydase-unstable hyaline cells, thrombocytoids, juvenile plasmatocytes and plasmatocytes I-IV.

[Cellular defense system of some synanthropic dipterans inhabitant of bacterially aggressive environment].

The hemocytic count and defense reaction within 4 families of higher Diptera: Tabanidae, Syrphidae, Muscidae and Sarcophagidae, whose larvae inhabit bacterially aggressive environment, were investigated. The least hemocytes types (3) were revealed in Tabanidae and Syrphidae larvae--prohemocytes, plasmatocytes and prophenoloxydase-containing unstable hyaline cells (oenocytoids). In Sarcophaga crassipalpis and Musca domestica stable hyaline cells and thrombocytoids or podocytoid-like cells can be added to this set. At the time of pupariation in Sarcophaga, new generation of prohemocytes is segregated into the hemolymph, which form small round or spindle-shaped hyaline cells. So, the number of plasmatocyte types in Sarcophaga increase to six. Typical to Calliphoridae juvenile plasmatocytes in the members of investigated families are absent. Among the one hemocyte type morphology also can vary, especially in unstable prophenoloxydase hyaline cells. In Drosophila there are crystal cells containing in the cytoplasm paracrystalloidal inclusions. In Calliphoridae there are big hyaline cells with homogenous cytoplasm producing circumferential bubbles. Both in Sarcophaga and Tabanidae they contain in their cytoplasm big globules. However in Sarcophaga they rapidly disintegrate, while in Tabanidae are maintained unchanged during hours. In Muscidae and Syrphidae prophenoloxydase extrusion occurs very early and these cells obtain pycnotic nuclei and very liquid cytoplasm with strings of granules. Thrombocytoids in Musca larvae are represented by big flattened anucleated irregular cytoplasm and "naked" nuclei and cytoplasmic fragments often with fan-like projections. Plasmatocytes in all species studied are the cells with pronounced phylopodies. In larvae they contain cytoplasmic catabolic inclusions and in pupa--ragments of apoptotic tissues. Clearance of hemolymph from alien particles in Sarcophagidae and Muscidae occur by thrombocytoides, while in Tabanidae by plasmatocyte nodulation. A differing case is Syrphidae whe-e charcoal injection produce depletion of hemolymph both from particles and all types of hemocytes. So the specimen of different higher Diptera families can use different schemes of cellular defense reaction.

A novel allergen Tab y 1 with inhibitory activity of platelet aggregation from salivary glands of horseflies.

BACKGROUND: Horsefly sting causes allergic reactions in human body. However, our knowledge on horsefly allergens remains poor. OBJECTIVES: To identify the novel horsefly allergens and characterize their properties. METHODS: A native allergen protein Tab y 1 (apyrase) was purified from the salivary glands of the horsefly Tabanus yao Macquart by gel filtration and ion exchange chromatography. Its sequence was determined by Edman degradation and cDNA cloning. Its allergenicity was assessed by immunoblotting for specific IgE, basophil activation test, skin prick test (SPT), and competitive enzyme-linked immunosorbent assay (ELISA). RESULTS: Tab y 1 showed a single diffusion band of 70 kDa on SDS-PAGE. Seventy percent (7/10) of patients with horsefly allergy tested positive to Tab y 1 in SPT; sera from 81% (30/37) of patients reacted to Tab y 1 on western blots. Purified Tab y 1 reduced approximately 42% sera IgE reactivity to horsefly salivary gland extract on a competitive ELISA. Tab y 1 upregulated the expression of CD63 and CCR3 on passively sensitized basophils by up to approximately 4.9-fold. Tab y 1 also showed enzymatic activity to hydrolyze ATP and ADP, and potent antiplatelet aggregation and antithrombotic activities. CONCLUSION: The current work identified a novel major allergen of horsefly, Tab y 1, with antiplatelet aggregation and antithrombotic activities, which implicates Tab y 1 in helping horseflies suck host blood, meanwhile causing allergy in their human hosts.

Purification and characterization of two new allergens from the salivary glands of the horsefly, Tabanus yao.

BACKGROUND: Horsefly bite can cause allergic reactions in humans. There is no information about allergenic horsefly proteins. OBJECTIVES: The current work aims to purify and characterize IgE-binding proteins from horsefly salivary glands. METHODS: Two IgE-binding proteins, Tab a 1 and Tab a 2 with molecular weight of 26 and 35 kd, respectively, were purified and characterized from 60,000 pairs of horsefly salivary glands of Tabanus yao, respectively. Their primary sequences were determined by Edman degradation and cDNA cloning. Their allergenicity was examined using enzyme-linked immunosorbent assay (ELISA), ELISA inhibition tests, and immunoblots. RESULTS: Immunoblotting demonstrated IgE binding by 32 and 34 of 37 (86.5% and 91.8%) subjects' sera to Tab a 1 and Tab a 2, respectively. They were identified as an antigen 5-related (Ag 5) protein and hyaluronidase, respectively. ELISA inhibitions of serum IgE reactivity to the horsefly salivary gland extract (SGE) using purified Tab a 1 and Tab a 2 were significant (about 45%). In addition, these proteins showed some IgE-binding capacity to sera of subjects with wasp sting allergy. CONCLUSIONS: We have first identified and characterized two IgE-binding proteins, Tab a 1, an Ag 5-like protein and Tab a 2, a hyaluronidase, from the horsefly salivary glands. They appear to be of importance for the allergic reactions induced by horsefly bite. These allergens are thus not only found in stinging but also found in hematophagous insects. These results also provided support for the presence of the so-called wasp-horsefly syndrome (WHS).

[Immune system of the blowworm Calliphora vicina (diptera, calliphoridae) as a source of medicines].

Study of mechanisms of cellular and humoral immunity of larvae of the blowworm Calliphora vicina has revealed three groups of pharmacologically active substances perspective for use in medicine--alloferons, allostatins, and antimicrobial peptides. Alloferons--the family of peptides of C. vicina stimulating selectively the cytotoxic activity of natural killers of the evolutionary ancient group of immunocompetent cells playing the key role in the system of mammalian antiviral and antitumor immunity. Alloferons are used in medicine for treatment of herpes-viral infections and viral hepatitis B. Allostatins-synthetic peptides combining structural characteristics of alloferons and some mammalian immunologically active proteins. Allostatins, like alloferons, produce stimulating effects on cytotoxic activity of natural killers and interferon production, but differ from them by pronounced adjuvant properties--the ability to enhance immune recognition of alien antigens. At present, allostatins are used to increase resistance of skin and mucosae to viral infections; in future, they might also find use in immunotherapy of cancer and other diseases. Another protein and peptide group perspective for use in medicine serve as antimicrobial peptides in immune response of larvae C. vicina. The study of the agent containing defensins, cecropins, diptericins, and praline-enriched C. vicina peptides shows the drugs of this type to be of great interest as the tool for treatment and prevention of infections caused by antibiotic-resistant bacteria.

Transmission blocking vaccines to control insect-borne diseases: a review

Insect-borne diseases are responsible for severe mortality and morbidity worldwide. As control of insect vector populations relies primarily on the use of insecticides, the emergence of insecticide resistance as well to unintended consequences of insecticide use pose significant challenges to their continued application. Novel approaches to reduce pathogen transmission by disease vectors are been attempted, including transmission-blocking vaccines (TBVs) thought to be a feasible strategy to reduce pathogen burden in endemic areas. TBVs aim at preventing the transmission of pathogens from infected to uninfected vertebrate host by targeting molecule(s) expressed on the surface of pathogens during their developmental phase within the insect vector or by targeting molecules expressed by the vectors. For pathogen-based molecules, the majority of the TBV candidates selected as well as most of the data available regarding the effectiveness of this approach come from studies using malaria parasites. However, TBV candidates also have been identified from midgut tissues of mosquitoes and sand flies. In spite of the successes achieved in the potential application of TBVs against insect-borne diseases, many significant barriers remain. In this review, many of the TBV strategies against insect-borne pathogens and their respective ramification with regards to the immune response of the vertebrate host are discussed.

Immunoregulatory peptides from salivary glands of the horsefly, Tabanus pleskei.

Horseflies are economically important blood-feeding arthropods and vectors for several pathogenic microorganisms. Horseflies rely heavily on pharmacological propriety of their saliva to get blood meal and suppress immune reactions of hosts. Few reports cover immune suppressants from horsefly salivary glands. Three immunoregulatory peptides named immunoregulin TP1-3 have been identified and characterized from salivary glands of the horsefly Tabanus pleskei (Diptera, Tabanidae). Immunoregulin TP1 could inhibit the secretion of interferon-gamma (IFN-gamma), monocyte chemoattractant protein (MCP-1) and increase the secretion of interleukin-10 (IL-10) induced by lipopolysaccharide (LPS) in mouse splenocytes. IL-10 is a suppressor cytokine of T-cell proliferation and cytokine responses. IL-10 can inhibit the elaboration of pro-inflammatory cytokines. In the current studies, Immunoregulin TP1 inhibited the IFN-gamma and MCP-1 secretion possibly by upregulating the IL-10 production, and finally might facilitate the blood-feeding of this horsefly. The current works will help understand the molecular mechanisms of the ectoparasite-host relationship.

Cutaneous manifestations in Hymenoptera and Diptera anaphylaxis: relationship with basal serum tryptase.

OBJECTIVES: To compare the clinical presentation of systemic anaphylaxis to Hymenoptera and Diptera with regard to basal serum tryptase (BT) and to evaluate mastocytosis in patients with elevated tryptase. PATIENTS AND METHODS: The medical records of 140 patients with a history of a systemic reaction to venom were retrospectively reviewed. Symptoms and severity of anaphylaxis and BT were recorded. Most patients with elevated tryptase were screened for mastocytosis: a dermatological examination with a skin biopsy was performed in 19 cases and a bone marrow biopsy in 14 cases. RESULTS: Tryptase was elevated in 23 patients. These patients reported fewer usual skin reactions (urticaria in 26.1% of cases with raised tryptase vs. 76.1% of cases with normal tryptase), more flushing (52.2% vs. 4.3%) and frequently did not present skin reaction (26.1% vs. 9.4%). They presented a more severe reaction (mean grade of severity: 3.48 vs. 2.69). Mastocytosis was diagnosed in seven patients with elevated tryptase: indolent systemic mastocytosis in six cases and cutaneous mastocytosis without systemic involvement in one case. In five cases, mastocytosis was previously undiagnosed. Lesions of cutaneous mastocytosis, diagnosed in five patients, consisted of urticaria pigmentosa in all cases and were often inconspicuous. CONCLUSION: These results demonstrate particular clinical features of the allergic reaction in patients with elevated BT and the higher frequency of mastocytosis in this population. In patients with a severe anaphylactic reaction without urticaria, but with flushing, tryptase should be assayed and an underlying mastocytosis should be considered.

An immunoregulatory peptide from salivary glands of the horsefly, Hybomitra atriperoides.

Horseflies are economically important blood-feeding arthropods and also a nuisance for humans, and vectors for filariasis. They rely heavily on the pharmacological propriety of their saliva to get blood meal and suppress immune reactions of hosts. Little information is available on horsefly immune suppressants. By high-performance liquid chromatography (HPLC) purification coupling with pharmacological testing, an immunoregulatory peptide named immunoregulin HA has been identified and characterized from salivary glands of the horsefly of Hybomitra atriperoides (Diptera, Tabanidae). Immunoregulin HA could inhibit the secretion of interferon-gamma (IFN-gamma) and monocyte chemoattractant protein (MCP-1) and increase the secretion of interleukin-10 (IL-10) induced by lipopolysaccharide (LPS) in rat splenocytes. IL-10 is a suppressor cytokine of T-cell proliferative and cytokine responses. IL-10 can inhibit the elaboration of pro-inflammatory cytokines. Immunoregulin HA possibly unregulated the IL-10 production to inhibit IFN-gamma and MCP-1 secretion in the current experiments. This immunosuppression may facilitate the blood feeding of this horsefly. The current works will facilitate to understand the molecular mechanisms of the ectoparasite-host relationship.

Mapping the peptide and protein immune response in the larvae of the fleshfly Sarcophaga bullata.

We chose the larvae of fleshfly Sarcophaga bullata to map the peptide and protein immune response. The hemolymph of the third-instar larvae of S. bullata was used for isolation. The larvae were injected with bacterial suspension to induce an antimicrobial response. The hemolymph was separated into crude fractions, which were subdivided by RP-HPLC, gel electrophoresis, and free-flow electrophoresis. In several fractions, we determined significant antimicrobial activities against the pathogenic bacteria Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa. Among antimicrobially active compounds we identified dipeptide beta-alanyl-L-tyrosine, protein transferrin, and two variants of peptide sapecin. We also partially characterized two novel antimicrobially active polypeptides; odorant-binding protein 99b, and a peptide which remains unidentified.

Nitric oxide synthase activity in tissues of the blowfly Chrysomya megacephala (Fabricius, 1794)

Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and humoral components. Cellular responses are mediated by hemocytes, and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In mammals, nitric oxide synthases (NOSs) are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of NO-producing systems in invertebrates have revealed functional similarities between NOS in this group and vertebrates. We attempted to localize NOS activity in tissues of naïve (UIL), yeast-injected (YIL), and saline-injected (SIL) larvae of the blowfly Chrysomya megacephala, using the NADPH diaphorase technique. Our findings revealed similar levels of NOS activity in muscle, fat body, Malpighian tubule, gut, and brain, suggesting that NO synthesis may not be involved in the immune response of these larval systems. These results were compared to many studies that recorded the involvement of NO in various physiological functions of insects.

Maggot excretions/secretions inhibit multiple neutrophil pro-inflammatory responses.

There is renewed interest in the use of maggots (Lucilia sericata) to aid in healing of chronic wounds. In such wounds neutrophils precipitate tissue damage rather than contribute to healing. As the molecules responsible for the beneficial actions of maggots are contained in their excretions/secretions (ES), we assessed the effects of ES on functional activities of human neutrophils. ES dose-dependently inhibited elastase release and H(2)O(2) production by fMLP-activated neutrophils; maximal inhibition was seen with 5-50 microg of ES/ml. In contrast, ES did not affect phagocytosis and intracellular killing of Candida albicans by neutrophils. Furthermore, 0.5 microg of ES/ml already inhibited neutrophil migration towards fMLP. ES dose-dependently reduced the fMLP-stimulated expression of CD11b/CD18 by neutrophils, suggesting that ES modulate neutrophil adhesion to endothelial cells. ES did not affect the fMLP-induced rise in [Ca(2+)](i) in neutrophils, indicating that ES act down-stream of phospholipase C-mediated activation of protein kinase C. In agreement, ES inhibited PMA-activated neutrophil functional activities. ES induced a rise in intracellular cAMP concentration in neutrophils and pharmacological activators of cAMP-dependent mechanisms mimicked their inhibitory effects on neutrophils. The beneficial effects of maggots on chronic wounds may be explained in part by inhibition of multiple pro-inflammatory responses of activated neutrophils by ES.

Still waiting for the toll?

Multicellular organisms including invertebrates and vertebrates live in various habitats that may be aquatic or terrestrial where they are constantly exposed to deleterious pathogens. These include viruses, bacteria, fungi, and parasites. They have evolved various immunodefense mechanisms that may protect them from infection by these microorganisms. These include cellular and humoral responses and the level of differentiation of the response parallels the evolutionary development of the species. The first line of innate immunity in earthworms is the body wall that prevents the entrance of microbes into the coelomic cavity that contains fluid in which there are numerous leukocyte effectors of immune responses. When this first barrier is broken, a series of host responses is set into motion activating the leukocytes and the coelomic fluid. The responses are classified as innate, natural, non-specific, non-anticipatory, non-clonal (germ line) in contrast to the vertebrate capacity that is considered adaptive, induced, specific, anticipatory and clonal (somatic). Specific memory is associated with the vertebrate response and there is information that the innate response of invertebrates may under certain conditions possess specific memory. The invertebrate system when challenged affects phagocytosis, encapsulation, agglutination, opsonization, clotting and lysis. At least two major leukocytes, small and large mediate lytic reactions against several tumor cell targets. Destruction of tumor cells in vitro shows that phagocytosis and natural killer cell responses are distinct properties of these leukocytes. This has prompted newer searches for immune function and regulation in other systems. The innate immune system of the earthworm has been analyzed for more than 40 years with every aspect examined. However, there are no known entire sequences of the earthworm as exists in these other invertebrates. Because the earthworm lives in soil and has been utilized as a successful monitor for pollution, there are studies that reveal up and down regulation of responses in the immune system after exposure to a variety of environmental pollutants. Moreover, there are partial sequences that appear in earthworms after exposure to environmental pollutants such as cadmium and copper. There are now attempts to define the AHR receptor crucial for intracellular signaling after exposure to pollutants, but without linking the signals to changes in the immune system. There are several pathways for signal transduction, including JAK/STAT, TOLL, TRAF PIP3, known in invertebrates and vertebrates. For resistance to pathogens, conserved signal transduction components are required and these include a Toll/IL-1 receptor domain adaptor protein that functions upstream of a conserved p38 MAP kinase pathway. This pathway may be an ancestral innate immune signaling pathway found in a putative common ancestor of nematodes, arthropods and even vertebrates. It could also help us to link pollution, innate immunity and transduction in earthworms.

Comparison of Oestrus ovis metabolic and somatic antigens for the immunodiagnosis of the zoonotic myasis oestrosis by immunoenzymatic probes.

Control of zoonosis implies reduction of infected animal hosts, and the first measure consists of a suitable and accurate detection test. An experimental study for determining the most appropriate antigen (metabolic or somatic) to be used in the detection of the oestrosis (Oestrus ovis) zoonotic myasis by means of immunoenzymatic probes was carried out. A flock of 23 uninfected goats was maintained under field conditions to allow their infection in Sassari (Sardinia, Italy). Caprine were bled monthly and serum samples processed by means of an iELISA. After comparing these results to the chronobiology of O. ovis, we proved that the IgG humoral response against the metabolic antigens increased only during the period of real risk of infestation (when adults fly, from May to September), whereas the absorbances against the somatic products were positive from the beginning of the study (in January, prior to infection). We concluded that the excretory/secretory products are most useful and suitable for the immunodiagnosis of oestrosis in goats, because a direct relation between the development of O. ovis and the IgG humoral response is possible, allowing a more accurate diagnostic.

Cytotoxic activity of blowfly Calliphora vicina hemocytes.

The cytotoxic activity of hemocytes isolated from larvae of the blowfly Calliphora vicina was tested using human myelogenous leukemia K562 cells as target. Both single cell and cytotoxicity assays demonstrated that the hemocytes recognize the K562 cells as nonself, firmly attach to their surface and induce target destruction in a manner resembling the effect of mammalian cytotoxic lymphocytes. The cytotoxic activity increased dramatically in the course of larval metamorphosis and was considerably higher shortly before the onset of pupariation, compared to the activity of human peripheral blood or mouse spleen lymphocytes. In insects, the cytotoxic hemocytes may take part in defense against eukaryotic parasites and in the elimination of aberrant self cells, as well as in developmental processes such as metamorphosis.