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Effect of Hypodermin A, an enzyme secreted by Hypoderma lineatum (Insect Oestridae), on the bovine immune system
Abstract
The absence of any inflammatory reaction around the first instar larvae (L1) of Hypoderma sp. in previously uninfested cattle suggested that these larvae may escape the non-specific defence system of the host. Immunosuppression had been noted during an experimental infestation. The aim of this work was to determine more precisely the potential role of hypodermin A (HA), an enzyme secreted by the larvae, in this immunosuppression. HA was found to have no effect on unstimulated lymphocytes from naive cattle but could influence the response of these cells to mitogens. In calves, injection of HA was accompanied by a decrease in the lymphocyte proliferative response to mitogens. This immunodepression lasted only for the duration of enzyme injections. In cattle, when HA is added, the antigen-dependent proliferative response increased significantly after 1 week of injection and disappeared 2 weeks after the end of the injection period. Finally, the rate of production of anti-HA antibodies increased at the same rate for calves and cows, and achieved a similar level. These results suggest that HA significantly modified the lymphoproliferative response for naive cattle and, to a lesser extent, immune cattle during the time of administration only.
... HA and HB induce cleavage of the α and β chains of complement component 3 (C3) in naive bovine sera [27]. HA is also directly involved in the inhibition of lymphocyte proliferation in animals affected by hypodermosis [28,29] and the cleavage of bovine IgG molecules into Fab and Fc fragments, with a reduction in the biological activity of these components [30]. ...
Background
Wohlfahrtia magnifica is an obligatory parasite that causes myiasis in several warm-blooded vertebrates. Adult females deposit the first-stage larvae directly onto wounds or natural body orifices (e.g., genitalia) of the host, from where they quickly colonize the host tissue and feed on it for development. The infestation of W. magnifica can lead to health issues, welfare concerns, and substantial economic losses. To date, little is known about the molecular mechanisms of the W. magnifica-causing myiasis.
Results
In this study, we collected parasitic-stage larvae of W. magnifica from wounds of naturally infested Bactrian camels, as well as pupae and adult flies reared in vitro from the wound-collected larvae, for investigating the gene expression profiles of the different developmental stages of W. magnifica, with a particular focus on examining gene families closely related to the parasitism of the wound-collected larvae. As key proteins related to the parasite-host interaction, 2049 excretory/secretory (ES) proteins were identified in W. magnifica through the integration of multiple bioinformatics approaches. Functional analysis indicates that these ES proteins are primarily involved in cuticle development, peptidase activity, immune response, and metabolic processes. The global investigation of gene expression at different developmental stages using pairwise comparisons and weighted correlation network analysis (WGCNA) showed that the upregulated genes during second-stage larvae were related to cuticle development, peptidase activity, and RNA transcription and translation; during third-stage larvae to peptidase inhibitor activity and nutrient reservoir activity; during pupae to cell and tissue morphogenesis and cell and tissue development; and during adult flies to signal perception, many of them involved in light perception, and adult behavior, e.g., feeding, mating, and locomotion. Specifically, the expression level analysis of the likely parasitism-related genes in parasitic wound-collected larvae revealed a significant upregulation of 88 peptidase genes (including 47 serine peptidase genes), 110 cuticle protein genes, and 21 heat shock protein (hsp) genes. Interestingly, the expression of 2 antimicrobial peptide (AMP) genes, including 1 defensin and 1 diptericin, was also upregulated in the parasitic larvae.
Conclusions
We identified ES proteins in W. magnifica and investigated their functional distribution. In addition, gene expression profiles at different developmental stages of W. magnifica were examined. Specifically, we focused on gene families closely related to parasitism of wound-collected larvae. These findings shed light on the molecular mechanisms underlying the life cycle of the myiasis-causing fly, especially during the parasitic larval stages, and provide guidance for the development of control measures against W. magnifica.
... The detection of the circulating antibodies through ELISA has become renown and facilitated the eradication of this menace from various regions of the world [9,10]. The similarities in the immune response to bovine hypodermosis caused by the larvae of Hypoderma spp and other myiasis-causing larvae are considered to be useful for the immunodiagnosis of hypodermosis in various mammals [11][12][13][14][15][16][17][18]. However, reliable and sensitive immunological methods, such as ELISA, for the diagnosis of hypodermosis, is strongly recommended [12,19,20]. ...
Przhevalskiana silenus (warble fly) grubs cause myiasis in goats, in mountainous and semi-mountainous areas and different regions in Pakistan, and cause substantial losses to live-stock. The palpation method for detecting warble flies generally neglects the infestation inten-sity; therefore, the development of a reliable and efficient diagnostic technique is extremely necessary. This study compared three indirect enzyme-linked immunosorbent assay (ELISA) methods for detecting anti-P. silenus antibodies using the hypodermin C (HyC) purified from Hypoderma spp. larvae collected in cattle (local isolate, Microbiology Laboratory, PMAS-Arid Agriculture University, Rawalpindi), the crude antigen from the first instar stage of P. silenus, and a commercial Bovine Hypodermosis Antibody ELISA kit (IDEXX Laboratory), for accurately estimating the seroprevalence of goat warble fly infestation (GWFI) in the Pothwar plateau, Punjab, Pakistan. The ELISA with the crude antigen of P. silenus proved very sensitive and spe-cific, 91.07% and 93%, respectively. The optical density exhibited a monthly variation, and the antibody titer began increasing from June, continually increased from July to December, and gradually decreased thereafter until March. The study confirmed the endemic status of GWFI in the Pothwar region and identified that ELISA based on the crude antigen of P. silenus was a more sensitive and specific immunodiagnostic method for determining seroprevalence, and could be employed for initiating nationwide eradication campaigns.
... Hypoderminae origin HyC is a member of collagenolytic enzymes related to the trypsin family. The HyC is primarily secreted by L1 larvae to degrade the collagen at physiological conditions while entering the host tissue [14][15][16] , whereas HyA and HyB serve as immunomodulators to suppress host immune response and promote larval survival in the host 17,18 . The molecule of HyC has been characterised as a major immunodominant antigen and suitable candidate for detecting Hypoderma specific antibodies from cattle and other ruminants [19][20][21] . ...
Goat warble fly infestation (GWFI) is a subcutaneous myiasis caused by larvae of Przhevalskiana silenus , an insect belonging to the order Diptera . The diagnosis of GWFI is challenging in the early larval instars (L1 and L2) as they are occult under the skin and hair coat causing prolonged economic loss in form of meat and hide damage. This necessitates early diagnosis for disease control at herd level and its prophylactic management to prevent economic losses. Hypodermins, a class of serine proteases from Hypoderminae subfamily have been used as serodiagnostic antigens for the past four decades for diagnosis of warble fly myiasis. In this study,the immunodominant antigen Hypodermin C (HyC) from P. silenus has been recombinantly expressed in E. coli and immunogenic characterisation of expressed protein was done. The protein shows hallmark residues in conserved cysteine and catalytic triad typical of serine proteases along with similar profile of immunoreactivity towards Hypoderminae infestation. The present study reports an optimised indirect-ELISA based on recombinant HyC derived from P. silenus for early diagnosis of GWFI. The optimised indirect ELISA provides a sensitive and specific immunodiagnostic for mass surveillance of the GWFI with diagnostic specificity and sensitivity of 96% and 100%, respectively and not showing any cross reactivity against other important parasitic and bacterial diseases of goats. This study presents the first report of indirect ELISA based on recombinant Hypodermin C antigen derived from P. silenus for the serosurveillance of goat warble fly disease .
... Pourtant, la migration larvaire est une agression multifocale qui devrait déclencher les mécanismes de défense de l'hôte, notamment la migration des neutrophiles, élément clé des systèmes de défense précoce.L'objectif de ce travail est d'étudier l'action des hypodermines, enzymes sécrétées par le parasite au cours de sa migration, sur cette inhibition. En effet, ces dernières sont connues pour inhiber différents systèmes de l'hôte, comme le complément(Boulard et Ben Charif, 1984;Boulard, 1989) ou le système immunitaire spécifique(Chabaudie et Boulard, 1991 ).Cette étude s'est faite sur deux fonctions essentielles du neutrophile : le chimiotactisme par le test de migration sous agarose et l'adhésion par l'étude de l'expression du CD18 en cytométrie de flux. HA (hypodermine A), à de faibles concentrations (20 f dml) inhibe significativement le chimiotactisme des neutrophiles de vaches, tandis que HB (hypodermine B) et HC (hypodermine C), à de plus fortes concentrations l'activent. ...
... Drinking water treatments of insect growth regulators generally do not prevent cattle grub larvae from reaching backs of cattle, but may prevent adults from enclosing from pupae, thus preventing reproduction. An insecticide- (Fisher et al., 1991(Fisher et al., ) 1991 HA antigen alone or with adjuvants Hypoderma spp No significant protection (Chabaudie and Boulard, 1991) 2011 ...
Cattle hypodermosis (warble fly infestation, WFI) is an economically important disease in livestock throughout the world. Larvae of Hypoderma spp. cause subcutaneous myiasis of domesticated and wild ruminants. The important species in cattle are Hypoderma bovis and Hypoderma lineatum whereas, Hypoderma diana, Hypoderma actaeon and Hypoderma tarandi, affect roe deer, red deer, and reindeer, respectively. Hypoderma crossi infects sheep and goat. Adults of the cattle grub are commonly known as heel flies, warble flies, bomb flies or gad flies. The larval stages of Hypoderma bovis and Hypoderma lineatum are commonly called ‘ox warbles’. The biology of hypodermosis is complex in nature. The parasitic larval stage of Hypoderma spp. spend about one year in domesticated as well as in the wild animals, while the adult free-living stage lasts only for few days. The disease causes heavy economic losses to animal production like milk and leather industry. It can also affect the general health status as well as the immune system of the body of the diseased animals. The early diagnosis of hypodermosis is of great importance for planning treatment and eradication program. The control measures of the disease have been efficiently practiced and consequently this disease is controlled at national level in many European countries. Keeping in view the importance of the disease, the present review intends to underscore and provide up to date information about the overall aspect of hypodermosis in animals with the existing literature available with special emphasis on hypodermosis on cattle.
... Drinking water treatments of insect growth regulators generally do not prevent cattle grub larvae from reaching backs of cattle, but may prevent adults from enclosing from pupae, thus preventing reproduction. An insecticide- (Fisher et al., 1991(Fisher et al., ) 1991 HA antigen alone or with adjuvants Hypoderma spp No significant protection (Chabaudie and Boulard, 1991) 2011 ...
Cattle hypodermosis (warble fly infestation, WFI) is an economically important disease in livestock throughout the world. Larvae of Hypoderma spp. cause subcutaneous myiasis of domesticated and wild ruminants. The important species in cattle are Hypoderma bovis and Hypoderma lineatum whereas, Hypoderma diana, Hypoderma actaeon and Hypoderma tarandi, affect roe deer, red deer, and reindeer, respectively. Hypoderma crossi infects sheep and goat. Adults of the cattle grub are commonly known as heel flies, warble flies, bomb flies or gad flies. The larval stages of Hypoderma bovis and Hypoderma lineatum are commonly called 'ox warbles'. The biology of hypodermosis is complex in nature. The parasitic larval stage of Hypoderma spp. spend about one year in domesticated as well as in the wild animals, while the adult free-living stage lasts only for few days. The disease causes heavy economic losses to animal production like milk and leather industry. It can also affect the general health status as well as the immune system of the body of the diseased animals. The early diagnosis of hypodermosis is of great importance for planning treatment and eradication program. The control measures of the disease have been efficiently practiced and consequently this disease is controlled at national level in many European countries. Keeping in view the importance of the disease, the present review intends to underscore and provide up to date information about the overall aspect of hypodermosis in animals with the existing literature available with special emphasis on hypodermosis on cattle.
Goat warble fly infestation (GWFI) is a subcutaneous myiasis caused by larvae of Przhevalskiana silenus , an insect belonging to the order Diptera. The diagnosis of GWFI is challenging in the early larval instars (L1 & L2) as they are occult under the skin and hair coat causing prolonged economic loss in form of meat and hide damage. This necessitates early diagnosis for disease control at herd level and its prophylactic management to prevent economic losses. Hypodermins, a class of serine proteases from Hypoderminae subfamily have been used as serodiagnostic antigens for the past four decades for diagnosis of warble fly myiasis. In this study, immunodominant antigen Hypodermin C (HyC) from P. silenus has been recombinantly expressed in E. coli and immunogenic characterisation of expressed protein was done. The protein shows hallmark residues in conserved cysteine and catalytic triad typical of serine proteases along with similar profile of immunoreactivity towards Hypoderminae infestation. The present study reports an optimised indirect-ELISA based on recombinant HyC derived from P. silenus for early diagnosis of GWFI. The optimised indirect ELISA provides a sensitive and specific immunodiagnostic for mass surveillance of the GWFI with diagnostic specificity and sensitivity of 96% & 100%, respectively and not showing any cross reactivity against other important parasitic and bacterial diseases of goats. This study presents the first report of indirect ELISA based on recombinant Hypodermin C antigen derived from P. silenus for the serosurveillance of goat warble fly disease.
Myiasis refers to the infestation of live human and vertebrate animals with dipterous larvae. Recent advances in understanding the specific and nonspecific immune responses of hosts to infestation by myiasis-causing larvae and the immunological strategies evolved by larvae against the host are the matter of great interest. Therefore this review provides information for better understanding the host-parasite relationships .It also explains the immunology and immunopathology of these larvae, the target response and their relation in the practical implications of immunological knowledge for diagnostic and immunization strategies.
Myiasis refers to the infestation of live human and vertebrate animals with dipterous fly larvae. Recent advances in understanding the specific and nonspecific immune responses of hosts to infestation by myiasis-causing larvae and the immunological strategies evolved by larvae against the host are the matter of great interest. Therefore this review provides information for better understanding the host-parasite relationships. It also explains the immunology and immunopathology of these larvae, the target response and their relation in the practical implications of immunological knowledge for diagnostic and immunization strategies.
Purified enzymes of Hypoderma lineatum (Insecta, Oestridae), were assayed for their proteolytic activity on bovine C3 in normal cattle sera. The products of cleavage by these serine proteases (hypodermins A, B, and C), were analysed by electrophoresis in SDS polyacrylamide gels followed by immunoblotting. The enzymatic attack was initially directed at the alpha polypeptide chain by hypodermin A at a concentration of 1 μg/ml of serum and by hypodermin B at 5 μg/ml. The generated peptides differed in their molecular size from those produced during natural degradation of C3 in a control serum by physiologically relevant enzymes. Hypodermin A, at a concentration of 1 μg/ml, also caused a cleavage of the β chain. At 5 μg/ml, hypodermin A induced total degradation of the C3 molecule. Hypodermin B (5 μg/ml) starts splitting C3 near cleavage sites of factor I. Bovine C3 appears to be highly sensitive to hypodermins A and B in normal sera. Apparent molecular sizes and alignment of the bovine C3 cleavage products are presented schematically. Hypodermin C, a collagenolytic enzyme, had no effect on C3 in normal sera. The biological consequences for the immunopathological reactions associated with hypodermosis are discussed.
There has been controversy as to whether complement augments immune responses or inhibits them. In this article John Weiler and his colleagues discuss recent evidence that complement fragments can inhibit immune responses that depend upon cellular proliferation.
Splenocytes from rats infected with Taenia taeniaeformis showed an early decreased proliferative response to the mitogen concanavalin A with larval growth. When larvae culture supernatant (in vitro product) was added to normal rat splenocytes, there was a decrease in the proliferative response to concanavalin A and phytohemagglutinin. The ability of infected rat splenocytes to produce interleukin-2 was decreased with larval growth. Addition of in vitro product to culture medium significantly depressed interleukin-2 production by normal as well as infected rat spleen cells. Culturing normal rat splenocytes with in vitro product for 3 days induced a suppressor cell population. When these cultured cells were admixed with fresh normal rat splenocytes plus concanavalin A, the proliferative response of the fresh cells was significantly reduced. The present results suggest that in vitro product secreted by larvae in hepatic cysts causes subversion of the cellular immune response in the host. Induction of a suppressor cell population could suppress interleukin-2 production which may lead to the inhibition of differentiation and proliferation of specific cytotoxic T lymphocytes.
1. 1. An enzyme with a collagenolytic activity has been isolated by chromatography on SE Sephadex from the first instar larvae of Hypoderma lineatum. 2. 2. The purified collagenase of molecular weight around 16,000 and of isoelectric point pH 3.5, cleaves the collagen molecule in two fragments of 3 4 1 4 at neutral pH. 3. 3. This enzyme is slightly inhibited by diisopropylfluorophosphate but specific trypsin or chymotrypsin inhibitors have no effects, neither do EDTA, cyanide, or p-chloromercuribenzoate.
The collagenase from the larvae Hypoderma lineatum, with a molecular weight of 24000 and isoelectric point of 4.1, was obtained in homogeneous form by ion-exchange chromatography. It is stoichiometrically inhibited by diisopropylfluorophosphate. On the other hand it is unaffected by ethylenediaminetetraacetate, P-chloromercuribenzoate, dithiothreitol, N-tosyllysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone and ovomucoid trypsin inhibitor. The enzyme which degrades native collagen in its helical parts, has a specific activity on thermally reconstituted collagen fibrils of 150 μg collagen degraded × min-1× (mg enzyme)-1 at 37°C. It hydrolyses casein but has no esterolytic activity characteristic of trypsin, chymotrypsin nor elastase. It has no action on the synthetic peptide 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucyl-glycyl-l-prolyl-d-arginine. The amino acid composition of Hypoderma collagenase indicates a distinct similarity with the serine proteinases of the trypsin family and with another athropode serine collagenase, that of the fiddler crab Uca pugilator. This suggests that eucaryotic collagenases with digestive rather than morphogenic function represent a new category of members of the trypsin family.
In and effort to identify factors important in the pathogenesis of filarial disease, studies of the cellular and humoral immune responses to the parasitic nematode Wuchereria bancrofti were carried out on thirthy-nine individuals in a region of the Pacific where filariasis is endemic. Blood lymphocytes from twenty-six patients with chronic filariasis (especially that associated with persistent microfilaraemia) gave only minimal responses to filarial antigens when challenged in vitro in a lymphocyte transformation assay. In contrast, brisk responses to these same antigens were elicited in cultures of cells from a control population of thirteen exposed but not infected individuals living in the same area, the greatest responses being found in uninfected children less than 10 yr old. The poor cellular responsiveness of infected individuals was filaria antigen-specific, as reactivity to tuberculin (PPD) and streptococcal (SK-SD) antigens was normal and equal in all groups. Furthermore, the immunologic deficit was limited to cell-mediated immune responses and appeared unchanged 2 wk after treatment of the patients with the antifilarial drug diethylcarbamazine. We interpret our findings as evidence for a state of antigen-specific cellular unresponsiveness in patients with this chronic parasitic infection and speculate that this immunologic deficit may be of fundamental importance in the pathogenesis of filarial disease.
We tested ability of 11 different lectins to stimulate DNA synthesis in bovine peripheral blood lymphocytes, using a wide range of experimental conditions. Five of the 6 lectins which induced DNA synthesis (concanavalin A, succinyl-concanavalin A, phytohemagglutinin M, pokeweed mitogens and lipopolysaccharide) did so under conditions similar to those optimal for stimulation of murine lymphocytes. The other lectin (peanut agglutinin) stimulated normal bovine lymphocytes whereas it does not stimulate normal mouse, rat, guinea pig or human lymphocytes. The binding of 5 different lectins to bovine peripheral blood lymphocytes was measured by fluorescence microscopy. Four of the lectins bound to various proportions of cells. Double labeling experiments using a rhodamine-labeled goat anti-bovine immunoglobulin reagent and fluorescein-labeled lectins showed that both peanut agglutinin and soybean agglutinin bound to lymphocyte populations which were negative for surface immunoglobulin. The majority of lymphocytes negative for surface immunoglobulin bound peanut agglutinin, indicating that this lectin may bind specifically to bovine 'T' lymphocytes.
Normal spleen cells of CBA mice or Fischer rats were cultured with mitogens or allogeneic cells, together with various substances of Schistosoma mansoni origin, and thymidine uptake was measured. The proliferation (DNA synthesis) of normal lymphocytes was inhibited by the incubation product of the parasite as well as by cell-free supernatant of schistosome culture. Inhibition was obtained only when active materials were added at the beginning of the culture. Both T and B cell proliferation were inhibited. The inhibitory activity found in cell-free supernatant suggested the release by the parasite of some factor(s) interfering with lymphocyte proliferation. Moreover, serum from rats infected by S. mansoni inhibited lymphocyte proliferation also. The inhibitor(s) appeared heat resistant, dialyzable and of low molecular weight (500-1000). Incubation of normal spleen cells with S. mansoni inhibitor(s) did not enhance the release of nonspecific suppressor cell factor. The inhibition of product(s) released by the parasite could explain part of the immunosuppression status found in schistosomiasis.