Hypodermin C Research Articles

Assesment of Hypoderma infestation in a wild population of Cervus elaphus from mountains Atlantic ecosystems in southwestern Europe (Spain)

Hypodermosis in Cervus elaphus was studied in the Riaño Regional Hunting Reserve, Province of León, north-western Spain. One hundred and ten red deer were examined for the presence of warble fly larvae. They were analyzed by PCR analysis of the COI region of mt-DNA and identified as Hypoderma actaeon. The prevalence of larvae was 42.7% with a mean intensity of 12.5 ± 18 (range 1–80) warbles/deer infested. The distribution of larvae in the infested animals showed an aggregated/overdispersed pattern (aggregation index = 25.84), where the larvae are not randomly or uniformly distributed, but strongly aggregated among their hosts. Larvae were found in all three states. First and second-instars were observed mainly in the autumn until the end of winter (November-March) and third-instars in late winter until mid-spring (March–May). The adult animals and the males had a higher prevalence than the young and the females, finding statistically significant differences only according to the sex of the animals. Seasonal variations were observed in the prevalence with the highest number of infested animals in winter and autumn, but not in terms of the mean intensity of parasites. Additionally, we assessed the presence of anti-Hypoderma antibodies in serum by means of indirect ELISA tests, using a crude larval extract (CLE) and a purified fraction the hypodermin C (HyC) obtained from first instars of Spanish isolates of Hypoderma lineatum (cattle). These findings confirm that H. actaeon is widely distributed in northern Spain, and provide new information about its chronobiology in mountainous Atlantic ecosystems from southwestern Europe.

Comparative Analysis of Different ELISA Methods for the Serodiagnosis of Przhevalskiana silenus Infestation in Goats

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 livestock. The palpation method for detecting warble flies generally neglects the infestation intensity; 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 specific, 91% 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.

First report of Przhevalskiana silenus derived recombinant hypodermin C based indirect ELISA for serodiagnosis of goat warble fly myiasis

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.

Hypodermin C improves the survival of kidney allografts

Although immunosuppressive therapies have made organ transplantation a common medical procedure worldwide, chronic toxicity is a major issue of long-term treatment. One method to improve such therapies is the application of immunomodulatory agents from parasites, such as Hypoderma lineatum (Diptera: Oestridae). Hypodermin C (HC) is an enzyme secreted by H. lineatum larvae, and our previous study showed that recombinant HC could degrade guinea pig C3 and inhibit the complement pathway in vitro, suggesting potential activity for inhibiting transplant rejection. However, such properties have not been fully demonstrated in vivo. In this study, we investigated the impact of HC on a fully MHC-mismatched, life-sustaining, murine model of kidney allograft rejection using B6 donors and BABL/c (HC transgenic or wild-type) recipients. Kidney grafts were analyzed by histology, immunohistochemistry and western blotting. The results suggested that HC could effectively inhibit kidney allograft rejection. These findings suggest HC is a promising strategy to improve the survival of human implants.

Use of a crude extract or purified antigen from first-instar cattle grubs,Hypoderma lineatum, for the detection of anti-Hypodermaantibodies in free-ranging cervids from southern Spain

During the 2003-2005 hunting seasons, a total of 120 Cervidae, including 39 red deer (Cervus elaphus hispanicus) and 81 fallow deer (Dama dama), were examined for subcutaneous myiasis. Animals were shot from January to June in southern Spain. Specific antibodies against Hypodermatinae (Diptera: Oestridae) were detected by indirect enzyme-linked immunosorbent assay (iELISA) using a crude larval extract (CLE) and a purified antigen [hypodermin C (HC)] obtained from first instars of Hypoderma lineatum (De Villers) (Diptera: Oestridae). Hypoderma actaeon Brauer was the only species detected in this study, which represents the first confirmation of this species in fallow deer from Spain. The overall prevalence of animals presenting subcutaneous larvae (14.2%) was considerably lower than the prevalences determined by iELISA with CLE (43.3%) and HC (40.0%). Red deer showed a higher prevalence of Hypoderma than fallow deer. The concordance between larval examination during the hunting season and iELISA using both antigens was low, whereas the concordance between the CLE and HC ELISAs was good. Larval antigens obtained from H. lineatum constitute a good tool for the diagnosis of H. actaeon in Cervidae, especially when the hunting season does not coincide with the maximum presence of larvae on the back.

Evaluation of an antigen capture ELISA for the early diagnosis of Hypoderma lineatum in cattle under field conditions

An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions.

Dynamics of antibodies against hypodermin C in reindeer infested with the reindeer warble fly, Hypoderma tarandi

Serum samples from 25 reindeer ( Rangifer tarandus tarandus) were assayed for antibody against hypodermin C (HyC) using an ELISA. Nineteen animals were calves (born in 1998, 1999 or 2001) and six were adults (3–10 years old at first blood collection). The samples were collected over periods of 4 months (calves born in 2001) or 27 months (adults and calves born in 1998 and 1999), the latter encompassing three Hypoderma tarandi infestation seasons. The calves received antibodies against HyC from their mothers, either by placental transfer or through the ingestion of colostrum. The low level at 3 h postpartum compared to the high level 3 days after birth in one calf suggests that the antibodies are transferred through colostrum. The levels of antibody of maternal origin decreased rapidly and reached low levels by mid-July, which coincides with the onset of the major Hypoderma ovipositioning season in this region. The calves thus did not appear to be protected by antibody against HyC when they were exposed to H. tarandi infestation for the first time. Antibody levels increased following infestation and reached a maximum during November or December, which coincides with when the H. tarandi larva stops migrating after it has reached the site under the skin of the back of the host and develops further. Levels declined thereafter and reached a nadir during the following summer. After the subsequent re-infestation, the increase in levels occurred at least 1 month earlier than with the first infestation. Levels remained elevated throughout the year after repeated infestations. This implies that the antibodies persist after the annual exit of mature larvae from the animal, and after larvae have been killed by application of ivermectin. Levels in adults, however, declined significantly with age, and levels were significantly lower in animals that were 4–11 years old than in 1-year-old animals during the same 1-year period. This supports the contention that the functional capacity of the immune system declines gradually with age. The study demonstrated that HyC is potentially useful for serological diagnosis of hypodermosis in reindeer, but the persistence of antibodies complicates interpretation of antibody-based surveillance programme data in all cases other than first-time exposure.

Detection of antibodies to Hypoderma lineatum in cattle by Western blotting with recombinant hypodermin C antigen

The cDNA encoding the entire mature hypodermin C (HC) of Hypoderma lineatum was cloned and expressed in Escherichia coli as a glutathione S-transferase fusion protein using pGEX vector. The recombinant HC protein (rHC) was tested by Western blotting to detect antibodies to H. lineatum in cattle. Western blotting with rHC as antigen clearly differentiated between H. lineatum-infested cattle sera and normal cattle sera. Forty-six out of forty-eight serum samples from cattle in Central Mongolia were positive, whereas all 30 serum samples from cows in Hokkaido, Japan, were negative by Western blotting. The result of Western blotting was identical to that of a previously developed enzyme-linked immunosorbent assay. These data demonstrated that Western blotting, with rHC expressed in E. coli, might be a useful method for the diagnosis of cattle hypodermosis.

Sequencing and gene expression of hypodermins A, B, C in larval stages of Hypoderma lineatum

The cDNAs of hypodermins, enzymes secreted by the larvae of the parasitic fly Hypoderma lineatum, were sequenced. Four cDNA clones were isolated, one encoding hypodermin A (HA), one encoding hypodermin C (HC), and the two others encoding proteins related to hypodermin B (HB). The amino acid sequences deduced from the nucleotide sequences confirmed that these enzymes are serine proteases. HA and one of the HB proteins had potential N-linked glycosylation sites. Analysis of hypodermin protein, RNA and DNA at different larval stages indicated that protein overexpression is regulated transcriptionally for HA and HB, and by transcriptional and DNA amplification for HC.

In vitro and ex vivo responses of bovine lymphocytes to hypodermin C, an enzyme secreted by Hypoderma lineatum (Insect Oestridae)

The aim of this work was to determine if Hypodermin C (HC), an enzyme secreted by Hypoderma spp. larvae during their migration, is able to depress the lymphocyte response to mitogens and antigens. HC, when added to the lymphocyte culture, was able to influence the response of naive-calf lymphocytes to phytohemagglutinin. Secondly, injections of HC to uninfested and previously-infested cattle did not modify the proliferative response of the lymphocytes of these animals to mitogens. Thirdly, injections of HC to infested cattle did not negatively influence the specific humoral and cellular responses.