Use of a crude extract or purified antigen from first-instar cattle grubs, Hypoderma lineatum, for the detection of anti-Hypoderma antibodies 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.

[The first case of Hypoderma tarandi-associated human myiasis in Greenland]. / Første tilfælde af Hypoderma tarandi-associeret myiasis på Grønland.

We present the first case of human myiasis in Greenland caused by the warble fly Hypoderma tarandi. Noticing a persisting, migrating, stinging facial sensation, a female patient eventually extirpated a larva from her upper eyelid, and the larva was confirmed to species level using DNA-based methods. A high prevalence of H. tarandi in reindeer, the main reservoir host, and increasing populations of musk ox may imply an increased risk of zoonotic transmission. Although diagnosis is challenging, rapid intervention is crucial to reduce the risk of ophthalmomyiasis.

Myiasis presenting as cellulitis of the cheek.

A case of myiasis due to the northern cattle grub (Hypoderma bovis) in a 2 1/2-year-old girl presenting as cellulitis of the cheek and periorbital cellulitis is detailed in this report. A literature review of similar cases reported over the past five years is included.

Myiasis due to Hypoderma lineatum infection mimicking the hypereosinophilic syndrome.

Myiasis is the infestation of live humans with larvae of Diptera (true flies). This report describes a protracted illness caused by infestation with Hypoderma lineatum, resembling the hypereosinophilic syndrome. A 35-year-old man had a 9-month multisystemic illness with pronounced eosinophilia, pleuritis, pericarditis, and myositis. Treatments including glucocorticoids did not alter the disease. Diagnostic studies included computed tomography, 2-dimensional echocardiography, leukocyte count, surgical biopsy of skin and muscle, blood immunoglobulin levels, and blood chemistry. Myiasis was recognized when a worm emerged from the patient's skin; after a second worm emerged, the patient's symptoms disappeared rapidly. Other determinations included IgE and IgG levels specific for H lineatum, Western blot, and immunofluorescence for eosinophil major basic protein; IgG antibodies to H lineatum decreased after emergence of the worms. The patient's symptoms mimicked the hypereosinophilic syndrome but resolved when the myiasis became apparent. Specific serologic analyses can identify infected patients, and ivermectin may be useful as treatment.

Ophthalmomyiasis caused by the reindeer warble fly larva.

Two boys with ophthalmomyiasis caused by the first instar larva of the reindeer warble fly Hypoderma tarandi are reported. Both were 9 years old and came from the coast of northern Norway. One had ophthalmomyiasis interna posterior and one eye had been removed because of progressive pain and blindness. Histological examination showed the remains of a fly larva. The second boy had ophthalmomyiasis externa with a tumour in the upper eyelid, and histological examination showed a warble with a well preserved larva. Identification of the parasite in the histological material was based on the finding of cuticular spines and parts of the cephalopharyngeal skeleton identical with those of the first instar larva of H tarandi.

Comparison of the use of secretory and somatic antigens in an ELISA for the serodiagnosis of hypodermosis.

An antigen was prepared from metabolic products which were produced by maintaining first instar larvae of Hypoderma lineatum in RPMI tissue culture medium for 48 h. Three major proteins were identified in the secretory products and were characterised in terms of their molecular weights and iso-electric points. The antigen compared favourably with a soluble extract of larvae when used in an enzyme-linked immunosorbent assay (ELISA) against a panel of control sera and 2000 bovine sera collected from farms in Andalucia, Spain.

A competitive ELISA for the serodiagnosis of hypodermosis.

Hypodermosis is a parasitic disease of cattle caused by Hypoderma lineatum and H. bovis. It is an important health and welfare problem of infested cattle and a cause of considerable economic loss. In live animals, detection can be either by direct clinical examination of infested cattle and palpation of second and third stage larvae in the back or by the use of serological methods. This paper describes a competitive ELISA for the detection of antibodies to Hypoderma species in cattle sera. It has been validated using a small panel of 40 samples from clinically positive cattle and 200 samples from clinically negative (unexposed) cattle from a warble free area. Sensitivity and specificity calculated from this panel were 100% and 92% respectively at 2 SD from the mean or 92.5% and 98.5% at 3 SD from the mean. The use of serology and direct clinical examination of affected cattle, are discussed as methods for monitoring hypodermosis. The competitive ELISA should prove a useful adjunct to the standard sandwich ELISA, particularly in the resolution of samples which display non-specific binding properties.

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.

Use of pooled serum or milk samples for the epidemiological surveillance of bovine hypodermosis.

An enzyme-linked immunosorbent assay (ELISA) was used on pooled serum and milk samples to determine whether hypodermosis could be detected where a larger sero-epidemiological survey was required. This study was undertaken to assess the potential of this assay for testing sera on milk samples, pooled from 10 cows, and determining the period of the year when detection was optimal. The sensitivity of the assay was determined by increasingly diluting a positive serum with pooled negative sera, from 1:10 to 1:100. The diagnostic lower limit of the assay requires at least two serological reactors within a herd of 100. The kinetic development and depletion of anti-Hypoderma antibody of individual and pooled sera or milk from 30 cows was evaluated from November to July. Anti-Hypoderma antibody levels of two groups of 8 calves, one control and one teated with ivermectin (Ivomec), were tested from October to June. These preliminary results indicate that an ELISA assay on serum or milk samples pooled from 10 cows can be used between February and April to evaluate the prevalence of hypodermosis within cattle herds in France, demonstrating the feasibility of using pooled serum already collected for bovine leucosis testing.

Screening of commercial milk samples using ELISA for immuno-epidemiological evidence of infection by the cattle grub (Diptera: Oestridae).

Pasteurised and ultra high temperature (UHT)-treated milk were tested over the period November 1997-February 1998 in order to evaluate the applicability of an hypodermosis ELISA test on commercial milk samples. Milk samples from six dairy companies were analysed and the development of anti-Hypoderma antibodies recorded for the period from September 1998 to June 1999. For one dairy (no. 3) bulk milk samples were also obtained from the tankers, transporting milk away from the farms, and analysed; the results were compared to the parasitological status of the cows whose milk had been collected on the farms. Out of 32 pasteurised samples tested, 23 were positive, exceeding the cut-off (22%), while 9 samples were negative. UHT milk was always negative. The antibody levels in milk samples from four of the six companies were highest during January and decreased gradually during February to May 1999. The bulk milk samples also had high antibody levels and 47.2% of milk producing cows had lesions of hypodermosis. It was concluded that testing commercial milk for antibodies is an efficient way of detecting the presence of hypodermosis in cattle, especially in those countries for which no data on this disease are available.

Detection of circulating hypodermin C: an antigen capture ELISA for diagnosis of cattle grub (Diptera: Oestridae) infestations.

An antigen capture assay for the detection of circulating hypodermin C was developed for diagnosis of hypodermosis. A murine monoclonal antibody to recombinant hypodermin C was raised using rapid immunization and a one-step hybridization-cloning technique. A highly reactive, specific monoclonal antibody was tested using sera spiked with known quantities of purified, native hypodermin C or with recombinant hypodermin C. Sensitivity of 96.4% and specificity of 95.6% for the antigen capture assay was assessed using a panel of sera from animals unexposed to cattle grubs and from cattle with palpation proven cattle grub infestations. Data from this panel of sera was used to establish the cut-off OD for further testing. The kinetics of circulating hypodermin C was assessed using the assay in three groups of cattle artificially infested with 50, 100 or 200 first instar Hypoderma lineatum. Antigen was first detected approximately 6 weeks after infestation. The amount of antigen detected increased in each group of animals reaching peaks at different times in each group. Levels of antigen fell quickly following arrival of grubs at the back and completion of the molt to second instar.

Enzyme-linked immunosorbent assay for the detection of antibodies to Hypoderma bovis in cattle.

An adaptation of the enzyme-linked immunosorbent assay has been shown to detect antibodies in the circulation of cattle infected with Hypoderma bovis. Strong positive reactions were obtained from all infected cattle, even those which harboured only one larva. A strong cross reaction was observed in sera taken from cattle infected with H lineatum, but not in cattle infected with Fasciola hepatica or Ostertagia ostertagi.

The prevalence of bovine hypodermosis in Greece.

Hypodermosis is a common parasitic disease of great importance on cattle production and breeding worldwide. In Greece, no reliable data are available regarding the extent of this infection and therefore, as expected, no national or regional control programme is being applied. In order to cover this lack of information, a total of 4200 cattle blood sera were collected from different parts of the country and tested for hypodermosis using the ELISA method. The prevalence of the infection was found to be 37.4%, varying from place to place between 0.7 and 78.8%. The range of the infection per flock was from 0 up to 100%.

Comparative study of anti-Hypoderma antibody kinetics in sera, single and bulk milk samples of naturally infested cattle.

The investigation was carried out in Basilicata region (Southern Italy) from October 1997 to June 1998. Fifteen dairy cows bred in semiconfined conditions on a farm with a history of hypodermosis were sampled once a month for sera and milk; bulk milk from these animals was also collected monthly from the farm's tanker. Samples were tested for anti-Hypoderma spp. antibodies (Abs) with an ELISA technique and clinical parasitological examination was carried out monthly from January to July on all the animals in order to detect grubs. Blood and single and bulk milk samples yielded similar antibody kinetics and patterns in accordance with results obtained in previous immunological surveys in Italy. All animals were warbled in the spring time. November-January was confirmed to be the most suitable period for seroepidemiological survey for weather conditions in Southern Italy. The ELISA test proved once again to be very useful because it is simple to perform and cost effective. Either blood or milk samples may be used for epidemiological surveys; bulk milk may be very useful for the preliminary detection of hypodermosis on farms or in areas where there is no data available on the diffusion of the disease.

Serodiagnosis of goat warble fly infestation by Przhevalskiana silenus with a commercial ELISA kit.

Cross-reactivity between Hypoderma lineatum antigen and anti-Przhevalskiana silenus antibodies has been demonstrated by an ELISA technique. To evaluate the applicability of a commercial ELISA kit for the immunodiagnosis of goat warble fly infestation, different dilutions of serum and conjugate were tested, the development of antibody to P silenus in naturally infested goats was studied, and the results were compared with an ELISA technique using an antigen extracted from the first instar larvae of H lineatum. The best results were obtained with a serum dilution of 1:50; with both techniques the highest antibody concentration were recorded in October, November and December. In view of the confirmed cross-reactivity between H lineatum antigen and anti-P silenus antibodies, and the simplicity and rapidity of the assay, the commercial ELISA kit can be considered as a useful tool for the diagnosis of goat warble fly infestation.

An intradermal test to detect latent warble (Hypoderma spp.) infection in cattle.

An intradermal test was developed to screen cattle for infection with the first-instar larvae of the warble flies Hypoderma lineatum and H. bovis. The diagnostic antigen, prepared from the first-instar larvae of H. lineatum, produced a distinct dermal reaction in cattle infected with the first-instar larvae of either species, but not in cattle in which the infection could not be confirmed later either on necropsy or by the appearance of warbles in the back. The reaction to the antigen was unpredictable in cattle with warbles in their backs. The diagnostic property of the antigen was also demonstrated in rabbits and guinea pigs artificially sensitized to the antigen.

[Neuromeningeal hypodermyiasis complicated by hydrocephaly. Value of nuclear magnetic resonance imaging]. / Hypodermose neuroméningée compliquée d'hydrocéphalie. Intérêt de l'imagerie par résonance magnétique nucléaire.

A 9-year old country boy developed blepharitis with inflammation of the face and, 1 month later, eosinophilic meningitis with paralysis of 3 limbs and of an abducent nerve. Nuclear magnetic resonance imaging of the central nervous system disclosed a lesional signal beneath the floor of the 4th ventricle, which was compatible with the presence of a larva of fly. Treatment with thiabendazole was tried, and the clinical signs regressed. Six months later, an asymmetrical hydrocephalus due to obstruction of Monroe's foramen by an inflammatory granuloma was discovered. Human hypodermyasis, due to migration in tissues of larvae of flies, is not rare in cattle-breeding areas. Neuromeningeal disorders are observed in 12% of the cases, consisting of eosinophilic meningitis sometimes associated with neurological deficit or seizures. Such complications as intracerebral haematoma or meningeal haemorrhage may occur during the usually benign course of the disease.

[Serological diagnosis of an infestation caused by the early developmental stages of botfly larvae (Oedemagena tarandi) in reindeer]. / Serologicheskaia diagnostika invazii, vyzyvaemoi rannimi stadiiami razvitiia lichinok podkozhnogo ovoda (Oedemagena tarandi) severnykh olenei.

Undertaken investigations have shown that the antigen prepared from larvae of O. tarandi is diagnostically effective and stricktly specific only to this infection in the reaction of indirect hemagglutination (RIHA). The experiments have proved the possibility of practical use of RIHA as a principal method of early diagnosis of infection of reindeer caused by O. tarandi.

[Development of the ELISA kit for the detection of Hypoderma bovis antibodies in cattle. I. Development of components]. / Opracowanie zestawu diagnostycznego do wykrywania przeciwcial anty-Hypoderma bovis u bydla testem ELISA. I. Uzyskanie komponentów.

The aim of these investigations was to prepare the antigen and control sera for the ELISA. Positive control sera were obtained from 163 cows showing clinical symptoms of H. bovis infection and negative control sera from 60 cows free from the infection. The antigen for the ELISA was obtained from H. bovis L1 larvae. Three basic fraction were isolated from whole excreted/secreted antigen by elution from gel and by preparative isoelectrofocusing. Using the Western-Blotting method it was demonstrated that Hy A (22-24 kDa and pI 4-5) had the best antigenic property. As can be seen from these investigations the developed ELISA kit for the detection of H. bovis antibodies in cows is highly specific and sensitive.