John H. Pruett

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 patients skin; after a second worm emerged, the patients 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 patients 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.

Induction of intradermal skin reactions in the bovine by fractionated proteins of Hypoderma lineatum

Protein species found in crude extracts of Hypoderma lineatum (Villers) 1st-instar gullet-stage larval homogenates were fractionated by chromatography and further identified by conventional polyacrylamide gel electrophoresis (PAGE) and SDS/PAGE techniques. Seven major fractions were resolved by ion exchange chromatography. Conventional PAGE of the crude antigen preparation revealed a minimum of 14 protein species, while SDS/PAGE revealed 3 major protein species. The ability to elicit cutaneous reactivity by isolated proteins was determined by the intradermal skin test. An immediate-type hypersensitivity reaction was elicited in the skin of vaccinated and previously infested animals by the components of the 4 protein fractions tested. Only one protein fraction, fraction 4, elicited an apparent delayed reaction at 48 h in vaccinated and previously infested animals. A non-specific background reaction was elicited in control animals by the fractionated proteins and was most notable between 1- and 4-h post-injection.

Antigen-specific lymphocyte proliferative responses in vaccinated and Hypoderma lineatum-infested calves

Cattle infested with the common cattle grub, Hypoderma lineatum (Villers) develop specific humoral antibodies and a cellular immune reaction, defined by delayed-type hypersensitivity, to purified H. lineatum proteins. This investigation was designed to study the antigen-specific bovine lymphocyte response to hypodermin A (HyA), a serine protease of larval first-instar H. lineatum. Calves were vaccinated with either native or denatured HyA, and challenge-infested with H. lineatum. The kinetic development of a cellular immune response to HyA was monitored during vaccination and infestation. The HyA-specific responses were highly variable and weak during vaccination and infestation. Although HyA-specific lymphocyte blastogenic responses were observed, no correlation was noted between the magnitude of antigen-specific, peripheral lymphocyte proliferation and larval mortality. In striking contrast to responses observed during infestation, intense HyA-specific lymphocyte responses were observed with 3 calves 6 months after recovery from infestation. In addition, those responses were further heightened by a 250 micrograms booster injection of pure HyA.

Post-blood meal analysis of Stomoxys calcitrans (L.) haemolymph for the presence of bovine immunoglobulin G

Abstract Pooled haemolymph samples collected from adult stable fly, Stomoxys calcitrans , following a blood meal, were analyzed for the presence of bovine immunoglobulin G. Western-blot analysis, utilizing anti-sera specific for bovine immunoglobulin G, with a demonstrated sensitivity of 1 ng/fly, failed to detect immunoglobulin G in the haemolymph. This finding does not rule out the possibility that subdetectable, but still biologically active, levels of immunoglobulin G enter the haemolymph. However, based on our estimates of the size of a blood meal and the amount of immunoglobulin G in bovine blood, we calculate that if it passes the gut wall into the haemocoel it is much less than 1% of the amount ingested. In contrast, small amounts of bovine serum albumin, a protein larger than immunoglobulin G subunits and fab or fc fragments, does pass into the haemolymph at detectable levels. This research has important implications from the standpoint of induced immunity to bloodsucking flies.

Effects of adjuvants on bovine humoral and cellular responses to hypodermin A

Hypodermin A, a serine protease of the first-instar larva of the common cattle grub, Hypoderma lineatum (Villers), when formulated with complete Freunds adjuvant and administered to naive calves, will elicit protective immunity defined by an increase in in vivo larval mortality. This study evaluated two veterinary acceptable adjuvants, alhydrogel and amphigen (alone and in combination), for suitability as an adjuvant for hypodermin A. Complete Freunds adjuvant (CFA) is not an acceptable adjuvant for use because of adverse reactions at the injection site. The veterinary acceptable adjuvants were not as effective as CFA in inducing an antibody response as detected in the peripheral circulation. Of the adjuvants evaluated, the mixture of alhydrogel and amphigen induced the highest serum antibody response to hypodermin A. All adjuvants evaluated induced comparable immediate-type skin test responses, and the mixture of alhydrogel and amphigen was most comparable with CFA in terms of delayed-type skin reaction and resultant cellular infiltration at the reaction site. Although the mixture of alhydrogel and amphigen, when compared with CFA, did not elicit comparable levels of responsiveness in all parameters tested, the overall performance of the mixture suggests it to be worthy of further efficacy investigation in a vaccine formulation with hypodermin A.

Shared epitopes between the soluble proteins of Hypoderma lineatum and Hypoderma bovis first instars.

Cattle are known to acquire immunological resistance to hypodermyiasis by repeated exposure to both species of cattle grubs, Hypoderma lineatum (Villers) and Hypoderma bovis (L.). Vaccination of cattle with purified proteins of H. lineatum, particularly hypodermin A, is known to protect cattle against hypodermyiasis by this species. The development of a protective recombinant vaccine against both species using hypodermin A isolated from H. lineatum would require that immunological epitopes be shared by complementary proteins in H. bovis. The purpose of this study was to investigate the soluble proteins of H. bovis first-instars for shared epitopes with H. lineatum. Soluble H. lineatum and H. bovis first-instar larval proteins were resolved by nondenaturing polyacrylamide electrophoresis, blotted onto nitrocellulose paper, and probed with selected polyclonal cow, polyclonal rabbit, and mouse monoclonal antisera. Considerable cross-reactivity was demonstrated by antibodies in the serum of an H. lineatum-infested cow as 6 of 10 resolved H. bovis proteins were bound by the antibodies. The most common shared epitope(s) was associated with hypodermin C, a collagenolytic protease. Hypodermin A shared epitope(s) were noted on 1 prominent H. bovis band (HB1-2). Hypodermin B, a prominent protein in H. lineatum, did not appear to share epitopes with H. bovis proteins. Shared epitopes between H. bovis proteins and hypodermins A and C of H. lineatum would suggest that cross-protection of cattle against H. bovis can be expected by vaccination with recombinant proteins of H. lineatum.

Antibodies of antigens of Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) in the sera of infested sheep: a serological technique for the detection of screwworm myiasis.

Antibodies to screwworm, Cochliomyia hominivorax (Coquerel), antigens are found in serum of previously infested host sheep and for at least one month after infestation. ELISA provides a serological technique for detecting previous exposure to screwworm in noninfested hosts. The technique is adaptable to a field situation and has potential as a method for screening noninfested hosts for indirectly estimating infestation rates.

EVALUATION OF METHODS FOR THE ISOLATION OF HIGH QUALITY RNA FROM BOVINE AND CERVINE HIDE BIOPSIES

Abstract:  Molecular investigations of the ruminant response to ectoparasites at the parasite–host interface are critically dependent upon the quality of RNA. The complexity of ruminant skin decreases the capacity to obtain high quality RNA from biopsy samples, which directly affects the reliability of data produced by gene expression experiments. Two methods for isolating total RNA from skin were compared and the use of 4M guanidinium isothiocyanate (GITC) during frozen storage of the specimens was evaluated. In addition, the best procedure for RNA isolation from bovine skin punch biopsies was also tested on white-tailed deer skin biopsies. Skin biopsy punches were collected and frozen prior to pulverization for RNA isolation. Total RNA quantity and integrity were determined by spectrophotometry and capillary electrophoresis technology, respectively. Significantly increased total RNA yield (P < 0.05) and higher integrity (P < 0.05) were obtained with a TRI Reagent® isolation method. Freezing and subsequent storage of bovine skin punch biopsies in 4 M GITC did not affect the amount or integrity of total RNA recovered by either RNA isolation method. However, quantity and integrity of total RNA extracted with the TRI Reagent method were again significantly higher than with the alternate technique, confirming it as the superior method. The TRI Reagent isolation method also yielded high quality total RNA from white-tailed deer skin punch biopsies, suggesting the usefulness of this method for obtaining RNA of a quality suitable for gene expression studies in other ruminant species.

Multiple transcripts encode glucose 6-phosphate dehydrogenase in the southern cattle tick, Rhipicephalus (Boophilus) microplus

Glucose 6-phosphate dehydrogenase (G6PDH) is an enzyme that plays a critical role in the production of NADPH. Here we describe the identification of four transcripts (G6PDH-A, -B, -C, and -D) that putatively encode the enzyme in the southern cattle tick, Rhipicephalus (Boophilus) microplus. The genomic DNA that is spliced to produce G6PDH-A and -B is 8,600–9,000 bases in length and comprises 12 exons. Comparison of the R. microplusG6PDH gene structure with those available from insects and mammals revealed that the tick gene is most like that of humans. Detection of the four transcripts was evaluated by quantitative RT-PCR using template from larvae, unfed adult females and males, salivary gland tissues from 2- to 3-day-fed adult females and males, and salivary gland tissue of 4- to 5-day-fed adult females. The G6PDH-A and -C transcripts were present in all templates, and both displayed induced expression in salivary gland tissue of fed, adult females but not matched males. The G6PDH-D transcript was detected only in unfed adults and in larvae, a stage in which it was most abundant relative to the other three transcripts. The G6PDH-B transcript, while detectable in all templates, was of low copy number suggesting it is a rare transcript. Induced expression of G6PDH-A and G6PDH-C in fed females may play a role in the tolerance of oxidative stress that is induced upon feeding, and the transcript abundance in fed females may be a function of bloodmeal volume and the time adult females spend on the host relative to adult males.