Robert B. Grieve

Characterization of novel fucosyl- and tyvelosyl-containing glycoconjugates from Trichinella spiralis muscle stage larvae

The monosaccharide composition of an affinity-purified family of antigenically-related Trichinella spiralis larval glycoproteins was determined by gas chromatography/mass spectrometry. This group of 6 major glycoproteins, designated TSL-1, originates in the muscle stage (L1) larval stichosome. They are present on the L1 surface and in excretory/secretory products of L1 larvae, are stage-specific, and are highly immunodominant. The glycosyl composition of the TSL-1 antigens was remarkable in 2 respects: (1) fucose accounted for 36 molar percent of the glycosyl residues; and (2) a 3,6-dideoxyhexose was identified, which accounted for at least 24 molar percent of the glycosyl residues. Previously, 3,6-dideoxyhexoses have been found only in certain Gram-negative bacterial lipopolysaccharides and in ascaroside alcohols (ascarylose) of Ascaris eggs. The 3,6-dideoxyhexose found in the TSL-1 antigens also was found in ES. This Trichinella sugar has been chemically identified as a 3,6-dideoxyarabinohexose, the same as found in Ascaris eggs. However, the absolute configuration of the TSL-1 sugar is D-(tyvelose), not L-(ascarylose) as is found in Ascaris eggs. Methylation analysis indicated that the TSL-1 3,6-dideoxy-D-arabinohexose was present entirely as non-reducing terminal residues. Approximately 83% of the fucose was also present as non-reducing terminal residues, with the remaining fucose found as 3,4-linked branched residues.

Immune-mediated adherence of eosinophils to Toxocara canis infective larvae: the role of excretory-secretory antigens.

Summary The participation of Toxocara canis larval excretory‐secretory antigens in immune‐mediated adherence was determined in vitro. Adsorption of immune sera with excretory‐secretory antigens removed some complement components, removed IgG antibody directed against larval surfaces, and abrogated all adherence observed with untreated immune serum. At least four antigens could be implicated in adherence, by Western blot analysis of adherence mediating sera.

Dirofilaria immitis: Proteases produced by third- and fourth-stage larvae

A model of cutaneous extracellular matrix was used to determine if live Dirofilaria immitis larvae secrete proteases which are active at physiological pH and capable of degrading macromolecules found in cutaneous tissue. After 72 hr, 100 third-stage larvae (L3) degraded 24% of the total matrix, while fourth-stage larvae (L4) degraded 10%. A sharp increase in the amount of matrix degraded by L3 corresponded with the onset of the molting process. L3 and L4 degraded comparable amounts of the glycoprotein and elastin components of the matrix, but molting L3 degraded nearly twice the amount of the collagen component (62% vs 35%). Characterization of proteases present in larval-soluble extracts and excretory-secretory products using synthetic substrates and protease inhibitors demonstrated cysteine-protease and metalloprotease activity. Cysteine protease activity was found in whole worm extracts of both L3 and L4. Metalloprotease was secreted at higher levels by molting L3, but was also secreted by L4. Partial separation of the metalloprotease by size-exclusion chromatography indicated that the molecular weight of the native enzyme was in the 49-54 kDa range. The cysteine protease activity was demonstrated in fractions corresponding to 34-39 kDa. The biological function of the D. immitis larval proteases remains to be conclusively determined; however, these data suggest that they are involved in degradation of components of cutaneous tissue and in the molting process.

Serologic diagnosis of zoonotic pulmonary dirofilariasis

Four symptomatic and four asymptomatic patients with histologically confirmed zoonotic pulmonary dirofilariasis caused by Dirofilaria immitis (dog heartworm) were evaluated serologically. Five patients had diagnostic indirect hemagglutination titers to D. immitis and six had positive findings by enzyme-linked immunosorbent assay. Of the two patients that had nondiagnostic titers by both enzyme-linked immunosorbent assay and indirect hemagglutination, one had an encapsulated necrotic adult worm that appeared to have been dead for some time, and the serum specimen for the second patient had been obtained five months following surgical removal of the granuloma. These findings suggest good sensitivity for these serologic methods in active cases, but declining antibody titers and decreased sensitivity following worm death. In general, cross-reactivity of the enzyme-linked immunosorbent assay with serum from patients with other nonfilarial parasitic infections or neoplasms was not observed.

Molecular characterization of a Dirofilaria immitis cDNA encoding a highly immunoreactive antigen

Dirofilaria immitis, a filarial nematode, is the causative agent of canine and feline heartworm disease. Previous research has demonstrated that immunity to D. immitis can be induced in dogs by repeated chemical abbreviation of infections while the parasite is a fourth-stage larva. Sera obtained from dogs immunized in this manner has been effective in passively transferring larval killing and stunting. These immune sera, by comparison to nonimmune sera from infected cohorts, recognize a number of unique D. immitis antigens, some of which are larval specific. In this study immune dog sera were used to screen a D. immitis larval cDNA expression library. Three overlapping cDNA clones, Di22, Di18 and Di16, were obtained that encode a portion of a large molecule, greater than 150 kDa, that is composed of multiples of a 399-bp repeat. This protein when immunoblotted with antibody against a recombinant expressed Di22 fusion protein is found in larval as well as adult extracts and excretory-secretory products, and is seen as a series of ascending subunits, each approximately 15 kDa larger than the previous one. This antigen is highly immunogenic, as evidenced by the strong reactivity of the recombinant expressed Di22 fusion protein with sera from immune dogs, microfilaremic dogs and infected amicrofilaremic dogs. While the function of this antigen is unknown it has significant sequence similarity with an allergen found in Ascaris.

Analysis of Toxocara canis larval excretory-secretory antigens: physicochemical characterization and antibody recognition.

Toxocara canis larval excretory-secretory antigens (TEX) were resolved by gradient pore polyacrylamide gel electrophoresis and analyzed using silver, periodic acid-Schiff, and immunoperoxidase stains. At least 15 bands between 29 and 94 kilodaltons (kDa) were detected by silver stain, all of which were recognized by antibodies in serum of a patient with visceral larva migrans. Immunoperoxidase stain detected an additional band at 92 kDa and 4-6 others above 200 kDa. Periodic acid-Schiff stain also detected the high molecular weight components, but did not detect constituents of approximately 53 and 57 kDa. Immunoperoxidase stain using antibody from the vitreous fluid of an ocular larva migrans patient detected 2 TEX components, approximately 76 and 80 kDa. Antigens were compared with respect to batch of larvae and age of larvae in culture. Qualitative differences that correlated with batch were found in the number of constituents above 200 kDa, and in 1 component of 78 kDa. Qualitative differences were noted in many minor components, some of which appeared to correlate with age of larvae in culture. Major TEX constituents were recognized consistently by antibody, regardless of batch or age of larvae. Total protein production per larva was approximately 8 ng/day, and was consistent over time. There was no evidence of neutral proteases in TEX.

Antibody to interleukin 5 prevents blood and tissue eosinophilia but not liver trapping in murine larval toxocariasis

Mice previously sensitized by infective‐stage larvae of the canine nematode, Toxocara canis, trap large numbers of challenge larvae within the liver; trapped larvae are found within eosinophilic granulomas. To investigate the role of eosinophils in this phenomenon we examined larval trapping in mice depleted of blood and tissue eosinophils by treatment with a monoclonal antibody (MoAb) (TRFK‐5) produced against recombinant murine interleukin 5 (rmIL‐5). Control mice received either an isotype‐matched control MoAb or PBS. On day 0 test mice were given a sensitization dose of 125 infective T. canis eggs. Test and challenge control mice received 500 infective eggs on day 28. All mice were killed on day 42 and larval numbers within the liver were determined. Liver samples were also collected for histopathological and morphometric examination. When compared to test mice treated with PBS or the isotype control, the level of circulating eosinophils in anti‐IL‐5‐treated test mice was reduced by 94–96% on days 14 and 27,99% on day 35, and 100% on day 42; the level of tissue eosinophils within liver granulomas on day 42 was reduced by 92–95%. The total area of inflammation within the liver was similar among all test groups. However, the highly eosinophilic infiltrates, present in control sections, were replaced in anti‐IL‐5‐treated mice by lymphocytes, macrophages, and foreign‐body giant cells. No difference was found in larval trapping between antibody‐treated groups. These findings suggest that the eosinophil is not necessary for liver trapping in murine larval toxocariasis.

Molecular cloning of a developmentally regulated protein isolated from excretory-secretory products of larval Dirofilaria immitis☆

Three proteins isolated from the excretory-secretory products (ES) of larval Dirofilaria immitis have been previously characterized and termed the 20, 22L and 22U kDa proteins. Two of the proteins (20 and 22L) were produced and released around the time of the third molt and were specifically recognized by immune dog sera. An amino acid sequence common to both proteins was used to synthesize a DNA probe to molecularly clone these molecules from a 48-h third stage larval cDNA library. The DNA sequence of the isolated clones encoded a 17.5 kDa protein with a 21 amino acid hydrophobic leader sequence that when removed yielded a 15.3 kDa protein starting with the N-terminal sequence obtained from the 20 kDa protein and containing all sequences obtained from tryptic peptides of the 20 and 22L kDa proteins. It was hypothesized that the 20 and 22L kDa proteins were the same, differing only by a 21 amino acid hydrophobic leader sequence which was later cleaved. The calculated molecular masses were consistent with those determined by reducing Tris-tricine SDS-PAGE. Expression of the protein without the leader sequence was accomplished in Escherichia coli. Antiserum raised against the expressed protein demonstrated the presence of the protein in L3 and L4, but not in adults or microfilariae. Expression of the protein with the leader sequence using a baculovirus system demonstrated processing of the signal sequence at the same time as found in larval D. immitis ES. Sera from dogs immune to infection were reactive with the D. immitis proteins expressed in either E. coli or insect cells.

Serodiagnosis of ocular toxocariasis: a comparison of two antigens.

This study was designed to compare the sensitivity and specificity of enzyme linked immunosorbent assay (ELISA) for the serodiagnosis of ocular toxocariasis using Toxocara canis embryonated egg antigen (TEE) and toxocara excretory-secretory or exoantigen (TEX) produced in vitro. TEE and TEX ELISA were comparably sensitive, but TEX ELISA was better able to discriminate between serum samples from patients with ocular toxocariasis and those from patients with retinoblastoma. In addition, preabsorption of sera with Ascaris suum embryonated egg antigen seemed to be essential to prevent false positive results with TEE ELISA but was not so critical for TEX-ELISA. Further studies are still required to standardise TEX for serodiagnosis.

Active and passive immunization of mice against larval Dirofilaria immitis.

The objective of this study was to determine if Dirofilaria immitis larvae would survive in diffusion chambers implanted in dogs and mice and secondly to determine if mice could be immunized against infection with D. immitis. Dirofilaria immitis third-stage larvae (L3) survived and grew in diffusion chambers implanted in dogs and mice for at least 3 wk. BALB/c mice, which were repeatedly infected with live L3, showed resistance to challenge infections. Dead L3, with or without adjuvants elicited no protective immunity. A correlation was found between the degree of immune protection seen in mice and antibody levels to soluble larval antigen but not to antibody levels to surface antigens. A monoclonal antibody was prepared that reacted with the surface of D. immitis and Onchocerca lienalis L3, but not to the surfaces of other stages and species of various filarial worms. When this antibody was administered to mice prior to challenge no significant reduction in larval survival was observed.