Epicutaneous sensitization with Dermatophagoides farinae induces generalized allergic dermatitis and elevated mite-specific immunoglobulin E levels in a canine model of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a cutaneous hypersensitivity associated with elevated levels of antigen-specific IgE, commonly to house dust mites (HDMs). It remains controversial as to whether sensitization and clinical disease are induced by cutaneous exposure to HDM. OBJECTIVES: The objectives of this study were to determine whether repeated applications of Dermatophagoides farinae slurry to intact skin of Maltese-Beagle atopic (MAB) dogs would result in the development of clinical signs or lesions resembling spontaneous canine AD, to determine whether repeated slurry applications would induce elevations in mite-specific IgE and/or IgG, and to determine whether mite antigens could be demonstrated within the dermis of application sites. METHODS: Dogs received weekly slurry applications to the axilla and groin, and were patch tested at 120 days, or were patch tested at days 1, 60 and 120, but did not receive further slurry applications. Skin biopsies and serum samples were obtained on days 1, 60 and 120. RESULTS: Pruritic dermatitis was seen in all dogs by day 60. D. farinae-specific IgE was elevated by day 60. Histologic examination of early application sites revealed mild, mononuclear perivascular dermatitis. Later application sites were characterized by a dense inflammatory infiltrate and oedema in both the dermis and the epidermis. Immunofluorescent staining confirmed the presence of D. farinae antigens in the dermis. CONCLUSIONS: This study demonstrated that epicutaneous application of HDM slurry to MAB dogs results in elevations of HDM-specific IgE, localized and generalized pruritic dermatitis resembling spontaneous canine AD, and histologic changes typical of IgE-driven inflammation. We feel that these results suggest that epicutaneous exposure to allergen may play an important role during both the sensitization and the perpetuation of AD, and provide support for the use of a canine model in the investigation of the pathogenesis of AD.

Evaluation of the clinical and allergen specific serum immunoglobulin E responses to oral challenge with cornstarch, corn, soy and a soy hydrolysate diet in dogs with spontaneous food allergy.

Fourteen dogs with known clinical hypersensitivity to soy and corn were maintained on a limited antigen duck and rice diet until cutaneous manifestations of pruritus were minimal (78 days). Sequential oral challenges with cornstarch, corn and soy were then performed. Subsequently, the dogs were fed a diet containing hydrolysed soy protein and cornstarch. Throughout the study period the dogs were examined for cutaneous manifestations of pruritus and, additionally, serum was collected for measurement of allergen-specific and total immunoglobulin (Ig)E concentrations. Intradermal testing with food antigens was performed prior to entry into the study and after 83 days. A statistically significant clinical improvement was measured between days 0 and 83. Significant pruritus was induced after oral challenge with cornstarch, corn and soy (P = 0.04, 0.002, 0.01, respectively) but not with the hydrolysed diet (P = 0.5). The positive predictive value of the skin test for soy and corn allergy was reduced after feeding a soy and corn free diet. Although increases in soy and corn-specific serum IgE concentrations were measured in individual dogs post challenge they were not statistically significant and could not be used to predict clinical hypersensitivity.

Management of drug-resistant cyathostominosis on a breeding farm in central North Carolina.

REASONS FOR PERFORMING STUDY: Possible anthelmintic resistance on a breeding farm where a rapid rotation anthelmintic programme had been implemented for 9 years was investigated. Cyathostomins resistant to fenbendazole and pyrantel were documented by faecal worm egg count reduction test (FWECRT). OBJECTIVES: To 1) manage small strongyle transmission in a herd of horses in which resistance to both pyrantel pamoate and fenbendazole was identified and thereby reduce the risk of clinical disease in the individual animal, 2) monitor the change in resistance patterns over time and 3) monitor the efficacy of ivermectin over the study period. METHODS: Targeted ivermectin treatment of horses on the farm was instituted for mature horses with faecal worm egg counts (FWEC) > 200 eggs/g (epg) and for horses < age 2 years with FWEC > 100 epg. RESULTS: Over a 30 month period, targeted ivermectin treatment achieved acceptable control in mares, as judged by FWEC, and improved control of patent cyathostome infection in consecutive foal crops. Egg reappearance time (ERT) after treatment with ivermectin was < 8 weeks in mares and foals more frequently in the second year of the study than in the first year. Numbers of anthelmintic treatments were reduced by 77.6 and 533% in the mare and foal group, respectively. CONCLUSIONS: Targeted ivermectin treatment may be an economically viable method of managing multiple drug resistant cyathostominosis. POTENTIAL RELEVANCE: Use of ivermectin should be monitored closely for development of resistance.

IgE is present on peripheral blood monocytes and B cells in normal dogs and dogs with atopic dermatitis but there is no correlation with serum IgE concentrations.

Blood was collected from 29 dogs, 14 with atopic dermatitis (AD) and 15 controls. Total serum IgE was quantitated. Peripheral blood monocytes were harvested and labeled with leucocyte markers and anti-canine IgE before analysis by flow cytometry. There was no statistically significant difference between the atopic and control groups when the mean number of cells in the monocyte (CD14), antigen presenting cell (CD1c) or B cell (CD21) populations were examined. However, the variation in cell numbers was significant and much greater in the atopic group for CD1c and CD14 labeled cells. The mean percentage of double labeled cells, CD1c/IgE and CD14/IgE was significantly lower in the atopic population compared with the controls. More variation was observed in the numbers of monocytes of atopic dogs (CD14/IgE) and antigen presenting cells (CD1c/IgE) of control dogs. The mean percentage of B cells expressing IgE was 65 and 51% in the atopic and control groups respectively which is greater than that reported in humans. There was no statistically significant difference. Total serum IgE concentrations were similar in each group and did not correlate with cell bound IgE in any of the leucocyte populations studied. Canine AD is associated with more variability in circulating monocyte numbers and lower numbers of monocytes expressing IgE than control dogs. Unlike in humans, there is no correlation between circulating and cell bound IgE. Furthermore, high levels of IgE in the dog may be related to a greater number of B cells in the circulation committed to IgE production.

Skin mast cell histamine release following stem cell factor and high-affinity immunoglobulin E receptor cross-linking in dogs with atopic dermatitis.

Stem cell factor (SCF) influences mast cell activation and inflammatory mediator release, and is elevated in tissues undergoing allergic inflammation. Wheal formation in response to the injection of SCF or anti-immunoglobulin (Ig)E antibody injection was compared between normal (n = 10) and nonlesional atopic (n = 10) canine skin. In situ SCF secretion was compared between lesional and nonlesional skin using immunohistochemistry. Histamine release by skin cell suspensions after stimulation with SCF, concanavalin A (ConA) or rabbit anticanine IgE antibodies was compared between normal and atopic dogs. All dogs exhibited strong responses to intradermal SCF injection at 10 and 50 ng mL(-1). Atopic dogs had significantly (P = 0.002) larger wheal responses to anti-IgE than normal dogs; but there was no difference in numbers of skin mast cells bearing IgE as detected by immunohistochemistry. Only atopic dogs exhibited interstitial deposition of SCF in both lesional and nonlesional skin specimens. Median histamine release stimulated by SCF in the absence of IgE from lesional skin cells was higher in atopic than normal dogs (P = 0.04). These experiments suggest that dermal SCF secretion could potentiate histamine release following IgE receptor cross-linking and thus, could be one of the explanations for the inherent mast cell hyperexcitability observed in canine atopic dermatitis.

Natural killer cell-dependent immunoglobulin G2a anti-bovine serum albumin (BSA) response elicited by high molecular weight dextran-BSA conjugates associated with dextran-mediated macrophage-natural killer cell interaction.

The roles of the interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) produced during natural killer (NK) cell interaction with macrophages (M phi) were investigated as the basis for the induction of immunoglobulin G2a (IgG2a) anti-bovine serum albumin (BSA) responses by high molecular weight dextran conjugated to BSA (HMW-DEX-BSA). BALB/c mice immunized with HMW-DEX-BSA produced significantly higher levels of both IgG1 and IgG2a anti-BSA than did mice immunized with BSA alone. Both IgG1 and IgG2a anti-BSA levels were higher in mice immunized with BSA conjugated to dextran of molecular weight (MW) 5 000 000-40 000 000 compared with dextran of MW 10,000-60,000. The enhancement of anti-BSA IgG2a levels but not of anti-BSA IgG1 levels was inhibited when free BSA was added to the HMW-DEX-BSA conjugate. NK cell depletion during HMW-DEX-BSA immunization of mice resulted in significantly lower anti-BSA IgG2a levels without affecting anti-BSA IgG1 levels. Naive splenocytes or M phi + NK cell co-cultures incubated with HMW-DEX or HMW-DEX-BSA produced higher IFN-gamma levels than splenocytes or co-cultures incubated with BSA alone. HMW-DEX stimulated both IFN-gamma and IL-12 production by M phi + NK cell co-cultures in a dose-dependent manner. DEX-induced IFN-gamma production by NK cells was dependent upon the presence of IL-12, and IL-12 production by M phi was dependent upon the presence of IFN-gamma in these co-cultures. Both M phi and NK cells bound DEX to their surfaces. These data demonstrate that BSA linked to HMW-DEX enhanced both T-helper-1- and T-helper-2-associated antibody responses to BSA. The results also indicate an IL-12-dependent positive feedback interaction between NK cells and M phi that supports a NK cell/IFN-gamma-dependent mechanism for enhancement of anti-BSA IgG2a antibody responses in mice immunized with HMW-DEX-BSA protein conjugates.

Identification of a collectin-like protein in pig serum that binds a component in perienteric fluid from Ascaris suum.

A collectin-like protein (CLP) of the acute phase protein family that binds the polysaccharides mannan and alpha-1-6 dextran was isolated from the serum of pigs infected with Ascaris suum. A monoclonal antibody generated against this protein and used to characterize the CLP revealed on SDS-PAGE and western blot analysis that the protein had a molecular weight of approximately 48 kDa under reducing conditions and greater than 100 kDa under nonreducing conditions. Enzyme-linked immunosorbent assay (ELISA) showed that the CLP bound to substances in the perienteric fluid of Ascaris suum (APF). Molecular weight fractionation of APF demonstrated that CLP binds primarily to APF substances of greater than 100 kDa. Binding of CLP to APF was partially blocked by phosphatidylinositol. This is the first report of a porcine CLP and the binding of a CLP to components of the common nematode Ascaris suum.

Food hypersensitivity reactions in Soft Coated Wheaten Terriers with protein-losing enteropathy or protein-losing nephropathy or both: gastroscopic food sensitivity testing, dietary provocation, and fecal immunoglobulin E.

The purpose of this study was to evaluate Soft Coated Wheaten Terriers (SCWTs) affected with protein-losing enteropathy (PLE) or protein-losing nephropathy (PLN) or both for allergy to food. We performed gastroscopic food-sensitivity testing, a provocative dietary trial, and measurement of fecal immunoglobulin E (IgE) in 6 SCWTs affected with PLE or PLN or both. Positive gastroscopic food-sensitivity test reactions were noted in 5 of 6 dogs. Positive reactions were found to milk in 4 dogs, to lamb in 2 dogs, and to wheat and chicken each in 1 dog. Adverse reactions to food (diarrhea, vomiting, or pruritus) were detected in all 6 dogs during the provocative dietary trial. Adverse reactions were found to corn in 5 dogs, to tofu in 3 dogs, to cottage cheese in 2 dogs, to milk in 2 dogs, to farina cream of wheat in 2 dogs, and to lamb in 2 dogs. Serum albumin concentrations significantly decreased and fecal alpha1-protease inhibitor concentration significantly increased 4 days after the provocative trial when compared with baseline values. Antigen-specific fecal IgE varied throughout the provocative trial, with peak levels following ingestion of test meals. We conclude that food hypersensitivities are present in SCWTs affected with the syndrome of PLE/PLN. Mild inflammatory bowel disease was already established in the 6 SCWTs of this report at the time of study, making it impossible to determine if food allergies were the cause or result of the enteric disease.

Association of elevated lymph node cell release of histamine and tumor necrosis factor with genetic predisposition to limb edema formation in dogs infected with Brugia pahangi.

Brugia pahangi infection in the canine rear limb results in marked lymphatic duct and popliteal lymph node pathologic changes. Limb edema is variably associated with infection and does not correlate well with duct or node lesions. To understand the mechanisms of limb edema, lymph node cells were collected by sequential biopsy following infection and examined for production of inflammatory mediators. Lymph node cells from a litter of dogs selectively bred with a high incidence of edema formation (82%) demonstrated spontaneously released histamine and prostaglandin E2 levels higher than those of closely related nonedema-forming dogs (0-20%) and/or control dogs. These edema-forming dogs also showed elevated release of tumor necrosis factor-alpha when cells were cultured with Brugia antigen. Toluidine blue staining of infected lymph node sections revealed that the edema-forming dogs had higher numbers of mast cells than infected lymph nodes of nonedema-forming dogs.

Auto IgG anti-IgE and IgG x IgE immune complex presence and effects on ELISA-based quantitation of IgE in canine atopic dermatitis, demodectic acariasis and helminthiasis.

Atopic dermatitis is a common allergic disease manifestation in dogs; however, there is no correlation between clinical disease and detectable total serum IgE. Auto antibodies of the IgG subclass against IgE may affect the detection of serum IgE by immunoassay and may be important in the regulation of IgE production by B cells. ELISA were developed to detect serum antibodies specific for IgE using a newly available canine monoclonal IgE of known antigen specificity, generated from a canine x murine heterohybridoma. To test for correlation of auto IgG anti-IgE levels with manifestation of atopic dermatitis, the sera from 101 atopic dogs were compared with sera from non-atopic dogs of various breeds, foxhounds manifesting clinical signs of demodectic acariasis and helminth parasitized random bred dogs for quantities of IgG anti-IgE measured in units/ml compared to a high titer standard serum. To test for serum effects on quantitation of IgE, known amounts of canine monoclonal IgE were added to various sera and measured by capture ELISA with detecting monoclonal antibodies specific for heat labile or heat stabile epitopes. Unheated sera from dogs manifesting clinical atopic dermatitis and helminth parasitized dogs had levels of IgG anti-IgE that were significantly lower than various breeds of dogs not manifesting dermatologic lesions and foxhounds manifesting demodectic acariasis. Heating sera at 56 degrees C for 3 h to denature the high affinity binding site on the IgE heavy chain caused a marked increase over non-heated sera in detectable IgG anti-IgE in almost all dogs. This increase was most profound in helminth-infected dogs and foxhounds manifesting demodectic mange with 7 fold increases each, respectively, and in atopic dogs with a 5 fold increase compared to 3 fold increases for clinically-normal springer spaniels and all soft coated wheaten terriers. The terriers demonstrated an association of lower heated serum values of IgG anti-IgE with manifestation of a familial syndrome of protein-losing enteropathy and protein-losing nephropathy. The ability of mouse anti-canine IgE monoclonal antibodies specific for either heat labile or heat stabile epitopes to detect canine monoclonal IgE added to sera in known amounts varied from serum to serum and at different concentrations of the same serum, but did not correlate with IgG anti-IgE values for these sera. The range of absolute levels of serum IgE in dogs showing little or no inhibition of detection of added IgE was < 0.5 ng/micromilligram to 2 micrograms/micromilligram. It was concluded that the increase in detectable IgG anti-IgE after heating sera indicates that IgG x IgE immune complexes are normally present in most dogs; however, the increase over uncomplexed IgG anti-IgE was most pronounced in dogs manifesting atopic dermatitis and demodectic acariasis. A quantitative comparison of IgG anti-IgE or IgG x IgE to total serum IgE was not made because the ability of monoclonal antibodies specific for either heat labile or heat stable epitopes on the IgE heavy chain to detect IgE added to serum, as well as innate serum IgE, was highly variable in different dilutions of serum from individual to individual.

Inhibition by platelets of in vitro blastogenic responses of canine PBMC by a PAF-dependent mechanism.

It was demonstrated that platelets can inhibit in vitro blastogenic responses by a platelet activating factor (PAF)-dependent mechanism. A procedure for the isolation of virtually platelet-free canine peripheral blood mononuclear cells (PBMC), using Ficoll density gradient centrifugation followed by Percoll density centrifugation, was developed to investigate the mechanism by which platelets inhibit the in vitro blastogenic response of PBMC. It was shown that PBMC purified on Ficoll gradients alone are contaminated with platelets and proliferate weakly compared with platelet-free PBMC purified on an additional Percoll gradient. Addition of platelets to PBMC cultures in the presence of PAF receptor antagonist resulted in a proliferative response similar in intensity to that of platelet-free PBMC cultures, whereas the addition of platelets to PBMC cultures in the absence of PAF receptor antagonist resulted in marked inhibition of the mitogen-induced proliferative response. Therefore, PAF is likely to be involved in the inhibition of in vitro proliferative responses of platelet-contaminated canine PBMC.

Canine IgE monoclonal antibody specific for a filarial antigen: production by a canine x murine heterohybridoma using B cells from a clinically affected lymph node.

Canine popliteal lymph node cells taken at the onset of clinical disease from a rear limb infected with the filarial nematode Brugia pahangi were fused with mouse myeloma cell line P3X63.Ag8.653 cells. Of the several canine immunoglobulin-producing clones from this fusion, one was found to produce canine IgE specific for a filarial nematode antigen. The cell line has undergone limiting dilution cloning six times over the past 3 years and continues to produce monoclonal antibody of the IgE subclass at a rate of greater than 3 mg/l. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the cell culture supernatant protein that bound to protein A beads, showed bands at molecular weights (MW) of approximately 75,000 and 25,000 that were characteristic of epsilon and kappa or lambda chains, respectively. A mouse monoclonal antibody specific for canine IgE bound the 75,000 MW band, as demonstrated by Western blot. Western blots of aqueous extracts of adult filarial nematodes demonstrated binding of the canine IgE monoclonal antibody to a single 35,000 MW peptide from B. pahangi but not Dirofilaria immitis; immunochemistry using frozen sections of adult worms, microfilariae and fourth stage larvae revealed focal binding of the monoclonal IgE to worm tissue adjacent to dorsal and ventral cords of only Brugia adults.

Modulation of cellular and humoral immunity, and disease manifestation during onset of patency in Brugia pahangi-infected dogs.

Recently, it has become possible to obtain as predicted disease manifestation in selectively bred dogs infected with the naturally occurring lymphatic nematode, Brugia pahangi. In this study, an attempt was made to correlate limb oedema with dynamic changes in immune cell responses occurring in the lymph node at the site of infection during onset of patency. Three litters of dogs were selectively bred; one for the expression of clinical disease, one for the lack of expression of clinical disease and one was of non-specific phenotype. Lymph node cells from 10 of 11 dogs showed a parasite-specific proliferative response at 4-6 weeks post-infection (p.i.), before the onset of patency. In six of 11 dogs, a loss of proliferative response to BpA in the infected node cells was detected around the time of onset of patency. In contrast, there was no reduction in the proliferative response to the mitogen phytohaemagglutinin (PHA). The proliferative response to BpA by unresponsive node cells could be restored by addition of substimulatory amounts of murine or human recombinant interleukin-2 (IL-2) to the culture medium. However, there was no correlation between the proliferative response of lymph node cells from infected limbs and the expression of clinical disease. Similarly, when in vitro parasite-specific antibody production by infected lymph node cells was examined, antibody production manifested by all dogs at 5 weeks p.i. was markedly changed at 8 weeks p.i., but these changes did not correlate with clinical disease. This lack of correlation indicates that the immune response to lymphatic filarial infection, as measured in this study, does not necessarily result directly in disease manifestation, and that other genetically determined factors may influence both the parasite-specific immune response and the clinical outcome of infection.

Dirofilaria immitis: detection of parasite-specific antigen by monoclonal antibodies in glomerulonephritis in infected dogs.

For the identification of circulating parasite antigens associated with immune-complex glomerulonephritis in dogs infected with Dirofilaria immitis, monoclonal antibodies (mAbs) were generated against adult worms. A total of 11 mAbs were selected for cloning because of their high productivity and their lack of cross-reactivity with Toxocara canis in indirect immunofluorescence tests. The ability of mAbs to detect circulating antigens was examined using an antigen-capture enzyme-linked immunosorbent assay. Of the 11 mAbs, only NAK-1, an IgG2a mAb, was capable of detecting circulating antigens in 75% of infected dogs. However, this mAb was highly species-specific in its detection of circulating antigens, since sera from dogs infected with other nematode parasites were negative. Furthermore, the mAb detected antigens at the same glomerular sites in which IgG and/or C3 were deposited. The antigen deposits were observed along capillaries and/or in mesangial cells. The epitope recognized by this mAb is probably a carbohydrate, as it remained stable for 1 h at 100 degrees C and was sensitive to periodate treatment. Two bands of 62 and 26 kDa, respectively, were detected on Western blots by the mAb when sera from dogs infected with D. immitis were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted.

Inhibition of antigen-driven proliferative responses and enhancement of antibody production during infection with Brugia pahangi.

Long standing Brugia pahangi infections in seven dogs, restricted to one rear limb popliteal lymph node and its afferent ducts, were monitored with regard to proliferative responses and antibody production specific for a PBS extract of B. pahangi (BpA) by cells from infected and uninfected lymph nodes and by PBL. Five of 10 dogs were negative for proliferative responses to BpA in node cells from infected limbs, yet they had positive PBL responses, and another was negative in both node cells and PBL. Production of BpA-specific antibody was detected in cultures of node cells from infected limbs of 9 of 10 dogs, but only in two cultures of node cells from uninfected limbs and not at all in PBL cultures. Three dogs with responsive node cells produced the least amount of anti-BpA antibody in culture. Injections of B. pahangi adult worm excretory/secretory products (ES), totaling 1 mg over 48 h, into the limb of the original infections in seven dogs, resulted in inhibition of Ag-driven proliferation by cell populations previously responsive to BpA. There was a loss of PBL responsiveness by all but one infected dog and a loss of node cell response by the two dogs previously responsive in infected and uninfected nodes. This loss of responsiveness lasted at least 28 days in three dogs. There was no evidence of suppression of responses to mitogens either before or after ES injection. In contrast, BpA-specific antibody production was greatly increased in node cells from infected limbs injected with ES. Similar injections into the uninfected limbs of two infected dogs produced no change of proliferative responses or of antibody production in the uninfected node. These results indicate that ES can modulate immune cell, Ag-driven proliferation, and simultaneously enhance antibody production in previously infected nodes. This may promote parasite survival by inhibiting cellular attack based on delayed-type hypersensitivity while directing immune responses toward production of antibodies that are less damaging to the adult helminth.

Selective breeding of dogs for segregation of limb edema from microfilaremia as clinical manifestations of Brugia infections.

Three generations of beagles were monitored for microfilaremia (mf) and clinical disease during repeated infection with Brugia pahangi and were selectively bred for offspring manifesting limb edema and low or amicrofilaremia. A high microfilaremic female mated to a high microfilaremic male produced 7 pups, 6 of which maintained mf greater than 1,000/ml for greater than 2 years after 5 monthly infections of 10 infective larvae each. An uninfected female mated to another high mf male produced 5 pups, 4 of which did not exceed 1,000 mf/ml 7 months after initiation of the repeating infection regimen; 1 of these remained amicrofilaremic after 2 additional challenges. Neither the parents nor the offspring from these matings manifested chronic limb edema. Two matings were conducted with offspring from the microfilaremic female by breeding siblings with the lowest mf and breeding siblings with the highest mf. The high mf siblings produced 4/5 offspring manifesting chronic limb edema (greater than or equal to 7 months duration) and either no mf (in 2 dogs) or less than 100 mf/ml after the repeating infection regimen. The lower mf siblings produced 5 offspring, all with greater than 1,000 mf/ml 6 months after the initiation of the repeating infection regimen; none manifested edema. Comparisons of IgG antibody levels, specific for extracts of adult worms, showed no consistent differences between these 2 litters of dogs that could be associated with limb edema or mf when monitored for 16 months; however, the onset of lymph node enlargement was much earlier in the group of dogs manifesting limb edema than in the other litter.

Taenia taeniaeformis: evasion of complement-mediated lysis by early larval stages following activation of the alternative pathway.

Activation of the alternative pathway of complement by T. taeniaeformis oncospheres and early stage metacestodes, although a factor in host defense against primary infection, does not directly lead to the killing of the parasite larvae observed prior to day 6 post-infection in innately resistant BALB/cByJ inbred mice. Immunogold labelling techniques clearly demonstrated tegument-associated C3 on in vitro-activated oncospheres incubated with non-immune mouse sera. However, C5, a protease necessary for the assembly of the membrane attack complex, was not detected. Early stage larvae cultured from in vitro-activated oncospheres escaped membrane damage and survived incubation in non-immune sera from both BALB/cByJ and taeniid-susceptible C3H/HeDub mice. Comparisons of cobra venom factor-treated and untreated C5-deficient B10.D2osn mice revealed no significant differences in parasite burden and local eosinophil infiltration at 6 days post-infection, suggesting that the terminal arm of the complement system is necessary for the previously reported role of complement in resistance to primary infection in BALB/cByJ and C3H/HeDub mice. An in vivo test of chemotaxis indicated that although both complement-intact mouse strains examined responded to intraperitoneal injections of inulin, there were lower numbers of eosinophils in C3H/HeDub mice than in BALB/cByJ mice, perhaps pointing to possible mouse strain differences in C5a generation/catabolism or eosinophil ability to respond to C5a. Lectin-binding studies showed an affinity of PNA for the exposed surface of taeniid oncospheres and 4-day post-infection metacestodes; however, binding of lectin to the carbohydrate moiety did not inhibit complement activation.

Effect of depletion of L3T4+ cells on resistance to early primary infection of mice with Taenia taeniaeformis.

The role of L3T4+ T lymphocytes in early primary infection with the metacestode of T. taeniaeformis was investigated by selective removal of these cells in vivo by parenteral injections with the rat monoclonal antibody (MAb) GK1.5 directed against the L3T4 molecule. Comparisons between treated and non-treated BALB/cByJ mice, normally resistant to infection with T. taeniaeformis, demonstrated that the treated mice had a greater percentage of viable parasites in the livers. Eosinophils were prominent in the region immediately surrounding parasite larvae in control mice, whereas treated mice showed virtually no eosinophil infiltration. Additionally, fewer tissue macrophages were evident near parasite larvae in the treatment group when compared to controls. The more susceptible C3H/HeDub strain mice demonstrated similar responses following treatment with the MAb, including diminished parasite killing and limited inflammatory cell infiltration. When C3H/HeDub mice were injected with the cytotoxic agent vinblastine sulfate, which has been shown to diminish Lyt-2+ suppressor cell activity, these mice remained unable to mount a strong local cellular response to the larval parasite. It is suggested that L3T4+ T lymphocytes play a crucial role in the innate resistance to T. taeniaeformis infection during the first 6 days post-infection. Effects seen following vinblastine treatment may be a result of drug-induced alterations in leukocyte chemotaxis, toxicity to other effector T cell populations, or a specific depletion of a functional Lyt-2+ T cell population that is required in addition to L3T4+ T cells for the expression of resistance to primary infection with T. taeniaeformis.

Protective immunity against Brugia malayi infective larvae in mice. II. Induction by a T cell-dependent antigen isolated by monoclonal antibody affinity chromatography and SDS-PAGE.

A mAb directed against filarial worm secretory/excretory product and reactive with Brugia malayi larval worm surface was used in conjunction with preparative SDS-PAGE to isolate protective Ag from extracts of adult B. malayi. The IgM mAb OVH bound to a repeating carbohydrate epitope present in adult, infective, and fourth stage larvae and microfilariae of B. malayi, and on the surface of fourth stage larvae. Ag bearing this epitope were also present in the sera of hosts infected with a variety of helminths, including Brugia, Onchocerca, Dirofilaria, and Paragonimus. Affinity chromatography of SDS extract of adult Brugia, using mAb OVH immobilized on agarose beads, isolated several Ag that separated into multiple protein staining bands on SDS-PAGE. In comparing SDS-PAGE-fractionated Ag from the crude SDS extract with fractionated mAb OVH-isolated Ag for the ability to protect BALB/c mice from challenge with B. malayi-infective larvae, it was found that of the mAb OVH-isolated Ag only those at a molecular mass of 26 to 32 kDa were protective while the original SDS extract yielded protective Ag at the following molecular mass: greater than 200, 170 to 200, 40 to 44, 33 to 36, 23 to 28, 20 to 22, and 17 to 19 kDa. Although Ag isolated by mAb OVH were highly protective, they failed to induce high antibody levels against the immunogen or SDS extracts compared to crude SDS extract immunized mouse sera, as determined by immunoblot and ELISA. Transfer of nylon wool non-adherent T cells from BALB/c mice immunized with the 26- to 28-kDa fraction of mAb OVH-isolated Ag to naive mice just before challenge with infective larvae of B. malayi resulted in a 70% reduction in larvae recovered 14 days after challenge.

Protective immunity against Brugia malayi infective larvae in mice. I. Parameters of active and passive immunity.

Protective immunity against infective larvae of Brugia malayi was studied in different strains of mice using various sources of antigens. The following strains of mice were susceptible to infective larvae development for 2 weeks after primary ip challenge: BALB/c, C3H/HeJ, C3H/NeN, C3H/HeJms, C57BL/6Jms, and DDD. In comparison to gerbils, BALB/c mice developed stronger resistance to infective larvae after immunization with irradiation attenuated larvae or with killed microfilariae (mf). However, killed mf failed to enhance resistance in C3H/HeJ mice, although C3H/HeN mice were strongly protected and C3H/HeJms mice were protected to a lesser degree by this antigen. Extracts of mf with phosphate buffered saline and sodium dodecyl sulfate both induced high levels of resistance in BALB/c mice. Transfer of resistance from BALB/c mice immunized with attenuated infective larvae to naive mice was accomplished at a high level at protection with nylon wool nonadherent spleen cells (T cells) but not with adherent cells treated with anti-Thy 1.2 serum and complement. In contrast, sera from immunized mice were much less protective.