Bruce Hammerberg

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.

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.

Auto IgG anti-IgE and IgG × 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.

Synthetic IgE peptide vaccine for immunotherapy of allergy

An immunotherapeutic vaccine for allergy was produced by designing IgE-based synthetic peptide immunogens and selecting them for functional immunogenicity. The vaccine targets the binding site on IgE for the high affinity receptor Fc epsilon RI, by active immunization. The peptide target site on IgE heavy chain was selected from among the amino acid sequences for the C epsilon 2, C epsilon 3, and C epsilon 4 domains. These were characterised by epitope mapping studies for cross-reactivity to IgE and functional antigenicity. A peptide, modified from positions 413-435 of a loop region of C epsilon 3 and subjected to conformational constraint, elicited anti-IgE antibodies that blocked IgE-mediated histamine release. It was immunopotentiated by linkage to a promiscuous T helper site to produce a wholly synthetic chimaeric immunogen. This immunogen was shown to induce polyclonal site-specific anti-IgE antibodies that obstruct binding to Fc epsilon RI, inhibit histamine release by IgE-sensitised basophils, inhibit passive cutaneous anaphylaxis, and do not signal degranulation. Immunized dogs experienced significant reductions in total serum IgE.

A canine model of cutaneous late-phase reactions : prednisolone inhibition of cellular and cytokine responses

Immunoglobulin E (IgE)‐mediated late‐phase reactions can be induced in atopic humans by intradermal injection of relevant allergens or anti‐IgE antibodies. The histology of these reactions resembles that of naturally occurring atopic dermatitis. Strikingly similar responses can be induced in dogs, suggesting that a canine model could prove valuable for preclinical investigation of drugs targeting late‐phase reactions. This study was designed to characterize the cellular, cytokine and chemokine responses after intradermal anti‐IgE injection in untreated and prednisolone‐treated dogs. Normal beagles were untreated or treated with prednisolone before intradermal injection of polyclonal rabbit anti‐canine IgE or normal rabbit IgG. Biopsies were taken before injection and 6, 24 and 48 hr after injection. Samples were evaluated by histological and immunohistochemical staining, as well as by real‐time quantitative polymerase chain reaction analysis. Dermal eosinophil and neutrophil numbers increased dramatically within 6 hr after injection of rabbit anti‐canine IgE, and remained moderately elevated at 48 hr. The numbers of CD1c+ and CD3+ mononuclear cells were also increased at 6 hr. The real‐time quantitative polymerase chain reaction demonstrated marked increases in mRNA expression for interleukin‐13 (IL‐13), CCL2, CCL5 and CCL17. Levels of mRNA for IL‐2, IL‐4, IL‐6 and IFN‐γ did not change within the limits of detection. Prednisolone administration suppressed the influx of neutrophils, eosinophils, CD1c+ and CD3+ cells, as well as expression of IL‐13, CCL2, CCL5 and CCL17. These data document the cytokine and chemokine responses to anti‐IgE injection in canine skin, and they demonstrate the ability of the model to characterize the anti‐inflammatory effects of a known therapeutic agent.

DIETHYLCARBAMAZINE-ENHANCED ACTIVATION OF COMPLEMENT BY INTACT MICROFILARIAE OF DIROFILARIA IMMITIS AND THEIR IN VITRO PRODUCTS

Microfilariae of Dirofilaria immitis and their products inhibited hemolytic complement in sera from various animal species. Inhibition of hemolytic complement activity, fluorescent antibody detection of specific complement proteins binding to microfilarial surfaces, and immunoelectrophoretic evaluation of complement protein C3 conversion demonstrated that (1) intact microfilariae depleted hemolytic complement activity maximally in the presence of diethylcarbamazine whereas the complement-fixing activity of microfilarial products was little affected by diethylcarbamazine; (2) complement proteins C3, properdin, and C5 bound to the cuticular surface of microfilariae; and (3) C3 was converted to a faster-migrating species during incubation with microfilariae or their products. The complement-depleting activity of in vitro products from viable microfilariae was soluble in 10% trichloroacetic acid, resistant to beta-elimination with 0.5 M NaOH, susceptible to treatment with 0.2 M periodate, and composed of 56% neutral sugar, 18% protein, 12% hexosamine, and 10% sulfate. The polysulfated, acidic mucopolysaccharide nature of the surfaces of microfilariae and the results presented here indicate that polyanionic components on worm surfaces or shed by microfilariae react with host complement proteins. This interaction may be enhanced by diethylcarbamazine and contribute to the pathology associated with microfilaremias.

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.

A comparison of the clinical manifestations of feeding whole and hydrolysed chicken to dogs with hypersensitivity to the native protein

Twenty-six dogs with known adverse food reactions were fed whole chicken for 14 days. From this group, 12 dogs with cutaneous manifestations following exposure to chicken meat were selected and randomly divided into two groups (n = 6). Each group was then fed hydrolysed chicken or hydrolysed soy for 14 days in a blinded crossover design with a 17-day washout period between each diet. Assessments of a CADESI (Canine Atopic Dermatitis Extent and Severity Index) score and pruritus were performed throughout the entire study, and combined in a global score (GS). Serum was collected weekly for the measurement of chicken- and soy-specific IgG and IgE. Dogs displayed the most severe clinical response when eating whole chicken compared to baseline (P < 0.001). The GS was significantly reduced in 11 of the 12 dogs when fed hydrolysed chicken were compared to those fed whole chicken (3.58 ± 2.81 versus 20.38 ± 14.65, P < 0.01). Serum immunoglobulin G and E responses were variable and did not show relationship with specific dietary exposure.

Levels of house dust mite-specific serum immunoglobulin E (IgE) in different cat populations using a monoclonal based anti-IgE enzyme-linked immunosorbent assay.

Levels of serum immunoglobulin E (IgE) specific for the house dust mites (HDMs) Dermatophagoides farinae (DF) and Dermatophagoides pteronyssinus (DP) in 58 cats with clinical signs suggestive of atopic dermatitis (allergic dermatitis cats), 52 cats with no history of allergic or immunological disease (nonallergic cats) and 26 specific pathogen-free (SPF) cats were measured using a monoclonal anti-IgE enzyme-linked immunosorbent assay. Reactivity to both native and reduced HDM allergens was compared. SPF cats had significantly lower levels of HDM-specific serum IgE than cats with allergic dermatitis and nonallergic cats. The difference in levels of HDM-specific IgE in the serum of cats with allergic dermatitis and nonallergic cats was significant for native DF allergen, but not for native DP allergen or reduced HDM allergens. The results suggest that DF in its native form may be a significant allergen in cats with allergic dermatitis. The clinical relevance of these reactions, however, remains to be proven.

THIRD COMPONENT OF COMPLEMENT, IMMUNOGLOBULIN DEPOSITION, AND LEUCOCYTE ATTACHMENT RELATED TO SURFACE SULFATE ON LARVAL TAENIA TAENIAEFORMIS

Cysticerci and strobilocerci of Taenia taeniaeformis were incubated with leucocytes from peritoneal washings of normal and T. taeniaeformis-infected rats in the presence of either normal sera or sera from infected rats. Leucocytes from infected and normal rats attached exclusively to the scolices but not the bladders of the larvae in the presence of serum from normal or infected rats. Heat inactivation at 56 C for 30 min destroyed the serum-mediated cell attachment. Histochemical staining of the larval taeniids with acid Alcian Blue demonstrated high concentrations of sulfated mucopolysaccharides on bladders that were not present on scolices. Immunofluorescent staining detected no difference in IgG deposition on the surfaces of bladders and scolices after incubation with rat sera in contrast to the markedly greater amounts of complement protein C3 found on scolices versus bladders. These results indicate that polysulfated substances on the bladder of this larval taeniid are associated with regional resistance to C3 deposition and leucocyte attachment.