Immitis-infected Dogs Research Articles

Detection of circulating immune complexes in dog sera by immune adherence hemagglutination method.

Circulating immune complexes (CIC) in the sera from 102 clinically healthy dogs and from 16 Dirofilaria immitis (D. immitis) infected dogs were measured by the immune adherence hemagglutination (IAHA) method. The level of CIC in the sera from healthy dogs was 28.2 +/- 29.1 micrograms/ml. There was no significant difference in the levels of CIC regarding sexes or ages. On the other hand, the level of CIC in the sera from D. immitis infected dogs was 230.3 +/- 117.1 micrograms/ml, which was significantly (p < 0.01) higher than that of healthy dogs. The results of this study confirm that IAHA method is one of the reliable means of detecting and/or diagnosing immune complex mediated diseases in dogs.

Immunohistological observations on pulmonary tissues from dogs infected with Dirofilaria immitis.

Pulmonary tissues from non-infected dogs, naturally Dirofilaria immitis-infected dogs and experimentally infected puppies, selectively necropsied after infection, were assessed using peroxidase-antiperoxidase (PAP) staining technology. Sequential sections of pulmonary tissue were PAP stained with anti-fresh D. immitis serum, anti-paraffin processed D. immitis serum, anti-dog immunoglobulin (IgG, IgG Fc, IgM) sera and anti-dog complement (C3) serum, and examined for antibody, complement and for D. immitis antigen. The extent of alveolar septal thickening was positively correlated with infection status. Cellular infiltration was most evident surrounding obstructed areas where D. immitis were in situ. Antigenic material (microfilariae, eggs, fragmented filariae) labelled by PAP was identified in the pulmonary arteries, alveolar capillaries and alveolar septa. Deposits of complement and IgG, presumably associated with immune complex formation, were also observed in some of the infected dogs. Identification of antigen, antibodies and complement associated with alveolar septal pathology suggested that immune mechanisms were active in its development.

Identification of Dirofilaria immitis-Infected Dogs with a Coagglutination Assay

The presence of Dirofilaria immitis excretory-secretory (ES) products was detected in the urine of infected dogs using a coagglutination assay. Urine samples from 30 naturally infected dogs were positive. Seventeen of them were microfilaremic, whereas 13 had become amicrofilaremic after receiving 2 courses of diethylcarbamazine. Urine samples from 20 dogs infected with other parasites, Dipetalonema reconditum (7), Toxocara canis (5), and Ancylostoma caninum (8), and urine samples from 20 healthy dogs were negative. The assay detected 200 ng/ml or more of ES products. This assay is simple, easy to perform with minimum training, and requires no equipment. Therefore it should be useful to detect canine filariasis under field conditions.

Parasite excretory-secretory antigen and antibody to excretory-secretory antigen in body fluids and kidney tissue of Dirofilaria immitis infected dogs.

Excretory-secretory (ES) antigens of adult Dirofilaria immitis were detected by enzyme-linked immunosorbent assay (ELISA) in serum and urine from each of 12 experimentally infected dogs. Excretory-secretory antigens in serum were first detected 154 days postinfection. Serum antibodies directed against parasite ES antigens were detected by ELISA in all dogs. Kidney tissue elution studies were performed in 10 dogs, and antibody and parasite ES antigens were demonstrated in each case. Antibody or parasite antigen was not detected in serum, urine, or kidney eluates from uninfected dogs. At peak concentrations of the ES antigens in serum, there were correlations with the number of adult D. immitis present in the dogs (r2 = 23.8, P less than 0.05) and with the antigen concentration in kidney eluates (r2 = 73.5, P less than 0.001). Peak serum antibody concentrations were not correlated with either the number of adult worms or the antibody concentrations in kidney eluates. This study suggests that detection of parasite antigens in urine may be an important diagnostic aid. In addition, the correlation between the concentration of D. immitis ES antigens in kidney tissue and in serum without a similar correlation between serum and kidney antibody concentrations suggests that D. immitis ES antigens adhere to kidney tissue.

Use of Parasite Antigen Detection to Monitor the Success of Drug Therapy in Dirofilaria immitis-Infected Dogs

Recently the authors developed a monoclonal antibody-based enzyme immunoassay for circulating Dirofilaria immitis antigen and demonstrated its utility as a diagnostic tool for canine dirofilariasis. In the present study, serum parasite antigen measurements were used to monitor the success of thiacetarsamide therapy in 2 controlled trials that involved 24 naturally infected dogs. Parasite antigen levels correlated significantly with adult worm burdens in untreated control dogs. Antigen levels fell dramatically by 8 wk after treatment in successfully treated dogs and were undetectable 12 wk after treatment in dogs that were parasitologically cured. Microfilarial counts exhibited seasonal periodicity in both treated and control dogs and were not useful in monitoring the success of adulticide therapy. Parasite antigen detection is quite useful in monitoring the efficacy of adulticide therapy for dogs infected with D. immitis. This approach may lead to improved clinical use of thiacetarsamide, and it should facilitate evaluation of new drugs for this important infection.

In vitro responses of bronchial spirals and parenchymal strips from healthy and heartworm-infected dogs

SUMMARY Bronchial spirals and pulmonary parenchymal strips from control (noninfected) and Dirofilaria immitis-infected dogs were tested for isometric contractile response to histamine and carbamylcholine. Responses of bronchial spirals from control dogs and from D immitis-positive groups did not differ in responses to histamine or carbamylcholine. Parenchymal strips from D immitis-positive dogs showed greater response to histamine than did those from controls; responses to carbamylcholine did not differ.

Dirofilaria immitis: Diethylcarbamazine-induced anaphylactoid reactions in infected dogs

The immunopathogenesis of the anaphylactoid Mazzotti reactions has been studied by comparing physiologic and immunologic aspects of diethylcarbamazine-induced shock in Dirofilaria immitis infected dogs with antigen induced anaphylaxis in infected and uninfected controls. Filarial antigen, specific host IgG antibody, and C1 and C3 complement levels were quantitatively measured over time in relation to the levels of histamine and prostaglandin D2 in the blood and changes in mean blood pressure. D. immitis antigen injected into uninfected dogs having no detectable IgG antibody to D. immitis or Toxocara canis produced a rapid drop in blood pressure that paralleled a drop in C1 and C3 levels and an increase in prostaglandin D2. Antigen injected into infected dogs with IgG antibody produced a similar drop in blood pressure and complement and increase in prostaglandin D2 which differed from the uninfected group only in the slower clearance of antigen from the blood. Diethylcarbamazine alone produced no measurable changes in blood pressure or complement in uninfected hosts. Diethylcarbamazine, however, administered into skin test positive infected dogs, produced a temporally slower but quantitatively similar loss in blood pressure, drop in complement, and increase in prostaglandin D2 and histamine to that induced by antigen injection. Complement activation and immune complex formation are initiated by antigen release, and subsequent vasoactive mediator release leads to shock with prostaglandin D2 being quantitatively higher in blood than is histamine.

Antigenic peptides on the surface of Dirofilaria immitis microfilariae

Various methods of radioiodination were employed to identify peptides on the surface of Dirofilaria immitis microfilariae. Optimum surface radiolabelling occurred with the lactoperoxidase-catalyzed reaction. Two major peptides of 16 and 14 kDa were labelled by this method. These peptides were soluble in Nonidet P-40, were not glycosylated, and showed no signs of disulfide linkages. These peptides were immunoprecipitated by sera from D. immitis-infected dogs, but not by sera from uninfected dogs or sera from dogs with potentially cross-reactive nematode infections. Analysis of the 14 and 16 kDa peptides by two-dimensional gel electrophoresis revealed that the 16 kDa peptide was a single unit with a pI of 5.25 whereas the 14 kDa band was composed of three individual peptides with pI values ranging from 5.6 to 6.1. Iodination by chloramine T resulted in the same panel of labelled peptides but suffered from poor efficiency of 125I incorporation. The viability of microfilariae labelled by the standard Bolton-Hunter method decreased by 50% following the reaction which resulted in the labelling of a variety of internal components.

Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs.

We recently reported that parasite antigens are detectable in the serum of Dirofilaria immitis-infected dogs by counterimmunoelectrophoresis (CIE). Hybridoma cell lines that produce monoclonal antibodies specific for these antigens were obtained by immunizing mice with a partially purified antigen preparation, fusing spleen cells with SP-2 myeloma cells, and screening cell culture supernatants for antibody by ELISA and CIE inhibition. Antibodies specific for two epitopes shared by the two major circulating parasite antigens were identified. Immunoperoxidase studies showed that the epitopes recognized by the monoclonals were widely distributed in D. immitis, but the female uterus and eggs were particularly strongly labeled. A monoclonal antibody-based ELISA was developed to measure parasite antigens in dog sera. Parasite antigens were detected in 45 of 46 sera from infected dogs but were absent in sera from uninfected dogs and sera from dogs infected with Dipetalonema reconditum. Serum antigen content was significantly correlated with the number of female worms recovered from infected dogs (r = 0.82, p less than 0.001). Antigenemia was first detected 6 mo after infection, and antigen levels remained fairly stable between 9 and 21 mo after infection. Parasite antigen detection with this monoclonal antibody-based ELISA appears to be superior to previously described diagnostic methods for canine dirofilariasis in terms of sensitivity, specificity, and relation to infection intensity.

Lesions in the liver and kidney of Dirofilaria immitis-infected dogs following treatment with ivermectin.

Six dogs with spontaneous heartworm disease were injected with a single dose of ivermectin. After 48 h of treatment, microfilariae counts were reduced by 92%-98% of pretreatment counts. In pretreatment biopsies examined by light and electron microscopy, microfilariae were unaltered in the sinusoids of the liver and also in the glomerular capillaries and interstitial blood vessels of the kidney. However, there was irregular thickening and dense deposits in the basement membranes of glomerular capillaries, along with a modest increase in mesangial cells and matrix. In post-treatment liver biopsies examined by light microscopy, there were numerous granulomas in the sinusoids which contained degenerated microfilariae. In post-treatment kidney biopsies there was moderate thickening of glomerular basement membranes along with pronounced proliferation of mesangial cells and matrix. Glomerular capillaries were partially or completely occluded by degenerated microfilariae. In addition, there were interstitial granulomas in the kidney. It was observed with the aid of electron microscopy that highly vacuolated and degenerated microfilariae were incorporated into granulomas in the liver sinusoids of post-treatment biopsies. In post-treatment kidney biopsies glomerular capillaries were usually occluded by degenerated microfilariae. Basement membranes were thickened and contained dense deposits. Mesangial cells and matrix were extensively increased. Interstitial granulomas in the kidney contained dead microfilariae.

Adverse drug reactions in the treatment of filarial parasites: Haematological, biochemical, immunological and pharmacological changes in Dirofilaria immitis infected dogs treated with diethylcarbamazine

Abstract In order to understand the mechanism of the shock reaction that can occur in Dirofilaria immitis -infected dogs, following treatment with diethylcarbamazine (DEC), a variety of blood parameters were measured. A decrease in red blood cell parameters occurred in reactive dogs, followed by an increase, suggesting a possible sequestration of erythrocytes followed by peripheral vasoconstriction. Changes in leucocytes were consistent with stress reactions. Thrombocytopenia occurred, but no evidence of coagulopathy was seen. Alanine aminotransferase and creatinine phosphokinase (especially CK1 and CK3) concentrations increased suggesting cell damage and possibly liver cell necrosis, possibly due to hypoxia. Complement was not activated during the reaction and there was no increase in circulating immune complexes. No changes in plasma concentrations or histamine, 5-hydroxytryptamine, kininogen or prostaglandin F metabolite were found. Bradycardia was not caused by changes in potassium or calcium ion concentrations. These results suggest that the shock reaction is not an anaphylactic Type 1 reaction and is not caused by mast cell degranulation or by immune complex activation of mediators. The most likely cause of the reaction is that DEC causes the liberation of substances from microfilariae or adult worms which are able to constrict the hepatic vein directly or via its sympathetic innervation.

Dirofilaria Immitis

Antimicrofilarial immunity was studied in Dirofilaria immitis-infected dogs in order to better understand amicrofilaremic filariasis in man. Sera from dogs with amicrofilaremic infections contained IgG antibodies specific for microfilarial surface antigens detectable by immunofluorescence and in vitro leukocyte adherence. In vivo immune mechanisms were studied by injecting 51Cr-labeled microfilariae (MF) into infected and uninfected dogs. Injected MF were concentrated in lung, liver, spleen, and kidneys of normal and microfilaremic dogs, but circulated throughout the 5-hour study period. In contrast, injected MF were rapidly cleared (15-30 min) from the blood of amicrofilaremic-infected dogs. Tissue radioactivity and histopathology indicated that injected MF were trapped and destroyed in the lungs of these dogs. Antibody-dependent clearance and destruction of MF is a potent anti-helminth effector mechanism in canine dirofilariasis. Similar events are likely to occur in amicrofilameric filariasis in humans.

Inhibition of the adverse reaction to diethylcarbamazine in Dirofilaria immitis-infected dogs by lodoxamide ethyl.

Lodoxamide ethyl, a new oral anti-allergy drug, was tested for its ability to inhibit the post-treatment adverse reaction to diethylcarbamazine (DEC) in Dirofilaria immitis-infected dogs. Lodoxamide was highly effective, as judged by the absence of both overt signs and characteristic post-DEC changes in platelet numbers, serum fibrinogen level, and serum transaminase (SGOT, SGPT) levels, in blocking the reaction in dogs with a microfilaremia level that would make them moderately reactive. Lodoxamide was less effective in blocking the reaction in dogs with microfilaremias greater than approximately 10,000 mf/ml, a level which is regularly associated with a severe post-DEC adverse reaction. Some dogs with this level of microfilaremia were fully protected, others partially protected, and others were afforded no protection. Two other agents tested, indomethacin and homochlorocyclizine, were found to have comparatively little or no blocking activity.

Rapid decrease of precipitating and reaginic antibodies in Dirofilaria immitis-infected dogs which develop severe adverse reactions following treatment with diethylcarbamazine.

Dirofilaria immitis-infected dogs were treated with diethylcarbamazine. Measurement of the level of IgG precipitating antibody was carried out by counterimmunoelectrophoresis and that of IgE by passive cutaneous anaphylaxis in pre-treatment and post-treatment serum samples. In dogs showing severe to moderate adverse reactions to the drug there were pronounced decreases of both antibodies to a "metabolic" antigen prepared from the medium in which adult female worms had been incubated. These antibody levels did not decline in non-reacting dogs. Precipitating antibody levels to somatic antigens extracted from adult worms and microfilariae remained unaltered in both reactive and non-reactive animals.