Microfilaremic Dogs Research Articles

Relationship between microfilaria count and sensitivity of the direct smear for diagnosis of canine dirofilariosis

Direct blood smear examination (using 0.05 ml of whole blood) detected 168 (80.9%) of 204 microfilaremic canine blood samples as determined by the modified Knott test for microfilariae (mff) of Dirofilaria immitis (using 1 ml of whole blood). Direct smear examination detected all of 134 microfilaremias greater than 50 mff ml −1, but only 31 of 70 (44.3%) microfilaremias having less than 50 mff ml −1. In a separate retrospective query of a database of 963 dogs with necropsy-confirmed heartworm infections, 834 (86.6%) were positive by the DiroCHEK heartworm antigen test, and 504 (52.3%) were microfilaremic by the modified Knott test. Only 2 (0.4%) of the microfilaremic dogs were DiroCHEK negative and another 18 (3.6%) were very weak positives. Although these microfilaremic dogs were not tested by direct smear, only one of the two DiroCHEK-negative and six of 18 weakly DiroCHEK-positive dogs had microfilaremias so low that a direct smear may have given a false negative result. Significant adverse reactions to either diethylcarbamazine or the macrolide endectocides have not been reported for microfilaremias less than 500 mff ml −1, thus substitution of the direct smear for a concentration test for mff, such as the modified Knott test or membrane filtration, does not appear to increase the risk of an unexpected adverse reaction to heartworm prophylactic drugs. Such a substitution results in only a very slight decrease (on the order of 0.1%) in the overall sensitivity of heartworm screening, provided a test for mff is run concurrently with an antigen test. If a test for mff is the only screening test used, then substitution of a direct smear for a concentration test may decrease the sensitivity of heartworm screening by nearly 20%, depending on the prevalence of low level microfilaremias in the population of dogs tested.

Molecular characterization of a calcium-binding protein from the filarial parasite Dirofilaria immitis

A full length D. immitis cDNA (nDiCal) encoding a protein with significant similarity to the calreticulin protein family was isolated from a 6-day fourth-stage larval cDNA expression library by immunoscreening, using serum from a rabbit immunized by repeated injection of small numbers of third-stage larvae. nDiCal is 1538 bp long and contains the 21 bp nematode splice leader sequence SL1 at the 5′ end. nDiCal encodes for a protein (pDiCal) with a predicted molecular mass of 46 kDa. pDiCal sequence analysis revealed similarities with calreticulin, a protein that typically resides in the endoplasmic reticulum. pDiCal possesses three consensus sequences of the calreticulin family of proteins: a neutral N-terminal region with a putative signal sequence; a proline- and tryptophan-rich P region; and a highly acidic C-terminal region. A 45Ca 2+-overlay assay showed that recombinant pDiCal (rDiCal) is a Ca 2+-binding protein. Antibodies to rDiCal identified a 56 kDa native antigen in all developmental stages including the excretory-secretory products derived from larvae and adult worms. Localization studies demonstrated the ubiquitous presence of pDiCal with intense expression in the hypodermis and syncitial muscle cells in both male and female adult worms. Labeling was also seen in the developing embryos within the uterus of the female worms. Sera from immune as well as chronically-infected microfilaremic dogs contained antibodies that bind rDiCal. In addition, immunoblot analysis showed that serum from a rabbit immunized with L3 cuticles reacted with rDiCal.

Antifilarial Effect of a Combination of Botanicals from Acacia auriculiformis and Centella asiatica on Canine Dirofilariasis

A mixture (1:1) of the ethanol extracts from the leaves of Centella asiatica and the funicles of Acacia auriculiformis was administered orally to pariah dogs naturally infected with Dirofilaria immitis at 0.04 mg/g body weight/d for 45 days. Two triterpenoid saponins, acaciaside A and B, isolated from the funicles of A. auriculiformis were combined with the leaf ethanol extract from C. asiatica (1:1) and tested at 0.04 mg/g body weight/d for 45 days on microfilaraemic dogs. There was an initial fluctuation in the microfilarial (mf) count in the blood following treatment with the mixture of crude extracts, and then the mf density decreased up to 99%. This suppression lasted for 120 days. When treated with the saponins mixed with the C. asiatica ethanol extract, there was an 86% reduction in mf density 30 days after the last date of treatment, and 68% reduction was maintained 120 days after the treatment.

Variation in protein concentrations in acute and chronic ascites in Dirofilaria immitis-infected dogs.

Microfilaremic dogs, developing ascites acutely following a reaction to diethylcarbamazine therapy, had similar protein concentrations in their ascitic fluid and plasma. In contrast, in dogs chronically infected with Dirofilaria immitis, the protein concentrations of ascitic fluid were found to be significantly lower than plasma protein concentrations. The acute development of ascites in such dogs is associated with high ascitic protein levels.

酵素抗体法改良型キットによる犬糸状虫成虫抗原の検出

The reliability of a newly devised serodiagnosis test kit aiming at more simple procedure with smaller sample volumes for detection of circulating adult heartworm antigen (enzyme immunoassay), was compared with that of a conventional kit. Forty-one of 42 heartworm-freedogs were identified correctly with both kits (specificity 97.6%). In 37 dogs with unmicrofilaremic infection, sensitivity of the new kit (45.9%) was not different from that of the conventional one (43.2%). Dogs with immature worm infection were negative with both the kits. In 35 microfilaremic dogs, detection errors of the new and conventional kits were 1 and 2 dogs (sensitivity: 97.1% and 94.2%), respectively.

Clinical, hematologic, and biochemical findings in dogs after induction of shock by injection of heartworm extract

Summary A crude, whole-body extract of female or male heartworms was injected iv into 28 dogs with and 22 dogs without heartworm (hw) infection. The female hw extract caused shock in 22 of 24 dogs with and 12 of 20 dogs without hw infection. The male hw extract induced shock in 4 of 4 dogs with and 1 of 2 dogs without hw infection. Prevalence of shock caused by female hw extract was significantly (P < 0.05) higher in dogs with than without hw infection; shock developed 5 to 30 minutes after hw injection. These signs were observed: marked decrease in blood pressure; collapse (initial collapse); paleness of mucous membranes; weak heart sounds; dyspnea; skin coldness; intestinal hyperperistalsis, and defecation; increases in rbc count, serum total protein concentration, serum osmolality, serum Na and blood glucose concentrations; and decreases in neutrophil, eosinophil, and platelet counts. Alanine transaminase, alkaline phosphatase, and lactate dehydrogenase activities increased substantially from the time of initial collapse to 24 hours after hw injection. Of 39 dogs with shock, 29 recovered from initial collapse, but 5 of the 29 subsequently collapsed again (secondary collapse), with bloody diarrhea followed by death. Of these 39 dogs, 6 died during initial collapse without bloody diarrhea, and 4 were euthanatized during initial collapse. It was confirmed that hw extract had, in fact, induced shock. These clinical, hematologic, and biochemical findings were fundamentally similar to those associated with shock resulting from administration of drugs, such as diethylcarbamazine and milbemycin D, in microfilaremic dogs with hw infection.

Immunoblotting analysis of somatic components of Dirofilaria immitis.

SDS-PAGE analysis of Dirofilaria immitis extracts demonstrated the complexicity of protein components of microfilariae, similar to that of adult male and female worms. Immunoblotting analysis using sera from microfilaremic and amicrofilaremic dogs with D. immitis infection suggest that antigenic components in the low molecular weight region may be related to the anti-microfilarial mechanism of the host.

Comparison of three diagnostic tests for Dirofilaria immitis in a low-incidence area.

was obtained, tests were repeated by a different technologist. Microfilaria identification using the alkaline phosphatase technique was done to differentiate between D. immitis and Dipetalonema reconditum on all samples in which microfilariae were observed. Clinical follow-up was obtained for 11 of the 12 dogs that had any positive test result by reviewing the medical record or discussing the case with the clinician. The results of the laboratory tests are summarized in Table 1. Samples from 1,347/1,359 dogs (99.12% of samples submitted) were negative for microfilariae and adult D. immitis antigen using both commercial kits. Microfilariae, subsequently identified as D. immitis, were observed in blood samples from 7 dogs. Four of these dogs also had adult D. immitis antigen detected by both kits. Two microfilaremic dogs were negative for D. immitis adult antigen with both kits. Blood from 1 dog that was positive for microfilariae was serologically positive for adult antigen with only 1 of the commercial kits. Five dogs were serologically positive for adult D. immitis antigen but negative for microfilariae. The 5 dogs that had positive tests for adult D. immitis antigen but negative filter tests were considered otherwise healthy. They had no evidence of circulatory or respiratory compromise. Adulticide treatment was recommended for each of these dogs, but owners chose to administer ivermectin or milbemycin oxime monthly to their pets. Owners were advised to observe their dogs carefully and to restrict their pets’ exercise for at least 2 days following the administration of the monthly heartworm preventative. Adverse reactions were not reported in any of these dogs. Six of the 7 dogs that tested positive for D. immitis microfilariae underwent treatment for the elimination of adult D. immitis and microfilariae, regardless of the results of the adult antigen test. Additional information could not be obtained for 1 dog that was positive on all 3 tests. Treatment in 1 dog that had positive results with all 3 tests was initiated as an emergency procedure when the dog developed acute respiratory distress. Two dogs that were microfilaremic were adult antigen negative using both kits, and 1 microfilaremic dog was antigen negative with 1 kit but antigen positive with the other. Such results are likely to occur if the adult worm burden is low. In these cases, the sensitivity of the tests for detection of adult antigen is decreased. 2,3,6,8,9 Based on physical examination and radiographs, the clinicians strongly suspected that 2 of these 3 dogs had few adult D. immitis in their right ventricles, and the dogs were treated with adulticides before beginning preventative therapy. The presence of adult D. immitis infection in the right ventricle in the third dog was questionable, yet the owner preferred to have the dog treated with adulticide before preventative therapy was begun. Although the number of infected dogs detected in this study was small, infection in 5 of 12 animals would have gone undetected if only a filter test, accompanied by acid phos

Clinical and laboratory changes after administration of milbemycin oxime in heartworm-free and heartworm-infected dogs

Summary Adverse reactions to oral administration of milbemycin oxime were investigated in heartworm (hw)-free and hw-infected dogs given either the minimal hw prophylactic dose (0.25 mg/kg of body weight) or the hookworm anthelmintic dose (0.5 mg/kg). In 12 hw-free control dogs treated with lactose excipient (100 mg/kg), abnormal signs were not observed. There were no differences between the 2 doses in prevalence of clinical signs of disease and laboratory test results. In 60 hw-free dogs (50 dogs administered the low dose, and 10 dogs given the high dose) and 46 nonmicrofilaremic hw-infected dogs (35 dogs administered the low dose, and 11 dogs given the high dose), only a transient and slight paleness of the visible mucous membranes, intestinal hyperperistalsis, or both were observed in some dogs. In 77 microfilaremic (mf) dogs (41 dogs administered the low dose, and 36 dogs given the high dose), weakness or loss of appetite was observed in 13 dogs (16.9%). Paleness of the visible mucous membranes was observed in 16 dogs (20.8%), intestinal hyperperistalsis was observed in 27 dogs (35.1%), and respiratory signs, such as mild labored respiration, were observed in 13 dogs (16.9%). Dullness of heart sounds was noticed in 4 dogs (5.2%). In 12 (9 dogs administered the low dose, and 3 dogs given the high dose) of 89 mf dogs (13.5%), adult heartworms migrated from the pulmonary arteries to the right atrium, causing signs of caval syndrome, including heart murmurs, jugular pulsations, and weakness. C and neutrophil counts increased slightly at 3 hours. Serum alanine transaminase, alkaline phosphatasIn hw-free dogs, rectal temperature tended to decrease gradually, and heart and respiratory rates were transiently decreased. Mean blood pressure, rbc count, serum total protein concentration, serum osmolality, and serum sodium (Na) and potassium (K) concentrations decreased 3 or 6 hours after administration. The total WBe, and lactate dehydrogenase activities did not change significantly. In hw-infected, nonmicrofilaremic dogs, trends in laboratory changes were almost the same as those in hw-free dogs. In mf dogs, circulating microfilaria count decreased starting at 6 hours after treatment. Heart rate, rbc count, and serum total protein concentration decreased transiently, similar to values in hw-free dogs. Rectal temperature, respiratory rate, and serum enzyme activities increased, and neutrophil and eosinophil counts, serum osmolality, and serum Na and K concentrations decreased. Changes in test results for dogs with hw migration were almost the same as those in mf hw-infected dogs. Heartworm-free and nonmicrofilaremic hw-infected dogs did not have clinically relevant problems after treatment with milbemycin oxime. Administration of milbemycin oxime does not involve risks for these dogs. However, reactions in mf dogs appear to be related to the death of microfilariae, and caution must be used when these dogs are treated with this compound.

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, > 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.

犬糸状虫抗原検出キットの評価と虫体摘出後の血中抗原価の変化

The sensitivity and specificity of a commercial heartworm (Dirofilaria immitis) antigen test kit were evaluated in 100 dogs with and without heartworm infections. All 25 microfilaremic heartworm-infected dogs, in which the number of heartworms ranged from 3 to 91, were identified correctly as positive (sensitivity: 100%). The test detected occult infection in 18 of 25 (72.0%) dogs. All heartworm-free dogs with (25 dogs) and without (25 dogs) roundworm (Toxocala canis) infection were indentified as negative (specificity: 100%). The accuracy in all 100 dogs tested was 93%. Changes in the circulating antigen levels after surgical heartworm removal were examined in 10 dogs using this kit. By surgical treatment, all heartworms were removed in 6 dogs, and removal efficiency ranged from 65.5 to 86.7% in the 4 other dogs. After heartworm removal, the circulating antigen levels declined in all dogs. The mean antigen level in 10 dogs was×730±1238 before removal, ×84±98 at 4 weeks after removal, ×36±39 at 8weeks after, and ×14±8 at 12 weeks after. There were no dogs in which the circulating antigen disappeared.

Prevention of adverse reactions following milbemycin D administration to microfilaremic dogs infected with Dirofilaria immitis.

Some adverse reactions such as shock-like reaction and dirofilarial hemoglobinuria (caval syndrome) occasionally occurred in microfilaremic dogs following milbemycin D (Milbe) administration. This study was carried out to seek the prevention of these adverse reactions. In two groups containing 16 and 9 dogs respectively which were administered either chlorpheniramine maleate (1 mg/kg) or indomethacin (2.5 mg/kg) simultaneously with Milbe (1 mg/kg), the incidence of clinical signs such as the pale color of the visible mucous membranes, respiratory disorders, caval syndrome and shock-like reaction as well as changes in clinical parameters such as RBC and WBC counts, WBC profile and serum total protein, were almost equal to that observed in the group administered Milbe alone. In 41 dogs administered prednisolone (1 mg/kg) simultaneously with Milbe (1 mg/kg), no shock-like reaction was observed. Changes in clinical parameters were different from those in the group administered Milbe alone, whereas some clinical signs of adverse reactions, including caval syndrome, were observed. These results indicated that prednisolone was effective for prevention of the shock-like reaction in microfilaremic dogs induced by Milbe.

犬糸状虫ミクロフィラリア寄生犬にイベルメクチンとジエチルカルバマジンを投与してみられる副作用の比較

The present study was designed to find out whether administration of ivermectin (IVM) to microfilaremic dogs causes the shock-like reaction that is well known as an adverse effect of diethylcarbamazine (DEC). Both IVM and DEC were given to the same dogs, and the occurrence of adverse reaction was compared between the two drugs. Thirty dogs, which had an average microfilaria count of 395 (11-3, 309)/20μl of peripheral blood, were used for the experiment. A single oral administration of IVM was given to each of the 30 dogs at the dose of 10μg/kg, and observation was continued for the following 7 days. In this period, there appeared no abnormality in clinical signs, WBC count, or in any of 13 items ofplasma biochemical test. There was no reduction in microfilaria count either after the administration. The DEC experiment was carried out after the IVM experiment was completed. A single oral administration of DEC-citrate was given to each of the 30 dogs at the dose of 6mg/kg. For the following 24 hours, the animals were observed for the appearance of clinical signs, and examined with the same test protocol as in the IVM experiment. Five dogs (16.7%) were judged to have suffered the shock-like reaction from one to three hours after the administration of DEC. In these dogs, the clinical signs were accompanied by an increase in WBC and marked rises in GOT, GPT and ALP, which indicated severe hepatic impairment. Even in the other 25 dogs that showed no clinical signs, similar changes were observed in the WBC and the biochemical test values, although the changes were milder.Thus, no abnormality associated with IVM administration was observed, even in the dogs that showed theDEC-associated shock-like reaction. Therefore, it appears that IVM has no tendency to cause the shock-like reaction which can occur following DEC administration in microfilaremic dogs.

Effects of heartworm and intestinal parasitic infections on hematology and peripheral lymph node cytology in louisiana dogs.

Hookworm infected dogs had higher blood basophil counts than hookworm negative dogs. Dogs with occult heartworm infection (i.e., without microfilaremia) had higher blood neutrophil counts than heartworm negative dogs. The percentage of eosinophils in peripheral lymph nodes was higher in heartworm infected, microfilaremic dogs than in both occult infected dogs and heartworm negative dogs. Heartworm or intestinal parasitic infection had no other significant effects on hematologic parameters or peripheral lymph node cytology.

Dirofilaria immitis: Identification and characterization of circulating parasite antigens

A method for the identification of circulating parasite antigens in filarial nematode infections was developed using canine infections with Dirofilaria immitis as a model. Filarial antigens ranging in molecular weight from 211 to 13 kDa were extracted from the sera of microfilaremic dogs by a solid phase immunobinding procedure and identified by immunostaining of Western blots. A major antigen of 104 kDa was selected for further characterization. The 104 kDa circulating antigen showed antigenic and biochemical identity with 104 kDa peptides found in extracts of adult male and microfilarial stages of the parasite. The 104 kDa peptide was antigenically stable under a variety of storage conditions. Its potential as a diagnostic target is discussed.

Stimulation of neutrophil respiratory burst and iodination reaction by opsonized microfilariae of Dirofilaria immitis.

It has been shown that the interaction of Dirofilaria immitis microfilariae (Mf) opsonized with sera from infected but amicrofilaraemic dogs (occult dogs) stimulated the respiratory burst and degranulation of neutrophils as measured by chemiluminescence and iodination. Sera from normal and microfilaraemic dogs gave either low level or non-significant reactions. Since the sera required were also those required for neutrophil-mediated cytotoxicity to D. immitis Mf in vitro, the results suggested that the products of oxygen reduction as well as the myeloperoxidase system could be involved in the killing of Mf by neutrophils. However, whether these pathways have a major role to play in neutrophil-mediated cytotoxicity to Mf is uncertain, as various chemical and enzymatic inhibitors of the products of the respiratory burst were unable to prevent or reduce cytotoxicity. Azide, which is a known inhibitor of the iodination reaction, also failed to reduce cytotoxicity.

Quantitative and qualitative changes in the humoral response of dogs through the course of infection with Dirofilaria immitis.

The present study compared the humoral response of dogs which developed microfilaremic or occult forms of dirofilariasis following experimental infection with Dirofilaria immitis L3 larvae. Quantitative analysis by ELISA revealed that antibody levels to adult somatic (AS), excretory-secretory (ES), and microfilarial (MF) antigens were highest during the patent phase of infection in dogs with either form of dirofilariasis. Patent sera from dogs destined for occult infections contained anti-AS and anti-MF antibody concentrations of 1,572 and 1,004 micrograms/ml, respectively, while microfilaremic-bound dogs contained 1,044 and 906 micrograms/ml, respectively. Chronic sera (430 days post infection) from occult dogs contained anti-AS and anti-MF antibody levels of 982 and 600 micrograms/ml, respectively, which were higher than in microfilaremic dogs. The antibody response to ES antigen was generally 10-fold less in absolute antibody concentrations at all time points tested. Immunoperoxidase staining of antigens transferred to nitrocellulose revealed the presence of several antigenic proteins which were recognized by occult, and to a lesser extent or not at all, by microfilaremic dogs. Sera drawn from occult-bound dogs 280 days post-infection, a time corresponding to microfilarial clearance (transition phase), contained higher antibody activity to microfilarial proteins weighing 47.5, 42.0, 34.2, and 22.4 kilodaltons compared to the microfilaremic dogs. This difference in antigen recognition became more apparent during the chronic phase of infection.

Liver Changes, Following Diethylcarbamazine Administration, in Microfilaremic Dogs Infected with Dirofilaria immitis

Livers of dogs which were microfilaremic due to infection with Dirofilaria immitis were studied after the administration of diethylcarbamazine. Most dogs had a severe shock-like clinical reaction. The main hepatic features were congestion and hemorrhage around the central vein and irregularly scattered foci of inflammation. The severity of the vascular reaction was associated with the number of microfilariae in the livers. Mast cells associated with the vascular reaction were not degranulated, suggesting that histamine-mediated anaphylaxis was not the cause of the reaction. The observations suggest that the mediator of the hepatic vascular reaction originates from the microfilariae and is influenced or activated by diethylcarbamazine.

Immunological and ultrastructural aspects of the cell-mediated killing of Dirofilaria immitis microfilariae.

Neutrophils in the presence of serum from dogs with occult Dirofilaria immitis infections were shown to be cytotoxic to D. immitis microfilariae recovered from the blood of microfilaraemic dogs. This cytotoxicity was correlated with the presence of IgM and IgG antibodies on the cuticular surface of microfilariae incubated in the sera from occult dogs. Such antibodies were not observed on the surface of microfilariae incubated in sera from microfilaraemic or normal dogs. The neutrophil attack was directed at the cuticular crypts, at which sites the worms appeared to be structurally most vulnerable because of the absence of the outer layer of the cuticle. The IgM antibodies were shown to be bound preferentially to these sites. Our data suggested that the neutrophil-mediated toxicity involved both hydrogen peroxide release and degranulation.

Immunoglobulin interactions with surfaces of sheathed and unsheathed microfilariae.

The sheath surface of Brugia pahangi microfilariae (mf) and the cuticle surface of Dirofilaria immitis mf were compared with regard to the ultrastructural arrangement of neutral and charged polysaccharides and binding of immunoglobulins from dog sera. Brugia pahangi: mf demonstrated large amounts of neutral sugar and negatively charged sugars just above the sheath surface, projecting some distance from the surface, in addition to a dense layer of sulphated material on the sheath surface. Microfilariae from high microfilaremic dogs showed no innately bound IgG or IgM when examined fresh from serum nor did they bind IgG or IgM from normal (NDS) or infected (IDS) dog sera after 48 h maintenance in RPMI 1640. Many of these mf did bind IgG and IgM from hyperimmune dog serum (ImDS) and these immunoglobulins were found binding at a distance from the sheath similar to that of the sugars. Dirofilaria immitis mf demonstrated much less neutral sugar at the cuticle surface, no negatively charged sugars and a diffuse, rather than dense, distribution of sulfated material extending from the cuticle. The majority of these mf showed innately bound IgG and IgM. After 48 h maintenance in RPMI 1640 D. immitis mf bound and shed IgG and IgM from NDS, high microfilaremia D. immitis IDS and D. immitis mf ImDS at distinctly different rates.