Indirect fluorescent antibody test in occult dirofilariasis.

Indirect fluorescent antibody titers to Dirofilaria immitis microfilaria (IFA-mf) and peripheral eosinophilia were recorded from 15 to 52 months in ten experimentally infected dogs with occult dirofilariasis (heartworm infection without microfilaremia). Five dogs which were experimentally sensitized with D immitis microfilaria did not exhibit microfilaremia after inoculation with infective-stage larvae. In three other dogs, microfilaremia suddenly ceased after 4 to 7 months. In these three dogs, antimicrofilarial antibodies were detectable by IFA-mf test as soon as microfilaremia ended. In the remaining two dogs, which exhibited spontaneous occult dirofilariasis, antibodies were detected at the end of the prepatent period of 6 months. The presence of adult worms was confirmed by angiocardiography. Significant IFA=mf titers (greater than or equal to 1:8) persisted after successful treatment with an adulticide. Reinfection of treated dogs reestablished occult dirofilarasis. Eosinophilia was present in all dogs and peaked at about 3, 6, and 9 months after they were inoculated with infective-stage larvae. At necropsy, the ten dogs harbored gravid, reproducing adult worms in the heart and pulmonary arteries.     

Prevalence and epidemiological aspects of Dirofilaria immitis in dogs from Kayseri Province, Turkey

This study was conducted to determine the prevalence of Dirofilaria immitis infection and to investigate the risk factors related to heartworm disease in dogs from Kayseri, Turkey. Blood samples were collected from 280 dogs from May 2005 to March 2006 and were examined by membrane filtration-acid phosphatase histochemical staining and antigen Elisa techniques to detect circulating microfilariae and antigens of D. immitis, respectively. Of the total of 280 dogs, 27 were positive for D. immitis with a prevalence value of 9.6%. In addition 29.6% of positive dogs determined to have occult D. immitis infections. D. immitis was the only canine filarial parasite present in the study area. The mean number of microfilariae in infected dogs was 4730+/-5479 per ml of blood. The highest heartworm prevalence were observed in 7-10 age group (28.6%) followed by 4-6 (17.1%) and 0.5-3 (4.8%) age groups. The differences between 0.5-3 and other age groups were found significant, whereas no statistically significant difference was observed between 4-6 and 7-10 age groups. The infection was more prevalent in males, larger breeds and the dogs not on prophylaxis. No statistically significant difference was observed between stray and owned dogs. Our results suggest that heartworm treatment and prophylaxis should be considered in Kayseri Province.     

Comparison of four serotests for the detection of Dirofilaria immitis infection in dogs

Three hundred two dogs were tested with 4 serotests for heartworm antigen (AG) or antibody (AB) and with the Knott test. The 4 serotests evaluated were an enzyme-linked immunosorbent assay (ELISA) for adult heartworm-specific AB (AB-ELISA), a quantitative, indirect immunofluorescent assay (IFA) for adult heartworm-specific AB (AB-IFA), an IFA test for microfilaria (MF)-specific AB (MF-IFA), and an ELISA for adult heartworm AG (AG-ELISA). The presence of heartworms was ascertained in all dogs by necropsy examination. Of 302 dogs, 20 (6.6%) had heartworms in the heart at necropsy. Of infected dogs, 9 (45%) had occult infections. Test sensitivities were 75%, 95%, 70%, and 75% for the AB-ELISA, AB-IFA, MF-IFA, and AG-ELISA, respectively. Test specificities were 85% (AB-ELISA), 77% (AB-IFA), 87% (MF-IFA), and 99% (AG-ELISA). The best agreement between serotest results and necropsy findings was obtained with the AG-ELISA (97%). The 4 serotests detected 86% (AB-ELISA), 100% (AB-IFA), 67% (MF-IFA), and 78% (AG-ELISA) of the dogs with occult heartworm infection. A significant (P less than 0.05) association between intestinal parasitism and positive heartworm test results was found with only AB-IFA. Seemingly, the Knott test, or some other concentration method for detecting circulating MF should be the first heartworm test performed. If the examination for MF is negative, the dog has clinical signs, and radiographic findings are suggestive of occult heartworm infection, then a serotest for adult heartworm AG is recommended.     

Diagnostic criteria for an enzyme-linked immunosorbent assay for occult heartworm disease: standardization of the test system in naturally exposed dogs

The development of criteria for interpreting and reporting the results of an occult heartworm enzyme-linked immunosorbent assay to practitioners is described. The antigen is a saline extract of adult female Dirofilaria immitis. The cutoff absorbance A400 nm values were estimated, using 106 dogs free of infection. Any A400 nm value less than 0.526 is considered negative and values greater than or equal to 0.784 are positive. Intermediate A400 nm values are interpreted as suspect. Absorbance values for serum samples from 13 client-owned amicrofilaremic dogs revealed a bimodal distribution consistent with presumptive diagnosis based on clinical signs, which indicates that the test may be used to support a diagnosis of occult heartworm disease. The present serotest, however, is unable to distinguish microfilaremic dogs from noninfected dogs. Serum from dogs infected with other common helminths failed to crossreact with the D immitis antigen, with the exception of Dipetalonema reconditum.     

Comparative efficacy of four commercially available heartworm preventive products against the MP3 laboratory strain of Dirofilaria immitis

A controlled laboratory study was conducted to evaluate the efficacy of four commercial products administered as a single treatment for the prevention of heartworm disease caused by Dirofilaria immitis in dogs. Forty-four commercially sourced Beagle dogs, 6-7 months of age, were received at the test site (Auburn University, Department of Pathobiology) on Study Day (SD) -72 to begin acclimation. On SD -30, each dog was inoculated subcutaneously with 100 infective, third-stage D. immitis larvae (MP3 strain, TRS Laboratories, Inc., Athens, GA). On SD -1, 40 dogs weighing 18.2-25.3 lbs were ranked by decreasing body weight and randomized to five groups of eight dogs each. On SD 0, the dogs assigned to Group 1 were treated orally with ivermectin/pyrantel pamoate chewable tablets, Group 2 dogs were treated orally with milbemycin oxime flavored tablets, Group 3 dogs were treated with selamectin topical solution, and Group 4 dogs were treated with imidacloprid/moxidectin topical solution. Group 5 dogs remained nontreated. Dosages for dogs in Groups 1-4 were based on the individual body weight of each dog and current labeled dose banding for each commercial product. All dogs were fasted overnight prior to treatment. Food was returned four hours after treatment. Animals were observed for abnormal clinical signs involving eyes, feces, respiration, behavioral attitude, locomotion/musculature, or skin conditions at prescribed intervals immediately after treatment and at twice daily intervals thereafter. On SD 90, whole blood was collected and tested for adult heartworm antigen. On SDs 119/120, the dogs were euthanized and subjected to necropsy examination for recovery of adult D. immitis and/or worm fragments. At necropsy, all 8 dogs in the nontreated group were infected with adult D. immitis (34-70 worms/dog, geometric mean (GM)=51.6 worms/dog). One or more adult D. immitis and/or worm fragments were recovered from 7 of 8 of the dogs each in Groups 1-3 (87.5% were heartworm positive). The respective GM worm burdens/dog for Groups 1-3 was 2.3, 2.4, and 2.3 which resulted in 95.6, 95.4 and 95.5% efficacy, respectively. No worms were recovered from any of the 8 dogs in Group 4 resulting in 100% efficacy.     

Can heartworm prevalence in dogs be used as provisional data for assessing the prevalence of the infection in cats?

Cats are considered a susceptible host for Dirofilaria immitis; however, increased host resistance is reflected by relatively low adult worm burdens in natural and experimental infections; the prolonged prepatent period (8 months); the low level and short duration of microfilaremia; and the short life span of adult worms (2-3 years). From April to September 2006, 212 cats and 608 dogs, all exposed for at least one transmission season, were screened for D. immitis infection in a multi-center study in the Po River Valley in northern Italy. Cats were initially evaluated by antibody testing; positive subjects were followed up by antigen testing and echocardiography (and necropsy if death occurred). The prevalence in dogs was 29% by a modified Knott test and antigen testing compared with a prevalence of 4.7% in cats by an antibody test; six of these infections (2.8%) were confirmed by the follow-up evaluations. This field study demonstrated that the prevalence of heartworm infection in cats in this area is within the expected limits of 9-18% of the prevalence in dogs. Antibody testing likely underestimates the real prevalence of D. immitis infection in cats. These results also emphasize the importance of preventive treatment in cats.     

Echocardiographic quantification of Dirofilaria immitis in experimentally infected cats

The safety of heartworm preventives in heartworm-positive cats has traditionally been evaluated using adult Dirofilaria immitis removed from infected dogs and surgically implanted into the cats. An alternate study model uses infective larvae to establish adult infections in cats. Unfortunately, the number of adult worms resulting from the latter method varies widely from none to more than 30, both unacceptable for studies of natural heartworm infection and for studies evaluating product safety in heartworm-infected cats. We sought to determine infection severity in experimental infections via echocardiography to reduce the chances of enrolling uninfected and heavily infected cats into a study. Eighty adult cats were each inoculated with 60 infective D. immitis larvae and maintained for 8 months to allow for the development of adult worms. Antigen and antibody testing, as well as echocardiographic imaging, were performed to confirm and estimate adult worm burdens. Approximately 8 and 12 months post-infection, echocardiographic examination was performed to confirm and enumerate adult D. immitis populations in the cardiovascular system. Worm burdens were stratified as 0, 1-3, 4-11, and > 11 adults, with 0 being considered uninfected and more than 11 considered too heavily infected to be relevant for anthelmintic studies. Cats with clinically relevant infections (1-10 adults) subsequently received multiple treatments with the investigational drug, and worm burdens were confirmed by necropsy 30 days following the final treatment. Worm burden estimated with echocardiography correlated well, but not precisely, with post-mortem counts (p < 0.001, r2 = 0.67). Echocardiography under-, over-, and exactly estimated heartworm burden 53%, 27%, and 22% of the time, respectively. Although the correct category (0-4) was determined by echocardiography in only 54% of cats, positive cats were distinguished from negative cats 88% of the time and the heaviest infections (> 11) were correctly categorized 95% of the time. Both false negative and false positive results were observed. We conclude that echocardiography is useful for detecting mature experimental heartworm infections, identifying cats that have rejected mature infection, and detecting very heavy heartworm burdens, but it is only moderately accurate in classifying lesser burdens. While echocardiography cannot be relied upon to consistently determine the exact heartworm burden in experimentally infected cats, it is useful in stratifying worm burdens for anthelmintic safety studies.     

The occurrence of Dirofilaria immitis in dogs in South Australia

Heart, lung and samples of blood from 230 dogs were examined for infections of filarial parasites. Dirofilaria immitis worms and microfilariae were detected in one dog. Blood samples from a further 1428 dogs were examined for microfilariae and 22 were found to be infected. Eighteen dogs were infected with D immitis microfilariae and four with Dipetolonema reconditum microfilariae. The histories were available for 18 of the dogs infected with heartworm and only seven dogs had not travelled outside South Australia. It was concluded that heartworm infection was endemic in South Australia but the apparent prevalence was only about 1%.     

Efficacy of selamectin in the prevention of adult heartworm (Dirofilaria immitis) infection in dogs in northern Italy

The efficacy of a novel avermectin, selamectin, was evaluated for the prevention of heartworm disease (adult Dirofilaria immitis infection) in 120 dogs (aged 9 months to 13 years at enrolment) presented as veterinary patients. The study was conducted at five veterinary practices in a heartworm hyperendemic region of northern Italy. Dogs were allocated randomly in a 2:1 ratio to treatment with either selamectin or ivermectin. Treatments were administered at monthly intervals for 6 months during the heartworm transmission season (May-November). Selamectin was applied topically in a single spot to the skin on each animal's back at the base of the neck in front of the scapulae as a unit dose that provided at least the minimum recommended dosage of 6mgkg(-1) (range, 6-12mgkg(-1)). Ivermectin (6microgkg(-1) of body weight) was administered orally at monthly intervals, in accordance with the manufacturer's product label recommendations. Study day 0 was defined individually for each dog as the day of first treatment administration. Efficacy was assessed on the basis of the absence of D. immitis microfilariae and adult heartworm (D. immitis) antigen in tests conducted on days 180 and 300. There were no adverse clinical signs arising due to treatment with selamectin and no drug-related mortalities. The prevention rate for D. immitis microfilariae and adult heartworm antigen was 100% for both selamectin and ivermectin. Thus, selamectin administered as a unit dose, providing at least the recommended minimum dosage of 6mgkg(-1), at monthly intervals during the heartworm transmission season was safe and 100% effective in the prevention of heartworm disease in dogs presented as veterinary patients.     

An investigation of the role of immunologic factors in anemia associated with canine heartworm disease

Direct Coombs' antiglobulin tests were performed on 80 dogs with patent Dirofilaria immitis infection and 170 dogs negative for microfilaria of D. immitis. Presence or absence of anemia was determined by hematocrit in 55 of the heartworm negative dogs and 68 of the dogs with heartworm disease. Heartworm infected dogs showed a higher incidence (37%) of anemia than noninfected dogs (14.5%). Anemia was most prevalent in two groups of dogs with heartworm infection, one group showing vena caval syndrome (91%) and the other occult dirofilariasis (62.5%). These latter two groups of dogs also showed a significantly higher number of positive Coombs' reactions at 37 degrees C than other dogs with heartworm disease and the noninfected dogs. The number of positive Coombs' reactions at 4 degrees C among the total of 80 dogs with heartworm infection was significantly higher than that for dogs without heartworm disease. However, there was no positive correlation between anemia and the outcome of the Coombs' test at either temperature. These findings do not suggest that immunologic factors play a major role in the pathogenesis of anemia in dogs with heartworm disease.     

Evaluation of a single injection of a sustained-release formulation of moxidectin for prevention of experimental heartworm infection after 12 months in dogs

OBJECTIVE: To evaluate the efficacy of a single injection of a sustained-release formulation of moxidectin in preventing heartworm (Dirofilaria immitis) infection for 12 months in dogs. ANIMALS: 14 healthy dogs. PROCEDURE: Group A (nontreated control dogs; n = 6) received sterile vehicle administered SC, and group B (treated dogs; n = 6) received a sustained-release formulation of moxidectin administered SC. All dogs were housed in a heartworm-endemic area for 11.5 months, and heartworm antigen and modified Knott tests were performed monthly. All dogs (including 2 additional control dogs [group C]) were then inoculated with infective-stage larvae (L3) of D. immitis, and 4.5 months later, all dogs were euthanatized and post-mortem examinations were performed. Adult D. immitis were counted and measured, and their age was estimated. RESULTS: All dogs in groups A and C were infected with young (4- to 4.5-month old) adult male and female D. immitis. No dogs in group B were infected with heartworms. CONCLUSIONS AND. CLINICAL RELEVANCE: The age of heartworms recovered suggests that infection was the result of experimental inoculation and not natural exposure to mosquitoes during the 11.5-month period the dogs resided in a heartworm-endemic area. A single SC injection of a sustained-release formulation of moxidectin was effective in providing protection against heartworm infection after 12 months in dogs. This formulation is a convenient method of heartworm prophylaxis that could eliminate the problem of poor owner compliance.     

Prevalence and distribution of Dirofilaria immitis in domestic dogs from Ankara and vicinity in Turkey

A total of 280 dogs in Ankara and vicinity were examined from November 2000 through December 2001 by the PetChek antigen ELISA and polycarbonate filter-acid phosphatase histochemical staining techniques for Dirofilaria immitis infection. Dirofilaria immitis (heartworm) infection was found in 26 dogs (9.3%), all of which were kept always outdoors and non prophylacted by their owners. The distribution of heartworm infected dogs was not uniform. Infection with D. immitis was correlated with differences in age and sex. But, no significant association was found.     

Epidemiological aspects of filariosis in dogs on the coast of Paraná state, Brazil: with emphasis on Dirofilaria immitis

The present study determined the prevalence and geographical distribution of Dirofilaria immitis and other filariae, from dogs in littoral areas of Paraná state, in Brazil. This survey spanned eight months, between 1998 and 1999, and was also designed to compare the efficacy of different tests for diagnosis of heartworm infection in that area. Blood samples were collected from 256 native-owned dogs distributed along the Paraná coastal area. Five diagnostic procedures were used: direct smear examination, the Knott's modified test, filtration assay, and two heartworm antigen detection kits. A follow-up imaging exam was performed to support the heartworm diagnosis. The imaging diagnosis included radiographic and ultrasonographic exams of six dogs that had positive results for the heartworm antigen detection kits, but showed different microfilarial burdens. The presence and severity of radiographic and ultrasonographic signs were compared with the results obtained in microfilariae detection and antigen tests. Diagnostic parasitology results indicated that 31.25% of the dogs were microfilaremic. Three different microfilariae were recovered: D. immitis, Dipetalonema reconditum, and the third (mf3) was not identified. D. reconditum was the species with the highest prevalence: 22.6%. In general, D. immitis prevalence was 5.47% (28.57% occult infections), but it varied along the coast and the range was from 0 to 20%. No correlation could be established between the overall scores for microfilarial counts (small or large numbers) and the severity of radiographic results or the likelihood of detecting filariae in the pulmonary artery using echocardiography. The finding of a different type of microfilaria (mf) suggested the existence of a third species in Paraná state, whose prevalence was 4.68%. These results show that to obtain a reliable diagnosis of heartworm infection, antigen detection kits are indicated. Knott's test or filtration should be performed to confirm microfilaremia and not for diagnosis of heartworm infection. Imaging tests support parasitology exams and add more about severity of infection. The northern areas, specially Guaraque?aba and Ilha das Pe?as, presented the highest number of heartworm-infected dogs.     

DISTRIBUTION AND DIAGNOSIS OF DIROFILARIASIS AND TOXOCARIASIS IN AUSTRALIA

SUMMARY: The prevalence of Dirofilaria immitis infection in dogs continues to increase in the temperate east coast zone of Australia (and is extending further south into New South Wales and Victoria). However, the infection rate has not changed in the tropics over the past 10 years where it would appear that a maximum infection rate of 90% occurs in a given Australian dog population. Twelve percent of Brisbane dogs had occult dirofilariasis and it is suggested that the proportion of occult infections was probably higher in the tropics. Dogs of all breeds appear equally susceptible to D. immitis with infection being more common in older male dogs. The level of microfilaraemia was, occasionally, proportional to the number of heartwprms per dog. Toxocara canis was present in about 75% of dogs from all areas studied except in Central Australia where the level of infection was much lower.
Immunodiagnosis of D. immitis and T. canis with high specificity and sensitivity was achieved by cyanogen bromide indirect fluorescent antibody and cell-mediated immunity tests using parasite antigens purified by affinity chromatography. These tests enabled occult dirofilariasis to be differentiated from unrelated canine cardiac and pulmonary failure. Such immunodiagnosis can aid in the early diagnosis of dirofilariasis particularly in situations where no circulating microfilariae can be detected.
The prevalence of serum antibody in man to purified Dirofilaria and Toxocara antigens was proportional to the incidence of respective canine infections at each location.     

Dirofilaria immitis and its potential mosquito vectors in central New York State.

Screening of 25,822 dog blood samples indicated approximately a 1% infection rate with Dirofilaria immitis. Dipetalonema reconditum microfilariae were found in approximately 2% of 1,876 feral dogs examined. Laboratory experimentation indicated that 6 of 10 local mosquito species examined allowed successful extrinsic incubation of D immitis. Indices of experimental infection indicated Aedes triseriatus and Anopheles quadrimaculatus were excellent hosts for D immitis, but other factors considered, Aedes vexans probably served as the primary vector of D immitis. Coquillettidia perturbans and Culex pipiens-restuans group were considered unsuitable hosts and thus unlikely vectors of canine heartworm. Natural filarial infections were found in An quadrimaculatus and Ae vexans.     

The enzyme-linked immunosorbent assay for detecting antibodies against Dirofilaria immitis in dogs

Antisera prepared in mice by injection of antigens from Dirofilaria immitis, Toxocara canis, Dipylidium canium and Fasciola hepatica and sera from Dirofilaria-infected and non-infected dogs were tested at different dilutions using an enzyme-linked immunosorbent assay (ELISA). For this ELISA, adult D. immitis antigen was fractionated by gel filtration methods and then absorbed with immunoadsorbent-fixed IgG fractions from mouse sera immunized with various parasites. The results indicated satisfactory discrimination between antisera to D. immitis and those to other parasites. A significant ELISA O.D. value was considered to be greater than or equal to 0.30 with a 1 : 250 dilution of the dog sera. However, the lack of significant differences between the O.D. value of microfilaraemic and amicrofilaraemic infections was observed. These facts suggest that the use of immunoadsorbent chromatography for canine dirofilariasis is especially useful for purifying antigens and eliminating cross-reactions against other parasitic infections, when immunological methods are used for serodiagnosis.     

Enzyme immunoassay of Dirofilaria immitis-specific IgE in infected dogs

An ELISA procedure was developed for monitoring the specific IgE response in dogs to Dirofilaria immitis infection. The results of this assay correlated well with, and appeared to be more sensitive than, the passive cutaneous anaphylaxis test. The IgE ELISA values of the positive reference serum and the passive cutaneous anaphylaxis test results showed that a serum to negative absorbance ration of 1.45 was statistically significant for discrimination and was used to evaluate the specific IgE response in the sera from 90 clinically diagnosed heartworm cases. This ELISA procedure was more sensitive, as it detected 78% of the 90 cases as compared to a detection rate of only 43-47% by IgG ELISA or IFA. Sera obtained from 23 experimentally infected dogs at 4-week intervals for 20 weeks post-infection, were assayed for D. immitis-specific IgE by ELISA. A group of the infected dogs was also treated with diethylcarbamazine during the course of infection. All the experimentally infected dogs developed a specific IgE response, with treated dogs generally responding earlier.     

Comparison of serologic tests for detection of antigen in canine heartworm infections

In 30 random-source dogs, we determined sensitivity and specificity of 5 serologic tests for detection of canine heartworm antigens. Seventeen of the dogs were infected naturally with adult Dirofilaria immitis, and 4 of the infected dogs were amicrofilaremic. The ability of the serologic tests to predict whether a dog was infected or uninfected (overall test accuracy) ranged from 73 to 97%. Sensitivity was not affected by circulating D immitis microfilariae, but was markedly influenced by the number of adult D immitis present. False-positive reactions were rare and were not associated with intestinal parasites or Dipetalonema reconditum microfilariae. Modifications of some of the test procedures were necessary to maximize test accuracy and reproducibility. These modifications and other technical details might limit the usefulness of some of the tests in a veterinary practice.     

Serologic prevalence of Dirofilaria immitis,Ehrlichia canis,and Borrelia burgdorferi infections in Brazil

Dogs infected with Dirofilaria immitis, Ehrlichia canis, or Borrelia burgdorferi may show nonspecific clinical signs or may be asymptomatic. In Brazil, E. canis and D. immitis infections are frequently diagnosed based on the presence of classical signs; however, serologic tests are seldom performed to confirm the presence of infection. To estimate the seroprevalence of these three canine diseases in Brazil, 2,553 dogs presented at veterinary practices for various tests, routine treatments, or examinations were evaluated by an in-office commercial ELISA test kit (SNAP 3Dx, IDEXX Laboratories). Each dog was examined by the veterinarian, and a whole-blood sample was collected and immediately tested for the simultaneous detection of B. burgdorferi and E. canis antibodies and D. immitis antigen. D. immitis infection was detected in 51 dogs (2.0%) and E. canis antibodies were present in 505 dogs 19.8%). Only one dog tested positive for B. burgdorferi antibodies.     

Two-dimensional echocardiography for diagnosis of dirofilariasis in nine dogs

Two-dimensional echocardiography (real-time ultrasonography) of the heart was used to detect adult Dirofilaria immitis in the right ventricle and atrium of a 2-year-old Labrador Retriever with occult heartworm disease and in 8 microfilaremic dogs. Adult worms were easily identified as parallel echogenic lines separated by a hypoechoic region. Images of worms fixed in formalin after recovery from dogs with experimentally induced heartworm infection had an identical appearance. The sensitivity of this imaging procedure was tested on the 8 dogs with naturally acquired infection, and worms were observed in the right ventricle of 7 of the dogs. Two-dimensional echocardiography appears to be a sensitive noninvasive procedure, which can be used in combination with thoracic radiography and serotests to improve accuracy of diagnosis of canine occult heartworm disease.