Heartworm Antigen Test Research Articles

Heat treatment and false-positive heartworm antigen testing in ex vivo parasites and dogs naturally infected by Dirofilaria repens and Angiostrongylus vasorum

BackgroundHeartworm antigen testing is considered sensitive and specific. Currently available tests are reported as detecting a glycoprotein found predominantly in the reproductive tract of the female worm and can reach specificity close to 100%. Main concerns regard sensitivity in the case of light infections, the presence of immature females or cases of all-male infections. Research and development have been aimed at increasing sensitivity. Recently, heat treatment of serum prior to antigen testing has been shown to result in an increase in positive antigen test results, presumably due to disruption of natural antigen–antibody complexes. Cross-reactions in dogs with both natural and experimental infections with Angiostrongylus vasorum and Spirocerca lupi have been reported, but cross-reactions with other helminths have not been extensively studied. In order to evaluate potential cross-reactivity with other canine and feline parasites, two studies were performed. Study 1: Live adults of Dirofilaria immitis, Dirofilaria repens, Toxocara canis, Toxocara cati, Dipylidium caninum, Taenia taeniaeformis and Mesocestoides spp. larvae were washed and incubated in tubes with saline solution. All worms were alive at the time of removal from the saline. Saline solutions containing excretory/secretory antigens were then tested for heartworm with six different, commercially available antigen tests. All results were evaluated blind by three of the authors. Study 2: Sera from dogs with natural infections by A. vasorum or D. repens, living in areas free of heartworm disease, were tested with the same tests before and after heat treatment (103 °C for 10 min).ResultsResults suggest that antigens detected by currently available tests are not specific for D. immitis. They may give positive results through detection of different parasites’ antigens that are normally not released into the bloodstream or released in a low amount and/or bound to antibodies. Tests may even detect antigens released by male D. immitis adult worms. D. repens appears to release more detectable antigens than the other worms studied.ConclusionsCross-reaction with A. vasorum and D. repens does occur in the field and could potentially occur with other helminths. Heat treatment decreases specificity by enhancing cross-reactivity.

Assessment of parasitological findings in heartworm-infected beagles treated with Advantage Multi\xae for dogs (10% imidacloprid\u2009+\u20092.5% moxidectin) and doxycycline

BackgroundAnecdotal reports support the position that the adulticidal heartworm treatment utilizing doxycycline and Advantage Multi®/Advocate® for Dogs (10% imidacloprid + 2.5% moxidectin) has successfully converted antigen-positive dogs to antigen-negative. To date, no controlled experimental studies have demonstrated the adulticidal efficacy of this treatment regimen. The aim of this study was to evaluate the parasitological and clinical efficacy of Advantage Multi® for Dogs (IMD + MOX) and doxycycline in heartworm-infected beagles.MethodsThis study utilized 16 dogs, 8 dogs in each of non-treated control and treated groups. A total of 16 adult Dirofilaria immitis (Missouri strain) were surgically transplanted into the jugular vein of each study dog. The treatment regimen of monthly IMD + MOX topically (per labeled dosage and administration) for 10 months and 10 mg/kg doxycycline BID orally for 30 days was initiated 30 days post-surgical transplant. Echocardiograms, radiographs, complete blood counts, clinical chemistry profiles, heartworm antigenemia and microfilaremia were evaluated every 4 weeks. Serum samples were assayed for heartworm antigen using the DiroCHEK® heartworm antigen test. The DiroCHEK® was performed according to the manufacturer’s recommendations and read using a spectrophotometer at 490 nm.ResultsAll dogs tested positive for the presence of heartworm antigen post-surgical transplant and prior to treatment. Heartworm antigen levels began declining in treated dogs 3 months post-treatment. Non-treated control dogs remained antigen-positive. No microfilariae were detected in treated dogs after 21 days post-treatment. At necropsy, adult heartworms were recovered from all non-treated control dogs with a range of 10–12 adult worms/dog for an average recovery of 10.6 adult heartworms/dog. In the IMD + MOX- and doxycycline-treated dogs, the range of adult heartworms recovered was 0–2 adult worms/dog, with five dogs having no adult heartworms present. The average adult heartworm recovery was 0.6/dog in the treated group. This treatment regimen demonstrated a 95.9% efficacy in eliminating adult heartworms (P < 0.0001).ConclusionsThis study demonstrated that this treatment regimen successfully eliminated D. immitis microfilariae by 21 days post-treatment, reduced heartworm antigen concentration over time, and had a 95.9% efficacy in the elimination of mature adult heartworms. Based on this study, we conclude that this treatment regimen is a relatively quick, reliable and safe option to treat canine heartworm infection as compared to other treatment regimens involving macrocyclic lactones, when the approved drug melarsomine dihydrochloride is unavailable, contraindicated or declined by an owner unable to afford the more costly treatment or concerned about the potential side effects.

ASSOCIATION BETWEEN POSITIVE CANINE HEARTWORM (DIROFILARIA IMMITIS) ANTIGEN RESULTS AND PRESENCE OF ACANTHOCHEILONEMA ODENDHALI MICROFILARIA IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

This study establishes a relationship between positive canine heartworm (Dirofilaria immitis) test results frequently observed in California sea lions (Zalophus californianus) and infection with the filarid nematode Acanthocheilonema odendhali. Four commercially available canine heartworm antigen tests were evaluated for cross-reaction with A. odendhali in California sea lions. Sera were tested from fifteen California sea lions with A. odendhali-associated microfilaremia, confirmed by blood smear, and with no evidence of D. immitis infection at necropsy. Ninety-five percent of tests were falsely positive for D. immitis. This study also determined that the prevalence of A. odendhali infection in stranded California sea lions from central California is approximately 23% by comparing the number of findings of mircofilaremia to the total number of California sea lions sampled at The Marine Mammal Center between 2005 and 2011, inclusive. Acanthocheilonema odenhali microfilaremia in California sea lions is likely to cross-react with canine heartworm antigen tests, and clinicians should interpret results with caution.

Prevalence of Dirofilaria immitis infection in dogs in Henan province, central China.

The heartworm Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariosis in dogs and cats, and also infects humans. However, there has been no study on dirofilariasis in dogs in central China. From March 2015 to February 2016, sera from 1176 randomly selected household dogs from Henan province, central China were examined for D. immitis antigen using the Canine Heartworm Antigen Test Kit. The overall seroprevalence of D. immitis in dogs in Henan province was 13% (155/1176). The prevalence was significantly higher in older dogs and dogs kept outdoors, compared to the younger ones and those sheltered indoors. No significant difference of prevalence was observed between sexes. The results suggest that the risk of exposure to D. immitis in dogs is high in Henan, and prophylaxis against the parasite is advisable to decrease the incidence of canine dirofilariosis in this region.

The prevalence and distribution of Dirofilaria repens in dogs in the Mazovian Province of central-eastern Poland.

The aim of this study was to determine the prevalence and distribution of canine dirofilariosis from different districts of the Mazovian Province in central-eastern Poland. 462 dogs aged from 1.5-14 years were examined for dirofilariosis. Blood samples were examined for the presence of microfilariae using the Knott method, as well as the method of Kingston and Morton, after centrifugation in haematocrit microtubes in order to determine the intensity of infection as the number of microfilariae in 60 microliters of blood. The species of microfilariae found were determined after staining on the basis of the morphological characters. Samples were also examined using Canine Heartworm Antigen Test Kit SNAP HTWM (IDEXX, USA) that allows the detection of circulating antigens of females of Dirofilaria immitis. The positive samples were examined using multiplex PCR assay for species confirmation. Microfilariae belonging to the species D. repens were found in the blood samples of dogs originating from the city of Warsaw and from 18 districts of Mazovian Province. The mean prevalence of that species observed in the province was 25.8%. The range of intensity counted with the number of microfilariae found in 60 microliters of blood amounted to between 1-150; median intensity 9 microfilariae. The highest prevalence, reaching 52.9% and the highest median intensity of infection reaching 28 microfilariae, was found in Radom district. The lowest prevalence, amounting to 4.2%, was registered in Grójec district; however, the lowest median intensity reaching 6 microfilariae was noted in Żyrardów district. The infected dogs had not been outside of Poland, which means that this is an autochthonous infection. Microfilariae and antigens of D. immitis were not found in any examined blood samples. Results of the presented study show that autochthonous dirofilariosis caused by nematodes D. repens commonly occurs in the area of the Mazovian Province.

Establishment of macrocyclic lactone resistant Dirofilaria immitis isolates in experimentally infected laboratory dogs.

BackgroundStrains of Dirofilaria immitis suspected of lack of efficacy (LOE) to macrocyclic lactone (ML) preventive drugs have been increasingly reported in dogs by practicing veterinarians since 2005 in the Lower Mississippi Delta region. If proven, and not controlled in the early stages, the emergence of ML drug resistance threatens to become a widespread problem in the US that may limit the effectiveness of current preventive drug treatment methods.MethodsTo validate practice reports, a statewide survey of Louisiana veterinarians was done to define the extent of the problem and identify focal ‘hotspots’ of reported ML LOEs using Geographic Information Systems (GIS) methods. The present study then utilized microfilariae (Mf) from two canine field cases from different state locations that fit criteria for a high index of suspicion of LOE against heartworms by ML drugs. Blood containing Mf from the canine field cases was used to infect and produce L3 in Aedes aegypti for experimental infection of two groups of dogs, each of which contained two laboratory dogs, one treated with prophylactic ivermectin (12 μg/kg) monthly for 6 months at twice the label dose (6 μg/kg), and one untreated control.ResultsBoth treated and untreated dogs from Group I and Group II developed patent D. immitis infections by 218 DPI and 189 DPI, respectively, as evidenced by a positive occult heartworm antigen test and microfilaremia by the Knott’s test. Mf counts gradually increased post-patency in test and control dogs. Infective larvae raised from microfilariae from the treated Group I dog were used to successfully establish a second generation isolate, confirming heritability of resistance in the face of a monthly ivermectin challenge dose of 24 μg/kg, given monthly for 3 months.ConclusionsThese experimental infection studies provide in vivo evidence of the existence of ML drug resistance in dogs infected by D. immitis L3 from suspect field LOE cases in the Lower Mississippi Delta. Results encourage further work on mechanisms underlying the emergence of ML resistance in D. immitis and development of evidence-based resistance management strategies for heartworm preventives in order to extend the useful life of current drugs.

Updated canine infection rates for Dirofilaria immitis in areas of Brazil previously identified as having a high incidence of heartworm-infected dogs.

BackgroundCanine heartworm infections were frequently diagnosed in Brazil before the new millennium. After the year 2000, the frequency of diagnosis showed a sharp decline; however, a few years later, new evidence indicated that the parasite was still present and that canine infection rates seemed to be increasing. Therefore, an updated survey of canine heartworm prevalence was conducted in several locations in south, southeast, and northeast Brazil.MethodsDogs from 15 locations having previously reported a high prevalence of heartworm infection were included in the survey according to defined criteria, including the absence of treatment with a macrocyclic lactone for at least 1 year. Blood samples from 1531 dogs were evaluated by an in-clinic immunochromatography test kit (Witness® Heartworm, Zoetis, USA) for detection of Dirofilaria immitis antigen. At each location, epidemiologic data, including physical characteristics and clinical signs reported by owners or observed by veterinarians, were recorded on prepared forms for tabulation of results by location, clinical signs, and physical characteristics.ResultsThe overall prevalence of canine heartworm infection was 23.1%, with evidence of heartworm-infected dogs detected in all 15 locations studied. There was a tendency for higher prevalence rates in environmentally protected areas, despite some locations having less-than-ideal environmental temperatures for survival of vector mosquitoes. Among physical characteristics, it was noted that dogs with predominantly white hair coats and residing in areas with a high (≥20%) prevalence of heartworm were less likely to have heartworm infection detected by a commercial heartworm antigen test kit than were dogs with other coat colors. In general, dogs older than 2 years were more frequently positive for D. immitis antigen than were younger dogs. Clinical signs of heartworm infections were rare or owners were unable to detect them, and could not be used for reliable prediction of the presence of heartworm.ConclusionsThese results indicate that the prevalence of D. immitis has increased in these areas of Brazil over the past few years. Small animal practitioners in these areas should include routine screening tests for heartworm infections in every dog’s annual evaluation protocol and make sure to have uninfected dogs on prevention.

Prevalence and distribution of Dirofilaria repens Railliet et Henry, 1911 in dogs in Poland.

In 2011-2013 1588 samples of dogs' blood were examined for dirofilariosis using Knott method, as well as the Kingston and Morton method. The species of microfilariae was determined on the basis of morphometric characteristics. Samples were also examined using the Canine Heartworm Antigen Test. Positive samples were examined using a multiplex PCR assay for species confirmation. Microfilariae belonging to the species D. repens were found in the blood samples of dogs from all the provinces of Poland. The mean prevalence of this species observed in Poland was 11.7%. The range of intensity of infection was counted using the number of microfilariae found in 60 microl of blood amounted to between 1 and 158, and the mean intensity was 18 microfilariae. Microfilariae and antigens of D. immitis were not found in any examined blood samples.

Heartworm ‘lack of effectiveness’ claims in the Mississippi delta: Computerized analysis of owner compliance – 2004–2011

A retrospective medical record review was conducted to identify factors from veterinary clinic medical records that may have contributed to suspected ineffectiveness of a heartworm preventive product. Patient records of 271 dogs, comprising 301 instances of positive heartworm antigen test results while the dogs were receiving heartworm preventive were evaluated. Nineteen veterinary practices in 17 counties and parishes in Arkansas, Louisiana, Mississippi, and Tennessee participated in the study. Records were selected by the veterinary clinics as representative of cases of suspected lack of effectiveness for a heartworm preventive, and for which an owner satisfaction claim had been filed with the manufacturer. Medical record data were entered into a software program, and a graphic representation was created to facilitate analysis of whether pet owners had purchased sufficient heartworm preventive for the dog to be compliant during the period when infection with Dirofilaria immitis could have led to the positive heartworm antigen test result for that patient (“window of infection”). In 243 (80.7%) cases, there was insufficient heartworm preventive purchased, leading to a gap in protection during the “window of infection”. In only five cases (1.7%) there were no purchase lapses or extenuating circumstances (underdosing of medication, multiple purchase gaps outside the established window of infection, or dogs have been diagnosed with heartworm infection more than once during the period studied). Half the cases were from multiple-dog households, and in many of these households, sharing of product between pets was acknowledged. In another 28% of the cases from multiple-dog households, more product was purchased than was needed for one dog, suggesting that the product was being shared between more than one pet. In most cases, there was at least one reason that a dog did not receive sufficient heartworm preventive product, placing the dog at risk of developing an infection with mature heartworms. Several actions were identified that veterinary clinics can take to improve heartworm disease prevention in their patients.

Outcome of a heartworm treatment protocol in dogs presenting to Louisiana State University from 2008 to 2011: 50 cases

Since 2008, the American Heartworm Society has recommended using a three-dose melarsomine protocol (a single intramuscular injection of melarsomine dihydrochloride at 2.5mg/kg, followed approximately 1 month later with two doses administered 24h apart) for all heartworm-positive dogs, with doxycycline given at 10mg/kg twice daily for 4 weeks prior to administration of melarsomine. To report the efficacy and side effects of this standard heartworm treatment protocol in 50 dogs presenting to our hospital from 2008 to 2011, information on the history, clinical, laboratory, and diagnostic imaging findings and treatment was obtained from medical records. When possible, additional follow-up information was obtained through telephone interviews with referring veterinarians and owners. Twenty-six dogs (52%) experienced minor complications, such as injection site reactions, gastrointestinal signs (vomiting, diarrhea, inappetance), and behavioral changes (lethargy, depression) during or after heartworm treatment. Twenty-seven dogs (54%) experienced respiratory signs (coughing, dyspnea) and heart failure attributed to progressive heartworm disease and worm death. Seven dogs (14%) died within the treatment period. Owners frequently reported behavioral changes, such as depression and lethargy, suspected to be secondary to pain. Fifty percent of owners surveyed indicated that, prior to the diagnosis, they either were not currently administering heartworm preventative, or they had recently adopted the dog from a shelter that did not administer preventatives. After treatment, 100% were administering heartworm preventatives to their pet. Eighteen dogs (36%) received a heartworm antigen test 6 months after adulticide therapy, 12 of which tested negative and six tested positive. Four of the dogs with a positive test at 6 months had negative tests 1 month later with no additional treatment. Adverse effects were common with the recommended protocol, but the majority of these were mild. Dogs in Class 1 (i.e., heartworm positive but otherwise largely lacking clinical evidence of disease) did not experience any major adverse effects or death.

Effects of Diagnostic Test Accuracy and Treatment Efficacy on the Occurrence of Suspected Failure of Heartworm Prophylaxis in Dogs

Food and Drug Administration Center for Veterinary Medicine (FDA/CVM) cited concern regarding failure of heartworm prophylaxis. The positive and negative predictive value of the heartworm antigen test is an estimate of the probability of adult heartworm infection. Assess the sensitivity, specificity, and predictive value of heartworm antigen tests. Explore the role of heartworm test accuracy and treatment with immiticide to generate reports of suspected failure of heartworm prophylaxis. Literature searches for published information on the accuracy of heartworm antigen tests and efficacy of immiticide for treatment of the adult heartworm. Weighted averages for heartworm antigen test sensitivity and specificity were 78.2 and 97.3%, respectively. Efficacy of immiticide by 2-injection or alternate dose protocols were 88.3 and 89.1%, respectively. Depending on prevalence, the positive predictive value of the heartworm antigen test ranged from 15 to 54% and negative predictive value from 99 to 99.9%. For a hospital testing 1,000 dogs per year, false-positive test results may vary from 24 to 27 dogs. If these dogs were on heartworm preventive, they may undergo treatment and be classified as prophylaxis failures. Ten percent of dogs who were treated and continued or placed on prophylaxis at the time of treatment may have adult heartworms when tested 1 year later and be presumed to represent failure of prophylaxis. When interpreting heartworm antigen test results, clinical signs, history, and regional prevalence of heartworm disease should be considered in estimating the predictive value of the test result. Limitations of test and treatment should be discussed with clients as part of the decision-making process.

Evaluation of a new in-clinic method for the detection of canine heartworm antigen

Canine heartworm is endemic in many parts of the world, and veterinarians rely on rapid in-clinic antigen tests to screen for this infection. Recently, an in-clinic, instrument-based rotor employing a colloidal gold agglutination immunoassay was launched in the marketplace (VetScan VS2 ® Canine Heartworm (HW) Antigen Test Kit; Abaxis, Inc.). Because of the widespread use of heartworm prevention and possible false negative test results in dogs with low heartworm burdens, the performance of the VetScan VS2 ® HW test and a commercially available in-clinic, membrane-based ELISA test (SNAP ® Heartworm RT Test; IDEXX Laboratories) was compared using samples from dogs with low heartworm burdens and/or low levels of circulating antigen. Ninety serum samples were evaluated using the two methods. Testing was performed according to the manufacturer's product insert by personnel blinded to sample status. The samples were derived from two populations: dogs with necropsy-confirmed heartworm status (40 with 1–4 female worms, 30 with no worms), and field dogs (20) confirmed positive for antigen by microtiter plate ELISA (PetChek ® Heartworm PF Antigen Test; IDEXX Laboratories). All 40 dogs with heartworms on necropsy were also confirmed to have circulating antigen by the PetChek HW ELISA. In necropsy-negative dogs ( n = 30), neither the VetScan VS2 HW nor SNAP HW tests detected heartworm antigen. Of the samples testing positive for antigen by PetChek HW ( n = 60), the VetScan VS2 HW and SNAP HW tests detected antigen in 15 and 56 samples, respectively. Percent agreement (plus 95% confidence interval) for each test relative to the PetChek HW qualitative result was 50% (40–60%) for VetScan VS2 HW and 96% (89–98%) for SNAP HW. Relative to the presence or absence of female worms at necropsy, agreement was 61% (50–72%) for VetScan VS2 HW and 99% (92–99.6%) for SNAP HW tests. It is clinically important that dogs with low heartworm burdens and/or low levels of circulating heartworm antigen be correctly identified by veterinarians in order to ensure prompt treatment, and the VetScan ® VS2 HW test does not appear to be as accurate as the SNAP HW or PetChek HW tests when performed on this subset of patients.

Monoclonal Gammopathy Associated With Heartworm Disease in a Dog

A 12-year-old, intact female, mixed Yorkshire terrier was evaluated for syncopal episodes, weakness, decreased appetite, and weight loss. Heartworm disease was diagnosed based on evidence of circulating microfilariae of Dirofilaria immitis on direct examination of blood smears and a positive SNAP heartworm antigen test. An immunoglobulin G (IgG) gammopathy, demonstrated by serum protein electrophoresis, was associated with heartworm disease in this dog. Response to treatment with both an adulticide and the microfilaricide ivermectin included remission of clinical signs and a decrease in the monoclonal gammopathy. To our knowledge, this is the first report of an IgG gammopathy associated with heartworm disease in the dog.

Incidence of positive heartworm antibody and antigen tests at IDEXX Laboratories: Trends and potential impact on feline heartworm awareness and prevention

Data from the IDEXX Laboratories Reference Laboratory Network were retrospectively examined for feline heartworm testing trends in testing frequency, geographic bias, and prevalence for the years 2000–2006. Examination of the data supports the commonly held view that veterinarians do not embrace heartworm disease testing or prevention in cats to the same degree they do in dogs. Despite significant awareness and adoption of heartworm testing and prevention in dogs, we hypothesized that heartworm testing rates are lower for cats than for dogs despite a significant prevalence of feline infection. This is important because a perceived low rate of infection in cats is likely to manifest in a low adoption of testing and prevention. In reality, the overall feline heartworm antigen-positive rate is significant – on average 0.9% over the period studied – and in some regions was estimated to be as high as 4.6%. This compares with an average canine heartworm prevalence rate of 1.2%, a feline leukemia virus prevalence of 1.9%, and a feline immunodeficiency prevalence of 1.0%. Based on the low rate of testing and these prevalence rates, practitioners are routinely missing cases of adult feline heartworm infections and the recently defined heartworm-associated respiratory disease (H.A.R.D). Increased antigen testing would result in detection of a significant number of positive cases. In addition, this population of infected cats would represent the “tip of the iceberg” relative to the greater number of cats that have early infection or are at risk for infection.

Computed tomography (CT) observation of pulmonary emboli caused by long-term administration of ivermectin in dogs experimentally infected with heartworms

Some studies have reported the adulticidal effect of long-term ivermectin (IVM) administration on adult heartworms in canines; however, there are no detailed reports on the course of the pulmonary artery embolism caused by the bodies of dead heartworms during the administration period. In this study, the pulmonary embolism caused over time by the dead worms was observed using computed tomography (CT). We subcutaneously inoculated 2 beagles with 100 infective third-stage larvae (L3) of Dirofilaria immitis. The dogs were orally administered a formulation containing 272 μg of IVM and 652 mg of pyrantel pamoate (Panamectin ® Chewables P272; Meiji Seika, Tokyo, Japan) at monthly intervals, beginning from 10 months after the subcutaneous inoculation. Along with IVM administration, periodic CT examination of the chest was performed. At 15 months after the initiation of IVM administration, the dogs were euthanized, the living heartworms were collected, and histopathological examination was performed. Starting from 1 month after the IVM administration, peripheral dilation of the pulmonary artery (suspected to be pulmonary embolism) and pneumonia were observed in the CT images; however, these findings improved over time. The appearance and disappearance of these lesions were observed in all the lobes during the IVM administration period. During this period, the clinical symptoms of pulmonary embolism were not recognized. After 1 month of IVM administration, chest radiographic examination revealed radiopaque lesions in 1 dog. Only some of the lesions detected by CT could be detected by radiography. Using echocardiography, heartworms were observed in the pulmonary arteries of both dogs from 6 months after subcutaneous inoculation to the end of the study period. Microfilaria disappeared from the peripheral blood at 1 month after IVM administration in 1 dog, and at 7 months in the other dog. The adult heartworm antigen test yielded positive results starting from 6 months after subcutaneous inoculation in 1 dog and after 7 months in the other dog; these results remained positive until the end of the study period. After the initiation of IVM administration, the ALP and CK levels were transiently elevated. The number of surviving adult worms collected at necropsy was 25 in 1 dog and 31 in the other. Histopathological examination revealed that the peripheral pulmonary artery dilation detected by CT was the embolus that resulted from the bodies of the dead heartworms. Moreover, vessel recanalization and inflammation along with lymphocyte infiltration around the vessels was observed. These results revealed that long-term IVM administration has a gradual adulticidal effect on heartworms in canines and embolism. From recovery findings showed pulmonary embolism in the CT image and histopathologic examination, long-term IVM administration can potentially be used for adulticidal treatment in clinical cases where it is difficult to perform surgical extirpation and administer arsenic therapy.

Are antigen test kits efficient for detecting heartworm-infected dogs at the southern distribution limit of the parasite in South America? Preliminary results

The aim of this study was to evaluate the performance of commercial heartworm antigen tests in dogs harbouring Dirofilaria immitis microfilariae near its distribution limit in South America. A total of 4934 blood samples of adult dogs from Southern Greater Buenos Aires were examined to detect circulating microfilariae in the buffy coat interface between December 2005 and April 2006. Microfilariae were detected in 88 (1.8%) blood samples and all the microfilariae observed were identified as D. immitis by acid phosphatase stain technique. In a first trial, 69 (78.4%) out of the 88 serum were positive by Speed ® Diro. Then, a new test was performed over 25 microfilariae-positive serum samples randomly selected among the 88 previously tested samples and using simultaneously Speed ® Diro, Witness ® Dirofilaria and Snap ® 3dx. This second trial showed identical results for the three different tests, in which 19 (76%) samples were positive. Therefore, more than 20% of microfilaremic dogs were antigen negative. The main hypothesis that could explain our finding is a low worm burden in the study area. According to our preliminary results, it is highly recommendable the complementary use of antigen tests and other procedures to obtain an accurate diagnostic near the distribution limit of the parasite.

Microfilaruria caused by canine dirofilariasis (Dirofilaria immitis): an unusual clinical presence

A 6-year-old, 18.2-kg, male mixed-breed dog was presented to Chulalongkorn Veterinary Teaching Hospital with clinical signs of hyperpnea, respiratory distress, vomiting, and anorexia. Blood and urine samples were collected for routine analysis. Microfilaremic examination and adult heartworm antigen test were conducted. Thoracic radiography was also performed. The clinical hematological and biochemical results demonstrated typical features of heartworm-infected dogs, including leukocytosis, thrombocytopenia, increases in alkaline phosphatase, alanine aminotransferase, and azotemia. Radiography showed typical findings of heartworm disease. Urinalysis revealed hematuria, pyuria, bacteriuria, hemoglobinuria, and proteinuria indicating lower urinary tract infection. Microfilaruria was detected in the urine sediment. They were identified morphologically as microfilariae of Dirofilaria immitis. The presence of microfilariae in the urine has not previously been described as a specific manifestation in dogs with dirofilariasis. The main objective of this article was to report new pathological findings in an extremely rare case of canine heartworm infection. However, its occurrence is presumably related to the inflammation or hemorrhage of the lower urinary tract and/or glomerulonephritis in this individual dog.

Evaluation of a covered-rod silicone implant containing ivermectin for long-term prevention of heartworm infection in dogs

To evaluate use of covered-rod (CR) silicone implants containing ivermectin for long-term prevention of infection with Dirofilaria immitisin dogs. 145 adult male and female dogs. Dogs received implants of different sizes, and ivermectin concentrations and serum ivermectin concentrations were monitored for 16, 57, and 56 weeks, respectively, in 3 preclinical dose selection studies. Ability of implants to prevent infection with D immitis was evaluated in 2 further studies; dogs were challenged with 50 infective third-stage larvae 52 weeks after implant administration and necropsied 145 days after challenge, and the total number of adult heartworms was counted. A field study was then undertaken in which client-owned dogs received an implant and plasma samples were collected at intervals until week 52 for ivermectin analysis and heartworm antigen determination. Use of the implants resulted in maintenance of an ivermectin concentration > or = 0.2 ng/mL for 12 months. In challenge studies, no treated dogs had adult heartworms, in contrast to untreated dogs, which all had adult heartworms at necropsy. In the field study, dogs treated with an implant had negative results of heartworm antigen testing for 12 months. The CR silicone implant containing 7.3 mg of ivermectin was 100% effective in preventing experimental infection with D immitislarvae and resulted in negative results for heartworm antigen in a field trial. This product has the potential to alleviate poor owner compliance with monthly prevention regimens.

Efficacy of Oral Tadalafil, a New Long-acting Phosphodiesterase-5 Inhibitor, for the Short-term Treatment of Pulmonary Arterial Hypertension in a Dog

Systolic pulmonary arterial hypertension (PAH) was diagnosed in a 15-year-old intact male Yorkshire terrier presented for progressive dyspnoea and coughing. Several examinations were performed (thoracic radiographs, faecal analysis, heartworm antigen test, tracheal fluoroscopy, abdominal ultrasound, complete blood cell count, urine and serum biochemistry) but the PAH remained of unknown origin. Despite medical treatment (diuretics and angiotensin-converting enzyme inhibitor), cardiovascular and respiratory signs dramatically worsened over a 1-month period, with several daily syncope, cyanosis and tachypnoea at rest requiring permanent oxygen therapy. Oral tadalafil (Cialis), a new long-acting phosphodiesterase-5 inhibitor, belonging to the same family as sildenafil (Viagra), was added to the background therapy. The condition of the dog improved quickly (< 24 h), and short-term follow up (7 days) showed a decrease in systolic pulmonary arterial pressure up to 26 mmHg concomitant with the disappearance of all respiratory and cardiac signs of PAH (cyanosis, syncope and tachypnoea). This case is of interest because it concerns the first reported short-term use of tadalafil in canine PAH. However, long-term studies with a large number of diseased animals are now required before prescription by general practitioners could be recommended.

Discrimination between six species of canine microfilariae by a single polymerase chain reaction

Canine dirofilariasis caused by Dirofilaria immitis is usually diagnosed by specific antigen testing and/or identification of microfilariae. However, D. immitis and at least six other filariae can produce canine microfilaremias with negative heartworm antigen tests. Discriminating these can be of clinical importance. To resolve discordant diagnoses by two diagnostic laboratories in an antigen-negative, microfilaremic dog recently imported into the US from Europe we developed a simple molecular method of identifying different microfilariae, and subsequently validated our method against six different filariae known to infect dogs by amplifying ribosomal DNA spacer sequences by polymerase chain reaction using common and species-specific primers, and sequencing the products to confirm the genotype of the filariae. We identified the filaria in this dog as D. repens. This is the first case of D. repens infection in the United States. Additionally, we examined microfilariae from five additional antigen-negative, microfilaremic dogs and successfully identified the infecting parasite in each case. Our diagnoses differed from the initial morphological diagnosis in three of these cases, demonstrating the inaccuracy of morphological diagnosis. In each case, microfilariae identified morphologically as A. reconditum were identified as D. immitis by molecular methods. Finally, we demonstrated that our PCR method should amplify DNA from at least two additional filariae ( Onchocerca and Mansonella), suggesting that this method may be suitable for genotyping all members of the family Onchocercidae.