Heartworm Antigen Test Research Articles

Platelet hyperresponsiveness and increased platelet-neutrophil aggregates in dogs with myxomatous mitral valve disease and pulmonary hypertension.

Pulmonary hypertension (PH) in dogs with myxomatous mitral valve disease (MMVD) is caused by increased pulmonary venous pressure. Thrombosis, vascular remodeling, and vasoconstriction mediated by platelets could exacerbate PH. Dogs with PH will exhibit a hypercoagulable state, characterized by increased platelet activation, platelet-leukocyte, and platelet-neutrophil aggregate formation. Eleven dogs (≥3.5 kg) diagnosed with MMVD and PH and 10 dogs with MMVD lacking PH. Prospective cohort ex vivo study. All dogs underwent echocardiographic examination, CBC, 3-view thoracic radiographs, and heartworm antigen testing. Severity of PH and MMVD were assessed by echocardiography. Viscoelastic monitoring of coagulation was assessed using thromboelastography (TEG). Platelet activation and platelet-leukocyte/platelet-neutrophil interactions were assessed using flow cytometry. Plasma serotonin concentrations were measured by ELISA. Unstimulated platelets from dogs with MMVD and PH expressed more surface P-selectin than MMVD controls (P = .03). Platelets from dogs with MMVD and PH had persistent activation in response to agonists. The number of platelet-leukocyte aggregates was higher in dogs with MMVD and PH compared with MMVD controls (P = .01). Ex vivo stimulation of whole blood resulted in higher numbers of platelet-neutrophil aggregates in dogs with MMVD and PH (P = .01). Assessment of hypercoagulability based on TEG or plasma serotonin concentrations did not differ between groups. Platelet hyperresponsiveness and increased platelet-neutrophil interaction occur in dogs with MMVD and PH, suggesting that platelets play a role of in the pathogenesis of PH. Clinical benefits of antiplatelet drugs in dogs with MMVD and PH require further investigation.

Evaluation of feline heartworm disease based on gross necropsy, serology, pulmonary histopathology, and radiographic evidence in adult shelter cats in northeastern Alabama

Background: Veterinary knowledge regarding feline heartworm has been increasing significantly over the past two decades. Necropsy surveys of shelter cats have shown feline adult heartworm infection prevalence to be 5–20% of the rate in unprotected dogs; however, other studies have shown feline heartworm antibody prevalence up to 33%, reflecting higher exposure rates and potential immature adult infections. Thus, the true prevalence of feline heartworm infection is likely underestimated due to the limitations of current diagnostic techniques, inadequate testing protocols, and the high likelihood of cats exhibiting transient clinical signs or dying without confirmation of infection. Diagnosing Feline Heartworm Disease (FHWD), also referred to as Heartworm Associated Respiratory Disease (HARD), is one of the conundrums of veterinary medicine. The purpose of this study was to evaluate and characterize the occurrence of Heartworm Associated Respiratory Disease [HARD] in shelter cats, naturally-infected with D.immitis.MethodsFifty shelter cats slated for euthanasia between December 2009 and June 2010 were investigated by gross necropsy, radiography, serology, and lung histopathology using techniques that have been established in experimental models of cat heartworm infection. The relationship between pulmonary vascular disease and serological markers for heartworm was also examined using correlations and statistical modeling. Serology included standard heartworm antigen test and a commonly used heartworm antibody test. Also included were heat-treated heartworm antigen test and two additional heartworm antibody tests previously evaluated on experimentally-infected cats.ResultsNone of the cats were heartworm antibody (HW Ab) positive on a commonly used HW Ab test used by many reference laboratories even though 20% of the study cats were heartworm antigen (HW Ag) positive on heat-treated samples. Two additional HW Ab test were positive on 26% and 22% of the study cats. The combination of heat-treated HW Ag, HW Ab tests, and histopathology indicated 34% of the study cats had HARD.ConclusionsUtilizing both, the above tests, and thoracic radiographs, enhanced the ability to predict vascular disease, possibly caused by infection with immature and adult heartworms and supported the premise that cats develop heartworm disease at the same rate as dogs.Graphical

Is the European badger a new host for Dirofilaria immitis? The first records in Greece

Dirofilaria immitis is a ubiquitous nematode parasite with zoonotic potential, transmitted by mosquitoes, that causes heartworm disease in various animal species. Dogs are the parasite’s typical final host, and wild carnivores represent the parasite’s reservoir in nature. Studies on D. immitis infections in wild animals are essential to assess infection pressure for domestic animals, and until now, there has been only one infection case reported in a European badger (Meles meles). The current report describes the first two European badger cases with cardiovascular dirofilariosis in Greece. Two adult male badgers were rescued in Heraklion and Chania, Crete Island, and admitted to “ANIMA -Wildlife Rehabilitation Centre” in Athens. The detailed clinical examination revealed that the first badger suffered from severe broncho-pneumonitis while the second one displayed clinical signs associated with severe brain trauma. Blood samples were taken for haematology and biochemistry analyses during their short hospitalisation period. In addition, different routine diagnostic tests were carried out, including heartworm antigen testing (ELISA) and the modified Knott’s test for microfilariae. Both badgers were positive in both tests. The animals died a few hours after their admission and the detailed necropsies followed, revealed the presence of three parasites in each animal’s right heart, morphologically identified as adults of D. immitis. These findings add the European badger in the list of additional potential reservoir hosts for D. immitis and highlight the potential role of wildlife for companion animals and human health.

Dirofilaria immitis Identified in Galapagos Sea Lions (Zalophus wollebaeki): A Wildlife Health and Conservation Concern.

The Galapagos sea lion (Zalophus wollebaeki), an endemic and endangered pinniped, faces an increasing threat due to infectious diseases related to domestic animals. Dirofilaria immitis, the parasite responsible for canine heartworm disease, is one such threat, as canine infections on the archipelago have been documented. We used a canine heartworm antigen test kit to analyze the blood from 25 juvenile Galapagos sea lions for D. immitis. Two (8%) sea lions tested positive for D. immitis antigen. Using morphologic and genetic assessments, we evaluated 20 filarial-like worms collected from within the heart of an adult male Galapagos sea lion during a previous routine postmortem examination. The intracardiac worms were morphologically consistent with adult D. immitis, and sequence analysis of targeted PCR amplicons confirmed their identity. This is the first report of D. immitis infection in Galapagos sea lions, which could become a major health problem for these pinnipeds. Further studies are necessary to confirm the level of threat from this parasite; however, widespread adoption of routine heartworm testing, prevention, and treatment in the canine population, and the control of mosquitos, could potentially reduce the disease impact on this endangered pinniped species.

Successful management of a persistent pneumothorax in a cat using an autologous blood patch pleurodesis

AbstractA 16‐year‐old, neutered, male cat presented to an emergency and specialty care facility for persistent pneumothorax. Bilateral thoracocentesis, biochemical and hematological evaluation, thoracic radiographs, abdominal ultrasound, heartworm antigen test and computed tomography scan were performed. No definitive cause of the pneumothorax was identified, making a diagnosis of spontaneous (idiopathic) pneumothorax most likely. Surgical exploration of the thorax was declined. Evacuation of the pleural space was performed for 4 days, without resolution of air accumulation. An autologous blood patch pleurodesis was performed on the fifth day. The pneumothorax resolved, and the patient was discharged from the hospital the following day. Recheck radiographs were performed 17 days after discharge, which confirmed resolution of pneumothorax. The patient was alive 1200 days after discharge, with no recurrent clinical signs. This case report demonstrates successful use of an autologous blood patch pleurodesis in a feline patient.

High prevalence of canine heartworm, Dirofilaria immitis, in pet dogs in south Texas, USA, with evidence of Aedes aegypti mosquitoes contributing to transmission

BackgroundThe canine heartworm Dirofilaria immitis, a filarioid nematode of dogs and other carnivores, is widespread in the USA and the world. Over 20 different mosquito species serve as intermediate hosts of D. immitis, but their contribution to transmission varies according to factors like host feeding patterns, geographic locations and climatic conditions. The yellow fever mosquito, Aedes aegypti, is a competent vector of D. immitis but is often dismissed as a vector of veterinary relevance given its anthropophilic feeding behavior. We evaluated the prevalence of D. immitis in pet dogs along the USA-Mexico border and assessed whether Ae. aegypti in the area are naturally infected with heartworm and are potentially acting as a vector.MethodsA total of 200 whole blood samples collected from pet dogs in the Lower Rio Grande Valley in south Texas from 2016 to 2019 were included in this study. Canine serum samples for D. immitis were tested using the DiroCHEK® Canine Heartworm Antigen Test Kit pre- and post-immune complex dissociations (ICD) and blood samples were tested using high-resolution melt (HRM) quantitative PCR (qPCR) and a probe-based qPCR. Additionally, mosquito specimens were collected and identified, and Ae. aegypti heads, abdomens and pools were tested using conventional PCR (cPCR) and HRM qPCR.ResultsOverall, heartworm prevalence in dogs aged > 6 months was 40.8% (64/157) when the results from all testing modalities were considered. Heartworm antigen was detected in 33.5% and 40.7% of the dogs using DiroCHEK® pre- and post-ICD, respectively. By molecular screening, 20.1% of dogs tested positive with probe-based qPCR, while only one tested positive with HRM qPCR. Of the Ae. aegypti abdomens from blood-fed Ae. aeygpti tested, 20 (21.7%) from mosquitoes that fed on dogs and four (7%) from those that fed on humans tested positive for heartworm. Among Ae. aegypti heads from blood-fed Ae. aeygpti, two (1.1%) were positive based on cPCR and four (2.5%) were positive based on HRM qPCR. No D. immitis DNA was detected in the 208 pools of whole bodies (358 individuals) of Ae. aegypti gravid females.ConclusionsOur study highlights a high prevalence of heartworm in dogs in south Texas and provides evidence that Ae. aegypti could be contributing to heartworm transmission in canine populations in this region.Graphical

Serodiagnostic difficulties and possibilities of heartworm disease in regions where both Dirofilaria immitis and Dirofilaria repens infections occur.

This retrospective study was performed on 71 dogs which had been admitted for heartworm screening or with clinical suspicion of heartworm disease. The examination methods included polymerase chain reaction (PCR) to identify Dirofilaria immitis and/or Dirofilaria repens infections and a heartworm antigen (Ag) test (VetScan). By using PCR, 26 dogs were found positive only for Dirofilaria immitis (Group 1), while 21 dogs for both D. immitis and D. repens (Group 2). Group 3 included 24 dogs with D. repens infection only according to the PCR results. The sensitivity of the VetScan Ag test for the Group 1 and 2 animals proved to be 97.7% (95% Blaker confidence interval; CI 89.0%-99.9%). The specificity of the VetScan Ag test, calculated from the results of Group 3, was found to be 66.7% (95% CI 45.6%-83.1%), which was lower than that reported from the USA, where D. repens does not occur. In cases when PCR results were positive for D. repens but negative for D. immitis, the occult dirofilariosis was the likely explanation for the positive D. immitis Ag tests. These observations highlight the importance of performing more Ag tests simultaneously in those areas where both Dirofilaria species are present.

Detection and cross-reaction of Dirofilaria repens using a commercial heartworm antigen test kit

Antigen enzyme-linked immunosorbent assay tests are widely used for the diagnosis of heartworm infection in dogs. While commercially-available heartworm antigen tests have high sensitivity and specificity, false-negative test results can occur in dogs with low worm burdens, female-only infections, or prior to patency. The use of immune complex dissociation (ICD) methods have demonstrated increased sensitivity in the detection of Dirofilaria immitis antigens and the reversal of false-negative antigen results. However, there are concerns pertaining to false-positive antigen results due to infections of other nematode parasites, especially post-ICD. Therefore, this study evaluated the effect of heat-treatment on serum samples of dogs experimentally-infected with Dirofilaria repens during the course of infection, to assess for potential cross-reactivity on heartworm antigen tests. Archived serum samples from three dogs experimentally-infected with D. repens were utilized. All samples were tested for cross-reactivity pre- and post-heat-treatment using the DiroCHEK® Heartworm Antigen test kit throughout infection (day -9 through 404 days post-infection; dpi). All heat-treated samples tested false-positive starting at 164 dpi and continuing through 404 dpi, thereby testing positive prior to patency. No cross-reactivity was observed for any dog at any time point prior to heat-treatment. Our results suggest that the ICD method decreased the specificity of heartworm antigen tests and caused cross-reactivity of serum from dogs experimentally infected with D. repens. In conclusion, heat-treatment of serum in areas co-endemic for D. repens and D. immitis has limited clinical value, and should be used with caution.

Development of a multiplex qPCR-based approach for the diagnosis of Dirofilaria immitis, D. repens and Acanthocheilonema reconditum

BackgroundDirofilaria immitis, D. repens and Acanthocheilonema reconditum are the main causative agents of zoonotic canine filariosis.MethodsWe developed a combined multiplex approach for filaria and Wolbachia detection using the 28S-based pan-filarial and 16S-based pan-Wolbachia qPCRs, respectively, involving a fast typing method of positive samples using triplex qPCR targeting A. reconditum, D. immitis and D. repens, and a duplex qPCR targeting Wolbachia of D. immitis and D. repens. The approach was complemented by a duplex qPCR for the differential diagnosis of heartworms (D. immitis and Angiostrongylus vasorum) and pan-filarial cox1 and pan-Wolbachia ftsZ PCRs to identify other filarial parasites and their Wolbachia, respectively. A total of 168 canine blood and sera samples were used to validate the approach. Spearmanʼs correlation was used to assess the association between filarial species and the strain of Wolbachia. Positive samples for both the heartworm antigen-test after heating sera and at least one DNA-positive for D. immitis and its Wolbachia were considered true positive for heartworm infection. Indeed, the presence of D. repens DNA or that of its Wolbachia as well as A. reconditum DNA indicates true positive infections.ResultsThe detection limit for Wolbachia and filariae qPCRs ranged from 5 × 10−1 to 1.5 × 10−4 mf/ml of blood. When tested on clinical samples, 29.2% (49/168) tested positive for filariae or Wolbachia DNA. Filarial species and Wolbachia genotypes were identified by the combined multiplex approach from all positive samples. Each species of Dirofilaria was significantly associated with a specific genotype of Wolbachia. Compared to the true positives, the approach showed excellent agreement (k = 0.98–1). Unlike D. immitis DNA, no A. vasorum DNA was detected by the duplex qPCR. The immunochromatographic test for heartworm antigen showed a substantial (k = 0.6) and a weak (k = 0.15) agreements before and after thermal pre-treatment of sera, respectively.ConclusionsThe proposed approach is a reliable tool for the exploration and diagnosis of occult and non-occult canine filariosis. The current diagnosis of heartworm disease based on antigen detection should always be confirmed by qPCR essays. Sera heat pre-treatment is not effective and strongly discouraged.

Comparative evaluation of field samples using 2 in-clinic assays for heartworm antigen detection in dogs

Dirofilaria immitis (heartworm) antigen testing is routinely performed in veterinary practices to detect canine heartworm infections. The purpose of this study was to evaluate the performance of two in-clinic assays to detect heartworm antigen on field samples from practices in heartworm endemic regions. Veterinary staff in 3 practices located in the Southern United States performed a side by side comparison of the SNAP® 4Dx® Plus Test (IDEXX) and the VETSCAN FLEX4® Test (Zoetis) on samples from canine patients presented for vector-borne disease screening. Assays were performed according to the manufacturer’s instructions. The remaining plasma sample was submitted for confirmatory testing using the PetChek® Heartworm Test (IDEXX) including immune complex dissociation (ICD) by heat treatment. A total of 232 samples were evaluated by the two in-clinic assays and PetChek Test. SNAP 4Dx Plus was significantly more sensitive for the detection of heartworm antigen in this study; sensitivity was 97.4 % for the SNAP 4Dx Plus test and 76.9 % for VETSCAN FLEX4 test (p < 0.01). The specificity of both tests was 99.5 %. This study reveals significant difference in detecting canine heartworm antigen in field samples.

Detection of Canine Vector-Borne Filariasis and Their Wolbachia Endosymbionts in French Guiana.

In French Guiana, canine heartworm disease is well known, but the diversity of filarial parasites of dogs remains largely unknown. A total of 98 canine blood samples from Cayenne and Kourou were assessed by a blood wet mount preparation, heartworm antigen test and molecular exploration of filarioid and Wolbachia DNAs, followed by a multiplex species-specific qPCR’s identification and a subsequent sequencing analysis. Thereafter, a phylogeny based on maximum likelihood was carried out to facilitate specific identification. Five dogs were microfilaremic. Heartworm antigens were detected in 15 (15.3%) dogs. Of these, six (6.1%) were considered as occult infections as neither microfilariae nor Dirofilaria immitis DNA were detected. The 11 (11.2%) D. immitis isolates corresponded to a low virulent strain. Six of the D. immitis isolates were positive for Wolbachia endosymbionts of D. immitis belonging to the clade C DNA. Acanthocheilonema reconditum DNA was detected in 3 (3.1%) samples. Of these latter, one was found co-infected with the Brugia sp. genotype and the DNA of the clade D of the Wolbachia endosymbiont of Brugia species. This latter was also detected in two filarioid DNA-free samples. Finally, two samples were positive for Cercopithifilaria bainae genotype, which is distinct from those identified in Europe. The present study highlights the urgent need to implement chemoprophylaxis associated with anti-Wolbachia drugs to control these potential zoonoses.

Comparison of acid- versus heat-treatment for immune complex dissociation and detection of Dirofilaria immitis antigen in canine plasma

Annual antigen testing is a mainstay for diagnosing infection with Dirofiliaria immitis in dogs; yet, it has been documented that some heartworm-infected dogs and cats test false-negative for antigen due to the presence of antigen-antibody complexes. Several studies have reported the use of heat as a reliable means of immune-complex dissociation (ICD) in recent years; however, the data regarding the use of acid as a reliable method of ICD for D. immitis detection are limited. The objective of this study was to compare an acid-based form of ICD to the more established and evaluated method of heat-based ICD in experimentally infected and non-infected dogs. Plasma from class A dogs experimentally infected ∼4 months prior with D. immitis (infected; n = 24) and dogs reared indoors with no history of exposure to mosquitoes (non-infected; n = 75) were evaluated for presence of D. immitis antigen (DiroCHEK® Heartworm antigen test kit). Each sample was divided into three aliquots for testing: [1] Control plasma (no acid- or heat-treatment), [2] acid-treated plasma (trichloroacetic acid (TCA), incubation, centrifugation for 5 min at 16,000 X g, buffer), and [3] heat-treated plasma (104 °C followed by centrifugation at 16,000 X g). Treatments for each aliquot were performed and tested in triplicate; results were determined both visually (color change) and by spectrophotometric analysis (optical density [OD] value). Of the 24 infected dogs, 0/24 tested positive for antigen in the absence of acid- or heat-treatment. Those same plasma samples following processing by either acid- or heat-treatment yielded 18/24 (75.0%) and 19/24 (79.2%) antigen-positive results, respectively. Of the 75 plasma samples from non-infected dogs, neither acid- nor heat-treatment of plasma caused any false-positive color changes or spectrophotometric values. These results indicate that acid as a means of ICD reliably allowed for the detection of D. immitis antigen in infected plasma while not inducing false-positive results in non-infected plasma samples.

Non-Arsenical heartworm adulticidal therapy using topical moxidectin-imidacloprid and doxycycline: A prospective case series

This prospective case series evaluated the adulticidal efficacy of topical 10 % moxidectin/2.5 % imidacloprid (M/I; Advantage Multi®, Bayer, Shawnee Mission, KS, USA) and doxycycline in dogs with naturally occurring heartworm infection (HWI). Twenty-two dogs with HWI whose owners declined melarsomine were treated with M/I at the preventive dosage twice monthly for 90 days then monthly thereafter and doxycycline (median [interquartile range; IQR] dosage 12.6 [12.0–16.1] mg/kg/day) for the first 15 days. Although strict activity restriction was not imposed, owners were asked to prevent their dogs from exercising strenuously. This protocol was referred to as the MOXY protocol. Antigen testing was performed every 30–60 days, until dogs had ‘no antigen detected’ (NAD). Twenty-one of the 22 dogs ultimately converted to NAD by 434 days (median [IQR]), 234 (179–303). One dog remained positive 701 days after MOXY initiation and was considered a treatment failure. All sera which converted to NAD on HW antigen testing were retested after heat-treatment. Twelve dogs had NAD on the heat-treated test on the same day as having their first NAD on the conventional test. Six of 9 dogs testing positive after heat-treatment were retested and all 6 had NAD on a heat-treated test within 2–3 months. Microfilaremia was cleared in all 8 dogs re-tested. Four dogs required treatment for cough, thought due to heartworm (HW) death, an average of 89 days after initiation of MOXY. This cough was most likely due to pneumonitis with heartworm-pulmonary thromboembolism. One dog required hospitalization for 24 -h and recovered fully with corticosteroid therapy and supportive care and 2 dogs were treated in an outpatient fashion with steroids. The MOXY protocol was tolerated and 96 % (21/22) of dogs converted to NAD, though 2 dogs required greater than 1 year to achieve this result. Nonaresenical-adulticide therapy may result in pneumonitis and heartworm-pulmonary thromboembolism at unpredictable times, potentially months after initiation of macrocyclic lactone therapy and exercise restriction should be considered when using a nonarsenical protocol. Although not currently recommended by the American Heartworm Society (AHS), non-arsenical strategies are in use and the goal of this study was to evaluate the efficacy, duration of therapy, and safety of an accelerated dosing protocol of M/I with doxycycline.

Assessing the potential cross-reactivity using a commercial heartworm ELISA kits of serum from dogs naturally infected with Onchocerca lupi

Onchocerca lupi is an emerging zoonotic parasite of dogs, endemic to the southwestern USA and areas of the Old World. Currently, there are no specific serological diagnostic tests able to detect O. lupi infection. Recent literature has demonstrated that commercially available heartworm antigen tests, despite being highly sensitive, may cross-react with infections by other filarid nematodes. There is no information on potential cross-reactivity of such tests in serum of dogs infected with O. lupi. Our objective was to assess serum samples of dogs naturally-infected with O. lupi for potential cross-reactivity before and after heat-treatment using a commercial heartworm ELISA kit. We obtained serum from 23 dogs naturally-infected with O. lupi. These dogs presented with ocular disease, and were consulted to schedule either surgical removal of ocular nodules due to infection or enucleation. Samples were tested in triplicate using the DiroCHEK® Heartworm Antigen Test kit (Synbiotics Corporation, Zoetis, Kalamazoo, MI, USA) following the manufacturers’ protocol pre- and post-heat-treatment. Samples were heat-treated using a dry heat block at 103 °C for 10 min and then centrifuged at 1818×g for 20 min. Out of a total of 23 dogs, 19 (82.6 %) had no antigen detected regardless of heat-treatment, three dogs tested positive before and after heat-treatment, and a single dog turned positive after heat-treatment. These three dogs that were positive before and after heat-treatment were confirmedly co-infected with Dirofilaria immitis by the veterinarians responsible for these cases, and we were unable to get the history or follow up with the dog that turned positive post-heat-treatment only. Our data suggest that O. lupi infections should not result in false-positives when using the DiroCHEK® in dog serum, before or after heat-treatment. Dogs with clinical ocular onchocercosis that test antigen-positive in DiroCHEK® are likely co-infected with D. immitis, and should be further tested, including evaluation of microfilariae in blood and diagnostic imaging. If heartworm infection is confirmed, the animals should be enrolled in the recommended treatment protocol in accordance to the guidelines of the American Heartworm Society or other local organizations.

Effects of milbemycin oxime, combined with spinosad, when administered orally to microfilaremic dogs infected with adult heartworms (Dirofilaria immitis).

OBJECTIVE To evaluate the safety of PO administration of a milbemycin oxime (MBO) and spinosad product to heartworm (Dirofilaria immitis)-positive microfilaremic dogs. DESIGN Randomized, blinded, complete block trial. ANIMALS 32 purebred Beagles with a patent heartworm infection. PROCEDURES Dogs ranked by sex and microfilaria counts (range, 398 to 1,980 microfilaria/mL) were assigned to 4 groups of 8 to receive 3 treatments PO at 28-day intervals beginning on day 0: placebo (control group) or spinosad-MBO tablets containing MBO at the upper end of the label dose range (0.75 to 1 mg/kg [0.34 to 0.45 mg/lb]; 1× group) or 3 (3× group) or 5 (5× group) times that dose. Blood samples were collected at various points for adult heartworm antigen and Knott tests. Necropsies were performed on day 65, and recovered adult heartworms were counted. RESULTS 1 control dog died from heartworm-associated complications. Other adverse events included mild, transient emesis (1 dog in each of the 1× and 5× groups and 3 dogs in the 3× group). Similar adult heartworm counts (range, 13 to 41) were obtained for all 4 groups. Results of blood antigen and microfilaria tests were positive throughout the study, with 1 exception in each of the 3× and 5× groups. Mean microfilaria counts increased with time in the control group, whereas reductions from baseline in treated groups ranged from 61.5% to 96.4%. CONCLUSIONS AND CLINICAL RELEVANCE The evaluated MBO-spinosad formulation caused no severe adverse events when administered PO to microfilaremic dogs. Although microfilaria counts decreased following treatment, repeated monthly MBO treatments were incompletely microfilaricidal, suggesting MBO should not be used as a microfilaricide.

Comparison of six commercial antigen kits for detection of Dirofilaria immitis infections in canines with necropsy-confirmed heartworm status

Patient-side test kits for detecting antigenemia in dogs associated with sexually mature female heartworms (Dirofilaria immitis) have been available for three decades, and these tests are continually updated and improved. To define the sensitivity (Se) and specificity (Sp) of contemporary antigen detection tests against cardiopulmonary D. immitis burden, we evaluated five patient-side kits—Anigen Rapid One Step® (Bio note), SNAP® 4Dx Plus Test Kit (IDEXX), WITNESS® Heartworm Canine Heartworm Antigen Test Kit (Zoetis), VetScan® Canine Heartworm Rapid Test (Abaxis), and Solo Step® CH Canine Heartworm Antigen Test (Heska), and one microplate ELISA (DiroCHEK®; Zoetis), using archived canine sera divided into five subclasses of female worms (0, 1–5, 6–20, 21–40, and >40). The patient-side tests were performed once, side-by-side according to each manufacturer’s protocol by personnel blinded to the D. immitis status of each dog. The overall Se and Sp of the patient-side kits was ≥97.5 and =94.0%, respectively. For samples from dogs with 1–5, 6–20, and 21–40 D. immitis, the Se was between 96 and 100%, with a slight increase in Se in dogs with ≥41 worms. The agreement between tests for all subclasses of D. immitis burden was between 99 and 100%. The Se and Sp for the ELISA compared with the necropsy results of dogs was 99 and 96%, respectively. Agreement between each patient-side test and the ELISA was between 97 and 100%. All commercially available tests can give practitioners excellent patient-side information, allowing them to make informed decisions on the need for additional diagnostic work-up before instituting new or continuing D. immitis prophylaxis.

Evaluation of heat-treating heartworm-positive canine serum samples during treatment with Advantage Multi\xae for Dogs and doxycycline

BackgroundThe use of heat-treatment in canine and feline serum has been hypothesized to break the formation of antigen-antibody complexes, thereby freeing the heartworm antigen allowing for detection by commercially available heartworm antigen kits. While studies have analyzed the effect of heat-treating serum and plasma samples in the detection of heartworm antigen, these studies have not utilized necropsy verified results for validation. This study evaluated the use of heat-treating serum samples in experimentally infected dogs during adulticidal treatment in comparison with necropsy adult heartworm recovery.MethodsAs part of a primary study, a total of 16 dogs were experimentally infected with 16 sexually mature adult heartworms using surgical transplantation, allocating 8 dogs in both the control and treated group. Treated dogs received 10 months of topical administration of Advantage Multi® for Dogs (10% Imidacloprid + 2.5% Moxidectin) every 4 weeks and 30 days of 10 mg/kg doxycycline BID. Blood samples were collected from all study animals prior to surgical transplantation of adult heartworms, on study days 0, 1, 3, 7, 14, 21, 28, and every 4 weeks thereafter for the duration of this study. Concentration of heartworm antigen was tested using the DiroCHEK® heartworm antigen test kit using serum samples both pre- and post-heat-treatment. Serum samples were heat-treated at 103 °C in a dry heat block for 10 min and centrifuging at 1818× g for 20 min.ResultsThere were a total of 4 instances (days 56, 140, 224 and 252) in 3 treated dogs in which a serum sample converted from negative for the detection of heartworm antigen prior to heat-treatment to positive for the detection of heartworm antigen post-heat-treatment. At necropsy, these dogs had no adult heartworms recovered and were all negative on antigen testing prior to and after heat treatment. There was 100% accuracy in the detection of either no infection, or 1–2 adult heartworm infections using the DiroCHEK in serum samples with and without heat-treatment at the time of necropsy.ConclusionsThe DiroCHEK accurately diagnosed all dogs with live adults recovered at necropsy as heartworm antigen positive and all those dogs with no live adults recovered at necropsy as heartworm antigen negative without the use of heat-treatment for samples taken on the day of necropsy. Therefore, these results indicate that the use of heat-treating serum samples did not provide data of any additional value in the diagnosis of heartworm-positive dogs receiving treatment in this study. Additionally, these results may indicate that the conversion of serum samples from negative to positive for the presence of heartworm antigen with heat-treatment may not always accurately diagnose live adult heartworm infections since no adult heartworms were recovered at necropsy for those dogs in which a conversion event occurred. These conversion events may be detecting residual antigen leftover after all adult worms have died or may even be detecting off- target antigens, which have been denatured during heat-treatment. While a necropsy was not performed at the time of the conversion events, no live adult worms were recovered from any of the dogs in which a conversion event occurred earlier in treatment.

Increasing incidence of Dirofilaria immitis in dogs in USA with focus on the southeast region 2013\u20132016

BackgroundA recent American Heartworm Society (AHS) survey on the incidence of adult heartworm infections in dogs in the United States of America showed a 21.7% increase in the average cases per veterinary clinic from 2013 to 2016. The analysis reported here was performed to see if heartworm testing results available via the Companion Animal Parasite Council (CAPC) aligned with the AHS survey and whether changes in heartworm preventive dispensing accounts for the increased incidence. The resistance of Dirofilaria immitis to macrocyclic lactones (MLs) has been previously reported.MethodsAn analysis of 7–9 million heartworm antigen tests reported annually to the Companion Animal Parasite Council (CAPC) from 2013 to 2016 was conducted and compared to the 2016 AHS survey. A state-by-state analysis across the southeastern USA was also performed. National heartworm preventive dispensing data were obtained from Vetstreet LLC and analyzed. All oral, topical and injectable heartworm preventives were included in this analysis, with injectable moxidectin counting as six doses.ResultsPositive antigen tests increased by 15.28% from 2013 to 2016, similar to the 21.7% increase reported by the AHS survey. Incidence in the southeastern USA increased by17.9% while the rest of USA incidence increased by 11.4%. State-by-state analysis across the southeastern USA revealed an increased positive test frequency greater than 10% in 9 of 12 states evaluated. During this time, the overall proportion of dogs receiving heartworm prophylaxis remained relatively unchanged. Approximately 2/3 of the dogs in the USA received no heartworm prevention each year.ConclusionThese CAPC data show the rate of positive heartworm tests increasing significantly (P < 0.0001) in the USA from 2013 to 2016, with a higher rate of increase in the southeastern USA than nationally. Only 1/3 of dogs in the USA were dispensed one or more doses of heartworm prevention annually by veterinarians, averaging 8.6 monthly doses/year. Veterinarians and pet owners should work together to follow CAPC and AHS guidelines to protect dogs from infection with D. immitis. Lack of preventive use and the emergence of heartworm resistance to MLs could both be impacting the increased rate of positive heartworm tests in dogs.

Comparative evaluation of commercially available point-of-care heartworm antigen tests using well-characterized canine plasma samples

BackgroundDirofilaria immitis is a worldwide parasite that is endemic in many parts of the United States. There are many commercial assays available for the detection of D. immitis antigen, one of which was modified and has reentered the market. Our objective was to compare the recently reintroduced Witness® Heartworm (HW) Antigen test Kit (Zoetis, Florham Park, NJ) and the SNAP® Heartworm RT (IDEXX Laboratories, Inc., Westbrook, ME) to the well-based ELISA DiroChek® Heartworm Antigen Test Kit (Zoetis, Florham Park, NJ).MethodsCanine plasma samples were either received at the Auburn Diagnostic Parasitology Laboratory from veterinarians submitting samples for additional heartworm testing (n = 100) from 2008 to 2016 or purchased from purpose-bred beagles (n = 50, presumed negative) in 2016. Samples were categorized as “positive,” “borderline” or “negative” using our established spectrophotometric cutoff value with the DiroChek® assay when a sample was initially received and processed. Three commercially available heartworm antigen tests (DiroChek®, Witness® HW, and SNAP® RT) were utilized for simultaneous testing of the 150 samples in random order as per their package insert with the addition of spectrophotometric optical density (OD) readings of the DiroChek® assay. Any samples yielding discordant test results between assays were further evaluated by heat treatment of plasma and retesting. Chi-square tests for the equality of proportions were utilized for statistical analyses.ResultsConcordant results occurred in 140/150 (93.3%) samples. Discrepant results occurred in 10/150 samples tested (6.6%): 9/10 occurring in the borderline heartworm (HW) category and 1/10 occurring in the negative HW category.The sensitivity and specificity of each test compared to the DiroChek® read by spectrophotometer was similar to what has been reported previously (Witness®: sensitivity 97.0% [94.1–99.4%], specificity 96.4% [95.5–100.0%]; SNAP® RT: sensitivity 90.9% [78.0–100.0%], specificity 98.8% [96.0–100.0%]). There were significant differences detected when comparing the sensitivities of the SNAP® RT and the Witness® HW to the DiroChek® among the 150 total samples (p = 0.003) and the 50 “borderline” samples (p = 0.001).ConclusionsIn this study, the sensitivity of the Witness® HW was higher than the sensitivity of the SNAP® RT when compared with the DiroChek® test results prior to heat treatment of samples.

Validation of immune complex dissociation methods for use with heartworm antigen tests

BackgroundAntigen testing is routinely used to diagnose canine Dirofilaria immitis infections. Immune complex dissociation (ICD) methods, which were employed in the original heartworm antigen tests to release antigen that was bound by endogenous canine antibodies, were discontinued with improvements in assay reagents. The purpose of this study was to evaluate different ICD methods for detection of heartworm antigen by microtiter plate ELISA and assess the performance in samples from pet dogs.MethodsThe original PetChek® Heartworm Test (IDEXX Laboratories, Inc.) utilized pepsin at an acidic pH for ICD prior to antigen testing. Performance and characteristics of the pepsin ICD method were compared with those for heat treatment (with and without EDTA) and acid treatment.ResultsAll four methods released complexed antigen in serum samples when tested using microtiter plate ELISA. Heat treatment required ≥600 μL of serum or plasma, whereas pepsin and acid methods needed only a 50-μL sample. Samples from 1115 dogs submitted to IDEXX Laboratories between 2014 and 2016 for investigation of discrepant heartworm results were evaluated with and without pepsin ICD using the PetChek Heartworm Test. Samples from 10% (n = 112) of the dogs were antigen positive with the ICD protocol only while 90% of the results remained unchanged. In a prospective study, antigen levels with and without ICD were evaluated for 12 dogs receiving pre-adulticide heartworm treatment with a macrocyclic lactone and doxycycline for 28 days. Serial samples revealed that three dogs had a reduction in detectable heartworm antigen within 4 weeks of initiating treatment. In these cases, heartworm antigen levels could be recovered with ICD.ConclusionsHeartworm antigen testing with ICD can be a valuable diagnostic tool for patients with discrepant results that have had intermittent use of a preventive, or have been treated with a macrocyclic lactone and doxycycline. Heartworm therapies may reduce antigen production and favor immune complexing in some dogs, resulting in false-negative results. Therefore, it is important to confirm positive heartworm antigen test results before initiating therapy.