Evaluation of diagnostic techniques for early detection of heartworm in experimentally infected dogs: identification of Dirofilaria immitis-derived microRNA in the initial 28 weeks post-inoculation.

BACKGROUND: Dirofilaria immitis, commonly known as heartworm (HW), is a parasitic nematode transmitted by various mosquito species, leading to heartworm disease (HWD) in dogs. Diagnosis of HW typically involves antigen or microfilariae detection, or visualization of adult worms through imaging or post mortem examination. Polymerase chain reaction (PCR) and micro RNA (miRNA) detection have been explored for HW diagnosis. METHODS: Three dogs, previously experimentally infected with HW, underwent blood sampling every 4 weeks for 7 months. Samples were assessed for antigen presence after heat treatment, PCR amplification, and microfilaria examination using Giemsa-stained thick smears. Additionally, whole blood aliquots underwent miRNA deep sequencing and bioinformatic analysis. RESULTS: Heartworm antigen was detectable after heat treatment at 20 weeks post-inoculation and via PCR at 24 weeks, with microfilariae observed in peripheral blood smears at 28 weeks. However, deep miRNA sequencing revealed that the miRNA candidate sequences are not consistently expressed before 28 weeks of infection. CONCLUSIONS: While ancillary molecular methods such as PCR and miRNA sequencing may be less effective than antigen detection for detecting immature larval stages in an early stage of infection, our experimental findings demonstrate that circulating miRNAs can still be detected in 28 weeks post-infection.

An Overview of Management Considerations for Mongolian Gerbils (Meriones unguiculatus), Cats (Felis catus), and Dogs (Canis familiaris) as Hosts for Brugia Infection.

Lymphatic filariasis is a mosquito-borne parasitic infection affecting an estimated 51.4 million people. Brugia malayi and Brugia pahangi are used in research because common nonprimate research species such as Mongolian gerbils (Meriones unguiculatus), cats (Felis catus), and dogs (Canis familiaris) can maintain the life cycle of these species of filarial nematodes. Although overall care and management of animals infected with Brugia spp. is relatively straightforward, there are some unique challenges and special considerations that must be addressed when managing a research colony infected with these parasites. In this review, we discuss our experience, share insight into biosafety and clinical management, and describe the expected clinical signs associated with Brugia infection in gerbils, cats, and dogs.

Effects of doxycycline dose rate and pre-adulticide wait period on heartworm-associated pathology and adult worm mass.

BACKGROUND: The American Heartworm Society canine guidelines recommend treatment with doxycycline prior to adulticide administration to reduce levels of Wolbachia and its associated metabolites, which are known to be a leading cause of pulmonary pathology. Studies have determined that doxycycline administered at 10 mg/kg BID for 28 days is an effective dose for eliminating Wolbachia, but what has not been determined is the clinical relevance of this elimination. The current guidelines also recommend a 30-day wait period following administration of doxycycline to allow for clearance of metabolites, such as Wolbachia surface protein, and for further reduction in heartworm biomass before administration of adulticide. Reducing the doxycycline dose and eliminating the wait period may carry practical benefits for the animal, client, and practitioner. METHODS: To investigate these treatment practices, Dirofilaria immitis adults were surgically transplanted into each of 45 dogs, which were divided into nine study groups of five dogs each. Seventy-five days after transplantation, two groups each were administered 5, 7.5, or 10 mg/kg BID doxycycline orally for 28 days and 6 µg/kg ivermectin monthly, with three untreated groups serving as controls. Study animals were necropsied and examined prior to treatment as well as 30 and 60 days post-treatment. RESULTS: Mean worm weight was unaffected by dosage but exhibited a significant increase at 30 days and significant decrease at 60 days post-treatment, including in control groups. Histopathology lesion scores did not significantly differ among groups, with the exception of the lung composite score for one untreated group. Liver enzymes, the levels of which are a concern in doxycycline treatment, were also examined, with no abnormalities in alanine aminotransferase or alkaline phosphatase observed. CONCLUSIONS: No consistent worsening of tissue lesions was observed with or without the AHS-recommended 30-day wait period, nor did reduced dosages of doxycycline lead to worsening of pathology or any change in efficacy in depleting worm weight. Mean worm weight did significantly increase prior to, and decrease following, the wait period. Future work that also includes adulticide treatment (i.e. melarsomine) will study treatment recommendations that may improve both animal health and owner compliance.

Evaluation of renal values during treatment for heartworm disease in 27 client-owned dogs.

BACKGROUND: Canine heartworm disease (CHD) caused by Dirofilaria immitis remains a common preventable disease with increasing incidence in some parts of the USA. The treatment guidelines of the American Heartworm Society (AHS) currently recommend monthly macrocyclic lactone administration, 28 days of doxycycline given orally every 12 h and three injections of melarsomine dihydrochloride (1 injection on day 2 of treatment followed 30 days later by 2 injections 24 h apart). Minocycline has also been utilized when doxycycline is unavailable. The systemic effects of CHD, which particularly impact cardiac and renal function, have been described, with infected dogs often experiencing renal damage characterized by an increase in serum concentrations of renal biomarkers. Although the AHS treatment protocol for CHD has been shown to be safe and effective in most cases, the potential for complications remains. No study as of yet has evaluated changes in symmetric dimethylarginine (SDMA), a sensitive marker of renal function, during treatment for CHD. The purpose of the present study was to evaluate renal function in dogs by measuring serum creatinine and SDMA concentrations during the adulticide treatment period. METHODS: Serum creatinine and SDMA concentrations were measured in 27 client-owned dogs affected by CHD at the following time points: prior to starting doxycycline or minocycline therapy (baseline), during doxycycline or minocycline therapy (interim), at the time of the first dose of melarsomine (first dose), at the time of the second dose of melarsomine (second dose) and at the dog's follow-up visit after treatment, occurring between 1 and 6 months after completion of therapy (post-treatment). Concentrations of creatinine and SDMA were compared between time points using a mixed effects linear model. RESULTS: Mean SDMA concentrations following the second dose of melarsomine were significantly lower (-1.80 ug/dL, t-test, df = 99.067, t = -2.694, P-Value = 0.00829) than baseline concentrations. There were no other statistically significant differences in the concentration of either biomarker between the baseline and the other time points in CHD dogs undergoing treatment. CONCLUSIONS: The results suggest that the current AHS protocol may not have a substantial impact on renal function.

The Domestic Dog as a Laboratory Host for Brugia malayi.

Of the three nematodes responsible for lymphatic filariasis in humans, only Brugia malayi is actively maintained in research settings owing to its viability in small animal hosts, principal among which is the domestic cat. While the microfilaremic feline host is necessary for propagation of parasites on any significant scale, this system is plagued by a number of challenges not as pronounced in canine filarial models. For this reason, we investigated the capacity in which dogs may serve as competent laboratory hosts for B. malayi. We infected a total of 20 dogs by subcutaneous injection of 500 B. malayi third-stage larvae (L3) in either a single (n = 10) or repeated infection events (125 L3 per week for four weeks; n = 10). Within each group, half of the individuals were injected in the inguinal region and half in the dorsum of the hind paw. To track the course of microfilaremia in this host, blood samples were examined by microscopy biweekly for two years following infection. Additionally, to identify cellular responses with potential value as predictors of patency, we measured peripheral blood leukocyte counts for the first year of infection. A total of 10 of 20 dogs developed detectable microfilaremia. Peak microfilaria density varied but attained levels useful for parasite propagation (median = 1933 mL-1; range: 33-9950 mL-1). Nine of these dogs remained patent at 104 weeks. A two-way ANOVA revealed no significant differences between infection groups in lifetime microfilaria production (p = 0.42), nor did regression analysis reveal any likely predictive relationships to leukocyte values. The results of this study demonstrate the competence of the dog as a host for B. malayi and its potential to serve in the laboratory role currently provided by the cat, while also clarifying the potential for zoonosis in filariasis-endemic regions.

X-treme loss of sequence diversity linked to neo-X chromosomes in filarial nematodes.

The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping in order to identify single nucleotide variants and insertions/deletions. In natural and laboratory Brugia populations, there is a lack of sequence diversity on chromosome X relative to the autosomes (πX/πA = 0.2), which is lower than the expected (πX/πA = 0.75). A reduction in diversity is also observed in other filarial nematodes with neo-X chromosome fusions in the genera Onchocerca and Wuchereria, but not those without neo-X chromosome fusions in the genera Loa and Dirofilaria. In the species with neo-X chromosome fusions, chromosome X is abnormally large, containing a third of the genetic material such that a sizable portion of the genome is lacking sequence diversity. Such profound differences in genetic diversity can be consequential, having been associated with drug resistance and adaptability, with the potential to affect filarial eradication.

The Course of Brugia malayi Microfilaremia in Experimentally Infected Cats.

As one of the causative agents of lymphatic filariasis in humans, Brugia malayi has been established as the laboratory model of choice for studying this infection owing to its viability in small animal hosts, with the domestic cat being significant among these. The usefulness of individual feline infections is highly dependent on the levels of circulating microfilariae in the blood; thus, characterizing the course of microfilaremia benefits our understanding of this model. In B. malayi-endemic regions, cats are also known reservoirs of infection, and describing microfilaremia in a controlled setting may improve transmission modeling. We followed the course of B. malayi infection in 10 experimentally infected cats from inoculation to ultimate resolution. Seven cats developed patency, with a peak microfilaria concentration of 6525/mL. In addition, to identify cellular responses with potential value as predictors of patency, we measured the peripheral blood leukocyte counts during the first 8 months of infection and tested for correlations with lifelong microfilaria production. No strong relationships were observed, though cell values did appear to shift with the maturation phases of the parasite. The data we present reflect the course of microfilaremia in an important laboratory model under controlled conditions.

A rapid, parasite-dependent cellular response to Dirofilaria immitis in the Mongolian jird (Meriones unguiculatus).

BACKGROUND: The Mongolian jird (Meriones unguiculatus) has long been recognized as a permissive host for the filarial parasite Brugia malayi; however, it is nonpermissive to another filarial parasite, canine heartworm (Dirofilaria immitis). By elucidating differences in the early response to infection, we sought to identify mechanisms involved in the species-specific clearance of these parasites. We hypothesized that the early clearance of D. immitis in intraperitoneal infection of the jird is immune mediated and parasite species dependent. METHODS: Jird peritoneal exudate cells (PECs) were isolated and their attachment to parasite larvae assessed in vitro under various conditions: D. immitis and B. malayi cultured separately, co-culture of both parasites, incubation before addition of cells, culture of heat-killed parasites, and culture with PECs isolated from jirds with mature B. malayi infection. The cells attaching to larvae were identified by immunohistochemistry. RESULTS: In vitro cell attachment to live D. immitis was high (mean = 99.6%) while much lower for B. malayi (mean = 5.56%). This species-specific attachment was also observed when both filarial species were co-cultured, with no significant change from controls (U(9, 14) = 58.5, p = 0.999). When we replicated these experiments with PECs derived from jirds subcutaneously infected with B. malayi, the results were similar (99.4% and 4.72% of D. immitis and B. malayi, respectively, exhibited cell attachment). Heat-killing the parasites significantly reduced cell attachment to D. immitis (mean = 71.9%; U(11, 14) = 7.5, p < 0.001) while increasing attachment to B. malayi (mean = 16.7%; U(9, 15) = 20, p = 0.002). Cell attachment to both species was reduced when larvae were allowed a 24-h pre-incubation period prior to the addition of cells. The attaching cells were identified as macrophages by immunohistochemistry. CONCLUSIONS: These results suggest a strongly species-dependent response from which B. malayi could not confer protection by proxy in co-culture. The changes in cell attachment following heat-killing and pre-incubation suggest a role for excretory/secretory products in host immune evasion and/or antigenicity. The nature of this attachment is the subject of ongoing study and may provide insight into filarial host specificity.

Clinical assessment of heartworm-infected Beagles treated with a combination of imidacloprid/moxidectin and doxycycline, or untreated.

BACKGROUND: Administration of moxidectin topically and doxycycline PO has been utilized experimentally as an alternative treatment for heartworm disease. However, clinical effects of this protocol remain poorly characterized. OBJECTIVE: To evaluate the clinical and postmortem findings associated with administration of doxycycline and monthly 10% imidacloprid + 2.5% moxidectin (IMD + MOX, Advantage Multi/Advocate) to Dirofilaria immitis-experimentally infected as compared to nontreated control dogs. ANIMALS: Sixteen purpose-bred, female, Beagle dogs. METHODS: Prospective, blinded, experimental study. Animals with surgically transplanted adult heartworms were randomized into 2 study groups of equal size: a nontreated control group (n = 8) and an IMD + MOX and doxycycline-treated group (n = 8). Randomization was performed using a complete block design according to circulating microfilarial concentrations, measured before treatment. Serum biochemical profiles, CBCs, thoracic radiographs and echocardiograms were performed prior to and 3 weeks after transplantation, and monthly for 10 months. Postmortem gross and histopathologic evaluations were performed. RESULTS: Compared to control animals, mean ± SD serum alanine aminotransferase (181 ± 203 U/L vs 33 ± 7 U/L; P < .0001) and alkaline phosphatase (246 ± 258 U/L vs 58 ± 19 U/L; P < .0001) activities were significantly higher in the treated group on day 28. Radiographic and echocardiographic evidence of heartworm disease was observed in both groups; however, no significant differences in these variables were noted between groups. Mean ± SD pulmonary arterial thrombus score was significantly higher in the treated vs nontreated group (3.9 ± 0.4 and 1.5 ± 2.1, respectively; P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: The treatment protocol was well-tolerated with no clinically relevant adverse effects for any variable evaluated during the observational period.

Canine gastrointestinal nematode transmission potential in municipal dog parks in the southeast United States.

Monthly canine parasite prophylactic products prevent not only adult heartworm infection, but also patent infections with specific gastrointestinal parasites. While most monthly products control and treat certain hookworm and roundworm infections, fewer are labeled for whipworm (Trichuris vulpis). Therefore, we hypothesized that fecal samples collected from municipal dog parks will have a greater prevalence of whipworm eggs compared to hookworm and roundworm eggs. In this study, canine fecal samples were collected from municipal dog parks in three southeastern states, with up to 20 fecal samples were collected from each park. A total of 200 fecal samples were obtained from dog parks in Georgia, North Carolina, and South Carolina. All fecal samples were examined for the presence of gastrointestinal helminths by a simple centrifugal flotation using sheather's sugar flotation solution. Of the 200 samples collected, 27% were positive for gastrointestinal helminths by fecal flotation. Of these infected fecal samples, 8.5%, 17%, and 1.5% contained whipworm, hookworm, and roundworm, respectively. However, the majority of hookworm-positive samples were collected from one park, whereas whipworm and roundworm samples were collected from multiple parks. These results could indicate that dogs are at risk of infection by all three parasites at dog parks, and that preventive strategies may need to be tailored not only to the specific region, but to specific infected dog parks.

Acetic acid as an alternative reagent in the modified Knott test.

The suitability of acetic acid as a safer alternative to formalin in the modified Knott test was evaluated for the diagnosis of canine heartworm (Dirofilaria immitis). Microfilaria concentration was measured by both methods and found to agree within reasonable limits (-5.84 % bias; -88.1-76.4 % limits of agreement). The level of agreement was lower when samples were prepared with a 24 h delay, but this was due to the formalin method tending to yield lower counts (-20.1 % bias; -90.5-50.2 % limits of agreement). Clearing the sample of hemoglobin improves readability and is a key feature of the modified Knott test. Hemolysis was significantly lower in the acetic acid method than the formalin method as measured by red blood cell count (6.83 × 106 and 8.79 × 106 cells/ml, respectively; p = 0.015) and absorbance at 415 nm (33.20 and 34.75, respectively; p < 0.001). Visual assessment, however, revealed little practical difference in readability. Finally, lengths of microfilariae were measured to ensure the validity of species identification by the acetic acid method; mean length was significantly shorter after acetic acid treatment (273 µm) than formalin treatment (316 µm; p < 0.001). Length reduction was also observed in acetic acid-treated Acanthocheilonema reconditum (254 µm versus 262 µm; p = 0.035), though these samples were stored prior to testing and are not directly comparable. We conclude that, while the readability of samples is similar for both methods, species differentiation must still be accomplished by other means. For most clinical purposes in determining the presence or absence of blood circulating microfilariae, however, acetic acid appears to be a suitable alternative to formalin in the modified Knott test.

Effects of diethylcarbamazine and ivermectin treatment on Brugia malayi gene expression in infected gerbils (Meriones unguiculatus).

Lymphatic filariasis (LF) threatens nearly 20% of the world's population and has handicapped one-third of the 120 million people currently infected. Current control and elimination programs for LF rely on mass drug administration of albendazole plus diethylcarbamazine (DEC) or ivermectin. Only the mechanism of action of albendazole is well understood. To gain a better insight into antifilarial drug action in vivo, we treated gerbils harbouring patent Brugia malayi infections with 6 mg kg-1 DEC, 0.15 mg kg-1 ivermectin or 1 mg kg-1 albendazole. Treatments had no effect on the numbers of worms present in the peritoneal cavity of treated animals, so effects on gene expression were a direct result of the drug and not complicated by dying parasites. Adults and microfilariae were collected 1 and 7 days post-treatment and RNA isolated for transcriptomic analysis. The experiment was repeated three times. Ivermectin treatment produced the most differentially expressed genes (DEGs), 113. DEC treatment yielded 61 DEGs. Albendazole treatment resulted in little change in gene expression, with only 6 genes affected. In total, nearly 200 DEGs were identified with little overlap between treatment groups, suggesting that these drugs may interfere in different ways with processes important for parasite survival, development, and reproduction.

Efficacy and side effects of doxycycline versus minocycline in the three-dose melarsomine canine adulticidal heartworm treatment protocol.

BACKGROUND: The American Heartworm Society currently recommends the use of a monthly macrocyclic lactone, a 28-day course of 10 mg/kg doxycycline BID, and the 3-dose protocol of melarsomine dihydrochloride for the treatment of canine heartworm disease. Doxycycline is necessary for the reduction of the bacterium Wolbachia, found in all heartworm life-stages. Previous price increases and decreasing availability prompted us to evaluate alternative tetracycline antibiotics, i.e. minocycline, for the reduction of Wolbachia during canine heartworm treatment. METHODS: Thirty-two heartworm-positive dogs were randomized to receive 10 mg/kg or 5 mg/kg of either doxycycline or minocycline for 28 days BID, for a total of 8 dogs per experimental group. All dogs received 6 months of Heartgard Plus® (ivermectin/pyrantel) and the 3-dose protocol of 2.5 mg/kg melarsomine dihydrochloride. Blood samples were collected prior to the initiation of treatment, every 7 days throughout tetracycline treatment, and then monthly thereafter until the dog tested negative for the presence of heartworm antigen. DNA was isolated from circulating microfilarial samples and qPCR was performed on each sample. RESULTS: A greater number of dogs in the 10 mg/kg doxycycline and minocycline treated groups experienced gastrointestinal side effects as compared to the 5 mg/kg doxycycline and minocycline treated groups. All eight dogs in the 10 mg/kg doxycycline-treated group tested negative for the presence of Wolbachia DNA by 28 days post-tetracycline treatment. A total of two dogs in both the 5 mg/kg doxycycline- and 10 mg/kg minocycline-treated groups and three dogs in the 5 mg/kg minocycline-treated group remained positive for the presence of Wolbachia DNA by the end of tetracycline treatment. CONCLUSIONS: No lung pathology was assessed in this clinical trial, therefore the clinical effect of the remaining Wolbachia DNA in the 10 mg/kg minocycline-, 5 mg/kg doxycycline- and 5 mg/kg minocycline-treated groups cannot be determined. Owner compliance in the proper administration of these tetracyclines may be impacted by the increased severe gastrointestinal side effects reported for the 10 mg/kg doxycycline- and minocycline-treated groups. We recommend that veterinarians prescribe the recommended 10 mg/kg doxycycline for canine heartworm treatment and reduce the dosage to 5 mg/kg in cases of severe gastrointestinal side effects in order to improve owner compliance in administration of medications.

Approaches to Canine Heartworm Disease Treatment Among Alumni of a Single College of Veterinary Medicine.

This descriptive study was designed to ascertain the current heartworm treatment strategies employed by veterinary graduates of a single college of veterinary medicine, to assess the frequency with which each of these treatment strategies is prescribed, and to report the motivation behind the use of these treatment strategies. A survey containing a combination of multiple-choice and open-ended questions was distributed via e-mail with an online link during 2013 to graduates of the University of Georgia College of Veterinary Medicine. Demographic data and opinions regarding treatment for cases of canine heartworm disease (HWD) were obtained, and motivation for recommending different treatment strategies was assessed. Nearly all 170 respondents (99%) indicated that they recommend melarsomine dihydrochloride for first-line treatment of canine HWD. Exercise restriction (80%) and monthly heartworm preventive (75%) were components of the treatment approach to HWD with no clinical signs. The majority of respondents (74%) indicated that when first-line treatment recommendations were declined, they endorsed long-term administration of ivermectin (i.e., "slow-kill" method) despite current American Heartworm Society guidelines that recommend against the use of long-term macrocyclic lactone administration for the monotherapy treatment of canine HWD. Respondents also indicated that owners' financial concerns frequently result in modification of HWD treatment. Routine inclusion of exercise restriction is commonly, but not universally, utilized and may represent an opportunity for improvement in the management of this disease. In addition, when first-line recommendations for heartworm disease treatment are declined, a two-dose melarsomine protocol instead of the slow-kill method should be considered.

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

BACKGROUND: The 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. METHODS: As 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. RESULTS: There 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. CONCLUSIONS: The 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.

A diagnostic algorithm for evaluating cases of potential macrocyclic lactone-resistant heartworm.

BACKGROUND: The emergence of macrocyclic lactone resistance in canine heartworm poses a substantial threat to what is currently the only effective, FDA-approved available method of prevention. Further study of the biotypes is necessary to understand the mechanism of resistance and evaluate novel prevention options. Identifying cases of drug-resistant infection remains problematic, however, especially when poor compliance and insufficient testing are concerns. Furthermore, a definitive demonstration of resistance requires experimental infection and treatment, which is prohibitively costly. METHODS: With the aim of identifying likely cases of macrocyclic lactone-resistant heartworm and preventing their continued spread, we describe an algorithm for determining the likelihood of drug resistance and appropriate treatment strategies for each case. RESULTS: This algorithm relies on the microfilarial suppression test (MFST), which has been used previously as an efficient and discrete measure of suspected resistance. By standardizing this method in a format that is readily available to practitioners, it could become possible to preliminarily survey the emergence and spread of resistance. CONCLUSION: Heartworm isolates identified through this method can be used in research to better understand macrocyclic lactone resistance so prevention strategies can be adapted.

Does evaluation of in vitro microfilarial motility reflect the resistance status of Dirofilaria immitis isolates to macrocyclic lactones?

BACKGROUND: Several reports have confirmed that macrocyclic lactone-resistant isolates of Dirofilaria immitis are circulating in the United States; however, the prevalence and potential impact of drug resistance is unknown. We wished to assess computer-aided measurements of motility as a method for rapidly assessing the resistance status of parasite isolates. METHODS: Blood containing microfilariae (MF) from two clinical cases with a high suspicion of resistance was fed to mosquitoes and the resultant L3 injected into dogs that were then treated with six doses of Heartgard® Plus (ivermectin + pyrantel; Merial Limited) at 30-day intervals. In both cases patent heartworm infections resulted despite the preventive treatment. Microfilariae isolated from these dogs and other isolates of known resistance status were exposed to varying concentrations of ivermectin in vitro and their motility assessed 24 h later using computer-processed high-definition video imaging. RESULTS: We produced two isolates, Yazoo-2013 and Metairie-2014, which established patent infections despite Heartgard® Plus treatments. Measurements of the motility of MF of these and other isolates (Missouri, MP3 and JYD-27) following exposure to varying concentrations of ivermectin did not distinguish between susceptible and resistant heartworm populations. There was some evidence that the method of MF isolation had an influence on the motility and drug susceptibility of the MF. CONCLUSIONS: We confirmed that drug-resistant heartworms are circulating in the southern United States, but that motility measurements in the presence of ivermectin are not a reliable method for their detection. This implies that the drug does not kill the microfilariae via paralysis.

Evaluation of the larval migration inhibition assay for detecting macrocyclic lactone resistance in Dirofilaria immitis.

Anthelmintics of the macrocyclic lactone (ML) drug class are widely used as preventives against the canine heartworm (Dirofilaria immitis). Over the past several years, however, reports of ML lack of efficacy (LOE) have emerged, in which dogs develop mature heartworm infection despite the administration of monthly prophylactics. More recently, isolates from LOE cases have been used to infect laboratory dogs and the resistant phenotype has been confirmed by the establishment of adult worms in the face of ML treatment at normally preventive dosages. Testing for and monitoring resistance in D. immitis requires a validated biological or molecular diagnostic assay. In this study, we assessed a larval migration inhibition assay (LMIA) that we previously optimized for use with D. immitis third-stage larvae (L3). We used this assay to measure the in vitro ML susceptibilities of a known-susceptible laboratory strain of D. immitis and three highly suspected ML-resistant isolates originating from three separate LOE cases; progeny from two of these isolates have been confirmed ML-resistant by treatment of an infected dog in a controlled setting. A nonlinear regression model was fit to the dose-response data, from which IC50 values were calculated. The D. immitis LMIA yielded consistent and reproducible dose-response data; however, no statistically significant differences in drug susceptibility were observed between control and LOE parasites. Additionally, the drug concentrations needed to paralyze the L3 were much higher than those third- and fourth-stage larvae would experience in vivo. IC50 values ranged from 1.57 to 5.56µM (p≥0.19). These data could suggest that ML resistance in this parasite is not mediated through a reduced susceptibility of L3 to the paralytic effects of ML drugs, and therefore motility-based assays are likely not appropriate for measuring the effects of MLs against D. immitis in this target stage.

Assessment of parasitological findings in heartworm-infected beagles treated with Advantage Multi® for dogs (10% imidacloprid + 2.5% moxidectin) and doxycycline.

BACKGROUND: Anecdotal 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. METHODS: This 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. RESULTS: All 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). CONCLUSIONS: This 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.