Canine Heartworm Research Articles

Exploring multiplex qPCR as a diagnostic tool for detecting microfilarial DNA in dogs infected with Dirofilaria immitis: A comparative analysis with the modified Knott’s test

Current recommendations to diagnose cardiopulmonary dirofilariosis in dogs caused by Dirofilaria immitis involves tandem antigen and circulating microfilariae tests. The modified Knott’s test is an important tool in heartworm diagnosis, allowing identification of circulating microfilariae. However, the subjective nature of the modified Knott’s test affects its accuracy and diagnostic laboratories usually do not provide a quantitative outcome. Quantitative enumeration of microfilariae enables clinicians to track treatment progress and acts as a proxy for detecting emerging macrocyclic lactone resistance. There is a need for better diagnostic tools suitable for routine use to efficiently and accurately quantify the presence of D. immitis microfilaremia. The aim of this study was to determine whether the quantitative modified Knott's test can be substituted by multiplex quantitative polymerase chain reaction (qPCR) targeting D. immitis and associated Wolbachia endosymbiont DNA in canine blood samples. To do this, genomic DNA samples (n = 161) from Australian dogs, collected as part of a previous 2021 study, were assessed in a TaqMan qPCR targeting DNA of D. immitis, Wolbachia sp. and Canis lupus familiaris. Of the 161 genomic DNA samples, eight were considered positive for D. immitis microfilariae. The qPCR assay demonstrated good efficiency (E = 90 to 110%, R2 > 0.94). Considering the performance and efficient use of bench time, this TaqMan qPCR assay is a suitable alternative to the modified Knott’s test for quantitative enumeration of microfilariae (Cohen’s kappa coefficient [κ]: κ = 1 using D. immitis qPCR marker, κ = 0.93 using Wolbachia qPCR marker). The qPCR data demonstrated a comparable result to that of the quantitative modified Knott’s test in a 2022 survey of D. immitis in Australian dogs (n = 23) before and after macrocyclic lactone (ML) administration. Improving the detection and diagnosis of canine heartworm infections will assist veterinarians in better managing and controlling disease outcomes and will be valuable for tracking the spread of ML resistance in Australia.

Dirofilaria immitis in Italian Cats and the Risk of Exposure by Aedes albopictus.

Background: Dirofilaria immitis, the agent of canine and feline heartworm disease (HWD), is a widespread mosquito-borne helminth. The true prevalence of HW infection in cats is likely underestimated due to the difficulty in establishing a definitive diagnosis. Aedes albopictus, a recognized competent vector for D. immitis, is currently considered the most invasive mosquito species worldwide and Italy presents the highest abundance of Ae. albopictus in Europe. Materials and Methods: The present study was aimed at evaluating the current seroprevalence of D. immitis antibodies in cats in Italy. Further, the ecological niche model (ENM) was applied to evaluate the potential future risk of feline HWD associated with the presence of Ae. albopictus. Results: Sera of 812 Italian cats were analyzed, and the average prevalence of D. immitis antibodies was 12%. Seropositivity was significantly associated with age (<6 years), whereas there was no association with sex or geographical area. Data obtained with the risk map showed that the highest risk of infection was found in northern inland areas and along coastal areas, whereas the lowest risk was identified at higher altitudes. The ENM correctly classified most of the areas where D. immitis seropositive cats were found, with 80.4% occurring in high and very high-risk areas. Conclusions: Results of the present study suggest that cats in Italy are exposed to D. immitis infection, and that routine prevention should be part of the general health care protocols in cats. Moreover, the resulting risk maps indicate that areas with a suitable habitat for Ae. albopictus may put cats at risk of exposure to D. immitis.

Usefulness of Tissue Doppler Imaging for the Evaluation of Pulmonary Hypertension in Canine Heartworm Disease

Background: Dirofilaria immitis is a nematode that produces proliferative pulmonary endarteritis in dogs due to direct contact of the adult parasites with the intima layer of the pulmonary arteries, leading to irreversible severe structural damage and sustained pulmonary hypertension (PH), which can produce severe cardiorespiratory disorders. The purpose of this study was to assess the diagnostic value of the echocardiography tissue Doppler imaging (TDI) in determining the presence of PH in dogs with heartworm disease. Methods: There were 116 heartworm-infected dogs with PH and 33 healthy dogs included in the study. Based on the right pulmonary artery distensibility index (RPADi) &lt; 29.5%, PH was present in 47.4% of infected dogs. Additionally, the animals were evaluated using other standard alternative echocardiographic measures to estimate PH. Moreover, a total of eight echocardiographic measurements were analysed using the TDI to determine its usefulness in diagnosing PH (E′, A′, S, E′:A′, global TDI, HRI-IVCT, HRI-IVRT, R-TEI). Results: The TDI measurements showed significant differences between dogs with and without PH, demonstrating a positive correlation with respect to the RPADi. In addition, cut-off values for the detection of PH with excellent sensitivity and specificity were found for E′:A′, global TDI, HRI-IVCT, HRI-IVRT and R-TEI. Conclusions: The TDI mode may be useful as an adjunct diagnostic method for the determination of PH in dogs with Dirofilaria immitis.

Efficacy and tolerability of the American Heartworm Society therapeutic protocol in dogs affected by heartworm disease without caval syndrome.

The American Heartworm Society medical protocol represents the current standard of therapy for canine heartworm disease without caval syndrome. However, data on the tolerability of this protocol are limited. This study aimed to describe efficacy and prevalence of possible treatment-related side effects in dogs with heartworm disease treated using the American Heartworm Society protocol. For this retrospective multi-centre cohort study, dogs diagnosed with classes 1 to 3 heartworm disease that completed the American Heartworm Society medical protocol were searched in four medical databases. Demographic, clinical, diagnostic, therapeutic and outcome data, including the number and type of possible treatment-related side effects, were retrieved. Thirty-five dogs were included. The median age and bodyweight were 6 years (1 to 13 years) and 17.3 kg (4.9 to 50 kg), respectively. Heartworm disease was classified as classes 1, 2 and 3 in 20 of 35, 11 of 35 and four of 35 dogs, respectively. In addition to the therapeutic recommendations of the American Heartworm Society, eight of 35 dogs underwent sedation to favour melarsomine administration, and 30 of 35 received ice at the injection site. After adulticide therapy, all dogs were hospitalised with cage rest [median time 12 hours (6 to 48 hours)]. All dogs survived the treatment. All dogs with long-term follow-up (32/35) became negative. Furthermore, treatment-related side effects were rare, mild and rapidly recovered without the need for supporting therapies; these included depression/lethargy (4/35 dogs), cough (2/35 dogs) and lameness, pain and gastrointestinal signs (1/35 dog each). The American Heartworm Society medical protocol is efficient and safe in dogs with classes 1 to 3 heartworm disease.

A preliminary investigation of canine heartworm infection rates in Western North Carolina

A preliminary investigation of canine heartworm infection rates in Western North Carolina

Genome structure and population genomics of the canine heartworm Dirofilaria immitis

The heartworm, Dirofilaria immitis, is a filarial parasitic nematode responsible for significant morbidity and mortality in wild and domesticated canids. Resistance to macrocyclic lactone drug prevention represents a significant threat to parasite control and has prompted investigations to understand the genetic determinants of resistance. This study aimed to improve the genomic resources of D. immitis to enable a more precise understanding of how genetic variation is distributed within and between parasite populations worldwide, which will inform the likelihood and rate by which parasites, and in turn, resistant alleles, might spread. We have guided the scaffolding of a recently published genome assembly for D. immitis (ICBAS_JMDir_1.0) using the chromosomal-scale reference genomes of Brugia malayi and Onchocerca volvulus, resulting in an 89.5 Mb assembly composed of four autosomal- and one sex-linked chromosomal-scale scaffolds representing 99.7% of the genome. Publicly available and new whole-genome sequencing data from 32 D. immitis samples from Australia, Italy and the USA were assessed using principal component analysis, nucleotide diversity (Pi) and absolute genetic divergence (Dxy) to characterise the global genetic structure and measure within- and between-population diversity. These population genetic analyses revealed broad-scale genetic structure among globally diverse samples and differences in genetic diversity between populations; however, fine-scale subpopulation analysis was limited and biased by differences between sample types. Finally, we mapped single nucleotide polymorphisms previously associated with macrocyclic lactone resistance in the new genome assembly, revealing the physical linkage of high-priority variants on chromosome 3, and determined their frequency in the studied populations. This new chromosomal assembly for D. immitis now allows for a more precise investigation of selection on genome-wide genetic variation and will enhance our understanding of parasite transmission and the spread of genetic variants responsible for resistance to treatment.

Relative diagnostic accuracy of point-of-care tests to rule-in Dirofilaria immitis infection in clinically suspect dogs: A systematic review and meta-analysis.

Canine heartworm, Dirofilaria immitis, can cause severe disease and sometimes death of the host. Associated clinical signs, lack of preventative usage and regional endemicity are unlikely sufficient by themselves to reach a definitive diagnosis. Several point-of-care (POC) diagnostic tests are commercially available to aid in-clinic diagnosis, however, there is variable diagnostic accuracy reported and no synthesis of published evidence. This systematic review aims at meta-analysing the likelihood ratio of a positive result (LR+) to inform the selection and interpretation of POC tests in practice to rule-in heartworm infection when there is clinical suspicion. Three literature index interfaces (Web of Science, PubMed, Scopus) were searched on November 11th, 2022, for diagnostic test evaluation (DTE) articles assessing at least one currently commercialised POC test. Risk of bias was assessed adapting the QUADAS-2 protocol and articles with no evidence of high risk of bias were meta-analysed if deemed applicable to our review objective. Substantial between DTE heterogeneity was investigated including potential threshold or covariate effects. A total of 324 primary articles were sourced and 18 were retained for full text review of which only three had low risk of bias in all four QUADAS-2 domains. Of the nine heartworm POC tests evaluated, only three, IDEXX SNAP (n DTEs = 6), Zoetis WITNESS (n DTEs = 3) and Zoetis VETSCAN (n DTEs = 5) could be analysed. Both WITNESS and VETSCAN DTEs showed substantial heterogeneity due to a putative threshold effect and no summary point estimates could be reported. SNAP DTEs showed acceptable heterogeneity, and a summary LR+ was estimated at 559.0 (95%CI: 24.3-12,847.4). The quality and heterogeneity of heartworm POC test DTEs was highly variable which restricted our summary of the diagnostic accuracy to only the SNAP test. A positive result from the SNAP test provides strong evidence of the presence of an infection with adult heartworm(s) in a dog patient and this test is warranted to rule-in clinical suspicion(s) in clinics. However, our review did not appraise the literature to assess the fitness of SNAP test, or any other POC tests, to rule-out heartworm infection in dogs without clinical suspicion or following heartworm therapy.

Droplet digital PCR as a tool to detect resistant isolates of Dirofilaria immitis

Prevention of canine heartworm disease, caused by Dirofilaria immitis, relies on macrocyclic lactones for which drug resistance is now a concern. Although genetic polymorphisms have been associated with resistance in D. immitis populations, the mechanism is still not well understood. The lack of reliable in vitro assays to detect resistance is a limitation for confirming resistance. Ten single nucleotide polymorphisms (SNPs) were previously clinically validated in D. immitis resistant isolates, using the MiSeq platform. This technique although useful for research studies is expensive and does not facilitate rapid detection of these markers in small numbers of clinical samples. We developed a droplet digital PCR protocol for detecting SNPs correlating with ML resistance. Specific primers and hydrolysis probes encompassing the wildtype and mutant alleles were designed to amplify the SNP targets from genomic DNA of different D. immitis isolates. Allele frequencies were determined and the suitability of the ddPCR assay was assessed and compared with MiSeq data. The ddPCR assay accurately detected and quantified alternate nucleotides in two isolates of reference, the ML-susceptible Missouri (MO) and ML-resistant JYD-34, at the previously identified SNP positions. The presence of the SNPs was also determined in additional isolates with known or putative susceptible or resistant phenotypes. We observed SNP1 and SNP2 are more predictive markers and appear suitable for rapid detection and monitoring of drug resistance. Our results suggested that ddPCR could be employed to distinguish infection due to actual genetic resistance from infection with susceptible parasites and also for rapid detection of isolates not only with ML susceptible and resistant genotypes but also mixed genotypes that correspond to heterogeneous isolates containing a mixed population of ML susceptible and resistant parasites. DdPCR may be a useful tool for conducting surveys, or assessments of individual isolates, for genetic evidence of resistance or developing resistance.

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.

CANINE HEARTWORM INFECTION AND PROPHYLAXIS USE AMONG PET CARETAKERS FROM THE CUMBERLAND GAP REGION OF TENNESSEE, USA.

The prevalence of infection with canine heartworm (CHW), Dirofilaria immitis, continues to increase across the United States, regardless of the availability of effective and affordable prophylactic products. Current reports of CHW prevalence as estimated by the Companion Animal Parasite Council (CAPC) are thought to under-represent the true magnitude of the issue because pet dogs that do not receive regular veterinary care are often excluded. This study estimated the prevalence of CHW in pet dogs and associated prophylaxis use in pet dogs in the Cumberland Gap Region with a combined doorstep diagnostic testing approach and caretaker survey. Dogs tested (n = 258) during the summers of 2018 and 2019 revealed a 2.3% (6/258) prevalence in the pet dog population with 33% (2/6) being microfilaremic. Questionnaire data from caretaker interviews revealed that 41.8% (108/258) of the dogs were not receiving CHW prophylaxis. Significant predictors of CHW prophylaxis use identified through logistic regression included pet caretaker awareness of CHW as an important health issue and the use of veterinary services in the year preceding participation in the survey. These results underscore the importance of veterinary-mediated client interaction to create risk awareness of CHW disease and association with prophylaxis compliance.

Fluralaner (Bravecto®) treatment kills Aedes aegypti after feeding on Dirofilaria immitis-infected dogs

BackgroundTransmission of canine heartworm (Dirofilaria immitis) from infected to naïve dogs is dependent on successful mosquito feeding and survival.MethodsTo determine whether treating heartworm-infected dogs with fluralaner (Bravecto®) limits the survival of infected mosquitoes, and potentially the transmission of D. immitis, we allowed female mosquitoes to feed on microfilaremic dogs and evaluated mosquito survival and infection with D. immitis. Eight dogs were experimentally infected with D. immitis. On day 0 (~ 11 months post-infection), four microfilaremic dogs were treated with fluralaner according to label directions while the other four were non-treated controls. Mosquitoes (Aedes aegypti Liverpool) were allowed to feed on each dog on days −7, 2, 30, 56, and 84. Fed mosquitoes were collected, and the number of live mosquitoes determined at 6 h, 24 h, 48 h, and 72 h post-feeding. Surviving mosquitoes held for 2 weeks were dissected to confirm third-stage D. immitis larvae; PCR (12S rRNA gene) was performed post-dissection to identify D. immitis in mosquitoes.ResultsPrior to treatment, 98.4%, 85.1%, 60.7%, and 40.3% of mosquitoes fed on microfilaremic dogs were alive at 6 h, 24 h, 48 h, and 72 h post-feeding, respectively. Similarly, mosquitoes fed on microfilaremic, non-treated dogs were alive 6 h post-feeding (98.5–100%) throughout the study. In contrast, mosquitoes fed on fluralaner-treated dogs 2 days after treatment were dead or severely moribund by 6 h post-feeding. At 30 and 56 days post-treatment, > 99% of mosquitoes fed on treated dogs were dead by 24 h. At 84 days post-treatment, 98.4% of mosquitoes fed on treated dogs were dead by 24 h. Before treatment, third-stage larvae of D. immitis were recovered from 15.5% of Ae. aegypti 2 weeks after feeding, and 72.4% were positive for D. immitis by PCR. Similarly, 17.7% of mosquitoes fed on non-treated dogs had D. immitis third-stage larvae 2 weeks after feeding, and 88.2% were positive by PCR. Five mosquitoes fed on fluralaner-treated dogs survived 2 weeks post-feeding, and 4/5 were from day 84. None had third-stage larvae at dissection, and all were PCR-negative.ConclusionThe data indicate that fluralaner treatment of dogs kills mosquitoes and thus would be expected to reduce transmission of heartworm in the surrounding community.Graphical

The seroprevalence of canine dirofilariosis in dogs in the eastern coastal areas of China

Background and objectivesThe present study was carried out to assess the presence of canine Dirofilaria immitis infection in pet dogs in China. Materials and methodsFrom October 2018 to November 2019, a total of 216 sera were collected from pet hospitals in Shandong, Jiangsu, Shanghai, Zhejiang, and Fujian regions of Eastern China. The sera were tested by using a commercial canine heartworm antibody ELISA test kit. Results70.8% of the pets had significant clinical symptoms resembled to heartworm infection; the overall dirofilariosis positivity found was 12.5% (27/216); Significant positive rates differences were observed between symptomatic and asymptomatic dogs (P < 0.05) (i.e. 15.7% and 4.7% respectively).The prevalence of infection in Shandong Province (15.5%) was the highest among the surveyed areas, but the difference among the geographic regions was not statistically significant (P > 0.05). Furthermore, the prevalence detected in summer (28.2%) was significantly higher than in other seasons (P < 0.05). In addition, no significant difference was observed between male and female sex (P > 0.05). ConclusionsAltogether, these results suggest that an epidemic of dirofilariosis exists in eastern coastal China, as such preventive measures should be taken to control the spread of dirofilariosis to reduce the risk of human and pet infection with heartworm.

Angiotensin-converting enzyme 2 in dogs with Dirofilaria immitis

BackgroundInfection by the canine heartworm, Dirofilaria immitis, causes significant cardiopulmonary disease, with progression impacted by increasing parasite numbers and duration of infection. The renin–angiotensin–aldosterone system (RAAS) is an important mediator of cardiac and pulmonary disease. Angiotensin-converting enzyme 2 (ACE2) mitigates the maladaptive effects of angiotensin II by converting it to angiotensin (1-7). We hypothesized that circulating ACE2 activity would be altered in dogs with high heartworm infection intensities relative to dogs without heartworms.MethodsFrozen serum samples (−80 °C) from 30 dogs euthanized at Florida shelters were analyzed for ACE2 activity using liquid chromatography–mass spectrometry/mass spectroscopy and a kinetics approach with and without an ACE2 inhibitor. A convenience sample of 15 dogs without heartworms (HW0) and 15 dogs with > 50 heartworms (HW>50) was included. Heartworm number and microfilariae presence were determined at necropsy. The effects of heartworm status, body weight, and sex on ACE2 were evaluated using regression analysis. Values of P < 0.05 were considered significant.ResultsAll HW0 dogs were D. immitis microfilariae-negative and all HW>50 dogs were D. immitis microfilariae-positive with a median adult worm count of 74 (minimum = 63, maximum = 137). The ACE2 activity of HW>50 dogs (median = 28.2 ng/ml; minimum = 13.6, maximum = 76.2) was not different from HW0 dogs (median 31.9 ng/ml; minimum = 14.1, maximum = 139.1; P = 0.53). The ACE2 activity was higher in dogs with high body weight (median 34.2 ng/ml minimum = 14.1, maximum = 76.2) than in dogs with low weight (median 27.5 ng/ml; minimum = 16.4, maximum = 139.1; P = .044).ConclusionsHeartworm infection did not impact ACE2 activity in shelter dogs with or without heartworms, but heavier dogs had higher ACE2 activity compared to lighter dogs. Comprehensive RAAS evaluation and additional clinical information would aid in understanding how ACE2 activity relates to the entire cascade and clinical status in dogs with heartworm disease.Graphical

Treatment of dogs with Bravecto® (fluralaner) reduces mosquito survival and fecundity

BackgroundMosquitoes serve as the vector of canine heartworm (Dirofilaria immitis), which represents a significant and persistent threat to canine health. A reduction in the longevity and/or reproductive success of mosquitoes that take a blood meal from fluralaner-treated dogs may consequently reduce the local transmission of heartworm and prevent new infections. A novel secondary effect of an oral formulation of the ectoparasiticide fluralaner (Bravecto®) against a laboratory strain of the mosquito Aedes aegypti, a potential major vector of canine heartworm, was investigated in this study.MethodsSix dogs were administered a single dose of fluralaner orally in the form of Bravecto® Chews (at the labeled fluralaner dose of 25 mg/kg body weight), while six control dogs received no treatment. Mosquitoes were fed on blood that was collected from each dog prior to treatment and weekly for 15 weeks post-treatment to assess the continued effects of fluralaner as its serum level decreased. Mosquito fitness was assessed by three parameters: rate of successful blood-feeding, survival, and egg laying.ResultsSuccessful blood-feeding rate was similar between control and treatment groups. In the fluralaner treatment, mosquito survival was significantly reduced within the first 24 h after blood-feeding, for the first 12 weeks post-treatment of the dogs (efficacy range = 33.2–73.3%). Survival of mosquitoes up until a potentially heartworm-infective timepoint (14 days post-blood-feeding) was significantly reduced in the fluralaner-treated group at several timepoints (1, 2, 5, 11, 12, 13, 14, and 15 weeks post-treatment; efficacy range = 49.4–91.4%), but was less consistently reduced at the other timepoints. Egg laying by mosquitoes was almost completely suppressed for the first 13 weeks following treatment of the dogs with fluralaner (treatment efficacy ≥ 99.8%).ConclusionsMosquitoes fed blood from fluralaner-treated dogs experienced a significant reduction in survival and fecundity. These findings support the potential for a reduction in heartworm transmission directly by lethal effects on the vector and indirectly through a reduction of the local vector population when mosquitoes are exposed to animals treated with fluralaner.Graphical

Further variation of the adulticide protocol for the treatment of canine heartworm infection: can it be even shorter and cost less?

BackgroundThis retrospective study evaluated modified three-dose melarsomine treatment protocols in a shelter setting and compared them to the American Heartworm Society (AHS)-recommended protocol.MethodsAs compared with the AHS protocol, the shelter protocols utilized doxycycline 10 mg/kg once daily (SID) or twice daily (BID), and varied the time from initiation of doxycycline (day 1) to the first melarsomine injection (M1). Dogs were retrospectively grouped based on the shelter’s current protocol (M1 on day 14; Group A) and the AHS protocol (M1 on day 60; Group C), allowing a week on either side of the target M1 day. Treatments that fell outside these ranges formed two additional treatment groups (Groups B and D). Respiratory complications were defined as respiratory signs requiring additional treatment, and were statistically compared for Groups A and C. New respiratory signs and gastrointestinal (GI) signs were compared between dogs receiving SID or BID doxycycline.ResultsOne hundred fifty-seven dogs with asymptomatic or mild heartworm disease at presentation were included. All dogs survived to discharge. There was no statistically significant difference between Groups A (n = 79) and C (n = 27) for new respiratory signs post-melarsomine (P = 0.73). The time to M1 for 14 dogs that developed new respiratory signs was a median of 19 days, compared with 22 days for 143 dogs without new respiratory signs (P = 0.2). Respiratory complications post-melarsomine were uncommon. New respiratory signs post-melarsomine occurred in 10/109 (9.2%) dogs receiving SID doxycycline and 4/48 (8.3%) dogs receiving BID doxycycline (P > 0.999). GI signs prior to M1 were recorded for 40/109 (36.7%) dogs receiving SID doxycycline and 25/48 (52.1%) receiving BID doxycycline (P = 0.08). Forty-four follow-up antigen test results were available; all tests performed > 3 months after the third melarsomine injection were negative.ConclusionsThis study provided support for initiating melarsomine after 14 days of doxycycline and for a lower doxycycline dose. Shorter and less expensive treatment protocols can increase lifesaving capacity and improve quality of life for shelter dogs by reducing the duration of exercise restriction and length of stay.Graphical

Points to Consider When Establishing and Rearing Culex Mosquitoes in the Laboratory.

Culex mosquitoes transmit several pathogens to humans and animals, including viruses that cause West Nile fever and St. Louis encephalitis and filarial nematodes that cause canine heartworm and elephantiasis. Additionally, these mosquitoes have a cosmopolitan distribution and provide interesting models for understanding population genetics, overwintering dormancy, disease transmission, and other important and ecological questions. However, unlike Aedes mosquitoes that produce eggs that can be stored for weeks at a time, no obvious "stopping" point exists in the development of Culex mosquitoes. Therefore, these mosquitoes require nearly continuous care and attention. Here, we describe some general considerations when rearing laboratory colonies of Culex mosquitoes. We highlight different methods so that readers may choose what works best for their experimental needs and laboratory infrastructure. We hope that this information will enable additional scientists to conduct laboratory research on these important disease vectors.

Echocardiographic Assessment of the Pulmonary Vein to Pulmonary Artery Ratio in Canine Heartworm Disease.

A total of 151 naturally heartworm-infected and 66 healthy dogs were included in the study. The presence/absence of PH was based on the right pulmonary artery distensibility index (RPAD index < 29.5%), and the PV:PA ratio was echocardiographically measured by the time-motion mode (M mode) and two-dimensional mode (2D mode). Other echocardiographic parameters were also assessed (pulmonary trunk to aorta ratio, tricuspid regurgitation pressure gradient, and AT:ET ratio). The results of the PV:PA ratio showed a highly positive correlation between the M and 2D modes (r = 0.928). The PV:PA ratio obtained by the M mode was identified as the strongest predictor for RPAD index (R2 0.628, p < 0.0001) with a good diagnostic accuracy (AUC = 0.99). The results of PV/PA by the 2D mode showed a similar prediction for the RPAD index (R2 0.606, p < 0.0001) with a good diagnostic accuracy (AUC = 0.98). Both of the 2D and M modes' PV:PA ratios decreased significantly with the presence of PH. A cut-off value of ≤0.845 showed high sensitivity and specificity for the M mode (97% and 94%, respectively) and the 2D mode (96% and 93%, respectively). The PV:PA ratio may be useful as a complementary diagnostic method for the estimation of moderate or severe PH in dogs with heartworm.

Prevalence of canine heartworm infection in Queensland, Australia: comparison of diagnostic methods and investigation of factors associated with reduction in antigen detection

BackgroundThe prevalence of Dirofilaria immitis infection in dogs is increasing globally and spreading into new areas. Prevalence of dirofilariosis in the state of Queensland, Australia, was as high as 90% before the introduction of macrocyclic lactones. Limited research on prevalence of D. immitis infection in dogs in Queensland has been reported in the last 30 years. Antigen testing is the most common method for detection of dirofilariosis but its accuracy is reduced by antigen getting trapped (blocked antigen) in immune complexes (ICs). The objectives of this research were to determine the prevalence of D. immitis infection in dogs from two geographical areas (Brisbane and Townsville) in Queensland, to determine the extent to which blocked antigen affects the validity of antigen testing, and to explore whether this was associated with microfilaraemia, location, age or sex.MethodsBlood samples from Brisbane (sub-tropical climate) and Townsville (tropical climate) shelter dogs were evaluated for the presence of D. immitis antigen before (conventional antigen testing, CAT) and after dissociation of ICs by heat treatment (antigen testing after heat treatment, ATHT), using a commercially available test. Microfilariae were detected using modified Knott’s test (MKT). Test proportions were compared with McNemar’s test and the association between antigen test-discordant results (positive for antigen after dissociation of ICs) and microfilaraemia, location, sex and age was modelled using logistic regression.ResultsDirofilaria immitis prevalence in dogs from Townsville (22% by CAT, 32.1% by ATHT and 16.7% by MKT) was significantly higher than in dogs from Brisbane (1.1% by CAT and MKT and 1.7% by ATHT) (P< 0.001). Dissociation of ICs allowed detection of significantly more D. immitis infected dogs than either conventional antigen testing or microfilariae detection, or the combined antigen and microfilariae detection (P< 0.001). The odds of dogs being positive for antigen after dissociation of ICs were significantly higher for microfilaraemic, 3–4-year-old female dogs from Townsville.ConclusionsThe high prevalence of infection with D. immitis in dogs from Townsville poses a health risk for local susceptible host species, including humans. Dissociation of ICs increases antigen detection and should be considered in dogs suspected of D. immitis infection but negative on routine testing.Graphical

Right Pulmonary Artery Distensibility Index in Heartworm Infected Dogs: Are the Different Methods Leading to Same Results?

Canine Heartworm Disease (HWD) is mainly a pulmonary vascular disease. The reduction of the Pulmonary Artery Distensibility (PAD) is an early index of pulmonary vascular disease. Echocardiographic evaluation of the Right Pulmonary Artery Distensibility index (RPADi) is calculated as the percentage change in diameter of the right pulmonary artery (RPA) between systole and diastole. Historically, two main methods have been used for RPADi calculation: The Venco method and Visser method; however, different hybrid methods have also been used by other authors. Therefore, it could be difficult for a clinician to decide which method to apply and how to interpret the results based on the reference values reported. The aim of this study was to compare the RPADi obtained by five different techniques (Venco classic, Venco modified, Visser classic, Visser modified 1, and Visser modified 2). The study design was a retrospective, single center, observational study. Forty-seven client-owned dogs were included. The measurements were performed off-line as an average of three consecutive cardiac cycles by a single investigator blinded to the dogs' diagnosis. The RPADi was satisfactorily obtained by all methods in all dogs. Intra-observer measurement variability was clinically acceptable both for systolic and diastolic measurements. Although the Bland-Altman test showed a statistical agreement between the various methods used to calculate the RPADi, these methods cannot be used interchangeably in a clinical setting. Instead, the measurement method and reference values should always be specified.

A rodent model for Dirofilaria immitis, canine heartworm: parasite growth, development, and drug sensitivity in NSG mice

Heartworm disease, caused by Dirofilaria immitis, remains a significant threat to canines and felines. The development of parasites resistant to macrocyclic lactones (ML) has created a significant challenge to the control of the infection. The goal of this study was to determine if mice lacking a functional immune response would be susceptible to D. immitis. Immunodeficient NSG mice were susceptible to the infection, sustaining parasites for at least 15 weeks, with infective third-stage larvae molting and developing into the late fourth-stage larvae. Proteomic analysis of host responses to the infection revealed a complex pattern of changes after infection, with at least some of the responses directed at reducing immune control mechanisms that remain in NSG mice. NSG mice were infected with isolates of D. immitis that were either susceptible or resistant to MLs, as a population. The susceptible isolate was killed by ivermectin whereas the resistant isolate had improved survivability, while both isolates were affected by moxidectin. It was concluded that D. immitis survives in NSG mice for at least 15 weeks. NSG mice provide an ideal model for monitoring host responses to the infection and for testing parasites in vivo for susceptibility to direct chemotherapeutic activity of new agents.