Identification and validation of a commercial cryopreservation medium for the practical preservation of Dirofilaria immitis microfilaria.

BACKGROUND: Dirofilaria immitis is a parasitic nematode transmitted by mosquitoes and the cause of heartworm disease in dogs and dirofilariasis in humans and other mammals. The parasite is endemic worldwide. Vector stage research requires a reliable supply of D. immitis microfilariae (mf). It is believed that cryopreserved mf would retain viability and provide a powerful tool for vector stage research. However, reports on cryopreservation of D. immitis mf are limited. Therefore, this study aimed to validate commercial cryopreservation media to establish a practical, convenient and reproducible storage procedure for D. immitis mf. METHODS: Six different commercially available cryopreservation media were compared with the traditional polyvinylpyrrolidone-dimethyl sulfoxide (PVP-DMSO) preservation solution. In vitro viability of purified D. immitis mf and mf-infected total blood was analyzed using a motility assay and propidium iodide staining. In vivo infectivity of Aedes aegypti mosquitoes with cryopreserved mf was assessed using a mosquito survival test and quantifying the number of third-stage larvae (L3) after 13 days post-infection. RESULTS: Purified mf cryopreserved in CultureSure showed the best viability when compared to mf cryopreserved in the remaining five commercially available media and PVP-DMSO. Viability of mf in mf-infected total blood cryopreserved in CultureSure varied with the ratio of infected blood to CultureSure. Optimum results were obtained with 200 µl mf-infected blood:800 µl CultureSure. CultureSure was also the optimum medium for cryopreserving mf prior to infectivity of A. aegypti. The number of L3 was approximately the same for CultureSure cryopreserved mf (3× concentrated solution) and non-cryopreserved fresh mf. CONCLUSIONS: CultureSure is an optimal commercial cryopreservation solution for the storage of D. immitis purified mf, mf-infected total blood, and mf used for in vivo mosquito experiments. Furthermore, this study describes an easy preservation method for clinical D. immitis-infected blood samples facilitating vector stage studies, as well as the study of macrocyclic lactone resistance in heartworms and the education of veterinarians.

The development of the dog heartworm is highly sensitive to sterols which activate the orthologue of the nuclear receptor DAF-12.

Prevention therapy against Dirofilaria immitis in companion animals is currently threatened by the emergence of isolates resistant to macrocyclic lactone anthelmintics. Understanding the control over developmental processes in D. immitis is important for elucidating new approaches to heartworm control. The nuclear receptor DAF-12 plays a role in the entry and exit of dauer stage in Caenorhabditis elegans and in the development of free-living infective third-stage larvae (iL3) of some Clade IV and V parasitic nematodes. We identified a DAF-12 ortholog in the clade III nematode D. immitis and found that it exhibited a much higher affinity for dafachronic acids than described with other nematode DAF-12 investigated so far. We also modelled the DimDAF-12 structure and characterized the residues involved with DA binding. Moreover, we showed that cholesterol derivatives impacted the molting process from the iL3 to the fourth-stage larvae. Since D. immitis is unable to synthesize cholesterol and only completes its development upon host infection, we hypothesize that host environment contributes to its further molting inside the host vertebrate. Our discovery contributes to a better understanding of the developmental checkpoints of D. immitis and offers new perspectives for the development of novel therapies against filarial infections.

An investigation of Dirofilaria immitis infection and its effects on mosquito wingbeat frequencies.

Each mosquito species has a different wingbeat frequency by which they attract mates. With just a brief recording (<1/10th of a second) these acoustic signatures can be analyzed to quickly determine if mosquitoes belong to a species that is known to transmit different pathogens. A recent study has shown that mobile phones are capable of capturing acoustic data from mosquito wingbeats. We examined wingbeat signatures and flight duration patterns of D. immitis infected and non-infected Aedes aegypti to determine if mobile phone recordings of wingbeat frequencies can be used to distinguish infected mosquitoes from non-infected ones. Female mosquitoes were recorded prior to and at various time points after feeding on infected or non-infected dog blood by placing individual mosquitoes into a collection vial and recording for 60 s using the Voice Memo app for iPhone 7 plus and 8. To uniformly analyze audio data, recordings were processed using a previously described automated algorithm in Python 3.0 to determine wingbeat frequency. A total of 1669 recordings were gathered, and mosquitoes were dissected to confirm the presence and number of D. immitis larvae. Our findings indicate that there was a significant effect on wingbeat frequency with an increasing number of L3 larvae. Specifically, as the number of L3, infective stage larvae increases, a decrease in wingbeat frequency is seen. However, there was no significant effect of increasing number of L1 or L2 larvae causing increasing wingbeat frequencies. The detection of a significant difference in wingbeat frequencies between mosquitoes harboring infective stage D. immitis larvae is unique and suggests the possibility of using wingbeat recordings as a tool for vector species and pathogen surveillance and monitoring.

Using population genetics to examine relationships of Dirofilaria immitis based on both macrocyclic lactone-resistance status and geography.

Prevention of infection with canine heartworm (Dirofilaria immitis) is based on the compliant administration of macrocyclic lactone (ML) drugs. Resistance to ML drugs is well documented in D. immitis; however, there remains a paucity of information on the spatial distribution and prevalence of resistant isolates. This project aims to improve understanding of ML-resistance by using a population genetic approach. We developed a large panel of microsatellite loci and identified 12 novel highly polymorphic markers. These 12, and five previously published markers were used to screen pools of microfilariae from 16 confirmed drug-susceptible, 25 confirmed drug-resistant, and from 10 suspected drug-resistant field isolates. In isolates where microfilarial suppression testing indicated resistance, Spatial Principal Component Analysis (sPCoA), Neighbor Joining Trees and Bayesian clustering all revealed high genetic similarity between pre- and post-treatment samples. Somewhat surprisingly, the Neighbor Joining tree and sPCoA generated using pairwise Nei's distances did not reveal clustering for resistant isolates, nor did it reveal state-level geographic clustering from samples collected in Georgia, Louisiana or Mississippi. In contrast, Discriminant Analysis of Principle Components was able to discriminate between susceptible, suspected-resistant and resistant samples. However, no resistance-associated markers were detected, and this clustering was driven by the combined effects of multiple alleles across multiple loci. Additionally, we measured unexpectedly large genetic distances between different passages of laboratory strains that originated from the same source infection. This finding strongly suggests that the genetic makeup of laboratory isolates can change substantially with each passage, likely due to genetic bottlenecking. Taken together, these data suggest greater than expected genetic variability in the resistant isolates, and in D. immitis overall. Our results also suggest that microsatellite genotyping lacks the sensitivity to detect a specific genetic signature for resistance. Future investigations using genomic analyses will be required to elucidate the genetic relationships of ML-resistant isolates.

Potential mosquito (Diptera: Culicidae) vectors of Dirofilaria immitis from residential entryways in Northeast Arkansas.

The nematode Dirofilaria immitis causes serious disease of canines in the United States. Transmitted by a variety of mosquito species, several studies have examined the prevalence of D. immitis in mosquitoes. However, no prevalence studies have focused on diurnal mosquitoes that could potentially enter homes. Mosquitoes were collected under doorway awnings at four residences to identify the diurnal blood-feeding mosquito species and prevalence of potential vectors harboring D. immitis in Jonesboro, Arkansas. Mosquitoes were collected from May to December 2016 using pheromone traps and aerial nets. Each mosquito was individually dissected and observed for larvae in the head, thorax, and abdomen to identify infective and non-infective larvae. Ten mosquito species from five genera were identified. Larvae, presumed to be D. immitis, were observed in five mosquito species, including Aedes albopictus, Anopheles quadrimaculatus, Culex pipiens quinquefasciatus, Culiseta inornata, and Psorophora columbiae and four genera, including Anopheles spp., Culex spp., Culiseta spp., and Psorophora spp. The diurnal mosquito, Ae. albopictus composed 3.4% of the total mosquitoes collected. An. quadrimaculatus and Anopheles spp. were the most abundant mosquitoes, composing 59.7 and 19.6% of the total mosquitoes collected, respectively. Of the 536 mosquitoes dissected, 60 (11.2%) were observed with D. immitis, and 33 (55.0%) of the heartworm-positive mosquitoes harbored infective (L3) larvae in the head or proboscis upon dissection. The high proportion of infective D. immitis larvae found in the mosquitoes aggregating under doorway awnings may increase the exposure of indoor dogs and cats to heartworm disease in northeast Arkansas.

Activation of the Toll pathway in Aedes aegypti blocks the development of emerging third-stage larvae of drug-resistant Dirofilaria immitis.

Dirofilaria immitis is the globally distributed agent of heartworm disease. Infection in canines causes debilitating disease that can be fatal if left untreated. Macrocyclic lactones can prevent heartworm disease in dogs, cats and ferrets by killing larvae before they develop into adult worms in the pulmonary artery. However, administration of prophylactic drugs to wild canids to prevent D. immitis infection is not feasible. Furthermore, a vaccine against heartworm is currently unavailable and drug resistant D. immitis have been identified, highlighting the need for new strategies to prevent parasite transmission. We recently established a method to block development of emerging third-stage larvae (eL3) from the mosquito Aedes aegypti by over-activating the Toll pathway, one of the major innate immune signaling pathways in mosquitoes. Our previous study used a drug-sensitive strain of D. immitis and it remains unknown if the strategy is effective against different D. immitis genotypes and, more importantly, if it would work against drug-resistant genotypes. The purpose of this study was to determine whether Toll pathway activation is capable of blocking eL3 development of D. immitis strains that are resistant to macrocyclic lactones. We infected mosquitoes with two independent strains of D. immitis previously confirmed as being resistant to macrocyclic lactones, and then activated Toll signaling by RNAi-mediated silencing of the pathway inhibitor, IκB/Cactus, and quantitatively measured eL3 development. Similar to the drug-sensitive strain, eL3 were strongly reduced by Toll activation in both drug-resistant strains. Furthermore, similar to the drug-sensitive strain, the reduction of eL3 in both drug-resistant strains suggests a defect in larval invasion of, or development in, the Malpighian tubules - the organ in the mosquito to which microfilariae migrate after ingestion and where the larvae undergo several developmental molts. In summary, Toll pathway activation blocks the development of three distinct D. immitis genotypes, including two different drug-resistant genotypes. If this strategy can be applied to heartworm vectors in the field, it may help reduce the spread of disease and is not predicted to favor the spread of drug resistance.

Chimeric symbionts expressing a Wolbachia protein stimulate mosquito immunity and inhibit filarial parasite development.

Wolbachia can reduce the capability of mosquitoes to transmit infectious diseases to humans and is currently exploited in campaigns for the control of arboviruses, like dengue and Zika. Under the assumption that Wolbachia-mediated activation of insect immunity plays a role in the reduction of mosquito vectorial capacity, we focused our attention on the Wolbachia surface protein (WSP), a potential inductor of innate immunity. We hypothesized that the heterologous expression of this protein in gut- and tissue-associated symbionts may reduce parasite transmission. We thus engineered the mosquito bacterial symbiont Asaia to express WSP (AsaiaWSP). AsaiaWSP induced activation of the host immune response in Aedes aegypti and Anopheles stephensi mosquitoes, and inhibited the development of the heartworm parasite Dirofilaria immitis in Ae. aegypti. These results consolidate previous evidence on the immune-stimulating property of WSP and make AsaiaWSP worth of further investigations as a potential tool for the control of mosquito-borne diseases.

Activation of mosquito immunity blocks the development of transmission-stage filarial nematodes.

Mosquito-borne helminth infections are responsible for a significant worldwide disease burden in both humans and animals. Accordingly, development of novel strategies to reduce disease transmission by targeting these pathogens in the vector are of paramount importance. We found that a strain of Aedes aegypti that is refractory to infection by Dirofilaria immitis, the agent of canine heartworm disease, mounts a stronger immune response during infection than does a susceptible strain. Moreover, activation of the Toll immune signaling pathway in the susceptible strain arrests larval development of the parasite, thereby decreasing the number of transmission-stage larvae. Notably, this strategy also blocks transmission-stage Brugia malayi, an agent of human lymphatic filariasis. Our data show that mosquito immunity can play a pivotal role in restricting filarial nematode development and suggest that genetically engineering mosquitoes with enhanced immunity will help reduce pathogen transmission.

Filarial infections in dogs in Cyprus, an apparently heartworm free island.

The current study investigated for the first time the occurrence of filarial infections in dogs in Cyprus. Blood samples of dogs from five districts of Cyprus (Lefkosia, Lemessos, Larnaka, Pafos and Ammochostos) were examined by the modified Knott's method and by serology, and the morphological classification of microfilariae was confirmed by molecular methods. A total of 200 dogs, 153 living in shelters and 47 owned dogs, were included in the study. Acanthocheilonema reconditum microfilariae were found in 9 (4.5%) samples and one (0.5%) sample was seropositive for D. immitis. No statistical significance was observed between microfilaraemic samples and the evaluated variables apart from the district (p = .005). The present study showed that dogs in Cyprus can be infected with blood circulating microfilariae and for the first time A. reconditum was reported as autochthonous infection in dogs in the country. No microfilariae of Dirofilaria spp. were detected. However, veterinarians should remain vigilant regarding Dirofilaria infections and should consider preventive protection to the animals, at least in case of travel in enzootic areas.

Further Characterization of Molecular Markers in Canine Dirofilaria immitis Infection.

Dirofilaria immitis is a common filarial parasite found in dogs and cats in the Americas, with the pathophysiological consequences of the infection differing somewhat between these 2 host species. Recent research efforts have been focused on determining if the microRNAs (miRNAs) released from adult Dirofilariae have a role as markers for distinguishing the intensity of adult worm infection, as well as determining the presence of new infections. This study expands previous work on 2 nematode miRNAs, miR34 and miR-71, by addressing their ability to discriminate between low and high D. immitis adult worm intensities in dogs. Serum samples were collected from 13 dogs, 8 of which carried known numbers of adult D. immitis at autopsy in their hearts and pulmonary vessels. Three groups of canine sera were created based on D. immitis burden: "control" (0 worms; 5 animals), "low intensity" (10-18 worms; mean ± SD = 12.3 ± 4.4; 4 animals), and "high intensity" (41-72 worms; mean 62.5 ± 15.1; 4 animals) groups. A qPCR analysis was performed on each sample to measure plasma levels of miR-34 and miR-71; however, no significant differences were observed between these groups in terms of levels of miRNAs, so the low- and high-intensity samples were then combined into a single "infected" category and compared to the "non-infected" controls. Copy numbers of both miR-34 and miR-71 were significantly higher in infected compared to uninfected animals ( P = 0.015 and P = 0.027, respectively). The Ct values of expression compared with the adult worm intensity for each miRNA revealed that both miR-34 and miR-71 significantly discriminate between the infected and non-infected groups ( P value < 0.0001 for both). These findings support the contention that miRNA 34 and miRNA 71, which are filarial-specific miRNAs, can both serve as biomarkers for the presence of D. immitis infection in dogs, but at this point they do not appear to reflect the actual intensity of adult parasites present.

Heartworm-associated respiratory disease (HARD) induced by immature adult Dirofilaria immitis in cats.

BACKGROUND: A controlled, blind research study was conducted to define the initial inflammatory response and lung damage associated with the death of immature adult Dirofilaria immitis in cats as compared with cats developing adult heartworm infections and cats on preventive medication. METHODS: Three groups of cats were utilized, 10 per group. All cats were infected with 100 third-stage (L3) larvae by subcutaneous injection. Group A cats were treated topically with selamectin (Revolution®; Zoetis) per label directions at 28 days post infection (PI) and once monthly for 8 months. Group B cats were treated orally with ivermectin (Ivomec®; Merial) at 150 µg/kg at 70 days PI, then every 2 weeks for 5 months. Group C cats were untreated PI. At baseline (Day 0) and on Days 70, 110, 168, and 240 PI, peripheral blood, serum, bronchial lavage, and thoracic radiographic images were collected on all cats. Upon completion of the study (Day 245), cats were euthanized and necropsies were conducted. RESULTS: Results were analyzed statistically between groups by ANOVA and by paired sample T testing for changes within the group over time. The selamectin-treated cats (Group A) did not develop radiographically evident changes throughout the study and were free of adult heartworms or worm fragments at necropsy. The heartworm life cycle was abbreviated with oral doses of ivermectin (Group B), shown by the absence of adult heartworms or worm fragments at necropsy. The early stage of immature adult worm in Group B cats, however, did induce severe pulmonary airway, interstitial, and arterial lung lesions, revealing that the abbreviated infection is a significant cause of respiratory pathology in cats. Cats in Groups B and C could not be differentiated based on radiographic changes, serologic antibody titers, complete blood count, or bronchoalveolar lavage cytology at any time point throughout the study. Eighty percent of cats in Group A and 100% of cats in Groups B and C became heartworm antibody positive at some time point post infection. CONCLUSIONS: The clinical implications of this study are that cats that become infected with immature adult heartworms may not develop fully mature heartworms and are only transiently heartworm antibody positive, but do develop Heartworm-Associated Respiratory Disease (HARD).

Development of Dirofilaria immitis and Dirofilaria repens in Aedes japonicus and Aedes geniculatus.

BACKGROUND: The mosquito-borne filarial nematodes Dirofilaria immitis and Dirofilaria repens primarily affect dogs but also cats, causing heartworm disease or subcutaneous dirofilariosis, respectively, and both may also cause zoonotic diseases in humans. Several mosquito species have been reported as competent vectors for these nematodes, but no data are available for the invasive mosquito species Aedes japonicus (Theobald, 1901). The objective of this study was to describe the development of both D. immitis and D. repens under standardised experimental laboratory conditions in mosquitoes. METHODS: For this purpose, both a laboratory strain and field-collected individuals of the invasive mosquito species Ae. japonicus and, for comparative purposes, a laboratory strain of Aedes geniculatus, a rare indigenous species sharing habitats with Ae. japonicus, and of the tropical species Aedes aegypti were used. Anticoagulated microfilariaemic blood was fed at a density of 3000 mf/ml to mosquitoes with a hemotek system. Blood-fed mosquitoes were incubated at 27 °C and 85% relative humidity, and specimens were dissected under the microscope at pre-set time points to observe developmental stages of both Dirofilaria species. Additionally, real-time PCRs were carried out in some microscopically negative samples to determine the infection rates. RESULTS: In field-collected Ae. japonicus infectious L3 larvae of both D. immitis and D. repens developed, rendering this mosquito species an efficient vector for both filarial species. Additionally, Ae. geniculatus was shown to be an equally efficient vector for both filarial species. Aedes japonicus mosquitoes from a laboratory colony were refractory to D. immitis but susceptible to D. repens, whereas Ae. aegypti was refractory to both filarial species. CONCLUSIONS: To our knowledge, Aedes japonicus was for the first time shown to be an efficient vector for both D. immitis and D. repens, indicating that this invasive and locally highly abundant species may contribute to a transmission of filarial worms. The data emphasize the necessity to perform vector competence studies with local mosquito populations as basis for risk assessments. We further demonstrated that detection of filarial DNA in a mosquito species alone does not allow to draw reliable conclusions with regard to its vector competence.

Geographic distribution of canine heartworm (Dirofilaria immitis) infection in stray dogs of eastern Romania.

A survey was conducted in the eastern part of Romania to assess the prevalence and geographical distribution of Dirofilaria immitis in dogs. Plasma samples were collected from 458 stray dogs hosted in shelters in 8 counties and tested serologically for the presence of heartworm. In addition, 45 blood samples from dogs of a shelter in Galati city were examined by the modified Knott and multiplex polymerase chain reaction (PCR) techniques. The immmunological assay showed a heartworm infection prevalence of 8.9% in the dogs. Optical density results for positive samples ranged between 0.217 and 2.683. Geographical information systems (GIS) were used to produce overlays of distribution maps of D. immitis prevalence and predictive maps based on temperature suitability. High prevalence of D. immitis was found in the central East up to the northern border of the country, i.e. Galati county (60%), followed by the counties of Vaslui (12.0%) and Iasi (7.7%). Out of 45 samples examined using the Knott test, 23 were positive for circulating microfilariae (51.1%), while 19 dogs were positive for D. immitis and 4 for both D. immitis and D. repens with the multiplex PCR test. The high prevalence for D. immitis shown in dogs in the Southeast (Galati, 42.2%) also by multiplex PCR gave strong support to the results achieved by the serological tests. The present study confirms the ability of GIS to predict the distribution and epidemiology of dirofilariosis in different geographical territories as has been already demonstrated by the empirical epidemiological data obtained at the continental, national and intraregional levels.

Dirofilaria immitis exhibits sex- and stage-specific differences in excretory/secretory miRNA and protein profiles.

The canine heartworm Dirofilaria immitis releases excretory/secretory molecules into its host and in culture. We report analyses of the types, amounts and stage-dependence of microRNAs and proteins found in D. immitis culture media recovered after incubating 800,000 microfilariae for 6days, 500L3 and 500L4 for 7days, as well as 40 adult females and 40 adult males for 48h, all separately. In addition, the presence of exosome-like particles was established by nanoparticle tracking analysis. Our results are in concordance with the D. immitis molecules previously detected in dog blood and in culture medium, but add additional insight into the sex- and stage-specificity of these processes. Of 131 miRNA candidates analyzed, none of the most abundant sequences was exclusively associated with one stage. Several isoforms of the nematode miR-100 family, miR-279, miR-71, were highly represented and overlapped substantially with the profile of heartworm miRNAs described from infected dog blood. lin-4 was primarily associated with males. We also report 4, 27 and 72 proteins in media from microfilariae, females and males, respectively. The only protein in common to all samples was actin, and only 9/88 proteins with a gene ontology description had not been reported in other studies of filarial secretomes. Exosomal proteins were well represented, dominated by cytoskeletal proteins, metabolic enzymes, zeta polypeptide, and chaperones.

PREVALENCE, GENETIC ANALYSES, AND RISK FACTORS ASSOCIATED WITH HEARTWORM (DIROFILARIA IMMITIS) IN WILD COYOTES (CANIS LATRANS) FROM FLORIDA, USA.

We detected heartworm (Dirofilaria immitis) in 37.2% of 212 coyotes ( Canis latrans ) collected from 28 counties in Florida, US, between February 2010 and April 2014. Adult coyotes had a higher prevalence (45.6% of 103) than juveniles (29% of 80), and there was no significant difference in prevalence between adult male and female coyotes. Adults demonstrated a higher prevalence of heartworm in northern counties (56% of 91) than in southern counties (23.1% of 121) and a higher prevalence in urban areas (58.1% of 31) than in rural areas (33.3% of 63). There was no significant difference in mean body weight between infected and uninfected adult males or females. Intensity ranged from 1 to 144 (x̄ =21.29, SD=23.89, n=77). Mixed-sex heartworm infections were found in 88.9% of positive hosts, where the female-to-male ratio of worms was 1.14:1 (n=27). No pathologic changes attributable to heartworm infection were seen. A subsample of heartworms was confirmed to be D. immitis by targeted PCR amplification and DNA sequencing. Of 25 samples submitted for genotyping eight sequence haplotypes were found, all of which were distinct from all publicly available sequences derived from heartworms collected from domestic dogs ( Canis lupus familiaris). These sequences revealed more polymorphisms than did previous samples of D. immitis, which provides a broader foundation for the possibility that the heartworms will develop resistance to anthelmintics.

[INFECTION OF BLOOD-SUCKING MOSQUITOES (DIPTERA: CULICIDAE) WITH DIROFILARIAE (SPIRURIDA, ONCHOCERCIDAE) IN THE TULA REGION].

Blood-sucking mosquitoes (n = 2277) collected in Tula and its Region in 2013-2014 were examined using a PCR assay for dirofilariae. A total of 12 species from 4 genera (Culiseta, Aedes, Ochlerotatus [foreign character] Culex) out of 18 found mosquito species were infected with Dirofilaria immitis and D. repens. The proportion of the infected mosquitoes was 2.5% (D. immitis, 1.5%; D.repens, 1%). According to preliminary data, the most efficient Dirofilaria vectors, in the Tula Region may be Ae. vexans, Ae. geniculatus, Och. cantans, and Cx. pipiens.

Morphological and molecular identification of Dirofilaria immitis from Jackal (Canis aureus) in North Khorasan, northeast Iran.

BACKGROUND & OBJECTIVES: The heartworm Dirofilaria immitis is an important mosquito-borne zoonotic nematode of domestic and wild mammals throughout the world, causing cardiopulmonary dirofilariasis. This parasite has been reported from carnivores in some provinces of Iran. However, in the present study, the occurrence of this filarial nematode is reported for the first time in wild canids of the North Khorasan Province, located in northeast Iran, based on morphological and molecular characteristics. METHODS: The carcasses of 45 golden jackals (Canis aureus), 16 foxes (Vulpes vulpes), 15 dogs (Canis familiaris), and one wolf (Canis lupus) were necropsied between 2013 and 2014. RESULTS: By gross examination, adult filarial nematodes were found in the cardiovascular system of four jackals (8.9%). The morphological characteristics of the recovered heartworms were compatible with D. immitis. DNA sequencing of the cytochrome c oxidase subunit I (cox1) gene of all four isolates was identical, showing 100% homology with several sequences registered in GenBank from other countries. No adult D. immitis was found in any of the other animals examined. INTERPRETATION & CONCLUSION: D. immitis is circulating in wildlife of the study area, suggesting the relevance of developing control programmes to prevent transmission of the disease to humans and domestic animals.

Fine-scale temperature fluctuation and modulation of Dirofilaria immitis larval development in Aedes aegypti.

We evaluated degree-day predictions of Dirofilaria immitis development (HDU) under constant and fluctuating temperature treatments of equal average daily temperature. Aedes aegypti mosquitoes were infected with D. immitis microfilariae and parasite development was recorded at set time points in dissected mosquitoes. Time to L3 development in Malpighian tubules and detection in mosquito heads was shorter for larvae experiencing a daily regime of 19±9°C than larvae at constant 19°C; larval development rate in Malpighian tubules was slower in fluctuating regimes maintained above the 14°C developmental threshold than larvae under constant temperatures. We showed that hourly temperature modeling more accurately predicted D. immitis development to infective L3 stage. Development time differed between fluctuating and constant temperature treatments spanning the 14°C development threshold, implicating a physiological basis for these discrepancies. We conclude that average daily temperature models underestimate L3 development-and consequently dog heartworm transmission risk-at colder temperatures, and spatiotemporal models of D. immitis transmission risk should use hourly temperature data when analyzing high daily temperature ranges spanning 14°C.