Immitis Microfilariae Research Articles

Goondapyrones A-J: Polyketide α and γ Pyrone Anthelmintics from an Australian Soil-Derived Streptomyces sp.

An investigation of ×19 soil samples collected under the auspices of the Australian citizen science initiative, Soils for Science, returned ×559 chemically dereplicated microbial isolates, of which ×54 exhibited noteworthy anthelmintic activity against either the heartworm Dirofilaria immitis microfilaria and/or the gastrointestinal parasite Haemonchus contortus L1-L3 larvae. Chemical (GNPS and UPLC-DAD) and cultivation (MATRIX) profiling prompted a detailed chemical investigation of Streptomyces sp. S4S-00196A10, which yielded new anthelmintic polyketide goondapyrones A-J (1-10), together with the known actinopyrones A (11) and C (12). Structures for 1-12 were assigned on the basis of detailed spectroscopic and chemical analysis, with preliminary structure activity relationship analysis revealing selected γ-pyrones >50-fold and >13-fold more potent than isomeric α-pyrones against D. immitis mf motility (e.g., EC50 0.05 μM for 1; EC50 2.7 μM for 5) and H. contortus L1-L3 larvae development (e.g., EC50 0.58 μM for 1; EC50 8.2 μM for 5), respectively.

Characterization of a unique catechol-O-methyltransferase as a molecular drug target in parasitic filarial nematodes.

Filarial nematodes cause severe illnesses in humans and canines including limb deformities and disfigurement, heart failure, blindness, and death, among others. There are no vaccines, and current drugs against filarial nematodes infections have only modest effects and are prone to complications. We identified a gene (herein called DiMT) encoding an S-adenosyl-L-methionine (SAM)-dependent methyltransferase with orthologs in parasite filarial worms but not in mammals. By in silico analysis, DiMT possesses catalytic sites for binding SAM and catecholamines with high affinity. We expressed and purified recombinant DiMT protein and used it as an enzyme in a series of SAM-dependent methylation assays. DiMT acted specifically as a catechol-O-methyltransferase (COMT), catalyzing catabolic methylation of dopamine, and depicted Michaelis Menten kinetics on substrate and co-substrate. Among a set of SAM-dependent methyltransferase inhibitors, we identified compounds that bound with high affinity to DiMT's catalytic sites and inhibited its enzymatic activity. By testing the efficacy of DiMT inhibitors against microfilariae of Dirofilaria immitis in culture, we identified three inhibitors with concentration- and time-dependent effect of killing D. immitis microfilariae. Importantly, RNAi silencing of a DiMT ortholog in Caenorhabditis elegans has been shown to be lethal, likely as a result of excessive accumulation of active catecholamines that inhibit worm locomotion, pharyngeal pumping and fecundity. Together, we have unveiled DiMT as an essential COMT that is conserved in parasitic filarial nematodes, but is significantly different from mammalian COMTs and, therefore, is a viable target for development of novel drugs against filarial nematode infections.

Australian Marine and Terrestrial Streptomyces-Derived Surugamides, and Synthetic Analogs, and Their Ability to Inhibit Dirofilaria immitis (Heartworm) Motility.

A bioassay-guided chemical investigation of a bacterium, Streptomyces sp. CMB-MRB032, isolated from sheep feces collected near Bathurst, Victoria, Australia, yielded the known polyketide antimycins A4a (1) and A2a (2) as potent inhibitors of Dirofilaria immitis (heartworm) microfilaria (mf) motility (EC50 0.0013-0.0021 µg/mL), along with the octapeptide surugamide A (3) and the new N-methylated analog surugamide K (4). With biological data suggesting surugamides may also exhibit activity against D. immitis, a GNPS molecular network analysis of a library of microbes sourced from geographically diverse Australian ecosystems identified a further five taxonomically and chemically distinct surugamide producers. Scaled-up cultivation of one such producer, Streptomyces sp. CMB-M0112 isolated from a marine sediment collected at Shorncliff, Qld, Australia, yielded 3 along with the new acyl-surugamides A1-A4 (5-8). Solid-phase peptide synthesis provided additional synthetic analogs, surugamides S1-S3 (9-11), while derivatization of 3 returned the semi-synthetic surugamide S4 (12) and acyl-surugamides AS1-AS3 (13-15). The natural acyl-surugamide A3 (7) and semi-synthetic acyl-surugamide AS3 (15) were shown to selectively inhibit D. immitis mf motility (EC50 3.3-3.4 µg/mL), however, unlike antimycins 1 and 2, were inactive against the gastrointestinal nematode Haemonchus contortus L1-L3 larvae (EC50 > 25 µg/mL) and were not cytotoxic to mammalian cells (human colorectal carcinoma SW620, IC50 > 30 µg/mL). A structure-activity relationship (SAR) study on the surugamides 3-15 revealed that selective acylation of the Lys3-ε-NH2 correlates with anthelmintic activity.

Development of a novel rodent model for dog heartworm microfilaremia using the severe-combined immunodeficiency mouse

Dirofilaria immitis is a mosquito-borne parasitic nematode that causes fatal heartworm disease in canids. The microfilariae are essential for research, including drug screening and mosquito-parasite interactions. However, no reliable methods for maintaining microfilaria long-term are currently available. Therefore, we used severe combined immunodeficiency (SCID) mice to develop a reliable method for maintaining D. immitis microfilaria. SCID mice were injected intravenously with microfilariae isolated from a D. immitis-infected dog. Microfilariae were detected in blood collected from the tail vein 218 days post-inoculation (dpi) and via cardiac puncture 296 dpi. Microfilariae maintained in and extracted from SCID mice showed infectivity and matured into third-stage larvae (L3s) in the vector mosquito Aedes aegypti. L3s can develop into the fourth stage larvae in vitro. Microfilariae from SCID mice respond normally to ivermectin in vitro. The microfilariae in SCID mice displayed periodicity in the peripheral circulation. The SCID mouse model aided in the separation of microfilariae from cryopreserved specimens. The use of SCID mice enabled the isolation and sustained cultivation of microfilariae from clinical samples. These findings highlight the usefulness of the SCID mouse model for studying D. immitis microfilaremia in canine heartworm research.

Prevention of heartworm infection in dogs using a combination of moxidectin, imidacloprid and praziquantel: evidence from a randomized clinical trial.

The aim of this study was to evaluate the efficacy of a topical combination of moxidectin 3.5%, imidacloprid 10% and praziquantel 10% for the prevention of Dirofilaria immitis (Leidy, 1856) infection in dogs. For this purpose, a randomized and controlled clinical trial was conducted between August 2021 and October 2022, in the municipality of Goiana, state of Pernambuco, north-eastern Brazil, where heartworm is highly prevalent. Of the 213 dogs initially sampled (baseline), 68 (31.9%) were positive for adult antigens (SNAP 4Dx Plus, Idexx) and/or microfilariae (modified Knott's test). On day 0, 140 negative dogs were randomly included in the treatment and control groups, 70 animals each. During the study, 60 dogs (34 treated and 26 untreated) were removed for different reasons. At the end of the study (day 360 ± 2), 36 treated and 44 untreated were sampled and included in the efficacy calculation. The efficacy against the development of adults and microfilariae was 84.7%, with only one treated dog being positive for adult antigens but negative for microfilariae. On the other hand, eight untreated dogs were positive for adultantigens and/or microfilariae, resulting in a significant difference in the number of positives between groups (Chi-square test = 4.706, df = 1, P = 0.0301). Remarkably, the efficacy against the appearance of D. immitis microfilariae was 100% (i.e., all treated dogs negative) and three untreated dogs were positive for microfilariae. The topical combination of moxidectin 3.5%, imidacloprid 10% and praziquantel 10% significantly reduced the risk of D. immitis infection in treated dogs as compared with untreated dogs, in a highly endemic area in north-eastern Brazil.

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.

“Slow kill” treatment reduces DNA damage in leukocytes of dogs naturally infected with Dirofilaria immitis

Parasitic diseases are considered to be a cause of oxidative stress which leads to oxidative damage of various molecules including DNA. This can result in mutations, replication errors, and genome instability. Therefore, aim of this study was to measure DNA damage induced by Dirofilaria immitis in the single cells such as dogs’ leukocytes using the comet assay. Also, we monitored the effects of antiparasitic treatment on mitigation of sensitivity to DNA damage in leukocytes treated with H2O2 using the in vivo and ex vivo comet assay. The whole blood samples from 34 dogs from Serbia were used, both males and females, from one to 13 years old, both pure and mixed-breeds. A rapid immunochromatographic test (Antigen Rapid Heartworm Ag 2.0 Test Kit, Bionote, Minnesota, USA) was used for the detection of D. immitis antigens. The modified Knott’s test and PCR were used in the aim of detecting D. immitis microfilariae in dogs’ blood, and evaluating the number of circulating microfilariae during the treatment. The genotoxicity evaluation showed that D. immitis infection resulted in DNA damage in naturally infected dogs, with the highest DNA damage occurring in the group of dogs with severe clinical signs. Treatment with ivermectin and doxycycline decreased DNA damage in leukocytes of dogs in all groups, as the intensity of infection decreased due to applied therapy. Ex vivo comet assay results showed that leukocytes exhibited decreased sensitivity to H2O2-induced DNA damage during treatment. The results of the modified Knott’s test and PCR in our study showed that treatment with ivermectin and doxycycline was successful in decreasing the average number of microfilariae during the time and at the end eliminating them from the dogs’ blood.

Elimination of Dirofilaria immitis Infection in Dogs, Linosa Island, Italy, 2020-2022.

On Linosa Island, Italy, Dirofilaria immitis infection has been hyperendemic in dogs and seroprevalent among islanders. In 2020, we implemented a heartworm disease elimination program on Linosa Island. Of 54 dogs tested for D. immitis antigen and microfilariae, 28 had positive results and received treatment with oral doxycycline twice daily for 4 weeks plus topical imidacloprid/moxidectin monthly for 12 months. The 26 dogs with negative results received monthly topical imidacloprid/moxidectin as preventive. During month 1, the number of microfilaremic dogs was reduced by 76.5%. From month 2 on, all animals were microfilariae negative, and during months 3 to 9, the number of antigen-positive dogs decreased progressively. Treatment of positive dogs coupled with chemoprophylaxis for noninfected dogs was effective, protecting them from new infections. The elimination program reduced the risk for human infection, representing a One Health paradigm. Monitoring and chemoprophylaxis are advocated to maintain the status of heartworm disease-free area.

Dogs infected by Dirofilaria immitis: a threat to the health of human and non-human animals in Rio de Janeiro, Brazil.

This study aimed to investigate the presence of Dirofilaria immitis microfilaremia in dogs from two regions of the state of Rio de Janeiro, Brazil, where heartworm infections are highly prevalent. Blood samples were collected from dogs aged > 12 months, independent of the use of preventatives. All samples obtained and analyzed using Knott's modified test by the investigators. A total of 133 blood samples were tested, and D. immitis microfilariae were detected in 29 of them, resulting in an occurrence of 21.8%. The percentage of dogs with microfilaremia detected raises concerns for pet families, one health professionals, and small animal practitioners. Microfilaremic dogs are the richest source of infection for the mosquitoes, increasing the risk of transmission. Therefore, the stakeholders in One Health must raise concerns regarding the health of wild animals, as wild canids and other species of wild animals are exposed to the risk of D. immitis infection. In addition, humans can get infected and develop human pulmonary dirofilariasis. In conclusion, the presence of dogs with microfilaremia potentiates opportunities for D. immitis transmission, exposing all animals, wild or domestic, human or non-human to the disease.

Multiple diagnostic tests demonstrate an increased risk of canine heartworm disease in northern Queensland, Australia

BackgroundCanine heartworm (Dirofilaria immitis) is a life-threatening infection of dogs with a global distribution. Information on the prevalence of D. immitis and associated risk factors for canine heartworm antigen positivity—and thus disease—in Australia is scarce or outdated. The current reference method for D. immitis diagnosis in dogs is via the detection of heartworm antigen in blood using commercially available microwell-based enzyme-linked immunosorbent assays (ELISAs). Heat treatment of canine plasma prior to testing has been suggested to increase test sensitivity. The aim of the current study was to estimate the prevalence of D. immitis in dogs confined to shelters in Queensland, Australia. The impact of heat treatment on antigen test results was also assessed.MethodsBlood samples (n = 166) were collected directly from dogs in seven shelters across Queensland (latitudinal span of approx. 1700 km) into EDTA blood collection tubes. A commercially available ELISA (DiroCHEK®) was used to detect canine heartworm antigen in untreated and heat-treated plasma. Whole blood was concurrently tested for the presence of microfilariae and D. immitis DNA using a modified Knott’s test and real-time PCR, respectively. Risk factors (age, gender, source, location) associated with the odds of positivity for canine heartworm were assessed using binary logistic regression models.ResultsA total of 16 dogs (9.6%; 95% confidence interval [CI]: 5.9–15.2%) were positive for canine heartworm based on combined test results. Heat treatment did not impact on the positivity of D. immitis antigen within samples (Cohen’s kappa = 0.98), but the optical density was significantly increased in paired plasma samples for D. immitis antigen-positive samples (Wilcoxon matched-pairs signed rank test, two-tailed P < 0.01). Location of the dog in a shelter in northern Queensland was the only risk factor significantly associated with the odds of a dog being more likely to be D. immitis antigen positive (odds ratio: 4.39; 95% CI: 1.26–13.51). All samples positive for the modified Knott’s test were also positive for D. immitis DNA by PCR.ConclusionsThis study demonstrated the presence of heartworm-positive dogs in shelters in Queensland, with positive animals significantly more likely to occur in northern Queensland than southern Queensland. Sustained testing for the presence of D. immitis microfilariae and antigen remain important diagnostic tools in areas with known and re-emerging canine heartworm activity.Graphical

Occurrence of Dirofilaria immitis in Stray Dogs from Nepal

Nematodes of the genus Dirofilaria are widespread vector-borne helminths (VBH) of veterinary relevance. Dirofilariosis caused by D. immitis is frequently diagnosed in dogs often showing a severe clinical condition known as heartworm disease. Assessing the distribution pattern of canine dirofilariosis is pivotal to undertake appropriate control measures and define the risk of infection in animals and humans. This study provides original data on the occurrence of D. immitis in naive stray dogs from Nepal. An epidemiological study was performed on stray dogs from Siddharthanagar, Lumbini region, Nepal. Alive animals were blood sampled and examined for microfilariae using direct microscopy, buffy coat centrifugation and modified Knott's method. In addition, hematobiochemical parameters, including packed cell volume (PCV), alanine aminotransferase (SGPT), aspartate aminotransferase (SGOT), alkaline phosphate (ALP), creatinine, blood urea nitrogen (BUN), urea and total protein were analysed, along with a clinical scoring (body temperature and respiratory rate). During the observation period, data on autopsies performed on stray dogs showing canine heartworm disease were also included. The overall prevalence of D. immitis in both alive and necropsied dogs was 22.49% (38/169). Out of 150 alive dogs, 29 (19.3%) were positive for D. immitis microfilariae. The prevalence of the infection varied according to the technique used, ranging from 16.0 to 19.3% based on direct blood smear microscopy and modified Knott's technique, respectively. The infection rate was significantly higher (p < 0.05) in dogs aging more than 3years. A positive correlation was observed between SGPT and SGOT and the presence of microfilariae (p < 0.05). Among the 19 dead stray dogs, 9 animals showed adult D. immitis nematodes. Results of this survey account for the presence of D. immitis in stray dogs from Nepal, thus supporting the existence of a local transmission cycle of diroflariosis in the region. Further investigations are warranted to accurately define the prevalence of the infection in the canine population and to undertake appropriate control measures to reduce the burden on the infection in dogs and, accordingly, the risk of transmission to humans.

A novel assay to isolate and quantify third-stage Dirofilaria immitis and Brugia malayi larvae emerging from individual Aedes aegypti

BackgroundMosquitoes transmit filarial nematodes to both human and animal hosts, with worldwide health and economic consequences. Transmission to a vertebrate host requires that ingested microfilariae develop into infective third-stage larvae capable of emerging from the mosquito proboscis onto the skin of the host during blood-feeding. Determining the number of microfilariae that successfully develop to infective third-stage larvae in the mosquito host is key to understanding parasite transmission potential and to developing new strategies to block these worms in their vector.MethodsWe developed a novel method to efficiently assess the number of infective third-stage filarial larvae that emerge from experimentally infected mosquitoes. Following infection, individual mosquitoes were placed in wells of a multi-well culture plate and warmed to 37 °C to stimulate parasite emergence. Aedes aegypti infected with Dirofilaria immitis were used to determine infection conditions and assay timing. The assay was also tested with Brugia malayi-infected Ae. aegypti.ResultsApproximately 30% of Ae. aegypti infected with D. immitis and 50% of those infected with B. malayi produced emerging third-stage larvae. Once D. immitis third-stage larvae emerged at 13 days post infection, the proportion of mosquitoes producing them and the number produced per mosquito remained stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12–14 post infection. Increased uptake of D. immitis microfilariae increased the fitness cost to the mosquito but did not increase the number of emerging third-stage larvae.ConclusionsWe provide a new assay with an associated set of infection conditions that will facilitate assessment of the filarial transmission potential of mosquito vectors and promote preparation of uniformly infectious third-stage larvae for functional assays. The ability to quantify infection outcome will facilitate analyses of molecular interactions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission.

Developmental regulation of Dirofilaria immitis microfilariae and evaluation of ecdysone signaling pathway transcript level using droplet digital PCR

BackgroundCurrent measures for the prevention of dirofilariasis, caused by the dog heartworm, Dirofilaria immitis, rely on macrocyclic lactones, but evidence of drug-resistant isolates has called for alternative approaches to disease intervention. As microfilariae are known to be in a state of developmental arrest in their mammalian host and then undergo two molts once inside the arthropod, the aim of this study was to look at the developmental regulation of D. immitis microfilariae that occurs in their arthropod host using in vitro approaches and to investigate the role of the ecdysone signaling system in this development regulation.MethodsDirofilaria immitis microfilariae extracted from dog blood were incubated under various culture conditions to identify those most suitable for in vitro culture and development of the microfilariae, and to determine the effects of fetal bovine serum (FBS), mosquito cells, and ecdysteroid on the development of the microfilariae. Transcript levels of the ecdysone signaling pathway components were measured with droplet digital PCR (ddPCR).ResultsIn vitro conditions that best promote early development of D. immitis microfilariae to the “late sausage stage” have been identified, although shedding of the cuticle was not observed. FBS had inhibitory effects on the development and motility of the microfilariae, but media conditioned with Anopheles gambiae cells were favorable to microfilarial growth. The transcript level study using ddPCR also showed that ecdysone signaling system components were upregulated in developing microfilariae and that 20-hydroxyecdysone increased the proportion of larvae developing to the sausage and late sausage stages in vitro.ConclusionsThe arthropod host environment provides cues required for the rapid development of D. immitis microfilariae, and the ecdysone signaling system may play an important role in filarial nematode developmental transitions. This study contributes to a better understanding of the developmental process of D. immitis microfilariae.Graphical

Current Situation of the Presence of Dirofilaria immitis in Dogs and Humans in Bucaramanga, Colombia.

The cardiopulmonary dirofilariosis caused by Dirofilaria immitis, is a vector-borne infection, which can be transmitted to humans. The main hosts are both domestic and wild canids. This species mainly occurs in tropical and subtropical climates, and temperature and humidity are the main factors that favor the presence and proliferation of culicid mosquitoes as vectors of the disease. There are few reports of cardiopulmonary dirofilariosis in dogs and humans in Colombia, a region with favorable climatic conditions which favors the presence of mosquitoes that act as vectors of the disease. Therefore, this study aimed to examine its current prevalence in dogs and the risk of human exposure to the disease in Bucaramanga, one of the most populated areas in Colombia located at the center of the country. Furthermore, its demographic and environmental characteristics could be useful as a study model for other similar locations and neighboring countries. Serum samples from 351 dogs and 506 humans from the Bucaramanga Metropolitan area were analyzed. All dog samples were analyzed by Knott's technique and tested with a commercial immunochromatographic to detect the presence of circulating antigens of D. immitis. Human samples were analyzed using a non-commercial ELISA test kit to detect IgG against the somatic antigens of adult D. immitis and Wolbachia. Positive results were further confirmed using western blot analysis. Thirty-eight dogs tested positive with a overall prevalence of 10.82%. Of these dogs, 18 showed D. immitis microfilariae, being 5.12% of the total population. The overall seroprevalence in humans was 6.71%; seroprevalence was significantly higher in individuals aged 16–34 years-old and in women than in men. To our knowledge, this study describes seropositivity to D. immitis for the first time in a Colombian human population located in the same area as that of dogs infected with D. immitis, which represents a potential threat to public health. In humans, age and gender can be considered risk factors for exposure to D. immitis.

Laboratory assays reveal diverse phenotypes among microfilariae of Dirofilaria immitis isolates with known macrocyclic lactone susceptibility status.

Prevention of canine heartworm disease caused by Dirofilaria immitis relies on chemoprophylaxis with macrocyclic lactone anthelmintics. Alarmingly, there are increased reports of D. immitis isolates with resistance to macrocyclic lactones and the ability to break through prophylaxis. Yet, there is not a well-established laboratory assay that can utilize biochemical phenotypes of microfilariae to predict drug resistance status. In this study we evaluated laboratory assays measuring cell permeability, metabolism, and P-glycoprotein-mediated efflux. Our assays revealed that trypan blue, propidium iodide staining, and resazurin metabolism could detect differences among D. immitis isolates but none of these approaches could accurately predict drug susceptibility status for all resistant isolates tested. P-glycoprotein assays suggested that the repertoire of P-gp expression is likely to vary among isolates, and investigation of pharmacological differences among different P-gp genes is warranted. Further research is needed to investigate and optimize laboratory assays for D. immitis microfilariae, and caution should be applied when adapting cell death assays to drug screening studies for nematode parasites.

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

BackgroundDirofilaria 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.MethodsSix 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.ResultsPurified 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.ConclusionsCultureSure 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.

Feasibility and comparative analysis of Dirofilaria immitis microfilaria freezing and fixation for student instruction and assessment of clinical parasitology skills

BackgroundDetection of D. immitis microfilaria (mf) is an important diagnostic skill in veterinary medicine and is critical to Day 1 veterinarians and technicians. Finding a supply of blood containing mf to teach the technique and formalin’s adverse environmental effects used in the diagnostic microscopic tests present a challenge.ResultsThis study evaluated the use of cryopreserved and recently drawn mf-infected blood along with two fixative reagents, acetic acid or formalin for mf detection. The specific aims included determining if veterinary students could 1) detect cryopreserved mf added to fresh blood using routine diagnostic testing and 2) detect morphological differences in the mf. The 236 students were kept blind from the sample status. The ability of the students to identify mf and the mf morphology were compared for the samples and fixatives evaluated. The results demonstrate using a combination of cryopreservation and acetic acid for teaching microfilaria diagnostic techniques is fleasible; however, the quality of the mf morphology is less than optimal when compared to freshly acquired mf containing blood. Compared to reference values, the mf demonstrated a decrease in size with each additional variable evaluated.ConclusionA majority (98.3%) of the 236 students correctly identified the presence of mf. Teaching laboratories could utilize cryopreserved mf-spiked donor blood in lieu of freshly collected mf-containing blood from a naturally or experimentally infected dog. Substitution of less hazardous chemicals for the fixative can be used. Finally, the change in size measurements provides a mechanism to ensure students can correctly measure mf as students are required to do verifiable measurements and cannot copy reference values from a text book since the cryopreservation and fixation methods cause the mf to measure smaller than textbook reference values.

Macrocyclic lactone anthelmintic-induced leukocyte binding to Dirofilaria immitis microfilariae: Influence of the drug resistance status of the parasite

The macrocyclic lactone anthelmintics are the only class of drug currently used to prevent heartworm disease. Their extremely high potency in vivo is not mirrored by their activity against Dirofilaria immitis larvae in vitro, leading to suggestions that they may require host immune functions to kill the parasites. We have previously shown that ivermectin stimulates the binding of canine peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) to D. immitis microfilariae (Mf). We have now extended these studies to moxidectin and examined the ability of both drugs to stimulate canine PBMC and PMN attachment to Mf from multiple strains of D. immitis, including two that are proven to be resistant to ivermectin in vivo. Both ivermectin and moxidectin significantly increased the percentage of drug-susceptible parasites with cells attached at very low concentrations (<10 nM), but much higher concentrations of ivermectin (>100 nM) were required to increase the percentage of the two resistant strains, Yazoo-2013 and Metairie-2014, with cells attached. Moxidectin increased the percentage of the two resistant strains with cells attached at lower concentrations (<10 nM) than did ivermectin. The attachment of the PBMCs and PMNs did not result in any parasite killing in vitro. These data support the biological relevance of the drug-stimulated attachment of canine leukocytes to D. immitis Mf and suggest that this phenomenon is related to the drug resistance status of the parasites.

Seroprevalence and current infections of canine vector-borne diseases in Nicaragua

BackgroundVector-borne diseases constitute a major problem for veterinary and public health, especially in tropical regions like Central America. Domestic dogs may be infected with several vector-borne pathogens of zoonotic relevance, which may also severely compromise canine health.MethodsTo assess the prevalence of canine vector-borne diseases in Nicaragua, 329 dogs from seven cities, which were presented to the veterinarian for various reasons, were included in this study. Dogs were examined clinically and diagnostic blood samples were taken for analysis of packed cell volume (PCV) and presence of microfilariae as well as antigen of Dirofilaria immitis and antibodies to Ehrlichia spp., Anaplasma spp. and Borrelia burgdorferi (sensu lato) by use of a commercially available rapid ELISA. To detect current infections, specific PCRs for the detection of E. canis, A. platys and A. phagocytophilum were carried out on blood samples of the respective seropositive dogs. Microfilaremic blood samples, as well as D. immitis antigen positive samples were further subjected to PCR and subsequent sequencing for filarial species identification.ResultsAntibodies against Ehrlichia spp. were present in 62.9% of dogs, while Anaplasma spp. seroprevalence was 28.6%. Antibodies against species of both genera were detected in 24.9% of dogs. Borrelia burgdorferi (s.l.) antibodies were not detected. Dirofilaria immitis antigen was present in six animals (1.8%), two of which also showed D. immitis microfilariae in buffy coat. In addition to D. immitis, Acanthocheilonema reconditum was identified by PCR and sequencing in two of four additional microfilaremic blood samples, which were tested negative for D. immitis antigen. Current E. canis infections as defined by DNA detection were present in 58.5% of Ehrlichia-seropositive dogs, while 5.3% of Anaplasma-seropositive dogs were PCR-positive for A. platys, 2.2% for A. phagocytophilum and 16.0% for both Anaplasma species. Current E. canis infection had a statistically significant negative impact on PCV, whereas no relationship between infection status and clinical signs of disease could be observed.ConclusionsThese results indicate that canine vector-borne diseases are widespread in Nicaragua and that dogs may constitute a reservoir for human infection with E. canis, A. phagocytophilum and D. immitis. Thus, the use of repellents or acaricides to protect dogs from vector-borne diseases is strongly recommended.

Prevalence of canine heartworm infection in Mississippi animal shelters

Understanding diagnosis of heartworm disease in the context of animal shelters’ needs and expectations is crucial for developing guidelines that specifically address the unique shelter population. Accurate and economical heartworm testing strategies are essential. However, current comprehensive guidelines for the management of heartworm disease in dogs are directed toward client-owned animals and do not address needs of animal shelters and other resource-scarce facilities. Additionally, testing recommendations do not take into account regional and local differences in heartworm prevalence across the United States that occur due to abiotic and biotic factors. The objective of this study was to determine the apparent prevalence of Dirofilaria immitis microfilaremia and antigenemia in dogs from Mississippi animal shelters. Further, we compare agreement between microfilaria and antigen testing in this population. We performed a cross-sectional study using canine serum and blood bank samples representative of the Mississippi shelter population. Microfilaria testing of whole blood included a blood smear and modified Knott test. Antigen testing of serum was performed using the DiroCHEK® antigen ELISA test. Analyses included descriptive and analytic statistics as well as Cohen’s kappa for test agreement. A total of 283 whole blood samples and 363 serum samples, representing 363 dogs from 18 shelters in 17 Mississippi counties, were utilized in this study. Sixty-four (22.6%) whole blood samples demonstrated D. immitis microfilariae on the modified Knott test and 125 (34.4%) serum samples had detectable D. immitis antigen. Increasing age and low body condition were associated with antigen-positive test results. Only age was associated with microfilaria-positive test results. There was moderate agreement between the antigen ELISA test and the modified Knott microfilaria test and poor agreement between the antigen ELISA and the blood smear. This study provides the first known report of the prevalence of D. immitis microfilaremia and antigenemia in Mississippi shelter dogs. We observed that prevalence of both microfilaremia and antigenemia was significantly higher in these sampled dogs compared to previous reports for the owned canine population in Mississippi. Heartworm infection presents unique management challenges for animal shelters. Knowledge of the expected prevalence in the area can be utilized for management decisions related to prevention, testing, and treatment of dogs in shelter populations.