Detection of Dirofilaria immitis via integrated serological and molecular analyses in coyotes from Texas, United States

Sobotyk, Caroline; Nguyen, Nathalia; Varner, Amanda; Saleh, Meriam N.; Hilton, Clayton D.; Tomeček, John M.; Esteve-Gasent, Maria D.; Verocai, Guilherme G.

doi:10.1016/j.ijppaw.2022.03.012

Cited by 13 publications

(18 citation statements)

References 40 publications

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“…The ELISA methods have shown higher sensitivity and specificity rates ranging from 85.7% to 100% depending on the number of mature female D. immitis present [3,10]. The use of molecular approaches for diagnosing heartworm infections has increased over the past years, including conventional PCR and qPCR targeting nuclear and mitochondrial genes [4,22,23]. The integration of molecular assays in the diagnosis of filarial infections has been shown to be an efficient and reliable screening tool to confirm the disease in intermediate and definitive hosts [23,24].…”

Section: Discussionmentioning

confidence: 99%

“…Genomic DNA was extracted and purified from 200 µl EDTA whole blood using the QIAmp DNA Blood Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions. All samples were screened for the presence of D. immitis DNA using a probe-based qPCR assay targeting a fragment of the cytochrome c oxidase subunit 1 ( cox1 ) of the mitochondrial DNA, with primers and probe previously described by Laidoudi et al [ 12 ] and protocol modified by Sobotyk et al [ 4 ]. A 166-base-pair cox1 fragment was amplified using the forward primer Fil.COI.749-F (5′-CAT CCT GAG GTT TAT GTT ATT ATT TT-3′) and reverse Fil.COI.914-R (5′-CWG TAT ACA TAT GAT GRC CYC A-3′), and TaqMan ® probe D.imm.COI.777-P (6FAM-CGG TGT TTG GGA TTG TTA GTG-MGB) designed by Laidoudi et al [ 12 ].…”

Section: Methodsmentioning

confidence: 99%

“…Real-time PCR reactions were performed on a QuantStudio 3 real-time PCR system (Applied Biosystems, Waltham, MA, USA) using the using TaqMan ® universal cycling conditions. Cycling conditions consisted of a hold stage at 95 °C for 20 s and 40 cycles of a two-step PCR stage at 95 °C for 1 s followed by 60 °C for 20 s [ 4 ]. All runs included a positive and a non-template control.…”

Section: Methodsmentioning

confidence: 99%

“…In dogs, D. immitis infection can result in clinical disease, including caval syndrome, hemolysis, liver and kidney dysfunction, heart failure, and death [3]. Due to the severity of canine heartworm disease and the increased prevalence rate over past decades, surveys have focused on the diagnosis and prevalence of D. immitis in dogs and wild canids that may serve as a reservoir host [4][5][6]. Despite the higher risk of complications and the increased risk of infection in endemic areas, canine heartworm disease is preventable.…”

Section: Introductionmentioning

confidence: 99%

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Probe-based qPCR as an alternative to modified Knott’s test when screening dogs for heartworm (Dirofilaria immitis) infection in combination with antigen detection tests

et al. 2022

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Background Current recommendations for diagnosis of Dirofilaria immitis infection in dogs rely on the detection of antigen produced largely by adult females coupled with the visualization of microfilariae (mf) in the circulation via a microfilaria detection test (MFDT). It is hypothesized that qPCR assays used in parallel with antigen detection tests will perform better in detecting mf than modified Knott’s test (MK), when combined with antigen detection. This study compares probe-based qPCR and MK techniques for mf detection used in parallel with the DiroCHEK® antigen test to screen for heartworm infection in shelter dogs. Methods Matching blood and serum samples were collected from 300 shelter dogs in Brazos and Harris counties, Texas, USA. Blood was assessed for the presence of mf via MK and the presence of D. immitis DNA by a species-specific probe-based qPCR assay. Serum samples were tested for the presence of heartworm antigen using DiroCHEK® before and after immune complex dissociation (ICD) via heat treatment. In addition, the performance of each diagnostic test was evaluated via Chi-square test, Cochran’s Q test, and post hoc analysis. Results Qualitatively, MK detected mf in 22.0% (66/300) of samples, 55 of which were morphologically identified as D. immitis and 11 as Acanthocheilonema reconditum. The range of heartworm mf was 28 to 88,803 mf/ml (median: 6627.5). Real-time PCR detected D. immitis DNA in 20.7% (62/300) of samples. Heartworm antigen was detected in 24.7% (74/300) of samples pre-ICD, and in 29.3% (88/300) post-ICD. When comparing tests, the Chi-square and McNemar’s tests showed that the difference between positive and negative proportions was statistically significant. The Cochran test showed the difference in the distributions of cases and non-cases was significant when individual tests were combined (χ2 = 62.3, df = 3, P < 0.0001) and when parallel methods were combined (χ2 = 43.1, df = 4, P < 0.0001). Conclusion Considering individual and combined test performances, practicality, and efficient use of bench time, this heartworm-specific probe-based qPCR method is a viable option as a mf detection test to be used in parallel with antigen tests for canine heartworm infection in diagnostic and research settings. Graphical Abstract

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Probe-based qPCR as an alternative to modified Knott’s test when screening dogs for heartworm (Dirofilaria immitis) infection in combination with antigen detection tests

et al. 2022

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“…According to yearly CAPC data from 2017-2021, the percentage of cats testing antigen-positive ranged from 1.45-3.85% and cats testing antibody-positive from 0.53-1.32% in Texas [26]. In addition to the existing risk of heartworm infection in an endemic area, susceptibility of various carnivore species is supported by epidemiological studies in wildlife and case reports in wild and captive species, many of which from Texas or in species that occur in Texas [5,7,10,12,36,37]. Moreover, many of the species represented in our sampling have been reported infected with heartworm or had antigen detected via commercial ELISA kits (Table 7).…”

Section: Discussionmentioning

confidence: 99%

Heartworm, Dirofilaria immitis, in carnivores kept in zoos located in Texas, USA: Risk perception, practices, and prevalence

Upton

Budke

Verocai

2022

Preprint

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Background Dirofilaria immitis, the causative agent of heartworm disease has long been established to affect both wild and domestic canids, felids, and mustelids. Recent literature demonstrates that additional families in the order Carnivora are also susceptible. The objectives of this study were to 1) better understand current practices surrounding heartworm prevention and diagnostics in zoological facilities located in the state of Texas, USA and 2) assess the seroprevalence of D. immitis infection among terrestrial carnivores kept in these facilities. Methods A questionnaire was completed by veterinarians or veterinary technicians representing 10 zoological facilities across Texas. This questionnaire was designed at the taxonomic family level, encompassing the 12 terrestrial carnivore families: Ailuridae, Canidae, Eupleridae, Felidae, Herpestidae, Hyaenidae, Mephitidae, Mustelidae, Prionodontidae, Procyonidae, Ursidae, and Viverridae. The second objective was achieved with the use of archival serum samples made available by six zoo facilities. Results Risk perception varied across facilities for every family, including among species belonging to Canidae. All facilities used monthly heartworm prevention in canids and felids, with more variation existing in the other families. The use of diagnostic testing and type and route of administration of preventive varied by facility, with oral ivermectin the most commonly used preventive. A total of 217 archival serum samples, belonging to 211 individual animals encompassing 11 families and 39 species, were tested with a commercial heartworm antigen ELISA test, pre- and post-immune-complex dissociation. A subset of samples was also assessed for presence of feline anti-heartworm antibodies using a commercial ELISA test. Two animals had antigen detected (0.95%), both Asian small-clawed otters from the same facility. Conclusions This study demonstrates that while the zoo veterinary community is aware of the risk and health impact of heartworm disease in canids and felids, there is still a great deal of uncertainty regarding the risks and ideal strategies for prevention in other carnivore families. The low prevalence found in zoo carnivores in Texas may serve as a baseline for future prevalence studies across the southern United States amid the emerging concern of macrocyclic lactone resistance.

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Dirofilaria immitis and Dirofilaria striata (Spirurida: Onchocercidae) detected in wild carnivores from Texas, United States

Ramos,

Hakimi,

Salomon

et al. 2024

International Journal for Parasitology: Parasites and Wildlife

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Detection of Dirofilaria immitis via integrated serological and molecular analyses in coyotes from Texas, United States

Cited by 13 publications

References 40 publications

Probe-based qPCR as an alternative to modified Knott’s test when screening dogs for heartworm (Dirofilaria immitis) infection in combination with antigen detection tests

Probe-based qPCR as an alternative to modified Knott’s test when screening dogs for heartworm (Dirofilaria immitis) infection in combination with antigen detection tests

Heartworm, Dirofilaria immitis, in carnivores kept in zoos located in Texas, USA: Risk perception, practices, and prevalence

Dirofilaria immitis and Dirofilaria striata (Spirurida: Onchocercidae) detected in wild carnivores from Texas, United States

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