07 May Development and Diagnostic Value of an ELISA-Based Method for Detecting Dirofilaria repens Infection in Dogs

Author: Iuliia Dakhno, PhD in Veterinary Medicine
Affiliation: Independent Researcher, USA
Correspondence: [email protected] | +1 (347) 778-1295

Abstract:
This article presents a methodologically validated ELISA approach for detecting Dirofilaria repens infection in dogs. Given the emerging risk of parasite introduction to the United States, especially through imported animals and climate-related changes in vector distribution, the proposed diagnostic tool has important applications in both veterinary and public health contexts.

Dirofilaria repens

Introduction
Dirofilaria repens is a filarial nematode responsible for subcutaneous dirofilariasis in domestic and wild canids, felids, and humans. Unlike Dirofilaria immitis, which causes cardiopulmonary disease and is endemic to North America, D. repens tends to reside in subcutaneous tissues, where it forms nodules or migratory swellings. D. immitis is widely studied and routinely screened, while D. repens remains underdiagnosed due to its subtle presentation and emerging zoonotic recognition (Genchi & Kramer, 2017; Capelli et al., 2018).

In dogs, infections often remain asymptomatic or present with mild dermatological signs (pruritus, alopecia, subcutaneous nodules). Microfilaremia is frequently absent, particularly in early or occult infections, making microscopic diagnosis unreliable. This underscores the need for serological tools, especially in endemic or high-risk areas (Medscape, 2023).

European studies highlight the northward spread of D. repens, attributed to climate change, pet migration, and suitable mosquito vectors (Aedes albopictus, Culex pipiens). Capelli et al. (2018) and Genchi & Kramer (2017) report increasing prevalence in central and northern Europe.

According to the CDC (2024), D. repens is not officially detected in the U.S., but the presence of competent vectors, pet travel, and a 2023 suspicious case (ASM Journal) suggest a real risk. Dantas-Torres & Otranto (2013) support the likelihood of introduction through global canine movement.

This study introduces a robust ELISA protocol for identifying D. repens exposure in dogs, validated on naturally infected sera and discussed as a tool for future surveillance and field-based diagnosis.

Materials and Methods
Sample Collection and Grouping
Sera were collected from three groups of dogs:
– Group 1: 40 dogs with confirmed D. repens infection (via microscopy or autopsy).
– Group 2: 20 clinically healthy dogs from non-endemic areas (negative controls).
– Group 3: 20 dogs infected with other helminths (D. immitis, Toxocara canis, Ancylostoma caninum).

Antigen Preparation
Adult D. repens worms were harvested from infected subcutaneous tissues in endemic regions of Ukraine. After homogenization and centrifugation, antigens were purified using gel filtration (Superose 12 HR 10/30). Peak II, which demonstrated the highest immunoreactivity, was selected for use.

ELISA Protocol
Plates were coated with purified antigen at 8.5 µg/mL in carbonate-bicarbonate buffer (pH 9.6), incubated overnight at 4°C, and blocked with 5% skim milk in PBS-Tween. Canine sera were diluted 1:100 and added in duplicate. After 1-hour incubation at 37°C, plates were washed and incubated with goat anti-dog IgG-HRP (1:10,000). The substrate (TMB) was applied, and absorbance was read at 450 nm.

Statistical Analysis
The cut-off OD value was calculated as the mean OD of the negative control group plus 2 standard deviations. Sensitivity, specificity, and predictive values were calculated. Inter- and intra-assay variability was assessed. Data were analyzed using GraphPad Prism 9.0.

Results
Overall Performance
– Sensitivity: 76.5%
– Specificity: 80.6%

OD Ranges:
– Group 1: 0.375–1.080
– Group 2: 0.090–0.265
– Group 3: 0.150–0.610

Group-wise Analysis Table:
Group | Samples | Positive | Negative | False Positives
——|———|———-|———-|—————–
1         | 40      | 31       | 9       | –
2         | 20      | 1        | 19      | 1
3         | 20      | 6        | 14      | 6

Discussion
The developed ELISA is a valuable addition to diagnostic protocols, especially for occult D. repens infections. Compared to microscopy, it offers enhanced detection for early-stage or latent infections. The use of native parasite antigens likely contributed to stronger immune recognition, but also led to some cross-reactivity, particularly with D. immitis.

The results are comparable to European findings (Capelli et al., 2018), where similar levels of sensitivity and specificity were reported. The increasing prevalence of D. repens in Europe due to environmental changes underscores the risk of its introduction to North America. Recent data from ASM (2023) describe a suspicious subcutaneous filarial infection in a U.S. patient. This is supported by Dantas-Torres & Otranto (2013), who emphasize the role of global pet movement in parasite spread.

Given the risk of importation and establishment in the U.S., ELISA-based tools are crucial. A portable test format could serve at borders, in shelters, and veterinary clinics.

Conclusions
The ELISA method described shows adequate sensitivity and specificity for detecting antibodies to D. repens in dogs. It supports early-stage diagnosis and could be developed into a portable diagnostic format. As climate change, travel, and global movement of pets increase, such tools become essential for controlling the spread of emerging parasitic threats.

References
 1. Capelli, G. et al. (2018). Recent advances on Dirofilaria repens in dogs and humans in Europe. Parasites & Vectors, 11(663). https://doi.org/10.1186/s13071-018-3205-x
 2. Genchi, C., & Kramer, L. (2017). Subcutaneous dirofilariosis (Dirofilaria repens): an infection spreading throughout the Old World. Parasites & Vectors, 10(Suppl 2), 517. https://doi.org/10.1186/s13071-017-2434-8
3. Centers for Disease Control and Prevention (CDC). (2024). About Dirofilariasis. https://www.cdc.gov/dirofilariasis/about/index.html
4. ASM Journal. (2023). Case of subcutaneous dirofilariasis in the U.S. Journal of Clinical Microbiology. https://journals.asm.org/doi/10.1128/jcm.00308-23
5. Dantas-Torres, F., & Otranto, D. (2013). Dirofilariosis in the Americas: a more virulent Dirofilaria immitis? Parasites & Vectors, 6(288). https://doi.org/10.1186/1756-3305-6-288
6. Medscape. (2023). Dirofilariasis: Practice Essentials. https://emedicine.medscape.com/article/236698-overview

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Last Updated on May 7, 2025 by Marie Benz MD FAAD