Seroprevalence and molecular surveillance of Dirofilaria immitis in shelter dogs from central New Mexico, US

Seroprevalence and molecular surveillance of Dirofilaria immitis in shelter dogs from central New Mexico, US | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Seroprevalence and molecular surveillance of Dirofilaria immitis in shelter dogs from central New Mexico, US Maureen A. Kelly, Alexa Starnes, Mary Schech, Erin Clarke, Rafael A. N. Ramos, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8614198/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Dirofilaria immitis , the causative agent of canine heartworm disease, is a mosquito-borne filarial nematode, endemic to most of North America. Clinical signs may include coughing, exercise intolerance, abnormal heart sounds, and lethargy, which can lead to heart failure and death in severe cases. For accurate diagnosis, the American Heartworm Society (AHS) recommends testing dogs annually for heartworm antigens and microfilariae detection. In this study, we assessed the prevalence and potential risk factors for D. immitis in dogs from Albuquerque, New Mexico, using antigen and microfilariae detection tests. Methods In 2023, we collected serum and whole-blood samples (n = 402) from dogs in Albuquerque, New Mexico, as part of a larger study to detect Onchocerca lupi. Serum samples were screened for D. immitis using a commercially available antigen-detection ELISA (DiroCHEK®, Zoetis) using both pre- and post-immune complex dissociation (ICD) via heat treatment. Following genomic DNA extraction, we also screened whole blood using a newly optimized probe-based real-time PCR (qPCR) assay. To assess the possible association between D. immitis presence and potential risk factors, including age, sex, breed group, and coat color, a univariate analysis was performed using either a Fisher's exact or a Chi-square test. Results Serum of 4 (0.99%) dogs tested antigen-positive pre-ICD, while 14 (3.47%) dogs tested antigen-positive post-ICD. We did not detect any positive dogs for D. immitis microfilariae by the probe-based qPCR. No risk factors were associated with the detection of the D. immitis antigen in this dog population. Conclusion Although microfilariae were not detected in this study, it is possible that infections were in the early stages of disease progression, with D. immitis detected in pre- and post-ICD via antigen testing. Low seroprevalence and no microfilariae DNA detected highlight the importance of using both pre- and post-ICD antigen detection alongside molecular-based microfilariae tests, particularly for companion animals living in low-endemic regions and for shelter populations that are less likely to be on heartworm prevention. Antigen detection canine heartworm disease Canis lupus familiaris microfilariae detection molecular diagnostics probe-based qPCR serology Figures Figure 1 Background Dirofilaria immitis is a vector-borne filarial nematode found worldwide and the causative agent of canine heartworm disease, with the potential to cause fatal outcomes, making it of great importance in veterinary medicine [ 1 – 3 ]. The life cycle of D. immitis lasts 6–7 months and requires a mosquito as an intermediate host, which transmits the infective stage (third-stage larvae) to susceptible definitive hosts [ 1 – 4 ]. Dogs infected with D. immitis exhibit a range of clinical signs, from mild signs such as coughing, exercise intolerance, and lethargy to more severe signs, including abnormal heart and lung sounds and caval syndrome, which can be fatal [ 1 – 4 ]. Due to the severity of the disease and the need for accurate diagnosis, the American Heartworm Society (AHS) recommends a serology-based test and a microfilariae detection test (MFDT) to confirm infection of D. immitis [ 1 – 5 ]. Serology-based testing, such as enzyme-linked immunosorbent assays (ELISA), can detect antigens produced by the reproductive tracts of at least three D. immitis adult females [ 6 – 11 ]. These commercially available tests are widely used in both clinical and research settings. Despite these serological assays demonstrating the highest diagnostic sensitivity and reliability to date, false negative results can still occur [ 12 – 15 ]. For example, in infections involving fewer than three females, male-only infections, or early parasitism involving only juvenile adults [ 10 , 16 , 17 ]. There is also a possibility of immune complex formation, which can be disrupted by heat treatment, allowing the release and detection of antigens [ 6 , 7 , 10 ]. DNA-based techniques for MFDTs, such as conventional PCR (cPCR), real-time PCR (qPCR), and droplet digital PCR (ddPCR), are becoming more widely available as send-out options from reference laboratories [ 18 – 24 ]. These techniques can aid in confirming D. immitis in suspected or inconclusive cases in clinical settings or in large-scale surveys that screen populations at risk, such as dogs, cats, and vectors [ 18 , 23 , 24 ]. Molecular assays exhibit high sensitivity and specificity, enabling the detection of only D. immitis infections, and are an essential tool for large-scale surveillance studies [ 18 , 23 , 24 ]. Currently, the AHS and other organizations (e.g., the Companion Animal Parasite Council (CAPC)) conduct active surveillance using clinical data on D. immitis to understand the nationwide impact, generating incidence and prevalence maps [ 2 , 25 ]. For AHS, each map estimates the overall incidence based on data collected at three-year intervals, and the most recent map reports the highest nationwide incidence to date, with the most cases reported in the southeastern United States (US) (Supplementary Data: Fig. S1 ) [ 2 ]. A similar estimated prevalence of D. immitis has also been reported in CAPC’s prevalence maps at the county, state, and national levels (Supplementary Data: Fig. S2) [ 25 ]. However, for both organizations, fewer cases are reported in the southwestern states, specifically New Mexico, which may be due to lower D. immitis endemicity in those states. This could also be due to limited data collection in these areas, finite reporting clinics, a lack of annual testing, or a limited number of established clinics, resulting in an underreported prevalence in these regions [ 26 , 27 ]. Nonetheless, active surveillance within at-risk populations, such as shelter dogs, is necessary to better understand the epidemiology of this potentially fatal parasite. Therefore, this study aimed to assess the prevalence and potential risk factors for D. immitis in dogs from Albuquerque, New Mexico, using recommended methods: a serological test and a molecular-based assay. Methods Sample Collection Between January and September 2023, both serum and whole blood samples (n = 402) were collected from shelter dogs in Albuquerque, Bernalillo County, New Mexico, as part of a larger epidemiological study to determine the prevalence of Onchocerca lupi [ 28 ]. Bernalillo County, one of the largest counties in the US, is home to the city of Albuquerque, with a warm-temperate, semi-desert climate [ 29 – 31 ]. All sample types were collected opportunistically prior to spay or neuter surgery in dogs, estimated to be older than 6 months [ 28 ]. Demographics were recorded at the time of sampling, including estimated age, sex, American Kennel Club (AKC) breed class, and coat color [ 28 ]. Once received, samples were aliquoted into 500 µL aliquots and stored at -20°C until processed for antigen and molecular testing. Antigen Evaluation Serum samples were evaluated using a commercially available ELISA antigen detection test (DiroCHEK®, Zoetis Inc., Kalamazoo, MI, USA) according to the manufacturer's instructions. Each sample was evaluated pre-ICD (no heat treatment) and post-ICD (heat treatment). For heat treatment, 250 µL of serum was incubated in a heat block at 103°C for 10 minutes, then centrifuged for 10 minutes at 16,000 RCF at room temperature [ 6 , 32 ]. Both pre- and post-ICD samples were processed and interpreted according to the manufacturer's protocol (Zoetis Inc., Kalamazoo, MI, USA). Once completed, each sample was assessed for visual color change within 5 minutes. If a color change occurred after five minutes, the sample was considered positive for D. immitis antigen. If a color change was not visible within the allotted time, the samples were recorded as “No antigen detected” (NAD). Results were also measured using a spectrophotometer (BioTek Synergy H1 Microplate Reader, Agilent, Santa Clara, California, USA) to quantify optical density (O.D.) at 490 nm [ 6 , 18 , 32 ]. DNA Extraction and qPCR Genomic DNA from each whole blood aliquot (500 µL) was extracted using the Maxwell® 48 RSC (Promega Corporation, Madison, Wisconsin, USA) with the Whole Blood DNA Kit (Promega Corporation, Madison, Wisconsin, USA) according to the manufacturer’s instructions. Samples were then screened for D. immitis using a previously published probe-based qPCR protocol [ 18 ], including a commercially available internal positive control (IPC) (VetMAX™ Xeno™ Internal Positive Control DNA, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). This assay targets a 166 bp fragment of the cytochrome oxidase subunit 1 ( cox1 ) region of mitochondrial DNA using primers (Fil.COI.749-F-5'-CATCCTGAGGTTTATGTTATTATTTT-3'; Fil.COI.914-R-5'-CWGTATACATATGATGRCCYCA-3') with a TaqMan® probe (FAM-5'-TATTTGGATTGCTGTATGGGG-3'-non-fluorescent quencher-MGB). All reactions were performed in a 20 µL reaction containing 1.5 µL of molecular-grade water, 0.5 µL (50 µM) per primer, 0.5 µL (20 µM) of probe, 10 µL 2x of TaqMan® Fast Advance Master Mix (Applied Biosystems, Waltham, Massachusetts, USA), 1 µL of VetMAX™ Xeno™ IPC - VIC™ Assay (ThermoFisher Scientific Inc., Waltham, Massachusetts, USA), 1 µL of VetMAX™ Xeno™ IPC (10 − 2.5 ) (ThermoFisher Scientific Inc., Waltham, Massachusetts, USA), and 5 µL of DNA template. All qPCR reactions were performed on a Quant-Studio 3 real-time PCR system (Applied Biosystems, Waltham, Massachusetts, USA). Following standard universal cycling conditions, which included a denaturation stage at 95°C for 20 seconds, followed by 40 cycles of a two-step PCR stage at 95°C for 1 second and 60°C for 20 seconds. All singlicate runs also included two positive controls and a no-template control. One positive control consisted of extracted DNA from an adult D. immitis specimen, confirmed both morphologically and molecularly. The second positive control used DNA from VetMAX™ Xeno™ IPC. For the no-template control, nuclease-free molecular water was used. Results of each qPCR were analyzed using Design & Analysis 2 software to determine the presence or absence of D. immitis within each sample (Applied Biosystems, Waltham, Massachusetts, USA). Data Analysis Statistical analysis was performed using STATA® version 19.5 BE-Basic Edition (College Station, TX, USA) to determine the association between demographic variables (e.g., age, sex, AKC breed group, and coat color) and the outcome ( D. immitis presence) for each diagnostic test. A univariate analysis was performed using either a Fisher’s exact or Chi-squared test, depending on the number of positives per test, with a p-value cut-off of p ≤ 0.05. Results The overall results of all diagnostic tests including antigen testing pre- and post-ICD, as well as probe-based qPCR, are summarized in Fig. 1 . Positivity rates of 0.99% (n = 4/402; 95% confidence interval (CI): 0.27–2.52) and 3.47% (n = 14/402; 95% CI: 1.91%–5.77%) were observed for pre-ICD and post-ICD, respectively. Table 1 shows all positive O.D. values for each dog, including both positive and negative control values. No dogs were positive for D. immitis microfilariae DNA via the probe-based qPCR. The univariate analysis did not reveal any potential risk factors (i.e., age, sex, AKC breed group, and coat color) associated with D. immitis antigen detection (Table 2 ). Table 1 Summary of the optical density (O.D.) values reported for all antigen-positive dogs from Albuquerque, New Mexico (n = 402). Sample No. O.D. Values of DiroCHEK® Pre-ICD Post-ICD Positive control Negative control 39 0.134 0.183 0.104 0.053 136* - 0.670 0.135 0.056 168 0.105 0.122 0.110 0.056 174 - 0.053 0.077 0.049 189 - 0.053 0.077 0.049 275 0.048 0.072 0.071 0.046 291 - 0.049 0.061 0.045 292 0.055 0.060 0.061 0.045 300 - 0.049 0.061 0.045 309* - 0.058 0.138 0.055 341 † - 0.074 0.136 0.060 343 † - 0.062 0.136 0.060 354 - 0.047 0.056 0.045 371 - 0.051 0.063 0.047 * Cercopithifilaria bainae co-infection; † Onchocerca lupi co-infection, as per Kelly et al. [ 28 ]. Table 2 Summary of demographic characteristics of the sampled dog population from Albuquerque, New Mexico (n = 402). Variable Total No. Dirofilaria immitis Pre-ICD Positive Dirofilaria immitis Post-ICD Positive No. (%) 95% CI Fisher’s exact No. (%) 95% CI Chi-square df p -value Age Juvenile (≤ 1 year old) 120 1 (0.8) 0.0—4.6 1.000 6 (5.0) 1.9—10.6 1.771 2 0.412 Adult (> 1–7 years old) 259 3 (1.2) 0.2—3.4 8 (3.1) 0.4—6.1 Senior (> 7 years old) 23 0 - 0 - Sex Female 193 2 (1.0) 0.1—3.7 0.658* 10 (5.2) 2.5—9.3 3.187 1 0.074 Male 209 2 (1.0) 0.1—3.4 4 (1.9) 0.5—4.8 American Kennel Club Breed Group Herding group 88 1 (1.1) 0.0—6.2 0.365 3 (3.4) 0.7—9.6 6.066 8 0.640 Hound group 16 0 - 0 - Non-sporting group 32 0 - 1 (3.1) 0.0—16.2 Sporting group 18 1 (5.6) 0.1—27.3 2 (11.1) 1.4—34.7 Terrier group 101 0 - 2 (2.0) 0.2—7.0 Toy group 92 1 (1.1) 0.0—5.9 5 (5.4) 1.8—12.2 Working group 41 1 (2.4) 0.0—12.9 1 (2.4) 0.0—12.9 Miscellaneous 12 0 - 0 - Unknown 2 0 - 0 - Coat Color Single Color 132 1 (0.8) 0.0—4.1 0.601* 4 (3.0) 0.8—7.6 0.119 1 0.729 Multiple Colors 270 3 (1.1) 0.2—3.1 10 (3.7) 1.8—6.7 Abbreviations: *1-sided Fisher’s exact; df: degrees of freedom; CI: Confidence Interval; Discussion In this study, a low seroprevalence (3.47%) for D. immitis was found in shelter dogs from Bernalillo County, New Mexico. Although considered low when compared to other highly endemic regions of the US, it is nearly six times higher than what is currently reported by CAPC in Bernalillo County (2023: 0.46%; 2024: 0.54%) and nearly 4 times higher at the state level for New Mexico (2023: 0.75%; 2024: 0.82%) [ 25 ]. The positivity reported by CAPC likely results from client-owned dogs receiving consistent veterinary care and monthly chemoprophylaxis, as the data collected by various veterinary diagnostic companies [ 25 ]. The increase in overall positive dogs observed over the past two years in both Bernalillo County and New Mexico may be due to a variety of factors, such as more clinics requesting and performing D. immitis diagnostics, which can lead to an increased rate, especially in communities that previously had low prevalence (e.g., New Mexico) [ 25 ]. Additionally, the possibility exists that re-screening of dogs previously confirmed positive for D. immitis that remain positive can affect the overall frequency reported, as these cases are not necessarily newly confirmed cases [ 1 , 2 , 25 ]. In large-scale epidemiological studies, shelter populations are commonly used to estimate the baseline prevalence of vector-borne parasites, including D. immitis , and play a significant role in local transmission by acting as reservoirs for vectors [ 1 , 2 , 9 , 11 ]. However, the lack of clinical history for dog populations in shelters poses challenges for epidemiological surveys, particularly when animals are translocated in response to natural disasters, import/export, or adoption events [ 1 , 2 , 9 , 23 , 26 , 27 , 33 , 34 ]. This was a limitation of this study, as clinical history was unavailable at the time of sampling [ 28 ]. The movement of heartworm positive dogs from highly prevalent areas to non-endemic regions has been previously documented [ 26 , 27 ]. For instance, in the state of Colorado, the effects of importing positive heartworm dogs contributed to the increase of seroprevalence from 0.5% in 2013 to 0.84% in 2017 [ 27 ]. Although increased prevalence has been observed in CAPC data and in AHS-reported incidence rates in New Mexico, animal translocation, especially in shelter populations, must be further evaluated in future investigations [ 2 , 25 ]. These dogs may also come from areas of the state with little epidemiological data available and/or suboptimal access to veterinary care, including Indian reservations. Of the 14 dogs identified as positive in the present study, four were positive in both the pre-ICD and post-ICD samples. Although we cannot correlate parasite burden with the ELISA kit's color intensity, it is worth noting that all four dogs showed higher O.D. values post-ICD than pre-ICD compared with the positive control [ 1 , 2 , 16 , 35 ]. Despite the high sensitivity of the antigen testing for diagnosing D. immitis , cross-reactivity can occur with other parasites, including Dirofilaria repens , Angiostrongylus vasorum , and Spirocerca lupi , when heat treatment is implemented [ 17 , 36 – 39 ]. To mitigate the potential effects of cross-reactivity, it can be beneficial to use molecular-based techniques in combination with microscopy-based approaches to ensure accurate results, though this may be more practical for research studies than for clinical practice. It should also be noted that these samples were collected as part of a previously published study focusing on the detection of O. lupi [ 28 ]. Two dogs (i.e., dogs 341 and 343) that were positive for D. immitis via post-ICD testing were also positive for O. lupi. This co-infection is likely, as O. lupi is a zoonotic filarial nematode endemic to the southwestern US [ 28 , 40 ]. It is unlikely that this was a false positive, as O. lupi does not cross-react in commonly used antigen tests [ 41 ]. In addition to O. lupi , the original study reported that 20 dogs were positive for Cercopithifilaria bainae (data not shown), a skin-dwelling filarial nematode transmitted by Rhipicephalus sanguineus [ 28 , 42 ]; in the present study, two additional dogs were also positive for D. immitis by post-ICD testing (i.e., dogs 136 and 309). Cercopithifilaria bainae has not yet been reported to cross-react with commercially available antigen tests for D. immitis , but additional data are necessary to refute this possibility. However, the likelihood decreases, given that the remaining C. bainae positive dogs (n = 18) did not yield a positive result pre- or post-ICD in this study, suggesting that false-positive results are less likely. Co-infections with blood- and cutaneous-borne filarial nematodes have been documented and considered in large-scale studies, especially in populations with unknown parasitic infections, such as domestic dogs and wild canids [ 10 , 28 , 42 ]. Within this study, we did not detect D. immitis positive dogs via probe-based qPCR, with nearly 1% (n = 4/402) positive via pre-ICD and 4% (n = 14/402) positive via post-ICD. Several possibilities could explain these discordant results. One possible cause could be that these dogs were examples of “occult infections”, where adult worms are present in the host with an absence of microfilariae circulating throughout the infected animal [ 2 , 7 , 10 , 43 , 44 ]. There are several examples of occult infections, such as the presence of juvenile adults that produce a detectable antigen but do not produce a significant number of microfilariae [ 2 , 7 , 10 , 43 , 44 ]. Another possibility is that mature adults are present but too few to reproduce microfilariae [ 2 , 7 , 10 , 43 ]. These occult infections suggest the detection was too early to confirm via MFDT. Additionally, given an unknown clinical history, there is a slight possibility that these positive dogs were recently treated with heartworm prevention, which cleared microfilariae from the circulation but did not treat the adult infection [ 2 , 16 , 21 , 43 ]. Nonetheless, in cases similar to this study, it is ideal to perform additional testing with these specific dogs to ensure an accurate diagnosis of D. immitis . Although we did not identify any significant risk factors associated with D. immitis , these results should be interpreted with caution and require additional epidemiological surveys in regions with reported low prevalence (i.e., southwestern US) [ 2 , 25 – 27 ]. This is the first epidemiological study to assess the prevalence of D. immitis in shelter dogs from Albuquerque, New Mexico, and reports a nearly sixfold higher (3.47%) prevalence than that reported in nationwide surveillance (0.54%) [ 2 , 25 – 27 ]. To further evaluate the increased prevalence of D. immitis , assessment of both environmental (i.e., mosquito species presence, temperature fluctuations) and societal factors (i.e., veterinary care availability, access to preventive measures) in highly endemic areas, such as the southeastern US, in real time for emerging endemic regions, including New Mexico [1–25, 45]. These future assessments will facilitate the development of control and prevention strategies to reduce the ongoing establishment of this fatal zoonotic filarial nematode. Declarations Acknowledgements We want to thank all those who assisted with this study at Albuquerque Animal Welfare. Author contributions MAK: technical writing – original drafter, data curation, laboratory optimization and diagnostic work, methodology validation, formal analysis, and statistical analysis. AS: technical writing – review and editing, data curation. MS: technical writing – review and editing, data curation. EC: technical writing – review and editing, data curation. RANR: methodology validation, technical writing – review and editing. HH: laboratory optimization and diagnostic work, methodology validation, technical writing – review and editing. PW: laboratory optimization and diagnostic work, methodology validation, technical writing – review and editing. GGV: Supervision, conceptualization, visualization, resources, funding, and technical writing – review and editing. Funding sources This research did not receive external funding. Data availability All data supporting the main conclusion of this study are included within the manuscript and provided as supplementary material. Ethical approval and consent to participate All biological samples collected complied with the animal use protocols approved by Texas A&M University’s Institutional Animal Care and Use Committee under the number 2022-0261. Consent to publication All authors consent to the publication of this manuscript. Competing interested The authors declare no competing interests. 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Companion Animal Parasite Council: Canine parasite prevalence maps (United States). https://capcvet.org/maps/#/ (2025). Accessed 11 August 2025. Barrantes Murillo DF, Moye A, Wang C. Heat treatment augments antigen detection of Dirofilaria immitis in apparently healthy companion dogs (3.8% to 7.3%): Insights from a large-scale nationwide survey across the United States. Pathogens. 2024;13 1:56; doi: 10.3390/pathogens13010056 . Drake J, Parrish RS. Dog importation and changes in heartworm prevalence in Colorado 2013–2017. Parasit Vectors. 2019;12 1:207; doi: 10.1186/s13071-019-3473-0 . Kelly MA, Clarke E, Hakimi H, Budke CM, Verocai GG. Prevalence of Onchocerca lupi in shelter dogs from an endemic region of the Southwestern USA. Parasit Vectors. 2025;18 1:335; doi: 10.1186/s13071-025-06988-5 . Cress JJ, Sayre, Roger, Comer, Patrick, and Warner, Harumi: Terrestrial ecosystems—isobioclimates of the conterminous United States: U.S. ecological survey scientific investigations map 3084, scale 1:5,000,000. https://pubs.usgs.gov/sim/3084/ (2009). United States Census Bureau: census county/townships, census designated places (CDP) and incorporated cities. https://edits.nationalmap.gov/apps/gaz-domestic/public/search/names/2409678 (2008). United States Census Bureau: QuickFacts Albuquerque city, New Mexico. https://www.census.gov/quickfacts/fact/table/albuquerquecitynewmexico/PST045223 (2023). Swartzentruber S, LeMonte A, Witt J, Fuller D, Davis T, Hage C, Connolly P, Durkin M, Wheat LJ. Improved Detection of Histoplasma Antigenemia following Dissociation of Immune Complexes. Clin. Vaccine Immunol. 2009;16 3:320–2; doi: doi: 10.1128/CVI.00409-08 . https://journals.asm.org/doi/abs/10.1128/cvi.00409-08. Little S, Braff J, Place J, Buch J, Dewage BG, Knupp A, Beall M. Canine infection with Dirofilaria immitis , Borrelia burgdorferi , Anaplasma spp., and Ehrlichia spp. in the United States, 2013–2019. Parasit Vectors. 2021;14 1; doi: 10.1186/s13071-020-04514-3 . Courtney C, Zeng Q. Predicting heartworm burdens with a heartworm antigen test kit. 1987. p. 387–390. Sobotyk C, Savadelis MD, Verocai GG. Detection and cross-reaction of Dirofilaria repens using a commercial heartworm antigen test kit. Vet Parasitol. 2021;289:109302; doi: 10.1016/j.vetpar.2020.109302 . Venco L, Manzocchi S, Genchi M, Kramer LH. Heat treatment and false-positive heartworm antigen testing in ex vivo parasites and dogs naturally infected by Dirofilaria repens and Angiostrongylus vasorum . Parasit Vectors. 2017;10 S2; doi: 10.1186/s13071-017-2444-6 . Aroch I, Rojas A, Slon P, Lavy E, Segev G, Baneth G. Serological cross-reactivity of three commercial in-house immunoassays for detection of Dirofilaria immitis antigens with Spirocerca lupi in dogs with benign esophageal spirocercosis. Vet Parasitol. 2015;211 3–4:303–5; doi: 10.1016/j.vetpar.2015.06.010 . Schnyder M, Deplazes P. Cross-reactions of sera from dogs infected with Angiostrongylus vasorum in commercially available Dirofilaria immitis test kits. Parasit Vectors. 2012;5:258; doi: 10.1186/1756-3305-5-258 . Roe CC, Holiday O, Upshaw-Bia K, Benally G, Williamson CHD, Urbanz J, Verocai GG, Ridenour CH, Nottingham R, Ford MA, Lake DP, Kennedy TA, Hepp CM, Sahl JW. Biting midges (Diptera: Ceratopogonidae) as putative vectors of zoonotic Onchocerca lupi (Nematoda: Onchocercidae) in northern Arizona and New Mexico, southwestern United States. Front Vet Sci. 2023;10:1167070; doi: 10.3389/fvets.2023.1167070 . de Oliveira CS, Savadelis MD, McLean NJ, Verocai GG. Assessing the potential cross-reactivity using a commercial heartworm ELISA kits of serum from dogs naturally infected with Onchocerca lupi . Vet Parasitol. 2020;280:109070; doi: 10.1016/j.vetpar.2020.109070 . Lineberry MW, Sundstrom KD, Little SE, Stayton EM, Allen KE. Detection of Cercopithifilaria bainae infection in shelter dogs and ticks in Oklahoma, USA. Parasit Vectors. 2020;13 1; doi: 10.1186/s13071-020-04089-z . Barrantes Murillo DF, Starkey L. Immune-complex dissociation for heartworm diagnosis. Today’s Vet Pract. 2024;14(5):Sept-Oct. Available from: https://todaysveterinarypractice.com/parasitology/immune-complex-dissociation-for-heartworm-diagnosis/ . Courtney CH, Zeng Q. Comparison of heartworm antigen test kit performance in dogs having low heartworm burdens. Vet Parasitol. 2001;96 4:317–22; doi: 10.1016/s0304-4017(01)00374-0 . Wang D, Bowman DD, Brown HE, Harrington LC, Kaufman PE, McKay T, Nelson CT, Sharp JL, Lund R. Factors influencing U.S. canine heartworm ( Dirofilaria immitis ) prevalence. Parasit Vectors. 2014;7 1:264; doi: 10.1186/1756-3305-7-264 . Additional Declarations No competing interests reported. Supplementary Files HWALQNMTables11526.docx HWALQNMSupplementaryFiles11526.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 30 Jan, 2026 Reviews received at journal 30 Jan, 2026 Reviews received at journal 28 Jan, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers invited by journal 24 Jan, 2026 Editor assigned by journal 24 Jan, 2026 Submission checks completed at journal 22 Jan, 2026 First submitted to journal 15 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8614198","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":581846780,"identity":"22c29c0b-f334-40a4-95d9-1244f9fbac5d","order_by":0,"name":"Maureen A. Kelly","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Maureen","middleName":"A.","lastName":"Kelly","suffix":""},{"id":581846781,"identity":"0085e834-997a-4e00-8009-1bbb1faf3063","order_by":1,"name":"Alexa Starnes","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Alexa","middleName":"","lastName":"Starnes","suffix":""},{"id":581846782,"identity":"105e8035-1228-442f-b16e-7548cdd88787","order_by":2,"name":"Mary Schech","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Mary","middleName":"","lastName":"Schech","suffix":""},{"id":581846783,"identity":"a5d92957-711e-44e2-891e-b5959cd269a9","order_by":3,"name":"Erin Clarke","email":"","orcid":"","institution":"Albuquerque Animal Welfare","correspondingAuthor":false,"prefix":"","firstName":"Erin","middleName":"","lastName":"Clarke","suffix":""},{"id":581846784,"identity":"5bb40646-37a1-41a7-86ac-690101c69fec","order_by":4,"name":"Rafael A. N. Ramos","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"A. N.","lastName":"Ramos","suffix":""},{"id":581846785,"identity":"d27fab42-a6dd-4132-8a5b-d7aad7448999","order_by":5,"name":"Hassan Hakimi","email":"","orcid":"","institution":"Kansas State University","correspondingAuthor":false,"prefix":"","firstName":"Hassan","middleName":"","lastName":"Hakimi","suffix":""},{"id":581846786,"identity":"3ad13ee8-3064-4f94-94b9-adc41d3d8082","order_by":6,"name":"Pabasara Weerarathne","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Pabasara","middleName":"","lastName":"Weerarathne","suffix":""},{"id":581846791,"identity":"9967b596-9557-4a11-b99f-b18df176601a","order_by":7,"name":"Guilherme G. Verocai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYBADHn5kngRRWiQbQFQCCVoYDA4Qq0U+IvnYhw81djLGx8+YbmD8YRdtcID54G0ePFoMb6Qlz5xxLJnH7EyO2Q2GhOTcDQfYkq3xauk5Y8zM28DMY3aDLQ2ohRmohcdMGr+W85+BWup5jGeAtdQDtfB/w6tFnr2HGajlMI+BBPMxoJbDIFvY8GoxYG8zZpxx7DiPxJnkYzcS0o7nzjzMZmw5B58tzcyPGT7UVNvztx9su/HBpjq373jzwxtv8NlyAJmXACKY8SgH29JAQMEoGAWjYBSMAgYAzcpJWcloeIoAAAAASUVORK5CYII=","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":true,"prefix":"","firstName":"Guilherme","middleName":"G.","lastName":"Verocai","suffix":""}],"badges":[],"createdAt":"2026-01-16 01:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8614198/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8614198/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101630248,"identity":"7d9b4564-4431-484e-8886-dcaa11bc05f9","added_by":"auto","created_at":"2026-02-02 05:25:23","extension":"tiff","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":161335,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of the results from pre- and post-ICD antigen detection and probe-based qPCR for \u003cem\u003eDirofilaria immitis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"HWALQNMFigure111526.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8614198/v1/676860cf3730dc521f5674f5.tiff"},{"id":101630600,"identity":"0cddb5d3-fd10-4106-a7b1-82714b5de1c1","added_by":"auto","created_at":"2026-02-02 05:25:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1180333,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8614198/v1/6019eb13-09ab-45e7-bb6b-37642d03fc5b.pdf"},{"id":101630250,"identity":"936f940d-a4cb-4934-a61c-885e2c85a48b","added_by":"auto","created_at":"2026-02-02 05:25:24","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":26614,"visible":true,"origin":"","legend":"","description":"","filename":"HWALQNMTables11526.docx","url":"https://assets-eu.researchsquare.com/files/rs-8614198/v1/0c27171787d6fea31d1f07e3.docx"},{"id":101630249,"identity":"df88f212-0018-4e22-bbcf-df43f277ea65","added_by":"auto","created_at":"2026-02-02 05:25:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":841070,"visible":true,"origin":"","legend":"","description":"","filename":"HWALQNMSupplementaryFiles11526.docx","url":"https://assets-eu.researchsquare.com/files/rs-8614198/v1/9ae77ca3d448cad81fd34557.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Seroprevalence and molecular surveillance of Dirofilaria immitis in shelter dogs from central New Mexico, US","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eDirofilaria immitis\u003c/em\u003e is a vector-borne filarial nematode found worldwide and the causative agent of canine heartworm disease, with the potential to cause fatal outcomes, making it of great importance in veterinary medicine [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The life cycle of \u003cem\u003eD. immitis\u003c/em\u003e lasts 6\u0026ndash;7 months and requires a mosquito as an intermediate host, which transmits the infective stage (third-stage larvae) to susceptible definitive hosts [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Dogs infected with \u003cem\u003eD. immitis\u003c/em\u003e exhibit a range of clinical signs, from mild signs such as coughing, exercise intolerance, and lethargy to more severe signs, including abnormal heart and lung sounds and caval syndrome, which can be fatal [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Due to the severity of the disease and the need for accurate diagnosis, the American Heartworm Society (AHS) recommends a serology-based test and a microfilariae detection test (MFDT) to confirm infection of \u003cem\u003eD. immitis\u003c/em\u003e [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSerology-based testing, such as enzyme-linked immunosorbent assays (ELISA), can detect antigens produced by the reproductive tracts of at least three \u003cem\u003eD. immitis\u003c/em\u003e adult females [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These commercially available tests are widely used in both clinical and research settings. Despite these serological assays demonstrating the highest diagnostic sensitivity and reliability to date, false negative results can still occur [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. For example, in infections involving fewer than three females, male-only infections, or early parasitism involving only juvenile adults [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. There is also a possibility of immune complex formation, which can be disrupted by heat treatment, allowing the release and detection of antigens [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDNA-based techniques for MFDTs, such as conventional PCR (cPCR), real-time PCR (qPCR), and droplet digital PCR (ddPCR), are becoming more widely available as send-out options from reference laboratories [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These techniques can aid in confirming \u003cem\u003eD. immitis\u003c/em\u003e in suspected or inconclusive cases in clinical settings or in large-scale surveys that screen populations at risk, such as dogs, cats, and vectors [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Molecular assays exhibit high sensitivity and specificity, enabling the detection of only \u003cem\u003eD. immitis\u003c/em\u003e infections, and are an essential tool for large-scale surveillance studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, the AHS and other organizations (e.g., the Companion Animal Parasite Council (CAPC)) conduct active surveillance using clinical data on \u003cem\u003eD. immitis\u003c/em\u003e to understand the nationwide impact, generating incidence and prevalence maps [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For AHS, each map estimates the overall incidence based on data collected at three-year intervals, and the most recent map reports the highest nationwide incidence to date, with the most cases reported in the southeastern United States (US) (Supplementary Data: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A similar estimated prevalence of \u003cem\u003eD. immitis\u003c/em\u003e has also been reported in CAPC\u0026rsquo;s prevalence maps at the county, state, and national levels (Supplementary Data: Fig. S2) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, for both organizations, fewer cases are reported in the southwestern states, specifically New Mexico, which may be due to lower \u003cem\u003eD. immitis\u003c/em\u003e endemicity in those states. This could also be due to limited data collection in these areas, finite reporting clinics, a lack of annual testing, or a limited number of established clinics, resulting in an underreported prevalence in these regions [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Nonetheless, active surveillance within at-risk populations, such as shelter dogs, is necessary to better understand the epidemiology of this potentially fatal parasite. Therefore, this study aimed to assess the prevalence and potential risk factors for \u003cem\u003eD. immitis\u003c/em\u003e in dogs from Albuquerque, New Mexico, using recommended methods: a serological test and a molecular-based assay.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample Collection\u003c/h2\u003e \u003cp\u003eBetween January and September 2023, both serum and whole blood samples (n\u0026thinsp;=\u0026thinsp;402) were collected from shelter dogs in Albuquerque, Bernalillo County, New Mexico, as part of a larger epidemiological study to determine the prevalence of \u003cem\u003eOnchocerca lupi\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Bernalillo County, one of the largest counties in the US, is home to the city of Albuquerque, with a warm-temperate, semi-desert climate [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. All sample types were collected opportunistically prior to spay or neuter surgery in dogs, estimated to be older than 6 months [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Demographics were recorded at the time of sampling, including estimated age, sex, American Kennel Club (AKC) breed class, and coat color [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Once received, samples were aliquoted into 500 \u0026micro;L aliquots and stored at -20\u0026deg;C until processed for antigen and molecular testing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntigen Evaluation\u003c/h3\u003e\n\u003cp\u003eSerum samples were evaluated using a commercially available ELISA antigen detection test (DiroCHEK\u0026reg;, Zoetis Inc., Kalamazoo, MI, USA) according to the manufacturer's instructions. Each sample was evaluated pre-ICD (no heat treatment) and post-ICD (heat treatment). For heat treatment, 250 \u0026micro;L of serum was incubated in a heat block at 103\u0026deg;C for 10 minutes, then centrifuged for 10 minutes at 16,000 RCF at room temperature [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Both pre- and post-ICD samples were processed and interpreted according to the manufacturer's protocol (Zoetis Inc., Kalamazoo, MI, USA). Once completed, each sample was assessed for visual color change within 5 minutes. If a color change occurred after five minutes, the sample was considered positive for \u003cem\u003eD. immitis\u003c/em\u003e antigen. If a color change was not visible within the allotted time, the samples were recorded as \u0026ldquo;No antigen detected\u0026rdquo; (NAD). Results were also measured using a spectrophotometer (BioTek Synergy H1 Microplate Reader, Agilent, Santa Clara, California, USA) to quantify optical density (O.D.) at 490 nm [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eDNA Extraction and qPCR\u003c/h3\u003e\n\u003cp\u003e Genomic DNA from each whole blood aliquot (500 \u0026micro;L) was extracted using the Maxwell\u0026reg; 48 RSC (Promega Corporation, Madison, Wisconsin, USA) with the Whole Blood DNA Kit (Promega Corporation, Madison, Wisconsin, USA) according to the manufacturer\u0026rsquo;s instructions. Samples were then screened for \u003cem\u003eD. immitis\u003c/em\u003e using a previously published probe-based qPCR protocol [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], including a commercially available internal positive control (IPC) (VetMAX\u0026trade; Xeno\u0026trade; Internal Positive Control DNA, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). This assay targets a 166 bp fragment of the cytochrome oxidase subunit 1 (\u003cem\u003ecox1\u003c/em\u003e) region of mitochondrial DNA using primers (Fil.COI.749-F-5'-CATCCTGAGGTTTATGTTATTATTTT-3'; Fil.COI.914-R-5'-CWGTATACATATGATGRCCYCA-3') with a TaqMan\u0026reg; probe (FAM-5'-TATTTGGATTGCTGTATGGGG-3'-non-fluorescent quencher-MGB). All reactions were performed in a 20 \u0026micro;L reaction containing 1.5 \u0026micro;L of molecular-grade water, 0.5 \u0026micro;L (50 \u0026micro;M) per primer, 0.5 \u0026micro;L (20 \u0026micro;M) of probe, 10 \u0026micro;L 2x of TaqMan\u0026reg; Fast Advance Master Mix (Applied Biosystems, Waltham, Massachusetts, USA), 1 \u0026micro;L of VetMAX\u0026trade; Xeno\u0026trade; IPC - VIC\u0026trade; Assay (ThermoFisher Scientific Inc., Waltham, Massachusetts, USA), 1 \u0026micro;L of VetMAX\u0026trade; Xeno\u0026trade; IPC (10\u003csup\u003e\u0026minus;\u0026thinsp;2.5\u003c/sup\u003e) (ThermoFisher Scientific Inc., Waltham, Massachusetts, USA), and 5 \u0026micro;L of DNA template. All qPCR reactions were performed on a Quant-Studio 3 real-time PCR system (Applied Biosystems, Waltham, Massachusetts, USA). Following standard universal cycling conditions, which included a denaturation stage at 95\u0026deg;C for 20 seconds, followed by 40 cycles of a two-step PCR stage at 95\u0026deg;C for 1 second and 60\u0026deg;C for 20 seconds. All singlicate runs also included two positive controls and a no-template control. One positive control consisted of extracted DNA from an adult \u003cem\u003eD. immitis\u003c/em\u003e specimen, confirmed both morphologically and molecularly. The second positive control used DNA from VetMAX\u0026trade; Xeno\u0026trade; IPC. For the no-template control, nuclease-free molecular water was used. Results of each qPCR were analyzed using Design \u0026amp; Analysis 2 software to determine the presence or absence of \u003cem\u003eD. immitis\u003c/em\u003e within each sample (Applied Biosystems, Waltham, Massachusetts, USA).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using STATA\u0026reg; version 19.5 BE-Basic Edition (College Station, TX, USA) to determine the association between demographic variables (e.g., age, sex, AKC breed group, and coat color) and the outcome (\u003cem\u003eD. immitis\u003c/em\u003e presence) for each diagnostic test. A univariate analysis was performed using either a Fisher\u0026rsquo;s exact or Chi-squared test, depending on the number of positives per test, with a p-value cut-off of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe overall results of all diagnostic tests including antigen testing pre- and post-ICD, as well as probe-based qPCR, are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Positivity rates of 0.99% (n\u0026thinsp;=\u0026thinsp;4/402; 95% confidence interval (CI): 0.27\u0026ndash;2.52) and 3.47% (n\u0026thinsp;=\u0026thinsp;14/402; 95% CI: 1.91%\u0026ndash;5.77%) were observed for pre-ICD and post-ICD, respectively. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows all positive O.D. values for each dog, including both positive and negative control values. No dogs were positive for \u003cem\u003eD. immitis\u003c/em\u003e microfilariae DNA via the probe-based qPCR. The univariate analysis did not reveal any potential risk factors (i.e., age, sex, AKC breed group, and coat color) associated with \u003cem\u003eD. immitis\u003c/em\u003e antigen detection (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of the optical density (O.D.) values reported for all antigen-positive dogs from Albuquerque, New Mexico (n\u0026thinsp;=\u0026thinsp;402).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eO.D. Values of DiroCHEK\u0026reg;\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePre-ICD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePost-ICD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePositive control\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNegative control\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.053\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e136*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.670\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.056\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.056\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.049\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.049\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.072\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.071\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.046\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e291\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.045\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e292\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.060\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.045\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.045\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e309*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.055\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e341\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.060\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e343\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.062\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.060\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.047\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.056\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.045\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e371\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.047\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*\u003cem\u003eCercopithifilaria bainae\u003c/em\u003e co-infection; \u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003cem\u003eOnchocerca lupi\u003c/em\u003e co-infection, as per Kelly et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of demographic characteristics of the sampled dog population from Albuquerque, New Mexico (n\u0026thinsp;=\u0026thinsp;402).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTotal No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDirofilaria immitis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePre-ICD Positive\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003e\u003cem\u003eDirofilaria immitis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePost-ICD Positive\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFisher\u0026rsquo;s exact\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo. (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eChi-square\u003c/p\u003e \u003cp\u003edf\u003c/p\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJuvenile\u003c/p\u003e \u003cp\u003e(\u0026le;\u0026thinsp;1 year old)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (0.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0\u0026mdash;4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e6 (5.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.9\u0026mdash;10.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e1.771\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e0.412\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdult\u003c/p\u003e \u003cp\u003e(\u0026gt;\u0026thinsp;1\u0026ndash;7 years old)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2\u0026mdash;3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e8 (3.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.4\u0026mdash;6.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSenior\u003c/p\u003e \u003cp\u003e(\u0026gt;\u0026thinsp;7 years old)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (1.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1\u0026mdash;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.658*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e10 (5.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.5\u0026mdash;9.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e3.187\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e0.074\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (1.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1\u0026mdash;3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4 (1.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.5\u0026mdash;4.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003eAmerican Kennel Club Breed Group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHerding group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0\u0026mdash;6.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003e0.365\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3 (3.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.7\u0026mdash;9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003e6.066\u003c/p\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e0.640\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHound group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNon-sporting group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1 (3.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0\u0026mdash;16.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSporting group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (5.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1\u0026mdash;27.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 (11.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.4\u0026mdash;34.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTerrier group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 (2.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.2\u0026mdash;7.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eToy group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0\u0026mdash;5.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 (5.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.8\u0026mdash;12.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWorking group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0\u0026mdash;12.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1 (2.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0\u0026mdash;12.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMiscellaneous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003eCoat Color\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSingle Color\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (0.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0\u0026mdash;4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.601*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4 (3.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.8\u0026mdash;7.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.119\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e0.729\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMultiple Colors\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2\u0026mdash;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10 (3.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.8\u0026mdash;6.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003cem\u003eAbbreviations: *1-sided Fisher\u0026rsquo;s exact; df: degrees of freedom; CI: Confidence Interval;\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, a low seroprevalence (3.47%) for \u003cem\u003eD. immitis\u003c/em\u003e was found in shelter dogs from Bernalillo County, New Mexico. Although considered low when compared to other highly endemic regions of the US, it is nearly six times higher than what is currently reported by CAPC in Bernalillo County (2023: 0.46%; 2024: 0.54%) and nearly 4 times higher at the state level for New Mexico (2023: 0.75%; 2024: 0.82%) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The positivity reported by CAPC likely results from client-owned dogs receiving consistent veterinary care and monthly chemoprophylaxis, as the data collected by various veterinary diagnostic companies [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The increase in overall positive dogs observed over the past two years in both Bernalillo County and New Mexico may be due to a variety of factors, such as more clinics requesting and performing \u003cem\u003eD. immitis\u003c/em\u003e diagnostics, which can lead to an increased rate, especially in communities that previously had low prevalence (e.g., New Mexico) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Additionally, the possibility exists that re-screening of dogs previously confirmed positive for \u003cem\u003eD. immitis\u003c/em\u003e that remain positive can affect the overall frequency reported, as these cases are not necessarily newly confirmed cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn large-scale epidemiological studies, shelter populations are commonly used to estimate the baseline prevalence of vector-borne parasites, including \u003cem\u003eD. immitis\u003c/em\u003e, and play a significant role in local transmission by acting as reservoirs for vectors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the lack of clinical history for dog populations in shelters poses challenges for epidemiological surveys, particularly when animals are translocated in response to natural disasters, import/export, or adoption events [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This was a limitation of this study, as clinical history was unavailable at the time of sampling [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The movement of heartworm positive dogs from highly prevalent areas to non-endemic regions has been previously documented [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For instance, in the state of Colorado, the effects of importing positive heartworm dogs contributed to the increase of seroprevalence from 0.5% in 2013 to 0.84% in 2017 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Although increased prevalence has been observed in CAPC data and in AHS-reported incidence rates in New Mexico, animal translocation, especially in shelter populations, must be further evaluated in future investigations [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These dogs may also come from areas of the state with little epidemiological data available and/or suboptimal access to veterinary care, including Indian reservations.\u003c/p\u003e \u003cp\u003eOf the 14 dogs identified as positive in the present study, four were positive in both the pre-ICD and post-ICD samples. Although we cannot correlate parasite burden with the ELISA kit's color intensity, it is worth noting that all four dogs showed higher O.D. values post-ICD than pre-ICD compared with the positive control [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Despite the high sensitivity of the antigen testing for diagnosing \u003cem\u003eD. immitis\u003c/em\u003e, cross-reactivity can occur with other parasites, including \u003cem\u003eDirofilaria repens\u003c/em\u003e, \u003cem\u003eAngiostrongylus vasorum\u003c/em\u003e, and \u003cem\u003eSpirocerca lupi\u003c/em\u003e, when heat treatment is implemented [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. To mitigate the potential effects of cross-reactivity, it can be beneficial to use molecular-based techniques in combination with microscopy-based approaches to ensure accurate results, though this may be more practical for research studies than for clinical practice.\u003c/p\u003e \u003cp\u003eIt should also be noted that these samples were collected as part of a previously published study focusing on the detection of \u003cem\u003eO. lupi\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Two dogs (i.e., dogs 341 and 343) that were positive for \u003cem\u003eD. immitis\u003c/em\u003e via post-ICD testing were also positive for \u003cem\u003eO. lupi.\u003c/em\u003e This co-infection is likely, as \u003cem\u003eO. lupi\u003c/em\u003e is a zoonotic filarial nematode endemic to the southwestern US [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. It is unlikely that this was a false positive, as \u003cem\u003eO. lupi\u003c/em\u003e does not cross-react in commonly used antigen tests [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In addition to \u003cem\u003eO. lupi\u003c/em\u003e, the original study reported that 20 dogs were positive for \u003cem\u003eCercopithifilaria bainae\u003c/em\u003e (data not shown), a skin-dwelling filarial nematode transmitted by \u003cem\u003eRhipicephalus sanguineus\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]; in the present study, two additional dogs were also positive for \u003cem\u003eD. immitis\u003c/em\u003e by post-ICD testing (i.e., dogs 136 and 309). \u003cem\u003eCercopithifilaria bainae\u003c/em\u003e has not yet been reported to cross-react with commercially available antigen tests for \u003cem\u003eD. immitis\u003c/em\u003e, but additional data are necessary to refute this possibility. However, the likelihood decreases, given that the remaining \u003cem\u003eC. bainae\u003c/em\u003e positive dogs (n\u0026thinsp;=\u0026thinsp;18) did not yield a positive result pre- or post-ICD in this study, suggesting that false-positive results are less likely. Co-infections with blood- and cutaneous-borne filarial nematodes have been documented and considered in large-scale studies, especially in populations with unknown parasitic infections, such as domestic dogs and wild canids [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWithin this study, we did not detect \u003cem\u003eD. immitis\u003c/em\u003e positive dogs via probe-based qPCR, with nearly 1% (n\u0026thinsp;=\u0026thinsp;4/402) positive via pre-ICD and 4% (n\u0026thinsp;=\u0026thinsp;14/402) positive via post-ICD. Several possibilities could explain these discordant results. One possible cause could be that these dogs were examples of \u0026ldquo;occult infections\u0026rdquo;, where adult worms are present in the host with an absence of microfilariae circulating throughout the infected animal [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. There are several examples of occult infections, such as the presence of juvenile adults that produce a detectable antigen but do not produce a significant number of microfilariae [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Another possibility is that mature adults are present but too few to reproduce microfilariae [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. These occult infections suggest the detection was too early to confirm via MFDT. Additionally, given an unknown clinical history, there is a slight possibility that these positive dogs were recently treated with heartworm prevention, which cleared microfilariae from the circulation but did not treat the adult infection [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Nonetheless, in cases similar to this study, it is ideal to perform additional testing with these specific dogs to ensure an accurate diagnosis of \u003cem\u003eD. immitis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAlthough we did not identify any significant risk factors associated with \u003cem\u003eD. immitis\u003c/em\u003e, these results should be interpreted with caution and require additional epidemiological surveys in regions with reported low prevalence (i.e., southwestern US) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This is the first epidemiological study to assess the prevalence of \u003cem\u003eD. immitis\u003c/em\u003e in shelter dogs from Albuquerque, New Mexico, and reports a nearly sixfold higher (3.47%) prevalence than that reported in nationwide surveillance (0.54%) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To further evaluate the increased prevalence of \u003cem\u003eD. immitis\u003c/em\u003e, assessment of both environmental (i.e., mosquito species presence, temperature fluctuations) and societal factors (i.e., veterinary care availability, access to preventive measures) in highly endemic areas, such as the southeastern US, in real time for emerging endemic regions, including New Mexico [1\u0026ndash;25, 45]. These future assessments will facilitate the development of control and prevention strategies to reduce the ongoing establishment of this fatal zoonotic filarial nematode.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to thank all those who assisted with this study at Albuquerque Animal Welfare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMAK: technical writing \u0026ndash; original drafter, data curation, laboratory optimization and diagnostic work, methodology validation, formal analysis, and statistical analysis. AS: technical writing \u0026ndash; review and editing, data curation. MS: technical writing \u0026ndash; review and editing, data curation. EC: technical writing \u0026ndash; review and editing, data curation. RANR: methodology validation, technical writing \u0026ndash; review and editing. HH: laboratory optimization and diagnostic work, methodology validation, technical writing \u0026ndash; review and editing. PW: laboratory optimization and diagnostic work, methodology validation, technical writing \u0026ndash; review and editing. GGV: Supervision, conceptualization, visualization, resources, funding, and technical writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the main conclusion of this study are included within the manuscript and provided as supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll biological samples collected complied with the animal use protocols approved by Texas A\u0026amp;M University\u0026rsquo;s Institutional Animal Care and Use Committee under the number 2022-0261.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interested\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A\u0026amp;M University, College Station, TX, 77843, USA\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eAlbuquerque Animal Welfare, Albuquerque, NM, 87112, USA\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eLaboratory of Parasitology, Federal University of the Agreste of Pernambuco, Garanhuns, PE, 55292-270, Brazil\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eCurrent affiliation: Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, 66506, USA\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcCall JW, Genchi C, Kramer LH, Guerrero J, Venco L. 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://todaysveterinarypractice.com/parasitology/immune-complex-dissociation-for-heartworm-diagnosis/\u003c/span\u003e\u003cspan address=\"https://todaysveterinarypractice.com/parasitology/immune-complex-dissociation-for-heartworm-diagnosis/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCourtney CH, Zeng Q. Comparison of heartworm antigen test kit performance in dogs having low heartworm burdens. Vet Parasitol. 2001;96 4:317\u0026ndash;22; doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0304-4017(01)00374-0\u003c/span\u003e\u003cspan address=\"10.1016/s0304-4017(01)00374-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang D, Bowman DD, Brown HE, Harrington LC, Kaufman PE, McKay T, Nelson CT, Sharp JL, Lund R. Factors influencing U.S. canine heartworm (\u003cem\u003eDirofilaria immitis\u003c/em\u003e) prevalence. Parasit Vectors. 2014;7 1:264; doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1756-3305-7-264\u003c/span\u003e\u003cspan address=\"10.1186/1756-3305-7-264\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Antigen detection, canine heartworm disease, Canis lupus familiaris, microfilariae detection, molecular diagnostics, probe-based qPCR, serology","lastPublishedDoi":"10.21203/rs.3.rs-8614198/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8614198/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eDirofilaria immitis\u003c/em\u003e, the causative agent of canine heartworm disease, is a mosquito-borne filarial nematode, endemic to most of North America. Clinical signs may include coughing, exercise intolerance, abnormal heart sounds, and lethargy, which can lead to heart failure and death in severe cases. For accurate diagnosis, the American Heartworm Society (AHS) recommends testing dogs annually for heartworm antigens and microfilariae detection. In this study, we assessed the prevalence and potential risk factors for \u003cem\u003eD. immitis\u003c/em\u003e in dogs from Albuquerque, New Mexico, using antigen and microfilariae detection tests.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn 2023, we collected serum and whole-blood samples (n\u0026thinsp;=\u0026thinsp;402) from dogs in Albuquerque, New Mexico, as part of a larger study to detect \u003cem\u003eOnchocerca lupi.\u003c/em\u003e Serum samples were screened for \u003cem\u003eD. immitis\u003c/em\u003e using a commercially available antigen-detection ELISA (DiroCHEK\u0026reg;, Zoetis) using both pre- and post-immune complex dissociation (ICD) via heat treatment. Following genomic DNA extraction, we also screened whole blood using a newly optimized probe-based real-time PCR (qPCR) assay. To assess the possible association between \u003cem\u003eD. immitis\u003c/em\u003e presence and potential risk factors, including age, sex, breed group, and coat color, a univariate analysis was performed using either a Fisher's exact or a Chi-square test.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSerum of 4 (0.99%) dogs tested antigen-positive pre-ICD, while 14 (3.47%) dogs tested antigen-positive post-ICD. We did not detect any positive dogs for \u003cem\u003eD. immitis\u003c/em\u003e microfilariae by the probe-based qPCR. No risk factors were associated with the detection of the \u003cem\u003eD. immitis\u003c/em\u003e antigen in this dog population.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eAlthough microfilariae were not detected in this study, it is possible that infections were in the early stages of disease progression, with \u003cem\u003eD. immitis\u003c/em\u003e detected in pre- and post-ICD via antigen testing. Low seroprevalence and no microfilariae DNA detected highlight the importance of using both pre- and post-ICD antigen detection alongside molecular-based microfilariae tests, particularly for companion animals living in low-endemic regions and for shelter populations that are less likely to be on heartworm prevention.\u003c/p\u003e","manuscriptTitle":"Seroprevalence and molecular surveillance of Dirofilaria immitis in shelter dogs from central New Mexico, US","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 05:24:49","doi":"10.21203/rs.3.rs-8614198/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-30T16:24:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-30T14:17:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T13:44:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104882759856463933095224910697260840987","date":"2026-01-27T08:49:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"270500917775523485690080934123845024639","date":"2026-01-26T14:17:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-24T15:29:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-24T15:25:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-22T05:38:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2026-01-16T00:53:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f45ddaeb-7f21-4544-9ec3-6b81df7ecd61","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-21T13:23:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-02 05:24:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8614198","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8614198","identity":"rs-8614198","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}