Introduction
In recent decades, the distribution of arthropod- and gastropod-borne diseases in companion animals has changed considerably (Fuehrer et al. Reference Fuehrer, Morelli, Unterköfler, Bajer, Bakran-Lebl, Dwużnik-Szarek, Farkas, Grandi, Heddergott, Jokelainen, Knific, Leschnik, Miterpáková, Modrý, Petersen, Skírnisson, Vergles, Schnyder and Strube2021). Factors such as climate change, urbanisation, habitat alterations, increased pet travel, and animal rehoming have facilitated the spread of these infections within endemic regions and their emergence in previously unaffected areas (Beugnet et al. Reference Beugnet and Chalvet-Monfray2013; Williams et al. Reference Williams, Buswell and Perisho2024; Wright et al. Reference Wright, Jongejan, Marcondes, Peregrine, Baneth, Bourdeau, Bowman, Breitschwerdt, Capelli, Cardoso, Dantas-Torres, Day, Dobler, Ferrer, Gradoni, Irwin, Kempf, Kohn, Krämer, Lappin, Madder, Maggi, Maia, Miró, Naucke, Oliva, Otranto, Pennisi, Penzhorn, Pfeffer, Roura, Sainz, Shin, Solano-Gallego, Straubinger, Tasker, Traub and Little2020). Consequently, effective diagnosis and control of canine parasitic infections have become increasingly important.
The nematode Angiostrongylus vasorum, also known as French heartworm, infects domestic dogs and wild carnivores, causing a life-threatening disease – angiostrongylosis. The clinical signs associated with A. vasorum infection in dogs can be highly variable, clustering typically into three primary categories: cardiopulmonary (difficulties in breathing: dyspnoea and tachypnoea) (Rinaldi et al. Reference Rinaldi, Cortese, Meomartino, Pagno, Pepe, Cringoli and Papparella2014), coagulopathies (hematomas and prolonged bleeding due to thrombocytopenia) (Morgan et al. Reference Morgan and Shaw2010), and neurological (ataxia, tremors, seizures, behavioural changes, and loss of vision) (Jang et al. Reference Jang, Parent, Hagen and Colwell2016; Morgan et al. Reference Morgan, Modry, Paredes-Esquivel, Foronda and Traversa2021). Early and accurate diagnosis is essential, as untreated infections may be fatal. The gold standard for diagnosing A. vasorum is the detection of larval stages in faeces using the Baermann technique. However, in-house patient-side antigen detection tests are commonly used for primary screening of this parasite (Liu et al. Reference Liu, Schnyder, Willesen, Potter and Chandrashekar2017; Morgan et al. Reference Morgan and Shaw2010).
Canine angiostrongylosis has been expanding across Europe, with increasing number of reports from previously non-endemic regions (Ferdushy et al. Reference Ferdushy, Kapel, Webster, Al-Sabi and Grønvold2009; Fuehrer et al. Reference Fuehrer, Morelli, Unterköfler, Bajer, Bakran-Lebl, Dwużnik-Szarek, Farkas, Grandi, Heddergott, Jokelainen, Knific, Leschnik, Miterpáková, Modrý, Petersen, Skírnisson, Vergles, Schnyder and Strube2021; Helm et al. Reference Helm, Morgan, Jackson, Wotton and Bell2010; Morgan et al. Reference Morgan, Modry, Paredes-Esquivel, Foronda and Traversa2021; Robbestad et al. Reference Robbestad, Jiménez-Meléndez, Robertson, Vatne, Hauback and Nerhagen2024). In Estonia, A. vasorum has been detected in wild canids (Laurimaa et al. Reference Laurimaa, Moks, Soe, Valdmann and Saarma2016a, Reference Laurimaa, Süld, Davison, Moks, Valdmann and Saarma2016b) but not in domestic dogs (Oborina et al. Reference Oborina, Mõttus and Jokelainen2021).
We report the first genetically confirmed case of Angiostrongylus vasorum infection in a domestic dog in Estonia and outline relevant diagnostic challenges.
Materials and methods
In February 2024, a 2-year-old female Beagle, imported from Poland 5 months earlier (September 2023), was presented to a veterinary clinic for investigation of a persistent cough. The precise acquisition location in Poland and the prior travel history of the dog were unknown. Thoracic radiographs revealed a mildly increased cardiac silhouette size and a bronchial lung pattern. Haematological and serum biochemical analyses were unremarkable. An initial empirical diagnosis of kennel cough with secondary pneumonia was made based on clinical presentation, followed by radiography and antimicrobial therapy. Despite treatment, the dog’s condition deteriorated over the following week, with worsening cough and development of haemoptysis. Therapy was adjusted by adding marbofloxacin and prednisolone.
A point-of-care antigen detection test (SNAP® 4Dx®, IDEXX, Westbrook, Maine, USA) was positive for Dirofilaria immitis, and treatment for canine heartworm disease was initiated, including strict exercise restriction, milbemycin oxime/afoxolaner, and doxycycline. The dog was subsequently referred to a veterinary cardiology specialist. Echocardiographic examination was unremarkable. A repeat SNAP® 4Dx® test remained positive. Given documented cross-reactivity between circulating antigens of A. vasorum and commercially available D. immitis antigen test kits, which may result in false-positive D. immitis test results (Schnyder et al. Reference Schnyder and Deplazes2012), and the progressive clinical signs, angiostrongylosis was considered as an alternative diagnosis.
An in-clinic antigen detection test specific for A. vasorum (Angio Detect, IDEXX, Westbrook, Maine, USA) repeatedly yielded invalid results. However, invalid Angio Detect test results have been described in dogs with high circulating A. vasorum antigen concentrations and are suspected to result from a hook effect (Barker et al. Reference Barker, Payne and Wilson2024). Blood samples were sent to LABOKLIN laboratory (Bad Kissingen, Germany) for further testing, including enzyme-linked immunosorbent assays (ELISA) for D. immitis and A. vasorum antigen, the ‘Canine Travel Profile Eastern Europe’ ELISA (detecting antibodies against Babesia canis, Anaplasma phagocytophilum, Rickettsia conorii/felis), and PCR assays for microfilariae of filarioid helminths and A. vasorum. Only the A. vasorum antigen test result was positive.
To expedite identification of the causative agent, haematological and faecal samples were submitted in parallel to the University of Tartu for genetic identification. DNA was extracted from two blood samples using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocols. From the two scat samples, DNA was purified with the Nucleospin DNA Stool kit (Macherey-Nagel, Düren, Germany). Initial species identification was performed by PCR amplification and sequencing (Table 1).
Primers for PCR amplification of mtDNA gene fragments of the cytochrome c oxidase subunit I (cox1) and large subunit of ribosomal RNA (LSU rRNA) for Angiostrongylus vasorum (Ang) and Dirofilaria spp. (Dir). The shorter primer pairs Ang1F-Ang2R (278 bp) and Dir1F-Dir2R (333 bp) were used for species identification, and the primer pair Ang3F-Ang3R (721 bp) for phylogenetic analysis
Table 1. Long description
The table consists of five columns and three data rows.
Row 1:
- Primer name: Ang 1 F and Ang 2 R.
- m t D N A locus: L S U r R N A.
- Primer sequence: G C G T G A G G A C A T T A A G G T A G C and G T A T G A T T T T C T G T T A A C T C T G G A.
- P C R product size: 278 b p.
- Reference: This study.
Row 2:
- Primer name: Ang 3 F and Ang 3 R.
- m t D N A locus: cox 1.
- Primer sequence: G A G A G T T C T A A T C A T A A G G A T A T T G G T A C and G C A A A A T C A A A A C A T A A A C C T C C G.
- P C R product size: 721 b p.
- Reference: This study.
Row 3:
- Primer name: Dir 1 F and Dir 2 R.
- m t D N A locus: cox 1.
- Primer sequence: G A T T G G T G G T T T T G G T A A T T G G A T G and C A A A C A A A C A T A C T A A T C T G A T C T A A A G T.
- P C R product size: 333 b p.
- Reference: Nõupuu et al. 2025.
The PCR reactions were carried out in a volume of 20 μL, using 1× HOT FIREPol MultiPlex Mix (Solis BioDyne, Tartu, Estonia), 0.25 μM of both primers and 10–50 ng of template DNA. PCR cycling was performed as follows: 95°C for 12 min, followed by 10 cycles of 95°C for 20 s, 55°C for 30 s (with annealing temperature reduced in each cycle by 0.5°C) and 72°C for 60 s; followed by 27 cycles of 95°C for 20 s, 50°C for 30 s, 72°C for 60 s; and finished with a final elongation step at 72°C for 2 min. Of the PCR product, 10 μL was used to examine the PCR result on a 1% agarose gel electrophoresis in 1× TAE buffer. The remaining 10 μL was used for PCR product purification with a mixture of one unit FastAP alkaline phosphatase and one unit of Exonuclease I (Thermo Scientific, Waltham, USA). The purification mixture was incubated at 37°C for 30 min and then at 80°C for 15 min to inactivate the enzymes. Both DNA strands were sequenced using the same primers as for the initial PCR. Sequencing was performed at the Core Facility of Genomics (Institute of Genomics, University of Tartu, Estonia). PCR and sequencing were successful with both A. vasorum-specific primer pairs (Ang) and negative with the D. immitis primer pair (Dir). Consensus sequences were assembled in the program Codon Code Aligner v.11.0.1 (https://www.codoncode.com/index.htm; CodonCode Corporation, Massachusetts, USA). Sequence quality was assessed by visual inspection of chromatograms, and mistakes were manually corrected.
Species identification (based on the LSU rRNA sequence) was performed by homology search with the program Nucleotide BLAST (nBLAST) (https://blast.ncbi.nlm.nih.gov).
Sequences were submitted to GenBank (accession codes PV093945 and PV093946).
To analyse relationships of the A. vasorum isolate from Estonia with those from different countries and hosts in Europe, a median-joining phylogenetic network was built, using the 694 bp sequence from the dog of this study (Est1) and 79 highly homologous sequences retrieved from GenBank. The network was inferred using program Network v.10.2.0.0 (Bandelt et al. Reference Bandelt, Forster and Röhl1999; http://www.fluxus-engineering.com, Fluxus Technology Ltd., 2004), with both indels and point mutations considered. Sequences were aligned in BioEdit v.7.7.1 (Hall Reference Hall1999), producing a dataset of 554 bp, corresponding to positions 105–658 in the complete mitogenome of A. vasorum (NC_018602). DnaSP v. 6.12.03 (Rozas et al. Reference Rozas, Ferrer-Mata, Sanchez-DelBarrio, Guirao-Rico, Librado, Ramos-Onsins and Sanchez-Gracia2017) was used to calculate Tajima’s D.
Results and discussion
The PCR primers (Table 1) designed to detect A. vasorum gave positive results for both scat samples (but negative for blood samples), whereas the genus-specific PCR for Dirofilaria spp. was negative for both blood and scat samples. The primer pair Ang1F-2R yielded a 276 bp sequence, corresponding to positions 3010–3285 in the complete mitogenome of A. vasorum NC_018602 from GenBank (Gasser et al. Reference Gasser, Jabbar, Mohandas, Schnyder, Deplazes, Littlewood and Jex2012). The primer pair Ang3F-Ang3R produced a 694 bp sequence, corresponding to positions 53–746 in NC_018602. Species identification, performed by homology search with the program Nucleotide BLAST, revealed 100% homology with several other sequences of A. vasorum, and a total of 79 sequences of the same species with homology greater than 95%. The next-best species match was A. costaricensis, with considerably lower homology (87%). Thus, the isolate from the dog in Estonia was unequivocally identified as A. vasorum.
The network analysis revealed (Figure 1) that the Estonian isolate shared the same haplotype with an isolate from a red fox (Vulpes vulpes) from the United Kingdom (Greater London area) and was closely related to several other haplotypes of the UK (fox, dog), Switzerland (fox), Ireland (dog), and Denmark (fox). The evolutionary network had a star-like structure with several divergent haplotypes and a statistically significant negative value of Tajima’s D (−1.98; P < 0.05), suggesting that the population of A. vasorum has expanded relatively rapidly after a bottleneck event.
Median joining network based on partial mtDNA cox1 gene sequences (554 bp), illustrating relationships of Angiostrongylus vasorum isolates from different countries and hosts in Europe. The dog isolate from Estonia (EST1) is depicted in a red rectangle and isolates from other countries (n = 79) in black circles. Country codes are according to ISO 3166-1 alpha-3 (CHE: Switzerland; DNK: Denmark; DEU: Germany; GBR: United Kingdom of Great Britain and Northern Ireland; NLD: Netherlands; PRT: Portugal; ROU: Romania). Small black rectangles represent median vectors (sequences not sampled or extinct). One bar equals one nucleotide difference; other differences are indicated by a number.
Figure 1. Long description
A radial diagram representing m t D N A c o x 1 gene sequences. The network is anchored by three large central nodes labeled A, B, and C.
Node A is the largest central hub, representing isolates from G B R fox-3x, C H E fox-2x, and R O U dog. Radiating from Node A are numerous black circular nodes representing various countries including C H E, D N K, D E U, G B R, N L D, P R T, and R O U.
Node B is located slightly southwest of Node A and represents G B R fox-2x and dog, C H E fox, R O U dog, and D N K dog. It connects to several fox and dog isolates from G B R and C H E.
Node C is further southwest, representing G B R fox-3x and C H E fox.
Directly between nodes B and C is a red rectangular node labeled E S T 1 dog and G B R fox.
Lines connect the nodes, with most representing a single nucleotide difference. Specific branches indicate higher differences, such as a long branch extending to the far West with a value of 4 leading to a G B R fox isolate, and a long branch extending North with a value of 2 leading to a G B R fox isolate. Small black squares along the lines represent median vectors for unsampled or extinct sequences.
Treatment with fenbendazole was initiated following the confirmation of the diagnosis, resulting in rapid clinical improvement. At one-year follow-up, the dog was clinically healthy with complete resolution of respiratory signs.
This report documents the first molecularly confirmed case of A. vasorum infection in a domestic dog in Estonia and provides evidence that canine angiostrongylosis may be emerging in the Baltic region.
The dog had been imported from Poland approximately five months prior to presentation. As A. vasorum is endemic in several regions of Central Europe (Fuehrer et al. Reference Fuehrer, Morelli, Unterköfler, Bajer, Bakran-Lebl, Dwużnik-Szarek, Farkas, Grandi, Heddergott, Jokelainen, Knific, Leschnik, Miterpáková, Modrý, Petersen, Skírnisson, Vergles, Schnyder and Strube2021), infection prior to importation cannot be excluded. However, the previous detection of A. vasorum in Estonian wild canids, the red fox ( Vulpes vulpes ; Laurimaa et al. Reference Laurimaa, Moks, Soe, Valdmann and Saarma2016a) and the raccoon dog ( Nyctereutes procyonoides ; Laurimaa et al. Reference Laurimaa, Süld, Davison, Moks, Valdmann and Saarma2016b), suggests that a local wildlife reservoir already exists. Given that Poland is endemic for A. vasorum and the dog was imported prior to presentation, pre-import infection appears more likely than local transmission. Whether this case represents an isolated importation or evidence of autochthonous transmission remains unclear. Systematic surveillance in domestic dogs and wild carnivores will be necessary to clarify the epidemiological status of A. vasorum in Estonia.
This case also underscores important diagnostic challenges in regions where angiostrongylosis is not routinely considered. Cross-reactivity between circulating antigens of A. vasorum and D. immitis (endemic in Estonia, Mõttus et al. Reference Mõttus, Mõtsküla and Jokelainen2024) in commercial point-of-care assays may result in false-positive heartworm test results (Schnyder et al. Reference Schnyder and Deplazes2012). In addition, repeated invalid results in species-specific antigen testing raise the possibility of a hook effect in cases of high antigenemia (Barker et al. Reference Barker, Payne and Wilson2024). Such diagnostic limitations may delay appropriate treatment and lead to unnecessary or inappropriate therapeutic interventions. In dogs presenting with respiratory signs and suspected heartworm infection but lacking echocardiographic evidence of D. immitis, angiostrongylosis should be considered as a plausible differential diagnosis. When clinical suspicion persists, serological findings should be supported by coprological examination and, where possible, molecular confirmation.
Climatic changes, increased movement of companion animals, and expanding wildlife reservoirs may facilitate further northward spread of A. vasorum in Europe. Veterinary practitioners in Northern and Eastern Europe should therefore include angiostrongylosis in the differential diagnosis of dogs presenting with respiratory, coagulopathic, or neurological signs, even in areas traditionally considered non-endemic. Failure to recognise the infection may result in delayed treatment and increased risk of severe or fatal outcomes.
Continued monitoring of domestic and wild hosts, combined with molecular epidemiological studies, will be essential to determine whether A. vasorum is becoming established in Estonia and to better understand its transmission dynamics in the Baltic region.
Acknowledgements
We thank the veterinarians involved in the management and treatment of the patient described in this report.
Financial support
The research has been financed by the Estonian Ministry of Education and Research and the University of Tartu (grants PRG1209, TK215, and PLTOM25917 to US).
Competing interests
The authors declare no competing interests.
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