Short Report
First molecular biologically confirmed case of Dirofilaria repens in a dog in Denmark
https://doi.org/10.21203/rs.3.rs-2438532/v1
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Dirofilariasis is spreading among dogs and humans in Europe with infections being established in many countries. In Denmark, at least one to two generations of Dirofilaria spp. can occur per year. Here, we describe the first molecular biologically confirmed case of D. repens infection in a dog in Denmark.
Dirofilaria
Emerging
dogs
Denmark
Zoonosis
Dirofilariasis is spreading among dogs and humans in Europe [1]. Dirofilaria immitis is the causative agent of human pulmonary dirofilariasis, and Dirofilaria repens causes human subcutaneous or ocular dirofilariasis [2]. At present, dirofilariasis is rarely diagnosed in Denmark, with less than 10 cases per year being registered in humans [3]. In Europe, Dirofilaria spp. are prevalent in Southern Europe and Mediterranean countries. However, D. immitis and D. repens are spreading north [2, 4, 5] with D. repens now being endemic in Poland, Ukraine, Germany, Austria, Hungary, the Netherlands, Estonia, Latvia and Lithuania [6]. Even as far as in Yakutsk, Siberia, located in the permafrost area, D. repens and D. immitis thrive [7].
Dirofilaria immitis and D. repens are nematodes with canids as their main definitive host, however, they are able to infect a wider range of vertebrate hosts including humans [2]. Adult worms live in the definitive host where they release microfilaria (first stage larvae, L1) into the bloodstream. Microfilaria are transmitted to the mosquito intermediate host during bloodmeals where they develop into the infective third stage larvae (L3). The L3 are transmitted to the definitive host when an infected mosquito feeds.
Several mosquito species can serve as intermediate host, including Aedes spp., Anopheles spp., and Culex pipiens [2]. Some of these species, e.g. Culex pipiens, Anopheles maculipennis and Aedes vexans, have been detected in Denmark or in areas very close to Denmark, such as the northern part of Germany, and the southern parts of Sweden [8, 9].
The development of infective Dirofilaria spp. L3 larvae in mosquitoes require time and the right temperature, but it has been estimated that at least one to two generations per year of Dirofilaria spp. can occur in Denmark [10]. Furthermore, dogs imported from endemic areas may harbor Dirofilaria spp., and in case of infection with D. repens, infected dogs may be asymptomatic for a long time, with adult worms and microfilaria living in infected dogs for up to four years [11].
Here, we communicate the detection of infection with D. repens in an imported dog in Denmark, a finding that according to the author’s knowledge is the first confirmed case in a dog in Denmark, and as cases of D. immitis infections in dogs have also been encountered in recent years in Denmark, this would seem to compel increased awareness of dirofilariasis.
A 6-year-old, castrated male mixed-breed dog, 22 kg, was brought to the University Hospital for Companions Animals, Department of Veterinary Clinical Sciences, Denmark for euthanasia as the owner was unable to keep the dog. Following euthanasia, the owner donated the dog to scientific and educational purposes. The owner reported that the dog was imported from Romania, and the owner had had the dog for three to four months. The owner had not observed any signs of disease in the dog, and neither had the dog been treated with any medications. Blood samples were collected in EDTA, heparin, and in serum tubes.
On subsequent haematological examination, multiple microfilaria were detected by microscopy of EDTA-blood in capillary tubes modified ad modum Collins [12]. In blood smears, microfilaria with morphology indicative of D. repens, i.e., short cephalic space terminated by a distinct pair of nuclei separate from the remaining somatic nuclei of the microfilaria [13], were observed (Fig. 1). Additional haematological examination revealed slightly increased erythrocyte count, haematocrit and haemoglobin, as well as a leukogram exhibiting a mild inflammatory pattern. Biochemical analysis was unremarkable. Infection with Dirofilaria spp. was suspected.
Visual inspection and manual palpation of the dog’s neck, torso, limbs and tail did not reveal any abnormal dermal or subdermal structures. The dog’s head could not be examined as it was removed for other studies. Upon incision, the four chambers of the heart and arteria pulmonalis were examined, and no macroscopic visible adult worms characteristic of D. immitis could be detected.
An EDTA-stabilized blood sample was submitted to Statens Serum Institut, Denmark, for PCR-analysis for filaroidal DNA; DNA was extracted using QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) following manufacturers protocol. The PCR used primers targeting the internal transcribed space 1 region and 5.8 ribosomal RNA gene with the forward primer FIL 18S and reverse primer FIL_5.8S_R after Mistry et al. [14] followed by Sanger sequencing using the PCR primers as sequencing primers.
The owner of the dog was contacted and informed of the finding and urged to seek medical advice for further information on aspects of dirofilariasis infections in humans.
To get a broader view of the occurrence on canine dirofilariasis, a search in the medical records database from 2016 to 2022 at the University Hospital for Companion Animals was conducted to retrieve information on the dogs previously diagnosed and/or treated for dirofilariasis.
PCR-analysis revealed DNA from D. repens. The obtained DNA sequence (Genbank accession no.: OQ091773) was 500 bp-long and showed 100% similarity to D. repens (Genbank accession no.: AB973229.1) with a complete coverage by nucleotide BLAST.
In addition to this first published case of D. repens infection in a dog in Denmark, the survey of patient medical records at the University Hospital for Companion Animals revealed that three dogs have been diagnosed and/or treated for dirofilariasis since 2016 (Table 1). All dogs were imported from endemic areas (Greece, Bulgaria, and Bosnia and Herzegovina) as part of dog rescue programs. All three dogs with D. immitis were diagnosed with a commercially available ELISA test (SNAP® 4Dx®, IDEXX, Westbrook, Maine) which affords detection of antigen of D. immitis, but not D. repens. The American Heartworm Society recommends that microfilaria testing be done in tandem with antigen testing in general, and that all positive D. immitis antigen tests should be confirmed by identifying circulating microfilaria, or by a different type of antigen test [15]. Microfilaria were only tested for and detected in one of the three dogs with suspected D. immitis infection, however, ultrasonographic findings were highly suggestive of an adult D. immitis worm in the right pulmonary artery in two dogs. One dog was euthanized at the time of diagnosis due to cost of treatment, one dog was treated according to the American Heartworm Society management protocol with melarsomine [15], and the owner of one dog declined the AHS management protocol due to costs. This dog was treated with imidacloprid and moxidectin, so-called “slow kill”, against medical advice. It is possible that other cases of dirofilariasis in dogs have been missed at the University Hospital, since screening for dirofilariasis is not performed on a routine basis. However, it is likely that other cases in Denmark may have been diagnosed by private veterinary practitioners, as the University Hospital have been contacted several times for advice on diagnosis, management, and treatment of dogs with D. immitis. Furthermore, since dirofilariasis is not notifiable in Denmark no official information on the prevalence in dogs are available.
| Dog 1 | Dog 2 | Dog 3 | |
|---|---|---|---|
| Time of diagnosis | September 2016 | July 2020 | October 2021 |
| Age at time of diagnosis | 1 year 6 months | 2 years 5 months | 1 year 5 months |
| Sex | Female, neutered | Male | Female, neutered |
| Breed | Medium breed mix | French bulldog | Great Pyrenees |
| Country of origin | Greece | Bosnia and Herzegovina | Bulgaria |
| Time in Denmark before diagnosis | 3 weeks | 1 year 10 months | 1.5 months |
| Clinical signs | Exercise intolerance, intermittent coughing, and gastroenteritis | Cutaneous swelling (likely unrelated to D. immits) | Decreased activity level and exercise intolerance |
| Diagnostics | Positive on D. immitis antigen test Evidence of a worm in the right pulmonary artery on echocardiography | Positive on D. immitis antigen test Microfilaria positive Evidence of a worm in the right pulmonary artery on echocardiography | Positive on D. immitis antigen test Evidence of a worm in the right pulmonary artery on echocardiography |
| Treatment and outcome | Initiated treatment with imidaclopid and moxidectin. Owner subsequently declined further treatment and opted for euthanasia of the dog | Owner declined treatment (AHS management protocol). Treated with imidacloprid and moxidectin. Subsequently lost for follow up | Successfully treated (AHS management protocol using melarsomine). Negative twice on D. immitis antigen test |
| AHS – American Heartworm Society | |||
The development to mature adult worms in the definitive host takes approximately 8 months for D. immitis, and 6 months for D. repens [16]. Larval development in the mosquito is temperature-dependent and takes 8–13 days at 27–30ͦC, 10–12 days at 24–26 ͦC, and 16–20 days at 22 ͦC [4]. Fortin and Slocombe [17] introduced the concept of the heartworm development unit (HDU) to explain the relationship between the development period and temperature in D. immitis. In a study on risk assessment for the endemisation of D. repens in a state in Germany, Sassnau and Genchi [18] defined a Development unit (DU) as the difference between the average daily temperature above 15°C, and the limit of 14°C, then counted degree days in excess of this threshold and, assuming that an infected mosquito does not survive 30 days in the wild [19], they defined an amount of at least 130 DUs in 30 consecutive days as the minimum for one larval development of D. repens. A study from 2009 reported that 1–2 Dirofilaria generations per year could develop in Denmark, and that during the past 15 years, there had been at least one occasion where the necessary 130 HDU was reached [10]. Local meteorological data from 2022 at Frederiksberg, Denmark [20], where the University Hospital for Companion Animals is located, are in line with this as there were 86 consecutive days with temperatures above 14°C with a total of 397.6 DU from June to September. Further, the relatively simple models like counting HDUs may underestimate the risk of larval development as they do not take influence of microclimate, such as “urban heat islands” or local biological dispositions and adaptations of the mosquitos etc., into account [15, 21, 22]
Increased movement of companion animals has been suggested as a main cause for spreading Dirofilaria spp. in Europe [1]. The dogs presented at the University Hospital were all imported and likely already infected upon arrival to Denmark. Of 4,681 dogs that had travelled to or been relocated to Germany from endemic areas 372 dogs (7.7%) were found to be microfilaremic via a modified Knott´s test [23]. Furthermore, a study from Norway reported that autoantibodies against D. immitis were detected in six blood samples from 80 stray dogs imported from Eastern Europe. This was of concern as competent mosquito species were also observed in Norway, and because there had been seven summers between 2000 and 2010 with sufficient temperatures to allow for larval development [24].
On the initiative of the Danish Veterinary and Food Administration, the Technical University of Denmark (DTU) made a report assessing the risk of transmission of vector borne disease from imported dogs [25]. According to the report, there is a risk of establishing a D. repens reservoir in Denmark. For both D. immitis and D. repens, there is a moderate zoonotic risk from dogs to humans, and a moderate impact on human health. D. immitis is considered a serious risk and D. repens a moderate risk to the health of dogs. To prevent establishment of a reservoir of microfilaremic canids it is suggested to test imported dogs for circulating microfilaria multiple times with a minimum of 6 months intervals, and to survey Danish mosquito species to document if the parasite is established [25]. The American Heartworm Society, in collaboration with the Association of Shelter Veterinarians, has developed a protocol and algorithm to minimize the risk of D. immitis transmission when transporting or relocating dogs [15]. This protocol may support veterinarians when advising dog owners of imported or relocated dogs from endemic areas.
In dogs, infection with D. repens can be asymptomatic for a long time [1], and adult worms and microfilaria may live up to four years in the definitive host [11]. Infected dogs can thus serve as silent reservoirs for several years, and together with the occurrence of competent mosquito vector species in Denmark, in addition to temperatures that may allow adult maturation, this is a cause for concern. This is further supported by a recent survey in Finland among medical doctors and veterinarians calling for increased medical and veterinary awareness on dirofilariasis [26]. If a reservoir of microfilaremia in dogs without the opportunity of proper veterinary care is first established, transmission is possible, and eradication is unlikely [15].
Heart Worm Development Unit
Development Unit
Acknowledgements
The authors wish to acknowledge Armina Babovic at The Veterinary Diagnostic Laboratory at the Department of Veterinary Clinical Sciences and the laboratories in the Department of Bacteria, Parasites, and Fungi at Statens Serum Institut (for skillful technical assistance. C. Rune Stensvold is thanked for his help with sequence analysis.
Funding:
Not applicable
Availability of data
The sequence is available in the GenBank database under the accession number OQ091773
Authors’ contributions
ALJ, AKHK, JFHL and JGHL conducted laboratory analyses and identified the infection caused by the filaroide nematode.
ALJ and AKHK performed postmorten examination of the dog.
JGHL, JLW, ASS and MBTH handled the clinical cases of dirofilariasis, performed the search in the medical records and reviewed the cases.
RPKDB identified the filaroide nematodes as D. repens by molecular methods.
All authors participated in drafting the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
A written owner consent form was obtained, and The Ethical Committee approved the project at the Department of Veterinary Clinical Sciences, SUND, University of Copenhagen (Approval number 2022-27).
Consent for participation
Not applicable.
Competing interests
The authors declare that they have no competing interests.
No competing interests reported.
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