Dirofilaria repens Microfilaremia in Three Dogs in Estonia

Introduction

In Europe, Dirofilaria repens is spreading northeast (Genchi et al. 2009, 2011, Simón et al. 2012, Kartashev et al. 2014). The previous northernmost available reports of likely autochthonous infections were from Latvia (Kirjušina et al. 2014), but imported cases have reached Norway (Sævik et al. 2014) and Finland (Evira 2014). Dirofilaria spp. are zoonotic, and canine infections cause public health concern.

A case of suspected D. repens has been described in Estonia (Järvis 2012). A 10-cm-long worm was found during surgery on a 5-year-old male dog without travel history outside Estonia. No reports of suspected or confirmed local human cases are available, and local mosquito vectors and potential sylvatic reservoir hosts have not been investigated for the presence of D. repens.

Case Reports

We diagnosed three dogs with microfilaremic D. repens infection among the 5494 dogs that visited the Small Animal Clinic of the Estonian University of Life Sciences, Tartu, Estonia, from December 2013 to November 2014.

The first case was a 12-year-old female German Shepherd Dog without travel history outside Estonia. The dog had a cystic soft tissue mass on the medial side of the hind leg. A blood smear evaluation revealed microfilaremia, and the dog was treated with a single dose of topical imidacloprid + moxidectin. Microfilariae were not found on a follow-up visit 17 days later. The dog had surgery to remove the mass but died during the postoperative observation period. The cause of death remained unspecified because a postmortem examination was not performed.

The second case was a 7-year-old entire male Newfoundland dog without travel history outside Estonia. The presenting complaint was poor cardiac systolic function and atrial fibrillation. On physical examination, enlargement and firm consistency of the right prescapular lymph node was noted. Microfilariae were detected in the fine needle aspirate of the reactive lymph node and in the blood smear. An immunochromatographic screening test (SNAP 4Dx, IDEXX Laboratories, Hoofddorp, the Netherlands) was negative for vector-borne pathogens, including Dirofilaria immitis antigen. A diagnostic PCR (Laboklin, Bad Kissingen, Germany) identified the microfilariae as D. repens. Selamectin spot-on, administered topically, and doxycycline tablets, administered orally, were added to the treatment plan. The dog remained clinically stable, and the enlarged lymph node decreased in size. However, approximately 4 weeks later, the condition of the dog suddenly deteriorated, and the dog died. The suspected cause of death was cardiovascular failure, but this remained unconfirmed because no postmortem examination was performed.

The third case was a 5-year-old entire male St. Bernard without travel history outside Estonia. The dog was a blood donor and routinely vaccinated and dewormed. The findings of a physical examination were unremarkable. Microfilaremia was noted on a microscopic evaluation of a blood smear. The dog was prescribed selamectin spot-on and doxycycline tablets, and its follow-up was planned.

The molecular analysis of the approximately 300- to 400-μm long microfilariae was performed from EDTA blood samples from the first two cases and a blood transfusion bag from the third. The microfilarial DNA was extracted from the blood by lysis (Lek-Uthai and Tomoen 2005). Species identification was based on amplifying partial mitochondrial sequences of 12S rDNA and cytochrome c oxidase subunit I (COI) using the primer pairs 12SF-12SR (Casiraghi et al. 2004) and COIintF-COIintR (Casiraghi et al. 2001), with minor modifications. For both PCR reactions, 1 U of DyNAzyme II DNA polymerase (Finnzymes OY, Espoo, Finland) was used, and the thermal profile for the COI-PCR was 94°C for 3 min, followed by 31 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 60 s, and a final extension at 72°C for 5 min.

Subsamples of the PCR products were visualized on a 1.5% agarose gel. The PCR products were treated with ExoSAP-IT (GE Healthcare, Buckinghamshire, UK), and the amplicons were sequenced bidirectionally (Haartman Institute, Helsinki, Finland). The sequences were edited and aligned using MEGA6 software and compared with GenBank entries using BLAST. In all three cases, both fragments (GenBank 12S, acc. no. KR780979; COI, acc. no. KR780980) were identical to several available D. repens sequences.

The described infections were likely autochthonous because the dogs reportedly had no travel history outside Estonia. Tartu is located at 58°23′N, 26°43′E, which is farther north than the northernmost latitude predicted to have suitable conditions for D. repens (Genchi et al. 2011), but where the threshold of 130 Heartworm Development Units has been reached (Genchi et al. 2009) and one or two yearly Dirofilaria generations were predicted for 2001–2011 and for 2030 (Kartashev et al. 2014).

D. repens reaches sexual maturity in 6–9 months postinfection, and the adults live for years (Simón et al. 2012). Thus, in Estonia, the earliest that an infected dog can contribute to the spread of the disease is the summer following infection. One of the cases was diagnosed in December of 2013, two were diagnosed in the summer of 2014, and all three dogs were microfilaremic at the time of diagnosis; therefore, these infections should date back to the summer of 2013 or earlier. Considering the age of the dogs, the time of the diagnoses, and the mosquito season, the two dogs diagnosed in the summer, at least, most likely contributed to further spread of the parasite before they were treated. Few treatment options were available, and off-label use was necessary (Jacsó et al. 2010, Sævik et al. 2014).

Microfilaremia was an unexpected or incidental finding in these three cases. During the year, three (0.05%, with a 95% confidence interval of 0.01–0.15) of the 5494 dogs that visited the clinic were diagnosed with D. repens. Blood smears are not routinely evaluated in dogs, and the infection is likely underdiagnosed due to low awareness.

Because pet dogs are within reach for medical interventions, whereas potential sylvatic hosts are not, diagnosing canine D. repens infections should be promoted among veterinarians. This is relevant particularly in Nordic and Baltic countries where dogs are not treated routinely with chemoprophylactic drugs against Dirofilaria spp. The local dog populations include susceptible reservoirs that, when infected and if not promptly diagnosed and treated, provide microfilariae to mosquitoes.

Conclusions

D. repens has spread farther north. Infection with D. repens should be added to the list of differential diagnoses for human and animal hosts that live in or have visited the northern parts of Europe.