Dirofilaria repens in dogs and humans in Lithuania

Abstract

Background: In Lithuania, the first case of canine subcutaneous dirofilariosis was recorded in 2010. Since then, an increasing number of cases of canine dirofilariosis have been documented in different veterinary clinics throughout the country. Human dirofilariosis was diagnosed in Lithuania for the first time in September 2011. However, to the authors' knowledge, there are no published data on the presence and prevalence of autochthonous dirofilariosis in dogs and humans in the country. The present study provides information about the predominant species and prevalence of Dirofilaria in dogs and describes the cases of human dirofilariosis in Lithuania. It also outlines PCR detection of the bacterial endosymbiont Wolbachia that contributes to the inflammatory features of filarioid infection.

Results: A total of 2280 blood samples and six adult worms from pet and shelter dogs were collected in the central and eastern regions of Lithuania in 2013-2015. Based on their morphological appearance, morphometric measurements and molecular analysis, all the adult nematodes were identified as Dirofilaria repens. The diagnosis of microfilariae in blood samples was based on blood smear analysis and Knott's test. The PCR and sequence analysis of the ribosomal DNA ITS2 region and cox1 gene confirmed the presence of D. repens. Overall, 61 (2.7%) of the 2280 blood samples were found to be positive for the presence of D. repens. The infection rate of D. repens was significantly higher in shelter dogs (19.0%; 19/100) than in pet dogs (1.9%; 42/2180) (χ² = 100.039, df = 1, P < 0.0001). Forty-nine DNA samples of D. repens-infected dogs were tested for the presence of the bacterial endosymbiont Wolbachia and, of these, 40 samples (81.6%) were found to be positive. Three ocular and six subcutaneous cases of human dirofilariosis were diagnosed in Lithuania in the period 2011-2018.

Conclusions: To the authors' knowledge, this is the first report of autochthonous D. repens infection in dogs and humans in Lithuania. The present data demonstrate that D. repens is the main etiological agent of dirofilariosis in Lithuania. The DNA of the filarioid endosymbiotic bacterium Wolbachia was detected in the vast majority of dogs infected with D. repens.

Keywords: Autochthonous; Dirofilaria repens; Dirofilariosis; Dogs; Humans; Lithuania; Wolbachia.

Fig. 1

Microfilariae (arrows) of D. repens in a a blood smear at 500× magnification and b the modified Knott’s test at 100× magnification

Fig. 2

Subcutaneous localisation of an adult D. repens worm (arrow) in a French bulldog during neutering surgery

Fig. 3

Subconjuctival localisation of a D. repens adult worm (arrow) in the human eye, patient no. 7 (Table 1)

Fig. 4

Phylogenetic tree of filarioid nematodes based on ITS2 rDNA sequences created using the maximum-likelihood (ML) method and bootstrap analysis of 1000 replicates. The representative sequences obtained in this study are marked with a black triangle

Fig. 5

A phylogenetic tree of filarioid nematodes based on cox1 gene sequences created using the maximum likelihood method and bootstrap analysis of 1000 replicates. The representative sequences obtained in this study are marked with a black square (MH469227: representative of sequences obtained from canine blood; MH469228, MH469229: sequences obtained from two adult nematodes)

Fig. 6

Neighbour-joining phylogenetic tree for the partial 16S rRNA gene of the Wolbachia endosymbiont. The phylogenetic tree was created using the Kimura 2-parameter model with a bootstrap analysis of 1000 replicates. Sequences with accession numbers were taken from GenBank for comparison. The identification source (host) and country codes are provided. Samples sequenced in the present study are marked with a black rhombus