Department of Eco-Epidemiology of Parasitic Diseases, Institute of Developmental Biology and Biomedical Sciences, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.
The Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 7610001, Israel.
Vet Parasitol. 2023 Mar;315:109882. doi: 10.1016/j.vetpar.2023.109882. Epub 2023 Jan 20.
Subcutaneous dirofilariosis is a fast-spreading infection of dogs, and occasionally of other carnivores and humans. Several factors contribute to its spread, including climate change, which facilitates development and survival of Dirofilaria repens in the mosquito vector. Movement/relocation of infected definitive hosts (dogs) from endemic regions to non-endemic regions is another possible cause of local emergence and the presence of a wide variety of wild reservoirs of the parasite may also contribute to its spread. The main aim of this study was to evaluate the genetic diversity of D. repens from different regions of Europe and to evaluate the spread of identified haplotypes and their geographic origin. A total of 95 D. repens isolates were obtained from Central and Eastern Europe (Poland, Belarus, Ukraine, Austria, Romania), NE Europe (Lithuania, Latvia, Estonia), Italy and Israel. All but two positive samples were obtained from the blood of dogs while one positive sample was obtained from an adult worm from a human case from the Lublin area in SE Poland and one sample was obtained from Anopheles plumbeus mosquito from Austria. Genetic diversity in D. repens isolates was evaluated by PCR amplification and sequencing of three genetic markers, including two mitochondrial genes (mtDNA): the cytochrome c oxidase subunit I (COI) and dehydrogenase subunit I (NADH). Additionally, the genomic marker, internal transcribed spacer 1 (ITS-1) was amplified and sequenced. Haplotypes were differentiated based on sequence alignments by identifying Single Nucleotide Polymorphism (SNPs) using DnaSP and Mega X. PopArt was used to construct a haplotype network including all identified haplotypes. Both mtDNA sequences (COI and NADH) were combined together for phylogenetic and network analyses. Altogether 18 haplotypes (DR1-DR18) were identified in combined mtDNA markers among 95 analysed samples. Haplotype DR1 was the most common encompassing 66 isolates: 42 isolates from Poland (41 from dogs and one from a human), 13 from Lithuania, 4 from Latvia, 2 from Ukraine and 5 from Romania. All other haplotypes grouped around haplotype DR1 separated by 1-5 SNPs, forming a star-like shape. Haplotype DR2 was the second most common haplotype, formed by six isolates from Romania. Interestingly, haplotype DR3 was represented only by four isolates from Israel. The remaining 15 haplotypes were represented by 1-4 isolates of different origins. Our study showed that only minor genetic diversity was found in D. repens since all isolates appear to have clustered in or branched out from haplotype DR1 with 1-5 SNP differences. The genetic diversity appears to be governed by geographic origin since isolates from neighbouring populations (countries) appear to share unique haplotypes while other populations that are geographically distant from individual haplotypes.
皮下包虫病是一种快速传播的犬类传染病，偶尔也会感染其他肉食动物和人类。多种因素促成了其传播，包括气候变化，这有助于蚊媒中的繁殖体在蚊子媒介中发育和存活。从流行地区向非流行地区转移感染的终末宿主（犬）也是当地出现的另一个可能原因，寄生虫的各种野生储主也可能有助于其传播。本研究的主要目的是评估来自欧洲不同地区的繁殖体的遗传多样性，并评估鉴定出的单倍型及其地理起源的传播。总共从中东欧（波兰、白俄罗斯、乌克兰、奥地利、罗马尼亚）、北欧（立陶宛、拉脱维亚、爱沙尼亚）、意大利和以色列获得了 95 个繁殖体分离株。除了两个阳性样本来自狗的血液外，其他所有阳性样本均来自波兰东南部卢布林地区的人类病例中的成虫，一个阳性样本来自奥地利的致倦库蚊。通过聚合酶链反应扩增和三种遗传标记（包括两个线粒体基因（mtDNA）：细胞色素 c 氧化酶亚基 I（COI）和脱氢酶亚基 I（NADH））的测序评估了繁殖体分离株的遗传多样性。此外，扩增并测序了基因组标记物，内部转录间隔区 1（ITS-1）。基于单核苷酸多态性（SNP），通过 DnaSP 和 Mega X 确定序列比对来区分单倍型。使用 PopArt 构建了包括所有鉴定出的单倍型的单倍型网络。将 mtDNA 序列（COI 和 NADH）合并在一起进行系统发育和网络分析。在 95 个分析样本中，在组合 mtDNA 标记中总共鉴定出 18 个单倍型（DR1-DR18）。单倍型 DR1 是最常见的，包含 66 个分离株：42 个分离株来自波兰（41 个来自狗，1 个来自人），13 个来自立陶宛，4 个来自拉脱维亚，2 个来自乌克兰，5 个来自罗马尼亚。所有其他单倍型围绕单倍型 DR1 聚集，通过 1-5 个 SNP 隔开，形成星状形状。单倍型 DR2 是第二常见的单倍型，由罗马尼亚的 6 个分离株组成。有趣的是，单倍型 DR3 仅由来自以色列的 4 个分离株组成。其余 15 个单倍型由不同来源的 1-4 个分离株代表。我们的研究表明，繁殖体的遗传多样性很小，因为所有分离株似乎都聚集在或从单倍型 DR1 中分支出来，仅存在 1-5 个 SNP 差异。遗传多样性似乎由地理起源决定，因为来自邻近种群（国家）的分离株似乎共享独特的单倍型，而其他种群则与单个单倍型在地理上相距甚远。
