De novo genome sequencing and comparative stage-specific transcriptomic analysis of Dirofilaria repens.

The zoonotic mosquito-borne filarial nematode Dirofilaria repens causes subcutaneous and ocular infections in dogs, cats and humans. From infected vertebrate hosts, microfilariae are taken up by mosquitoes and develop into infective L3. These are transmitted to new vertebrate hosts and develop over two further moults to adult worms. The aims of the project were (i) the de novo sequencing and annotation of the D. repens genome and (ii) comparative transcriptomic analyses of the two developmental stages, mf and L3. Genomic DNA was obtained from adult male D. repens. RNA was extracted from mf from naturally infected dogs and from L3 produced in Aedes aegypti mosquitoes fed on blood spiked with mf. The 99.59 MB genome was approximately 17% larger than that of the related species Dirofilaria immitis (dog heartworm) and contained 8.9% fewer predicted genes (10,357). Approximately 1.8% of identified proteins (206/11,262) could not be mapped to D. immitis. Out of these, six (2.9%) presented an ortholog in all other considered filarial nematodes (e.g. Loa loa) and Caenorhabditis elegans. A significantly higher number of D. repens proteins, compared with D. immitis, mapped to the filarial nematode L. loa, reflecting the similarity in biology of D. repens and L. loa. A total of 876 genes were differentially expressed, of which 591 could be annotated in UniProtKB/Swiss-Prot. In particular, 155 genes with a UniProtKB/Swiss-Prot annotation to C. elegans and filarial nematodes were upregulated in the L3 and 57 in the mf stage, respectively. Fifteen Gene Ontology Biological Processes were significantly enriched for the L3 group and 12 for the mf. To our knowledge these data provide the first insight into the differential gene expression profiles of this filarial nematode and can serve future investigations of metabolic processes and stage-specific diagnostics.

Recent advances on Dirofilaria repens in dogs and humans in Europe.

Dirofilaria repens is a nematode affecting domestic and wild canids, transmitted by several species of mosquitoes. It usually causes a non-pathogenic subcutaneous infection in dogs and is the principal agent of human dirofilariosis in the Old World. In the last decades, D. repens has increased in prevalence in areas where it has already been reported and its distribution range has expanded into new areas of Europe, representing a paradigmatic example of an emergent pathogen. Despite its emergence and zoonotic impact, D. repens has received less attention by scientists compared to Dirofilaria immitis. In this review we report the recent advances of D. repens infection in dogs and humans, and transmission by vectors, and discuss possible factors that influence the spread and increase of this zoonotic parasite in Europe. There is evidence that D. repens has spread faster than D. immitis from the endemic areas of southern Europe to northern Europe. Climate change affecting mosquito vectors and the facilitation of pet travel seem to have contributed to this expansion; however, in the authors' opinion, the major factor is likely the rate of undiagnosed dogs continuing to perpetuate the life-cycle of D. repens. Many infected dogs remain undetected due to the subclinical nature of the disease, the lack of rapid and reliable diagnostic tools and the poor knowledge and still low awareness of D. repens in non-endemic areas. Improved diagnostic tools are warranted to bring D. repens diagnosis to the state of D. immitis diagnosis, as well as improved screening of imported dogs and promotion of preventative measures among veterinarians and dog owners. For vector-borne diseases involving pets, veterinarians play a significant role in prevention and should be more aware of their responsibility in reducing the impact of the zoonotic agents. In addition, they should enhance multisectorial collaboration with medical entomologists and the public health experts, under the concept and the actions of One Health-One Medicine.

Subcutaneous dirofilariosis (Dirofilaria repens): an infection spreading throughout the old world.

BACKGROUND: Two main Dirofilaria species infect dogs: D. immitis and D. repens. While D. immitis has a worldwide distribution, D. repens is currently found only in Europe, Asia, and Africa. Adult D. repens are located in subcutaneous tissues of natural hosts where they survive for long periods of time. First-stage larvae, microfilariae, circulate in the peripheral bloodstream, where they are taken up by the mosquito intermediate hosts. Infected mosquitoes then transmit infective third-stage (L3) larvae to new hosts through the blood meal. In dogs, most infections are asymptomatic, although cutaneous disorders such as pruritus, dermal swelling, subcutaneous nodules, and ocular conjunctivitis can be observed. Currently, two factors have increased the concerns about this parasitic infection 1) its spread throughout the European countries and to other continents and its prevalence in dog populations, where in some cases it has overcome D. immitis; and 2) its zoonotic potential, which is much greater than that of D. immitis. RESULTS: Different hypotheses can be put forward to explain these concerns. First, climate change has allowed more favorable conditions for survival of culicid vectors. Second, accidental hosts such as humans may have a less efficient immune reaction against a parasite that is located in subcutaneous tissues, and thus less exposed to the host's immune response than, for instance, D. immitis. Furthermore, the absence of clinical signs in the majority of canine infections and the difficulty in diagnosing the infection, due to the lack of serologic tests and thus the reliance on the identification of microfilariae and differentiation from D. immitis to confirm the presence of the parasite, favor the further spread of this species. Finally, among the macrocyclic lactones currently used to prevent heartworm infection, only moxidectin has been found to be fully effective against the infective larvae transmitted by mosquitoes and partially effective (efficacy 96%) against adult D. repens in experimental studies. CONCLUSIONS: Dirofilaria repens infection is much more difficult than D. immitis to diagnose and control in the reservoir population (microfilaremic dogs). In addition, lack of familiarity with D. repens infection could lead to lack of vigilance underestimation for this parasite. The number of human cases in Europe and Asia is currently a serious public health concern. Medical doctors and veterinarians must collaborate closely for better control and surveillance of D. repens infection.

Development of Dirofilaria immitis and Dirofilaria repens in Aedes japonicus and Aedes geniculatus.

BACKGROUND: The mosquito-borne filarial nematodes Dirofilaria immitis and Dirofilaria repens primarily affect dogs but also cats, causing heartworm disease or subcutaneous dirofilariosis, respectively, and both may also cause zoonotic diseases in humans. Several mosquito species have been reported as competent vectors for these nematodes, but no data are available for the invasive mosquito species Aedes japonicus (Theobald, 1901). The objective of this study was to describe the development of both D. immitis and D. repens under standardised experimental laboratory conditions in mosquitoes. METHODS: For this purpose, both a laboratory strain and field-collected individuals of the invasive mosquito species Ae. japonicus and, for comparative purposes, a laboratory strain of Aedes geniculatus, a rare indigenous species sharing habitats with Ae. japonicus, and of the tropical species Aedes aegypti were used. Anticoagulated microfilariaemic blood was fed at a density of 3000 mf/ml to mosquitoes with a hemotek system. Blood-fed mosquitoes were incubated at 27 °C and 85% relative humidity, and specimens were dissected under the microscope at pre-set time points to observe developmental stages of both Dirofilaria species. Additionally, real-time PCRs were carried out in some microscopically negative samples to determine the infection rates. RESULTS: In field-collected Ae. japonicus infectious L3 larvae of both D. immitis and D. repens developed, rendering this mosquito species an efficient vector for both filarial species. Additionally, Ae. geniculatus was shown to be an equally efficient vector for both filarial species. Aedes japonicus mosquitoes from a laboratory colony were refractory to D. immitis but susceptible to D. repens, whereas Ae. aegypti was refractory to both filarial species. CONCLUSIONS: To our knowledge, Aedes japonicus was for the first time shown to be an efficient vector for both D. immitis and D. repens, indicating that this invasive and locally highly abundant species may contribute to a transmission of filarial worms. The data emphasize the necessity to perform vector competence studies with local mosquito populations as basis for risk assessments. We further demonstrated that detection of filarial DNA in a mosquito species alone does not allow to draw reliable conclusions with regard to its vector competence.

[(HUMAN DIROFILARIASIS: CLINICAL DIAGNOSTIC FEATURES ASSOCIATED WITH DIFFERENT DEVELOPMENTAL STAGES OF THE PATHOGEN)].

To seek medical advice is due to the time when a person has become infected during the infection transmission season (July-August) and the duration of development of the pathogen Dirofilaria (N.) repens in his/her body Clinical manifes- tations occurred in 61% of the patients 6-10 months after infection, as confirmed by the maximum body sizes (125-160 mm) of removed females which have reached puberty. PCR-based diagnosis in conjunction with microscopic studies improves the efficiency of identifying the patients and the species of the pathogens D.repens and D.immitis. The use of these methods for the first time in 2016 could confirm D.immitis infestation in a 14-month-old infant living in the Solnechnogorsk District, Moscow Region.

[CIRCULATION OF DIROFILARIA REPENS (RAILLIET ET HENRY, 1911) IN THE ARID ZONE OF SOUTHERN RUSSIA].

The prevalence of dirofilariasis was studied in dogs from the urban and rural areas in Southern Russia. The high prevalence of Dirofilaria repens infection were established among the dogs in the Republic of Kalmykia. The blood samples from 328 dogs were tested; D. repens infection was detected in 23.6 and 15.5% of the rural and urban dogs, respectively. The highest prevalence of D. repens infection was noted in of 4-6-year-old dogs. Dogs aged 0, 1-3, 4-6 7-9, and more 10 years were infected in 0, 26.3, 33.3, 29.4, and 28.5% of cases, respectively.

[CURRENT IDEAS ON OBLIGATE AND FACULTATIVE RELATIONSHIPS BETWEEN MAN AND THE DIROFILARIASIS PATHOGEN DIROFILARIA (N.) REPENS].

The ability of D. repens to complete its ontogenesis in man points to their obligate, rather than facultative rela- tionships. The fact that microfilariae are rarely found in human blood or are absent there may be associated with the removal of developing dirofilariae from humans in earlier than they achieve sexual maturity. Facultative ecological relationships to mosquitoes may be one of the reasons for limitation of human invasion cases. However, in long-standing microfilaremia in man (an obligate host), D.repens may take part in the epidemiological chain of dirofilariasis as a source of invasion.

[INFECTION OF BLOOD-SUCKING MOSQUITOES (DIPTERA: CULICIDAE) WITH DIROFILARIAE (SPIRURIDA, ONCHOCERCIDAE) IN THE TULA REGION].

Blood-sucking mosquitoes (n = 2277) collected in Tula and its Region in 2013-2014 were examined using a PCR assay for dirofilariae. A total of 12 species from 4 genera (Culiseta, Aedes, Ochlerotatus [foreign character] Culex) out of 18 found mosquito species were infected with Dirofilaria immitis and D. repens. The proportion of the infected mosquitoes was 2.5% (D. immitis, 1.5%; D.repens, 1%). According to preliminary data, the most efficient Dirofilaria vectors, in the Tula Region may be Ae. vexans, Ae. geniculatus, Och. cantans, and Cx. pipiens.

Dirofilaria repens and Dirofilaria immitis DNA findings in mosquitoes in Germany: temperature data allow autochthonous extrinsic development.

After the repeated demonstration of Dirofilaria repens infections in German dogs, D. repens and Dirofilaria immitis DNA was detected in mosquitoes trapped in 2011, 2012 and 2013 in northeastern and southwestern Germany within the framework of culicid monitoring projects. As temperature is the most important factor dictating the extrinsic development of dirofilarial larvae in the potential vector, data of the German Weather Service (DWD) were analysed for the regions where the positive mosquitoes had been collected. Based on the mean daily temperatures recorded by weather stations most closely to the collection sites of the mosquitoes, it can be concluded that the mosquitoes were trapped in time periods that allowed for the completion of the developmental cycle of the worms in the mosquitoes and a subsequent transmission to a vertebrate host. The results of this study confirm the principal climatic suitability of certain German regions for the establishment of natural dirofilarial transmission cycles. Moreover, the theoretical climatic considerations, together with findings of D. repens infections in German dogs and mosquitoes, strongly suggest that the continuing spread of at least D. repens from its traditional habitats in the Mediterranean has reached southwestern and northeastern Germany.

The detection of different Dirofilaria species using direct PCR technique.

A novel direct PCR assay for the detection of Dirofilaria spp. from EDTA blood, Knott test, FTA cards, adult filarial worms, skin nodules and Dirofilaria spp.-infected mosquitoes was tested. Larval and adult DNA of Dirofilaria spp. from FTA cards, from the mosquito vector and from worm fragments without prior DNA extraction was successfully obtained. As little as 3.11 larvae/100 µl blood on FTA cards could be detected. Thus, direct PCR is capable of directly detecting first larval stages in the blood, third larval stages in the mosquito vector and pieces of mature stages of Dirofilaria spp. The assay is a rapid, sensitive and cost-effective alternative to standard PCR.

Microfilarial periodicity of Dirofilaria repens in naturally infested dogs.

In the past years, canine and human cases of infestation by Dirofilaria repens (Spirurida, Onchocercidae) have been increasingly reported in several European countries. Subcutaneous dirofilariosis by D. repens may either be asymptomatic in dogs or may be characterized by subcutaneous nodules and other symptoms. Information on the periodicity of D. repens microfilariae in naturally infested animals is scant, and this might impair the accurate diagnosis of subcutaneous dirofilariosis and appropriate control plans. In the present study, eight dogs infested with D. repens were sampled twice daily at 12-h intervals for ten consecutive days, and the dog with the highest mean value of microfilariaemia was further sampled every 4 h for four consecutive days. The blood was microscopically and molecularly examined for microfilariae, and, additionally, negative samples were also subjected to a real-time PCR to evaluate the level of circulating DNA. The results demonstrated significant variations in circadian rhythms of D. repens larvae, with higher values of microfilariae per milliliter in the evening samples. A significant variation was also found at the individual level for the dogs with the highest values of microfilariaemia. All samples which were negative at the light microscopy and positive at the real-time PCR displayed levels of circulating parasite DNA <1 microfilaria per milliliter. Biological and clinical implications have been here discussed.