Parasitology Research

, Volume 112, Issue 7, pp 2647–2652

Qualitative risk assessment for the endemisation of Dirofilaria repens in the state of Brandenburg (Germany) based on temperature-dependent vector competence


    • Small Animal Practice
  • C. Genchi
    • Dipartimento di Scienza Veterinarie e Sanità PubblicaUniversità degli Studi di Milano
Original Paper

DOI: 10.1007/s00436-013-3431-2

Cite this article as:
Sassnau, R. & Genchi, C. Parasitol Res (2013) 112: 2647. doi:10.1007/s00436-013-3431-2


Climate change with an increase in average temperature may be responsible for propagation of temperature-dependent vectors and/or vector-associated pathogens in regions that were previously not endemic. The analysis of climate data of Brandenburg state (Germany) had showed that the extrinsic development of Dirofilaria repens, the causative agent of canine subcutaneous dirofilariosis, was possible from the years 2001 to 2012. This finding, associated to the movement of infected dogs and their relocation from endemic European areas to Germany makes possible a rapid endemization of the infection and highlight the risk for human health, being D. repens a frequent cause of zoonotic infections.


The nematode Dirofilaria repens is a parasite of the subcutaneous connective tissues of dogs, cats, and some old world carnivores. In dogs, D. repens is regarded as less pathogenic than Dirofilaria immitis, the causative agent of canine heartworm disease (McCall et al. 2008). However, during its patency, D. repens can cause clinical disease with the presence of nodular lesions and eczematous dermatitis in infected animals (Restani et al. 1962; Mandelli and Mantovani 1966; Rocconi et al. 2012).

After mating, the adult female worms release microfilariae (embryos) into the host bloodstream. Mosquitoes act as biological vectors. D. repens DNA has been shown by molecular methods in several mosquito genera, including Culex, Aedes, Anopheles, Armiger, and Mansonides (Favia et al. 1996; Cancrini et al. 2003; Lee et al. 2004; Cancrini et al. 2006). During the blood meal, microfilariae are taken up and migrate through the gut to the Malpighian tubules where they molt to infective stage (L3 larvae). The infective larvae leave the Malpighian tubules and migrate into the lower lip (labium) of the piercing proboscis. During the following blood meal, infected mosquitoes release infective larvae onto the skin of the host. Protected from drying by a droplet of haemolymph of the vector, larvae remain on the skin until actively penetrating through the microwound caused by the vector (McCall et al. 2008). In the final host, the larva develops in the subcutaneous connective tissue mainly on the trunk, extremities, and the head up to the adult stage (Webber and Hawking 1955). The prepatent period lasts about 27–34 weeks, and patency from 5 to 7 years (Weber and Hawking 1955; Anderson 2000).

In addition to the animal hosts, humans can be accidentally infected by D. repens and until the end of the twentieth century; the distribution of D. repens in Europe was mainly located in the Mediterranean area with foci in Italy, France, Greece, and Macedonia (Muro et al. 1999; Pampiglione et al. 2000; Dyachenko and Daugschies 2012). Outside Europe, D. repens is endemic in North Africa, Middle East, and parts of Southeast Asia (Pampiglione et al. 2000). Case reports of cutaneous dirofilariasis in dogs as well as in humans living in north Europe have usually been limited to imported “travel disease” (Jelinek et al. 1996; Kleiter et al. 2001; Keller et al. 2007).

In the last decade, autochtonous cases of human and canine infection have been found with increasing frequency in northeastern Europe (Svobodova et al. 2006; Babal et al. 2008; Auer and Suzani 2008; Szénási et al. 2008; Zarnowska-Prymek et al. 2008; Demiaszkiewicz et al. 2009; Mănescu et al. 2009; Kartashev et al. 2011). Overall, more than 1,000 human infections have been reported (Pampiglione and Rivasi 2000; Genchi et al. 2011; Simón et al. 2012). In most cases, the parasite was localized in subcutaneous nodules or in peri-, intra, or retroocular locations, although serious conditions mimicking cancer, tuberculosis, and psychoses have been reported (Pampiglione et al. 2000; Révész et al. 2008; Fleck et al. 2009; Fodor et al. 2009; Perret-Court et al. 2009; Poppert et al. 2009; Guzeeva et al. 2012). Apart from subconjunctival localization where the parasite can be easily observed and removed and the molecular analysis of infected tissues, other diagnostic tools are not fully reliable. The examination of histological sections of the infected tissues is not easy (Pampiglione et al. 2009) and serology is not specific (Genchi et al. 2011). As a consequence, cases of misdiagnosis and incorrect treatment are not infrequent.

Concerning the spread of canine infections towards previously non-endemic areas, of particular interest are 26 autochtonous cases. Five cases are from Karlsruhe/Baden-Wuerttemberg (Hermosilla et al. 2006; Pantchev et al. 2009a, b, 2011; Kershaw et al. 2009); 16 cases are from the Havelland, district in the state of Brandenburg near Berlin (Sassnau et al. 2009; Sassnau et al. 2012); 1 case is from The Netherlands (Overgaauw and van Dijk 2009); and 4 cases are from Austria (Duscher et al. 2009; Löwenstein and Spalling2009).

Along with humidity, temperature is one of the most important environmental factors that regulate larval development of D. repens in mosquitoes. Temperature dictates the time requirement for development of microfilariae to infective larvae. It has been shown that development takes 16–20 days at 22 °C, 10–11 days at 26 °C, and 8–13 days at 28–30 °C and there is a threshold of about 14 °C below which development does not proceed (Webber and Hawking 1955; Coluzzi 1964; Bain 1978; Fortin and Slocombe 1981; Cancrini et al. 1988; Genchi et al. 2009).

To describe the climate-dependent development of the extrinsic incubation period, Fortin and Slocombe (1981) introduced the heartworm development unit (HDU) for the close relative D. immitis. Development unit (DU) is defined as the difference between the average daily temperature, providing at least 15 °C, and the limit of 14 °C, the threshold below which the development will not proceed. The total environmental heat required for development may be expressed in terms of degree days in excess of this threshold. An amount of at least 130 DUs in 30 consecutive days is defined as the minimum for one extrinsic development. For D. repens, the development time of microfilariae to the infective stage at the different temperatures are quite similar: 8–13 days at 28–30 °C, 10–11 days at 26 °C, and 16–20 days at 22 °C (in Aedes aegypti, Aedes caspius, Aedes detritus, Aedes vexans, Anonopheles claviger, Anonopheles maculipennis, and Culex pipiens; Webber and Hawking 1955; Coluzzi 1964; Bain 1978; Cancrini et al. 1988). In Aedes albopictus, the development from microfilaria stage to infective larvae takes 14–18 days at 26 °C for D. immitis and 16–18 days for D. repens (Cancrini et al. 1995). Then, the model was applied to forecast D. repens spreading throughout Europe (Genchi et al. 2009).

Global climate change, with the consequence of an increasing heating of air, water, and soil, has been recognized since the publications of the IPCC in 2007 and the German Weather Service in 2012 (DWD 2012). This raises the question whether climate change is responsible for local changes making possible the extrinsic incubation of the parasite, with the risk of a possible endemisation in previously infection-free regions.

Materials and methods

To examine to what extent climate changes have an influence on the extrinsic incubation of D. repens in Germany and if it would forecast a spreading of the infection, data from the German Weather Service (DWD 2012) were evaluated.

The records of the average daily temperatures were obtained for the stations of Neuruppin [52.5° N, 12.5° E] (1961–2012), Potsdam [52.2° N, 13.0° E] (1960–2012), Angermünde [53.0° N, 13.6° E] (1960–2012) and Berge [52.4° N, 12.5° E] (1991–2012) in the state of Brandenburg (North Germany) and the weather station of Karlsruhe [49.0° N, 8.2° E] (1961–2012) in the state of Baden-Wuerttemberg (South Germany). Daily mean temperatures were used and the larval development unit (LDU) for D. repens was assessed as follows: at least 130 DU are necessary for one extrinsic development of microfilariae to infective larvae (Fortin and Slocombe 1981; Genchi et al. 2009, 2011). Because it is assumed that infected mosquitoes have a maximum life expectancy of 30 days (Slocombe et al. 1989), the 130 DU has to be achieved within 30 days (hereafter referred as 130 LDU/30).

The mean daily temperatures throughout May 1 and October 15, assumed as the potential local mosquito activity season, were stratified into three parts, from 1961 to 1970, 1982 to 1991, and from 2003 to 2012. Furthermore, the linear regression of the sum of day for each year with >130 LDU/30 in the window between May 1 and October 15 has been calculated. Data was generated with MS Excel (linear trend).


Mean temperatures

The analysis of the mean daily temperatures showed a rising trend during the years 1960–2012 for all the Brandenburg stations. The same trend was found in Karlsruhe where, however, the mean temperatures were constantly higher by about 1 °C in comparison with the other Brandenburg stations (Fig. 1).
Fig. 1

Daily mean air temperature (time window, 01 May to 15 October; years, 1960–2012)

The comparison of daily temperatures from 1961 to 1970, 1982 to 1992, and from 2003 to 2012 (time window, May 1 to October 15) for Brandenburg stations showed a rise from 14.9 to 15.9, 15.4 to 16.3, and 15.1 to 16.0 °C, respectively, with a mean increase of 1 °C. In the same periods, the increase of +1.2 °C (from 16.6 to 17.8 °C) was observed for Karlsruhe station (Fig. 2).
Fig. 2

Changes in mean air temperature (time window, 01 May to 15 October; years 1961–1970, 1982–1991, and 2003–2012)

Vector competence based on the extrinsic incubation period as a function of mean temperatures

The number of days with at least 130 LDU/30 in the interval 1961–2012 showed an increasing trend. For Brandenburg stations, the graphs of the 130 LDU/30 were close together. The requirement of not less than 130 LDU in 30 days was not fulfilled in 12 of 52 observed years in Neuruppin as a representative for the state of Brandenburg. In contrast to Brandenburg, no year with less than 130 LDU/30 was found in Karlsruhe (Fig. 3).
Fig. 3

Number of days per year with at least 130 LDU/30


Traveling and cross-border movement of pets throughout Europe has been greatly facilitated as a consequence of European harmonization rules and, compared with the past years, it is currently very common (Trotz-Williams and Trees 2003). Vaccination against rabies is required for pets, just as marking with a standard transponder and an official EU passport to identify pets vaccinated against rabies.

Unlike livestock animals, there are no transport restrictions or monitoring of cross-border movement in pets traveling into or from vector-born disease-endemic areas increasing the risk of the spreading of such infections (Fig. 4). Although the number of dogs traveling to southern Europe is unknown, the study of Hirsch and Pantchev (2008) showed that vector borne diseases are quite frequent in traveling dogs. Of 5,483 referred dogs for suspicion of “traditional travel sickness” in the years 2005 and 2006, the prevalence of leishmaniosis was about 18 %, about 13 % for ehrlichiosis, about 2.4 % for babesiosis, and 1.2 % of dogs were positive for D. immitis-circulating antigen (Hirsch and Pantchev 2008). Therefore, in one laboratory only, D. immitis infections were found within 2 years in 60 dogs. In a subsequent investigation (Pantchev et al. 2011), 8,545 samples of imported and traveling dogs were studied. One hundred twenty five dogs were microfilaraemic (1.46 %). Out of these, 73 Knott-positive samples were examined by PCR. In 35 cases, D. repens DNA could be detected. D. immitis was seen in 15 cases; and in 6 cases, a co-infection of D. repens and D. immitis was diagnosed. Similar results are reported from Naucke et al. (2011). In this retrospective study (2004–2009), 4,681 blood samples of pet dogs imported or traveled from Southern and Eastern Europe were examined. Circulating microfilariae were found in 372 blood samples (7.7 %) by the modified Knott test.
Fig. 4

Risk paths: possible routes for introduction of Dirofilaria infections

Among imported or relocated dogs from Southern and Eastern Europe to Central and Northern European countries, it is not uncommon to find animals of unknown origin. For these animals, frequently the costs do not always allow a reliable and comprehensive diagnostic evaluation to rule out the presence of imported infectious diseases. Dogs are often relocated by animal protection organizations from southern shelters and “killing stations” to Northern Europe. The risk to import pathogens with imports of infected pets must be considered because of the increased import volume of pets compared to the past.

In the case of canine cutaneous dirofilariosis, it is important to consider that there are three main obstacles to reliable diagnosis:
  • The long prepatent period (27–34 weeks) throughout which the infection cannot be detected.

  • Serological tests (as for D. immitis) do not exist, and definitive diagnosis can be done only throughout the detection of microfilariae in the blood stream; because microfilariae are subject to circadian rhythms, it is not always easy to find them to make a reliable diagnosis. Furthermore, not all the veterinarians and technicians are skilled in performing the Knott test, that is the recommended method for the identification of Dirofilaria species.

  • Clinical signs are not always present and sometime mimic other conditions.

Therefore, along with the long pre- and patent periods, many other factors can allow dogs to act as an active reservoir enhancing the risk for a possible endemisation of canine cutaneous dirofilariosis in previous free areas.

The data for the calculation of LDU/30 in the state of Brandenburg shows a turning point in the year 2001; and thereafter, until 2012, there was no year where the minimum requirement of 130 LDU/30 was not reached. Temperature is one of the major factors that dictate the mosquito vector ability to complete the extrinsic development of D. repens, and this was possible in the past 12 years in the state of Brandenburg as a result of climate change.

In the autumn of 2007, the first evidence of autochthonous canine cutaneous dirofilariasis in five dogs from a sled dog kennel in the Havelland, county of the state of Brandenburg, was found (Sassnau et al. 2009). Four years later, 11 new autochthonous cases were diagnosed in the same kennel in the winter 2011/2012 (Sassnau et al. 2012). The finding of these 16 autochthonous cases, the presence of mosquitoes (Kampen 2012) associated with sufficient temperature for larval extrinsic development and the increasing infection pressure due to traveling pets might made the endemisation of D. repens possible in the state of Brandenburg. Furthermore, infected vectors from endemic areas riding piggyback in trans European or global goods traffic can be seen as a risk factor. To note that, currently, the perception of infection risk is low in the region and the use of preventive strategies is quite unusual. In such a situation, the risk is not limited to dogs, but must include the risk for humans as an emerging zoonosis.

Copyright information

© Springer-Verlag Berlin Heidelberg 2013