Mixed infections of both species are not uncommon and have already been reported in several countries (10, 31–33) with a quite high prevalence e.g. Romania whereas reported in 2015, mixed infections represented 23.91% of all tested samples (34). Interestingly, the predominance of microfilariae of one of the species over the other has also been frequently observed, e.g. 7780 mf/ml and 427 mf/ml of D. immitis and D. repens, respectively (32, 35). A similar scenario has been observed in naturally co-infected dogs in Slovakia (personal communication, prof. Martina Miterpáková). Genchi et al. (36) observed this phenomenon in experimentally infected dogs and suggested that the interaction of both species may disrupt the progress of each other and pointed out that it may impact the further distribution of dirofilariasis in different regions. It is worth mentioning that some of the standard PCR protocols, especially based on duplex or multiplex format, were reported to fail in case of co-infections (35). As the reaction kinetics may be shifted towards only one of the products, the other may not be amplified. Thus, they should be used with caution and species-specific protocols should be promoted.
Another factor that may contribute to the less effective standard morphological/molecular examination is the consideration of the periodicity of the microfilariae in the bloodstream. Although the exact mechanism responsible for the circadian rhythm of the parasites is still puzzling, it is suggested that the dynamics of microfilaremia are related to the host's state and daily habits (activity, body temperature, oxygen/carbon dioxide pressure) and activity of the vectors in the individual region (35). A seasonal occurrence has also been noticed with the highest count of mf in the summer months (37–39). There are only a few researches on the periodicity of microfilaremia in dogs with mixed infections, but available data show that as the patent infection developed, both parasites present a common circadian rhythm. Peripheral microfilaremia was observed at all sampling times, with the highest peak at 1 am, whereas the lowest count of microfilariae was observed between 5–8 am. Interestingly, in two dogs even a zero count of one of the species was observed between 9 and 11 am (35). During phases with the lowest count microfilariae are assumed to be concentrated in the lung vessels (40). These results seem to be in line with our case. It was established that the blood for the differentiation of the infection was collected at 10.30 am, which may explain the low intensity of peripheral microfilaremia (200 mf/ml), which led to no D. immitis visible in the Knott's test.
In addition, a negative result of the antigen test and no sign of adult worms may indicate low worm burden infection (41), which also complies with AHS (American Heartworm Society) (42) and ESDA (European Society of Dirofilariosis and Angiostrongylosis) (43) directives. Followed by AHS, ”a negative antigen test result does not confirm that a dog is free of heartworm infection“ and further examination should be considered. No sign of adult parasites in echocardiography may also be misleading because ”in lightly infected dogs worms are often limited to the peripheral branches of the pulmonary arteries beyond the echocardiographic field of view“. In addition to their common locations, D. immitis worms were reported also in the pericardial sac, foramen oval, peritoneal cavity, brain, vena cava caudalis, and bronchus (44, 45). After all, this method can be crucial in the classification of the severity of the infection. As described, the patient did not show any specific heartworm symptoms, only occasional coughing and general lameness were observed. The infection should be considered “mild” and “Class 1 with low risk of thromboembolic complications”, according to AHS and ESDA directives, respectively (42, 43).
Here we report a first case of a dog naturally co-infected with D. repens and D. immitis in Poland. Although D. repens has been endemic for over 10 years, D. immitis has been reported rarely, previously in 2012 (28) and 2014 (29) based on an antigen test and necropsy, respectively. To our best knowledge, this is only the third case of heartworm infection in Poland. Although the country should not be considered even as pre-endemic yet, we conclude that heartworm disease may be underestimated and more individuals could have been undiagnosed or misdiagnosed. Especially if several factors strongly contribute to the spread of the disease.
The climate changes lead to the introduction of vectors competent for transmission but also expand the time of exposure to infection as more generations of Dirofilaria may occur in a single season. Larval development in mosquitoes strongly depends on the temperature and the fastest development was observed at 28–30°C, 8–9 days for D. immitis, and 9–13 for D. repens, whereas 14°C is a threshold, below which Dirofilaria will not evolve (46). Based on this knowledge the seasonal HW transmission model was formulated and can be used to predict Dirofilaria occurrence. Briefly, the model assumes that 130 HDUs (Heartworm Development Unit; calculated as a threshold temperature (14°C) subtracted from the average daily temperature) in 30 consecutive days are required for larvae to reach the infective stage (L3) (47–49). In recent years, occasional 130 HDUs were observed even in Northern-Europe countries like Sweden, Norway, Finland, and Denmark (2).
Human activities are also crucial for the transmission of the disease to non-endemic regions. Traveling with insufficiently protected and/or not properly examined pets contribute to the appearance of new outbreaks of the disease, that within a short period may lead to the endemisation in new areas. According to Fuerher et al. (2), > 30% of dogs in Poland are kept outside overnight, so they are at increased risk of mosquito bites.
Although the awareness of dirofilariasis increased in recent years and epidemiological data is being updated locally, still there are some research areas where the data is limited. One of them is molecular xenomonitoring which recently was improved in many European countries and several protocols have been described (50–52). In Poland, two studies conducted by Masny et al. (53, 54) estimated the infection rate (EIR) at 1.57% for D. repens in the Central part of Poland and no D. immitis or D. repens DNA was detected in vectors collected from Southern West part of Poland, respectively. Interestingly, xenomonitoring results did not correspond to the prevalence in local carnivorous populations. The authors warn that some of the protocols may lead to non-specific amplification of Setaria tundra DNA. Finally, it is worth noticing that all examined samples were collected between 2010 and 2012 and from only two single locations. Since this time, no more research was conducted in any other region of Poland. Although hundreds or even thousands of mosquitoes need to be examined to detect a single infected individual and xenomonitoring seems to be economically questionable, it provides better insights into the transmission risk and actual epidemiological status.
An important factor that may contribute to the uncontrolled spread of dirofilariasis, but unfortunately still neglected, are infections in free-living carnivores. Both Dirofilaria species infections were detected in foxes, jackals, wolves, or raccoon dogs in many regions of Europe. More recently even beech marten (55) and European badger (56) were considered as new reservoirs of dirofilariasis. Depending on the country, the overall prevalence in wild canids was 5.01% (DI); 3.01% (DI 1.62%, DR 1.39%), 3.92% (DI) in Serbia (57), Romania (58) and Hungary (59), respectively. In a recent study, Alsarraf et al. (60) report that the overall prevalence in Poland reached 3.13% which corresponds with the studies in other European countries. Interestingly, in neighboring Slovakia, D. repens infections were detected in 54.97–57.4% of examined foxes (61, 62). Moreover, only in the Tatry region (the natural borderline between Poland and Slovakia), the prevalence of foxes reached 24.6% (63). In Poland, the overall frequency of vulpine dirofilariasis caused by D. repens was 4.2% and 7% only in Central Poland (60). Instead of the quite low prevalence, we guess that wild canids, especially foxes, may play a big role in the spreading of the disease. In light of patent infections and lack of prevention or treatment practices, foxes provide constant access to microfilariae for new genera of mosquitoes, especially as they present rather nocturnal activity. Living in human-settled areas combined with a nomadic lifestyle and long-distance travels increase the risk of spreading to new regions. Therefore, inter-species transmission between dogs, wild animals, and humans sharing common territory cannot be neglected and should be scrupulously monitored.
As Poland is surrounded by at least two endemics (Slovakia, Ukraine) and one pre-endemic (Germany) country, we suppose that subsequent cases of both imported and autochthonous infections will be reported more frequently in the following years. Interestingly, in a recent study, Alsarraf et al. showed that genetic diversity among D. repens in populations seems to be related to geographical origin (16). Cultivation of this field of study may be a great contribution to the determination of the origin of infections and monitoring potential migration between populations. Unfortunately, there is still no data regarding the D. immitis haplotypes in Europe.