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Background    Dirofilaria immitis  and  Dirofilaria repens  are mosquito-borne zoonotic filarioids typically infecting dogs, causing a potentially fatal cardiopulmonary disease and dermatological conditions, respectively. The females are larviparous, releasing the larvae (microfilariae) into the bloodstream, which further develop in mosquito vectors. However, microfilaremia greatly fluctuates during a 24-h period. As the sampling time can greatly influence the accuracy of diagnosis, the aim of the present study was to assess the circadian periodicity of  D. immitis  and  D. repens  in naturally co-infected dogs in an endemic area of Romania and to investigate possible differences of periodicity between these two species.    Methods  Overall, four dogs harbouring natural co-infection with  D. immitis  and  D. repens  were selected and sampled every two hours for two consecutive days: two dogs in July 2014 and two in July 2015. At each sampling time, a 0.7 ml blood sample was taken. Modified Knott’s test was performed on 0.5 ml, and the remaining 0.2 ml were used for DNA extraction and molecular amplification, both in single and duplex PCR reactions. Microfilariae of both species were morphologically identified and counted in each collected sample, microfilaremia was calculated, and fluctuation was charted.    Results  The dynamics of microfilaremia showed similar patterns for both  Dirofilaria  species. In all four dogs,  D. immitis  was present at all sampling times, with several peak values of microfilaremia, of which one was common for all dogs (1 am), while minimum counts occurred between 5 and 9 am. Similarly, for  D. repens , one of the peak values was recorded in all dogs at 1 am, while minimum counts (including zero) occurred at 9 and 11 am. Single species-specific PCR reactions were positive for both  D. immitis  and  D. repens  in all collected samples, while duplex PCR failed to amplify  D. repens  DNA in many cases.    Conclusions  Both  Dirofilaria immitis  and  D. repens  microfilariae are subperiodic, following a similar variation pattern, with peak values of microfilaremia registered during the night in Romania. Duplex PCR fails to identify the infection with  D. repens  in co-infected dogs when the ratio of microfilaremia is in favour of  D. immitis .    Electronic supplementary material  The online version of this article (doi:10.1186/s13071-017-2055-2) contains supplementary material, which is available to authorized users.

Dirofilaria immitis and D. repens show circadian co-periodicity in naturally co-infected dogs