Abstract
BackgroundDirofilaria 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.MethodsOverall, 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.ResultsThe 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.ConclusionsBoth 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.
Highlights
Dirofilaria immitis and Dirofilaria repens are mosquito-borne zoonotic filarioids typically infecting dogs, causing a potentially fatal cardiopulmonary disease and dermatological conditions, respectively
Four of the dogs which had D. immitis and D. repens co-infection were enrolled in the experiment after receiving consent from the owner, as follows: dog 1, a 12 year-old castrated male and dog 2, a 4 year-old male were sampled in July 2014; dog 3, a 5 year-old male and dog 4, a 2 year-old male were sampled in July 2015
The present study further suggests the existence of a relatively stable pattern of the circadian periodicity of D. immitis and D. repens microfilariae in naturally co-infected dogs
Summary
Dirofilaria immitis and Dirofilaria repens are mosquito-borne zoonotic filarioids typically infecting dogs, causing a potentially fatal cardiopulmonary disease and dermatological conditions, respectively. A second species of zoonotic importance is D. repens, which resides in the subcutaneous tissues of the canine host and infection is frequently asymptomatic or associated with a series of dermatological conditions [2, 3]. Both species are regarded as zoonotic agents, but D. repens is more commonly reported in humans throughout Europe [4, 5]. In the case of both species, the number of microfilariae present in the peripheral blood fluctuates during the day, according to several potential factors, including geographic origin, and it is assumed that microfilariae concentrate in the lung vessels during the low peripheral microfilaremia phases [6]
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