Fig. 1
Gel electrophoresis of PCR products in a 1.5% agarose gel using Dirofilaria immitis-specific primers for ITS2 region (1a) and pan-filarioid primers for ITS2 region (1b). Lane M: MW marker; lane 1, sample 1: D. immitis DNA (dog); lane 2, sample 2: D. immitis DNA (jackal); lane 3, sample 3: D. immitis DNA (fox), lane 4, sample 4: positive control (D. immitis DNA); lane 5, sample 5: negative control.
Fig. 2
Neighbor-joining (NJ) phylogram generated from ITS2 and 5.8S (231 bp) sequences showing the relationships among the Dirofilaria immitis from Bulgaria and GenBank reference conspecific sequences. Onchocerca gibsonii (Og) was used as the outgroup. Numbers next to the branches indicate the bootstrap value calculated from 1,000 replicates. The evolutionary distances were computed using the Tamura 3-parameter method and are in the units of the number of base substitutions per site. Bulgarian isolates involved in the phylogram: D. immitis from domestic dog (DD-B), golden jackal (GJ-B), and red fox (RF-B). Geographical origins of the referenced isolates: POR - Portugal, TUR - Turkey, IRA - Iran, CHN - China, IND - India, TAI - Taiwan, BRA - Brazil, CHI - Chile. Hosts and vectors of the referenced isolates (their labels are in brackets following the country codes next to the phylogram): D - dog, RP - red panda, M - mosquito.
Fig. 3
Geographical distribution of Dirofilaria immitis in dogs (circles), red foxes (squares), golden jackals (rhombs) and wolf (triangle) recorded in the territory of Bulgaria. Orange marks represent D. immitis isolates analysed in the present study.
Table 1
Review on the occurrence of Dirofilaria immitis in dogs and wild-living carnivores in Bulgaria.
| Study year(s) | Host species (n) | Locality | Detection method | Prevalence % (ex/pos) | Reference |
|---|---|---|---|---|---|
| 1991 – 1996 | Dogs (341) | various regions | Knott´s test | 5.3 | Kanev et al. (1996) |
| 1998 | Dogs (20) | Stara Zagora | Necropsy | 10.0 | Gerogieva et al. (1999) |
| Dogs (258) | Knott´s test | 7.4 | |||
| HW antigen test | 7.4 | ||||
| Dogs (40) | Stara Zagora | Necropsy | 12.0 | ||
| 1997 – 1999 | Red foxes (78) | Necropsy | 5.2 | Georgieva et al. (2001) | |
| Golden jackals (45) | Necropsy | 4.4 | |||
| Wolves (18) | Necropsy | 5.5 | |||
| Dogs (487) | Knott´s test | 8.6 | |||
| 2001 – 2006 | Red foxes (113) | various regions | HW Necropsy antigen test | 9.2 3.0 | Kirkova et al. (2007) |
| Golden jackals (56) | Necropsy | 8.9 | |||
| Sofia | Knott´s test | 8.7 | |||
| 2011 | Dogs (240) | HW antigen test | 8.7 | Kostadinov (2012) | |
| Ruse | Knott´s test | 15.7 | |||
| HW antigen test | 15.7 | ||||
| 2012 – 2013 | Red foxes (87) | various regions | Necropsy | 27.6 | Mirchev et al. (2013) |
| 2015 | Dogs (167) | Stara Zagora | HW antigen test | 16.2 | Pantchev et al. (2015) |
| Dogs (27) | Burgas | (8/4) | |||
| Pazardzhik | (5/3) | ||||
| Silistra | (1/1) | ||||
| Sofia | (2/1) | ||||
| Red foxes (113) | Burgas | 26.7 | |||
| Pazardzhik | 44.4 | ||||
| Plovdiv | 40.9 | ||||
| Silistra | (5/2) | ||||
| Sliven | (9/3) | ||||
| 2012 – 2013 | Stara Zagora Haskovo | Necropsy | (8/2) (1/1) | Panayotova-Pencheva et al. (2016) | |
| Golden jackals (56) | Burgas | 29.1 | |||
| Pazardzhik | 58.7 | ||||
| Pleven | 27.8 | ||||
| Plovdiv | 48.1 | ||||
| Silistra | 44.4 | ||||
| Sliven | 18.2 | ||||
| Stara Zagora | (5/3) | ||||
| Haskovo | (4/4) | ||||
| Yambol | 14.3 | ||||
| 2013 – 2014 | Dogs (33) | Sofia | Knott´s test HW antigen test | 15.2 15.2 | Radev et al. (2016) |
| 2012 – 2017 | Dogs (367) | Stara Zagora | HW antigen test | 34.3 | Iliev et al. (2017) |
| 2017 – 2018 | Dogs (80) | Sofia | HW antigen test | 31.3 | Stoyanova et al. (2019) |