Advertisement

Parasites & Vectors

, 12:61 | Cite as

Species diversity, host preference and arbovirus detection of Culicoides (Diptera: Ceratopogonidae) in south-eastern Serbia

  • Ana Vasić
  • Nemanja Zdravković
  • Dragoș Aniță
  • Jovan Bojkovski
  • Mihai Marinov
  • Alexander Mathis
  • Marius Niculaua
  • Elena Luanda Oșlobanu
  • Ivan Pavlović
  • Dušan Petrić
  • Valentin Pflüger
  • Dubravka Pudar
  • Gheorghe Savuţa
  • Predrag Simeunović
  • Eva Veronesi
  • Cornelia SilaghiEmail author
  • the SCOPES AMSAR training group
Open Access
Research

Abstract

Background

Culicoides (Diptera: Ceratopogonidae) is a genus of small biting midges (also known as “no-see ums”) that currently includes 1368 described species. They are proven or suspected vectors for important pathogens affecting animals such as bluetongue virus (BTV) and Schmallenberg virus (SBV). Currently little information is available on the species of Culicoides present in Serbia. Thus, the aim of this study was to examine species diversity, host preference and the presence of BTV and SBV RNA in Culicoides from the Stara Planina Nature Park in south-eastern Serbia.

Results

In total 19,887 individual Culicoides were collected during three nights of trapping at two farm sites and pooled into six groups (Obsoletus group, Pulicaris group, “Others” group and further each group according to the blood-feeding status to freshly engorged and non-engorged). Species identification was done on subsamples of 592 individual Culicoides specimens by morphological and molecular methods (MALDI-TOF mass spectrometry and PCR/sequencing). At least 22 Culicoides species were detected. Four animal species (cow, sheep, goat and common blackbird) as well as humans were identified as hosts of Culicoides biting midges. The screening of 8291 Culicoides specimens in 99 pools for the presence of BTV and SBV RNA by reverse-transcription quantitative PCR were negative.

Conclusions

The biodiversity of Culicoides species in the natural reserve Stara Planina was high with at least 22 species present. The presence of C. imicola Kieffer was not recorded in this area. Culicoides showed opportunistic feeding behaviour as determined by host preference. The absence of SBV and BTV viral RNA correlates with the absence of clinical disease in the field during the time of sampling. These data are the direct outcome of a training programme within the Institutional Partnership Project “AMSAR: Arbovirus monitoring, research and surveillance-capacity building on mosquitoes and biting midges” funded by the programme SCOPES of the Swiss National Science Foundation.

Keywords

Culicoides spp. BTV SBV Host preference Serbia Capacity building Train the trainers concept 

Background

Culicoides (Diptera: Ceratopogonidae) is a genus of small biting midges (also known as “no-see ums”) that currently includes 1368 described species [1] in 32 subgenera [2]. They are important vectors of arboviruses of veterinary (bluetongue virus (BTV) [3, 4], Schmallenberg virus (SBV) [5], African horse sickness virus (AHSV) [3], epizootic haemorrhagic disease virus (EHDV) [6]) and medical importance (Oropouche virus) [7, 8]. Culicoides tend to blood-feed on and breed near domestic livestock and humans [9]. Culicoides-borne virus transmission in Europe is especially important for BTV which causes significant economic losses [10]. Even though Culicoides imicola Kieffer, one of the major BTV and AHSV vectors in Africa, southern Europe and Southeast Asia, seems to increase its distribution northwards [11], the expansion of BTV into Europe has enforced a re-evaluation of the importance of Palaearctic Culicoides species as competent vectors [12]. The role of other than C. imicola species was proven for the first time in Europe in studies from Italy [13, 14]. The predominant Obsoletus complex and Pulicaris complex were implicated in BTV transmission during the outbreak of BTV in northern Europe in 2006 [15]. It was postulated that infected Culicoides individuals were introduced by transport within ship-containers or by transport of live animals from endemic regions in Africa [15, 16]. It is also described that Culicoides introduction can occur via meteorological conditions (such as wind) [17]. In 2011, SBV has been reported for the first time in Europe in cattle from Germany and the Netherlands, causing disease with fever, decreased milk production, diarrhea and malformed newborn animals [5]. It rapidly spread through Europe in 2012 and 2013 [18], and re-emerged in Germany in 2014 with high sequence identity of the isolated virus genome to the first SBV sample implicating possible persistence of virus within the insect vectors [19].

The investigation of species occurrence, diversity, and abundance of the genus Culicoides in south-eastern Europe and the Balkan Peninsula started after the first introduction of BTV into Bulgaria in 1999 revealing the presence of Obsoletus complex specimens (75%) followed by Pulicaris complex (16%) [20]. Subsequent outbreaks of BTV occurred and entomological studies were done in Albania [21], Bosnia and Herzegovina [22], Croatia [23], the former Yugoslav Republic of Macedonia (FYROM), Montenegro and Serbia [21, 24]. Culicoides imicola was not captured or reported in any of the above-mentioned studies.

Since the outbreak of BTV serotype 9 in Serbia in 2002, the country was free of BTV until August 2014 when a new outbreak of BTV serotype 4 occurred [25]. The state monitoring programme in 2015 consisted of insect trapping, identification and detection of viral genome in Culicoides samples [25].

To contribute further to the knowledge on Culicoides in Serbia, the aims of our study were: (i) to identify Culicoides species present in the area of Stara Planina Nature Park (south-east Serbia); (ii) to identify host species for the local Culicoides population by DNA characterization; and (iii) to screen for BTV and SBV RNA in the collected Culicoides specimens.

Methods

AMSAR project concept

The Swiss National Science Foundation provided funding for the SCOPES (Scientific co-operation between eastern Europe and Switzerland) project No. 160429, “Arbovirus Monitoring, Surveillance and Research-capacity building on mosquitoes and biting midges (AMSAR)”. This project was a trilateral institutional partnership aiming at capacity building and spreading knowledge between partner institutions from Switzerland, Romania and Serbia during 2015–2017. The goal of the project was to provide training to young scientists in Romania and Serbia who would be able to continue working in the field of medical and veterinary entomology. The innovative “train the trainers” concept was used for the first time in this field and as a result, knowledge was widely shared and disseminated [Silaghi C. AMSAR: a capacity building project based on the “Train the trainers” concept. ESOVE 2016, 2 –7.10.2016, Lisbon, Portugal]. Thus, the authors of this paper were participants of the project involved in practical field and laboratory investigations.

Study area and description of stables

Stara Planina, located is south-eastern Serbia, is a nature reserve of the Ia protection category, i.e. strictly protected areas set aside to protect biodiversity and also possibly geological/geomorphical features, where human visitation, use and impacts are strictly controlled and limited to ensure protection of the conservation values according to the International Union for Conservation of Nature (IUCN).

The Stara Planina Nature Park is remote mountainous terrain (highest elevation Midzor peak, altitude 2169 m) with a high biodiversity of 1200 plant species (including 115 endemic, 100 strictly protected by State and 50 on the list of endangered species in Europe) and several animal species (116 butterflies, 46 amphibians and reptiles, 26 fish, 203 birds and 30 mammals) [26]. The autochthonous cattle breed “Busa” (approx. n = 100) is kept indoors at location A, in the area of Gornji Krivodol at an altitude of 886 m above sea level (43°6'37"N, 22°57'14"E) while location B is a private farm with two separate animal breeding locations for up to 50 goats indoors and up to 50 sheep outdoors in the village Kamenica at an altitude of 811 m (43°28'28"N, 22°21'21").

Insect collection

Insects were collected overnight in 70% ethanol using Onderstepoort Veterinary Institute (OVI) traps with UV light as source of attraction [27]. At each location, two traps were placed, one inside and the other outside of the stable. At location A, two overnight samplings (4th and 6th July 2016) were done, and at location B there was one night of sampling (5th July 2016). At the time of sampling, the morning temperatures at 6:00 h on 4th, 5th and 6th July 2016 were 14 °C, 16 °C and 16 °C and the maximum daily temperatures at 16:00 h were 26 °C, 29 °C and 32 °C, respectively. There was no atmospheric precipitation, the average relative humidity was 60% and wind velocity was up to 7.5 km/h [28].

Insect identification and sorting

All collected insects were separated into Culicoides specimens and other insects, which were discarded. From each trap, approximately 100 Culicoides individuals were randomly taken for morphological identification to the species level under a stereomicroscope with 10× and 20× magnification using the Interactive IIKC key [29, 30]. From the morphologically identified Culicoides specimens, the ones belonging to a species with an existing MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry) spectrum were species confirmed by MALDI-TOF MS. Most of the remaining morphologically identified Culicoides specimens were identified by PCR followed by sequencing in order to obtain final species identifications. Additionally, some of the Culicoides specimens were tested with both MALDI-TOF MS and sequencing.

All remaining Culicoides specimens from each night of trapping were separated into three groups (Obsoletus group, Pulicaris group and “Others” group) and further into freshly engorged and non-engorged forming a total of six groups. From each of the six groups from the six traps, up to 5 pools with up to 100 individuals each (depending on availability) were formed. The final total was 99 pools with altogether 8291 individual Culicoides (see below).

Identification of Culicoides species using molecular methods

DNA was extracted from the abdomens of the Culicoides with the GeneJET whole blood genomic DNA purification mini kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions for blood with the modification that insects were disrupted in phosphate-buffered saline (PBS) in a Tissue Lyser II (Qiagen, Hombrechtikon, Switzerland) with a 5 mm stainless steel bead at 30 Hz for one minute twice before 20 μl proteinase K was added to a total volume of 200 μl. The incubation at 56 °C was done overnight.

The quality and quantity of the obtained DNA was measured with Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA).

PCR was performed targeting a 585 bp region of the mitochondrial cox1 gene to identify the insect species. The multiplex PCR kit (Qiagen, Hombrechtikon, Switzerland) was used with the following primers: 0.5 μl of 100 μM C1-J-1718 mod (5'-GGA GGA TTT GGA AAT TGA TTTG-3') and 0.5 μl of 100 μM C1-N-2191 mod (5'-GTA AAA TTA AAA TAT AAA CTT CTGG-3') in final reaction volumes of 50 μl. A plasmid containing the target sequence of C. imicola was used as positive control and sterile water as a negative control [31].

The Qiagen MinElute kit (Qiagen, Hombrechtikon, Switzerland) was used to purify amplicons, and sequencing was done at Synergene (Schlieren, Switzerland). Chromatograms were quality checked and edited with Finch TV (finchtv.software.informer.com) and compared against the GenBank database using BLASTn [32] and BOLD [33]. Similarities higher than 97% were considered as a species match.

For MALDI-TOF mass spectrometry, head and thorax of insects were prepared as previously described [34]. Identification of specimens was done on a Mass Spectrometry Axima Confidence machine (Shimadzu-Biotech Corp., Kyoto, Japan) and the spectra were compared to the existing database [34].

Blood-meal identification

A two-step approach was used for host identification in blood-fed individuals. First, all samples were screened with a multiplex PCR approach based on cytb polymorphisms using 0.5 μl of 100 μM of primers UNIV2 (5'-TGA GGA CAA ATA TCA TTY TGA GGR GC-3'), CAPRA (5'-TTA GAA CAA GAA TTA GTA GCA TGG CG-3'), OVIS (5'-GGC GTG AAT AGT ACT AGT AGC ATG AGG ATG A-3') and BOVIS (5'-TTA GAT GTC CTT AAT GGT ATA GTA G-3') [35] in final volumes of 50 μl to detect blood meals on cow, goat and sheep. In the case of negative samples, these were tested with a generic PCR targeting the cytb gene (primers Cytbf 5'-GAG GMC AAA TAT CAT TCT GAG G-3' and Cytbr 5'-TAG GGC VAG GAC TCC TCC TAG T-3') followed by sequencing with the sequencing primer 5'-GGA CTC CTC CTA GTT TGT T5G G-3' as previously described [36]. Products were purified, sequenced and analysed as described above.

Pathogen detection by molecular methods

In order to determine RNA presence of SBV and BTV, RNA extraction was done from 99 pools of up to 100 Culicoides specimens the Gene JET RNA Purification kit (Thermo Fisher Scientific, Waltham, USA) following the manufacturer’s instructions. Insect tissue was disrupted in 300 μl lysis buffer with 3 mm bead in the Tissue Lyser II for 20–40 s at 30 Hz. Quality and quantity of RNA was measured with the Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA). No sample was excluded because of low RNA quantity or quality (quantity ranged from 1.03 ng/μl to 665.75 ng/μl; quality (A260/280 ratio) from 1.62 to 2.45).

For BTV RNA detection, the iTaq universal probes one-step kit (Bio-Rad, Hercules, USA) was used with primers BTV_IVI_F (5'-TGG AYA AAG CRA TGT CAA A-3'), BTV_IVI_R (5'-ACR TCA TCA CGA AAC GCT TC-3'), and the probe BTV_IVI_P (FAM-5'-ARG CTG CAT TCG CAT CGT ACG C-3'-BHQ1) as previously described [37].

For SVB RNA detection, the primers used were SBV-S-382F (5'-TCA GAT TGT CAT GCC CCT TGC-3'), SBV-S-469R (5'-TTC GGC CCC AGG TGC AAA TC-3') and the fluorogenic probe was FAM-5'-TTA AGG GAT GCA CCT GGG CCG ATG GT-3'-BHQ1 [5].

Results

Insect trapping

A total of 19,887 individual Culicoides specimens were collected during three nights of trapping in a total of six traps at two different locations (A and B). The total number of collected individuals per trap/night, their morphological grouping and blood-feeding status are shown in Table 1. Altogether, 6396 specimens (32.2%) were from the Obsoletus group, 1833 (9.2%) from the Pulicaris group, 11,649 (58.6%) from the “Others” group, and 9 damaged specimens (0.04%) could not be attributed to any group because group specific features were missing.
Table 1

Total number of collected Culicoides individuals by group and blood feeding status at two farm sites (A and B) in Stara Planina, Serbia

Location

Day of collection

Trap placement

Total no. of Culicoides collected

Obsoletus group

Pulicaris group

“Others” group

Culicoides spp.a

Total

n (%)

Engorged

n (%)

Non-engorged

n (%)

Engorged

n (%)

Non-engorged

n (%)

Engorged

n (%)

Non-engorged

n (%)

Engorged

n (%)

Non-engorged

n (%)

Engorged

n (%)

Non-engorged

n (%)

A: cattle

04.07.2016

Inside stable

187 (100)

18 (9.63)

169 (90.37)

1 (0.53)

97 (51.87)

1(0.53)

20 (10.70)

16 (8.56)

52 (27.81)

0 (0)

0 (0)

A: cattle

04.07.2016

Outside stable

3394 (100)

869 (25.60)

2525 (74.4)

131 (3.86)

1124 (33.12)

43 (1.27)

263 (7.75)

693 (20.42)

1136 (33.46)

2 (0.06)

2 (0.06)

A: cattle

06.07.2016

Inside stable

774 (100)

101 (13.05)

673 (86.95)

6 (0.78)

69 (8.91)

6 (0.78)

45 (5.81)

89 (11.50)

559 (72.22)

0 (0)

0 (0)

A: cattle

06.07.2016

Outside stable

10,435 (100)

3705 (35.5)

6730 (64.5)

411 (3.94)

1375 (13.18)

113 (1.08)

869 (8.33)

3181 (30.48)

4483 (42.96)

0 (0)

3 (0.03)

Subtotal A

  

14,790 (100)

4693 (31.73)

10,097 (68.27)

549 (3.71)

2665 (18.02)

163 (1.10)

1197 (8.09)

3979 (26.90)

6230 (42.12)

2 (0.02)

5 (0.04)

B: sheep and goats

05.07.2016

Inside stable

2694 (100)

1169 (43.39)

1525 (56.61)

648 (24.05)

861 (31.96)

99 (3.67)

137 (5.09)

421 (15.63)

526 (19.52)

1 (0.04)

1 (0.04)

B: sheep and goats

05.07.2016

Outside stable

2403 (100)

59 (2.45)

2344 (97.55)

26 (1.08)

1647 (68.54)

8 (0.33)

229 (9.53)

25 (1.04)

468 (19.48)

0 (0)

0 (0)

Subtotal B

  

5097 (100)

1228 (24.09)

3869 (75.91)

674 (13.22)

2508 (49.21)

107 (2.10)

366 (7.18)

446 (8.75)

994 (19.5)

1 (0.02)

1 (0.02)

Total

  

19,887 (100)

5921 (29.77)

13,966 (70.23)

1223 (6.15)

5173 (26.01)

270 (1.36)

1563 (7.86)

4425 (22.25)

7224 (36.33)

3 (0.01)

6 (0.03)

aCulicoides spp.: grouping was not possible because group specific features were missing due to samples damage

As shown in Table 1, 14,790 (74.4%) individuals were collected at location A (cattle farm). Higher numbers of Culicoides specimens (n = 13,829; 93.5%) were trapped outside the stables rather than inside (n = 961; 6.5%).

At location B (goat/sheep farm), 5097 (25.6%) individual Culicoides were collected. Interestingly, the number of Culicoides specimens trapped inside the stable (n = 2694; 52.85%) was similar to that sampled outside of the stable (n = 2403; 47.14%).

Overall 5921 (29.8%) individual Culicoides were freshly engorged (4693 at location A and 1228 at location B) and 13,966 (70.2%) were non-engorged (10,097 at location A and 3869 at location B).

Individual insect identification

Insect identification to the species level by morphology, MALDI-TOF MS and sequencing was completed on 592 Culicoides specimens, 393 from location A and 199 from location B (Table 2).
Table 2

Number of identified Culicoides to species level by MALDI-TOF mass spectrometry and PCR/sequencing per location in Stara Planina, Serbia. Some individuals identified by more than one method

Species

No. of identified individuals

Location A

Location B

Culicoides group

No. of identifications by MALDI-TOF

No. of identifications by sequencing

C. achrayi

2

2

0

“Others”

0

2

C. circumscriptus

3

3

0

“Others”

1

2

C. clastrieri

1

1

0

“Others”

0

1

C. deltus

5

2

3

“Others”

3

2

C. dewulfi

1

1

0

Obsoletus

0

1

C. fagineus

1

0

1

“Others”

0

1

C. fascipennis

71

49

22

“Others”

16

64

C. festivipennis

11

11

0

“Others”

7

4

C. furcillatus

11

3

8

“Others”

1

10

C. kibunensis

1

1

0

“Others”

0

1

C. lupicaris

31

21

10

Pulicaris

22

14

C. newsteadi

7

7

0

Pulicaris

1

6

C. obsoletus/scoticus

7

2

5

Obsoletus

0

0

C. obsoletus

69

39

30

Obsoletus

61

17

C. pallidicornis

4

4

0

“Others”

2

4

C. parotti

2

2

0

“Others”

0

2

C. picturatus

19

18

1

“Others”

0

19

C. pulicaris

3

2

1

Pulicaris

3

1

C. punctatus

11

11

0

Pulicaris

5

6

C. salinarius

2

2

0

“Others”

0

2

C. scoticus

150

61

89

Obsoletus

142

15

C. simulator

15

12

3

“Others”

0

15

C. subfasciipennis

10

10

0

“Others”

0

10

Culicoides spp.a

155

129

26

 

0

10

Total

592

393

199

 

264

209

aIdentification to Culicoides spp. done as a combination of morphological identification result, indefinite sequencing results (poor identity or ambivalent result) and MALDI- TOF mass spectrometry results. Specimens defined as C. obsoletus/scoticus were identified by morphological identification, while C.obsoletus and C. scoticus were confirmed by molecular methods which enables species identification

All 592 Culicoides specimens were morphologically identified and all specimens without a confirmed identification by molecular methods (PCR/sequencing and/or MALDI-TOF MS) were classified as Culicoides spp. (155 in total). Using MALDI-TOF MS technique we identified up to 264 specimens at the species level, whereas 209 specimens were identified by sequencing. Some specimens were identified with more than one method.

Altogether 22 Culicoides species were recorded in the examined locations. As shown in Table 2, C. deltus Edwards, C. fasciipennis Staeger, C. furcillatus Callot, Kremer & Paradis, C. lupicaris Downes & Kettle, C. obsoletus, C. picturatus Kremer & Deduit, C. pulicaris Linnaeus, C. scoticus and C. simulator Edwards, were present on both the cow farm (location A) and the sheep/goat farm (location B). Culicoides achrayi Kettle & Lawson, C. circumscriptus Kieffer, C. clastieri Callot, Kremer & Deduit, C. dewulfi Goetghebuer, C. festivipennis Kieffer, C. kibunensis Tokunaga, C. newsteadi Austen, C. pallidicornis Kieffer, C. parotti Kieffer, C. punctatus Meigen, C. salinarius Kieffer and C. subfasciipennis Kieffer were present only at location A, while C. fagineus Edwards was present only at location B.

Blood-meal identification

Out of 152 sequences of Culicoides samples for host preference analysis, 103 originated from location A while 49 were from location B. The blood hosts were identified in 14 Culicoides species and in 43 specimens identified as Culicoides spp. Alltogether 5 blood host species were confirmed. Overall, cows (Bos taurus) (n = 96), goats (Capra hircus) (n = 47), humans (Homo sapiens) (n = 6), sheep (Ovis aries) (n = 2) and common blackbirds (Turdus merula) (n=1) were identified as hosts (Table 3). Cows (n = 96) and blackbird (n = 1) were identified only at location A; sheep (n = 2) were identified only in location B, while goats were identified as hosts at location A (n = 2) and location B (n = 45). Human hosts were also found at location A (n = 4) and location B (n = 2).
Table 3

Identified blood hosts per Culicoides species and location

Culicoides species

Cow

Goat

Sheep

Blackbird

Human

C. achrayi

1

C. circumscriptus

C. fascipennis

31

7

1

C. festivipennis

1

C. furcillatus

2

1

C. lupicaris

4

2

C. newsteadi

1

C. obsoletus

5

7

1

C. obsoletus/scoticus

0

2

1

C. pallidicornis

2

C. picturatus

7

1

C. punctatus

4

C. salinarius

1

C. scoticus

1

18

2

C. subfasciipennis

5

1

Culicoides spp.a

33

9

1

Total

96

47

2

1

6

Total per location A

96

2

1

4

Total per location B

0

45

2

2

aIndividual insects which were morphologically confirmed to be Culicoides, but species identification could not be concluded by MALDI-TOF mass spectrometry and/or PCR/sequencing

Pathogen detection

A total of 8291 Culicoides were screened for BTV and SBV RNA presence. The number of individual Culicoides per group and feeding status is shown in Table 4. Out of 99 pools, of six different groups in total (Obsoletus group, n = 36; Pulicaris group, n = 20; “Others” group, n = 43) of which engorged (Obsoletus group, 14 pools; Pulicaris group, 5 pools; “Others” group, 17 pools) and non-engorged (Obsoletus group, 22 pools; Pulicaris group, 15 pools; “Others” group, 26 pools). Among the tested samples there were no positive results for BTV and SBV viral RNA by RT-qPCR (Table 4).
Table 4

Culicoides used for detection of BTV and SBV RNA in pools

Location, date and trap position

Total

N (n)

Obsoletus group

Pulicaris group

“Others” group

SBV RT-qPCR

BTV RT-qPCR

Engorged

N (n)

Non-engorged

N (n)

Engorged

N (n)

Non-engorged

N (n)

Engorged

N (n)

Non-engorged

N (n)

A: 04.07.2016 inside

5 (86)

1 (1)

1 (40)

0 (0)

1 (12)

1 (6)

1 (27)

Negative

Negative

A: 04.07.2016 outside

21 (1887)

2 (105)

5 (500)

1 (38)

3 (244)

5 (500)

5 (500)

Negative

Negative

A: 06.07.2016 inside

11 (665)

1 (5)

1 (54)

1 (3)

1 (39)

1 (56)

6 (508)

Negative

Negative

A: 06.07.2016 outside

25 (2403)

4 (306)

5 (500)

1 (97)

5 (500)

5 (500)

5 (500)

Negative

Negative

B: 05.07.2016 inside

22 (1134)

5 (500)

5 (500)

1 (96)

2 (132)

4 (399)

5 (489)

Negative

Negative

B: 05.07.2016 outside

15 (2116)

1 (13)

5 (500)

1 (4)

3 (219)

1 (7)

4 (391)

Negative

Negative

Total

99 (8291)

14 (930)

22 (2094)

5 (238)

15 (1146)

17 (1468)

26 (2415)

Negative

Negative

Abbreviations: N Total number of pools, n number of individual Culicoides

Discussion

In the past decade the Balkan Peninsula has encountered several outbreaks of BTV [24], and SBV activity was reported in 2013 [38]. Following these events, several studies were conducted in Bulgaria and Croatia to determine the abundance and species composition of Culicoides vectors [20, 23]. Even though there is a Culicoides monitoring programme [25] in Serbia, the data on abundance and species composition are scarce. The results of the Serbian Culicoides monitoring programme in 2015/2016 revealed the presence of Culicoides spp. from spring (April) to late autumn (December) [39]. Results of the present study showed that on both locations at Stara Planina Nature Park, Culicoides were present in large numbers, which is in correlation with the results of Culicoides collections in the neighbouring Bulgaria [20, 40]. The variation in number of collected Culicoides between two sampling dates at location A might have influence on the likelihood of collection of SBV- and BTV-positive specimens. This variation in numbers might have occurred due to altered microclimatic conditions between two sampling nights. Among the collected Culicoides, the morphological group “Others” was the most abundant (n = 11,649), followed by the Obsoletus group (n = 6396) and Pulicaris group (n = 1833). The highest number of individuals belonging to the Obsoletus group was recorded in Bosnia and Herzegovina [22], Croatia [23] and Romania [41]; however, this was not the case in our study. Since our study was completed in geographically close locations, we cannot generalize the group composition of Culicoides to larger territories in Serbia. Furthermore, in a study from Switzerland the group composition changed with different altitudes, revealing a higher abundance of Culicoides species that belong to “Others” at high altitudes [42].

We identified 22 Culicoides species. To our knowledge, there are no published data from neighboring countries (Greece, Croatia, and Bosnia and Herzegovina) on the presence of C. clastieri, C. deltus, C. lupicaris, C. picturatus, C. salinarius, C. simulator and C. subfascipennis, and C. clastrieri, C. lupicaris and C. picturatus were not recorded in Bulgaria. High Culicoides species diversity was recorded in Bulgaria with differences observed in species composition between two studies. This is probably due to habitat characteristics or availability of preferred blood hosts [20, 40]. In Croatia [23], the presence of C. circumscriptus, C. fasciipennis, C. fagineus, C. haranti Rioux, Descous & Pech, C. obsoletus, C. paolae Boorman, C. pulicaris, C. punctatus, C. scoticus and C. seavanicus Kieffer was determined and these results partially correlate with our findings. In Greece, 39 Culicoides species were found, and the findings differed according to the geographical area of the country [43]. Among these species, only 15 were found in our study, and 24 species detected in a study from Greece were not present in the sampled locations of Stara Planina Nature Park. Interestingly, C. impunctatus Goetghebuer was found in Bulgaria and Greece, but not in our study, possibly due to the sampling period. Our results did not show the presence of C. imicola, the main BTV vector in the Mediterranean basin, which is in agreement with findings in Albania [21], Bosnia and Herzegovina [22] and Bulgaria [20]. This implies the role of species other than C. imicola in the transmission cycle of BTV in the investigated locations in Serbia. Another study in northern Europe also identified that not a single specimen of C. imicola was detected amongst 100,000 Culicoides collected in France, Belgium and Luxemburg [44].

To the best of our knowledge, we describe the first data of host analysis for Culicoides in Serbia. Our results suggest for most species identified in this study have a mammophilic feeding behaviour, but interestingly, blood of a bird was recorded in one of the samples. Other domestic animals such as dogs and cats were also present at the sampling locations. The choice of animal host depends on intrinsic host preference of the insect species and host availability [45]. Opportunistic feeding tendencies in mammophilic biting midges for animals nearby were previously reported [35], which is also the observation in our study (location A - cow, location B - sheep and goat, as well as human hosts at both locations).

None of the tested Culicoides pools was positive for RNA of BTV and SBV. This finding is in relation with the absence of clinical cases in 2016 in the area of Stara Planina Nature Park (www.oie.int).

All results, discussions and conclusions presented here are a direct outcome from the capacity building project AMSAR based on the multiplying effect by “training-the-trainers” concept which has thus proven to be a successful scheme of capacity building in vector entomology.

Conclusions

The biodiversity of Culicoides species in Stara Planina Nature Park is high and at least 22 species are present. Culicoides imicola was not recorded in this area. Culicoides showed opportunistic feeding behaviour as determined by host preference. The absence of SBV and BTV viral RNA correlates with the absence of clinical disease in the field during the time of sampling.

Notes

Acknowledgments

We thank Jeannine Hauri, Anca Paslaru, Dr Felix Grimm (Institute of Parasitology, VetSuisse Faculty, Zurich, Switzerland) for technical assistance and training. We are also very thankful to Dr Samuel Furrer (Zoo Zürich, Switzerland) and Dr Andrea Vögtlin (Institute for Virology und Immunology (IVI), Bern, Switzerland) for scientific cooperation and to Dr Jolene Karlson (FLI, Greifswald, Germany) for thorough revision of the English language.

SCOPES AMSAR training group: 4Adriana Aniță, 4Ioana Alexandra Anton, 4Andrei Cimpan, 4Lavinia Ciucă, 4Luciana Crivei, 1Aleksandar Cojkić, 1Darko Davitkov, 1Vladimir Drašković, 1Bojan Gajić, 1Uroš Glavinić, 4Maria-Larisa Ivănescu, 8Mihaela Kavran, 4Andrei-Cristian Lupu, 4Raluca Mîndru, 4Daniela Porea, 1Radiša Prodanović, 3Oliver Radanović, 2, 4Cristian Răileanu, 5Stefan Raileanu, 1Marko Ristanić, 4Constantin Roman, 1Ljubodrag Stanišić, 8Slavica Vaselek, 1Miloje Đurić.

Funding

The project was financed by the SCOPES programme of the Swiss National Science Foundation (SNFS) and the Swiss Agency for Development and Cooperation (SDC).

Availability of data and materials

Data supporting the conclusions of this article are provided within the article. The datasets used and/or analysed during this study are available from the corresponding author upon reasonable request.

Authors’ contributions

CS designed and coordinated the project. JB, IP, GS and MM coordinated the implementation of the project in Serbia and Romania. CS and EV were trainers in Switzerland. AV, PS, ELO and DA were trainee-trainers in Switzerland. AV, NZ, DA, JB, MM, ELO, IP, DPu, PS, EV and CS were trainers in the training schools and participated in field trapping and laboratory examinations. DPe and DPu contributed to morphological identification of collected Culicoides. AV and NZ performed the RNA extractions and real-time PCRs. MN and VP performed MALDI-TOF data analysis. SCOPES AMSAR training group participated in all practical aspects of the study. AV and CS drafted the manuscript. All authors critically revised the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.
    Borkent A. Numbers of extant and fossil species of Ceratopogonidae. 2016. https://wwv.inhs.illinois.edu/files/4014/6785/5847/WorldCatalogtaxa.pdf. Accessed 2 Nov 2018.Google Scholar
  2. 2.
    Augot D, Mathieu B, Hadj-Henni L, Barriel V, Zapata Mena S, Smolis S, et al. Molecular phylogeny of 42 species of Culicoides (Diptera, Ceratopogonidae) from three continents. Parasite. 2017;24:23.CrossRefGoogle Scholar
  3. 3.
    Carpenter S, Veronesi E, Mullens B, Venter G. Vector competence of Culicoides for arboviruses: three major periods of research, their influence on current studies and future directions. Rev Sci Tech. 2015;34:97–112.CrossRefGoogle Scholar
  4. 4.
    Mellor PS. Replication of arboviruses in insect vectors. J Comp Pathol. 2000;123:231–47.CrossRefGoogle Scholar
  5. 5.
    Hoffmann B, Scheuch M, Höper D, Jungblut R, Holsteg M, Schirrmeier H, et al. Novel orthobunyavirus in cattle, Europe, 2011. Emerg Infect Dis. 2012;18:469–72.CrossRefGoogle Scholar
  6. 6.
    Maclachlan NJ, Zientara S, Savini G, Daniels PW. Epizootic haemorrhagic disease. Rev Sci Tech. 2015;34:341–51.CrossRefGoogle Scholar
  7. 7.
    Romero-Alvarez D, Escobar LE. Oropuche fever, and emergent disease from the Americas. Microbes Infect. 2018;20:135–46.CrossRefGoogle Scholar
  8. 8.
    Roberts DR, Hoch AL, Dixon KE, Llewellyn CH. Oropouche virus. III. Entomological observations from three epidemics in Para, Brazil, 1975. Am J Trop Med Hyg. 1981;30:165–71.CrossRefGoogle Scholar
  9. 9.
    Tabachnik WJ. Culicoides and the global epidemiology of bluetongue virus infection. Vet Ital. 2004;40:145–50.Google Scholar
  10. 10.
    Wilson A, Mellor P. Bluetongue in Europe: vectors, epidemiology and climate change. Parasitol Res. 2008;103:69–77.CrossRefGoogle Scholar
  11. 11.
    Guichard S, Guis H, Tran A, Garros C, Balenghien T, Kriticos DJ. Worldwide niche and future potential distribution of Culicoides imicola, a major vector of bluetongue and African horse sickness viruses. PLoS One. 2014;9:e112491.CrossRefGoogle Scholar
  12. 12.
    Wilson A, Mellor P. Bluetongue in Europe: past, present and future. Philos Trans R Soc Lond B Biol Sci. 2009;364:2669–81.CrossRefGoogle Scholar
  13. 13.
    Caracappa S, Torina A, Guercio A, Vitale F, Calabro A, Purpari G, et al. Identification of a novel bluetongue virus vector species of Culicoides in Sicily. Vet Rec. 2003;153:71–4.CrossRefGoogle Scholar
  14. 14.
    De Liberato C, Scavia G, Lorenzetti R, Scaramozzino P, Amaddeo D, Cardeti G, et al. Identification of Culicoides obsoletus (Diptera: Ceratopogonidae) as a vector of bluetongue virus in central Italy. Vet Rec. 2005;156:301–4.CrossRefGoogle Scholar
  15. 15.
    Carpenter S, Wilson A, Mellor PS. Culicoides and the emergence of bluetongue virus in northern Europe. Trends Microbiol. 2009;17:172–8.CrossRefGoogle Scholar
  16. 16.
    Mehlhorn H, Walldorf V, Klimpel S, Schmahl G. Outbreak of bluetongue disease (BTD) in Germany and the danger for Europe. Parasitol Res. 2008;103(Suppl. 1):S79–86.CrossRefGoogle Scholar
  17. 17.
    Jacquet S, Huber K, Pages N, Talavera S, Burgin LE, Carpenter S, et al. Range expansion of the bluetongue vector, Culicoides imicola, in continental France likely due to rare wind-transport events. Sci Rep. 2016;6:27247.CrossRefGoogle Scholar
  18. 18.
    Afonso A, Abrahantes JC, Conraths F, Veldhuis A, Elbers A, Roberts H, et al. The Schmallenberg virus epidemic in Europe - 2011–2013. Prev Vet Med. 2014;116:391–403.CrossRefGoogle Scholar
  19. 19.
    Wernike K, Hoffmann B, Conraths FJ, Beer M. Schmalleberg virus recurrence, Germany, 2014. Emerg Infect Dis. 2015;21:1202–4.CrossRefGoogle Scholar
  20. 20.
    Purse BV, Nedelchev N, Georgiev G, Veleva E, Boorman J, Denison E, et al. Spatial and temporal distribution of bluetongue and its Culicoides vectors in Bulgaria. Med Vet Entomol. 2006;20:335–44.CrossRefGoogle Scholar
  21. 21.
    Lika A, Mersini K, Crilly J. Note on the distribution and abundance of Obsoletus Complex and Pulicaris Complex (Diptera: Ceratopogonidae) in southern Albania. XVIII International Congress of Mediterranean Federation of Health and Production of Ruminants. Perugia: FeMeSPRum-Mediterranean Federation of Health and Production of Ruminants; 2009.Google Scholar
  22. 22.
    Omeragic J, Vejzagic N, Zuko A, Jazic A. Culicoides obsoletus (Diptera: Ceratopogonidae) in Bosnia and Herzegovina-first report. Parasitol Res. 2009;105:563–5.CrossRefGoogle Scholar
  23. 23.
    Listes E, Bosnic M, Lojkic M, Cac Z, Cvetnic Z, Madic J, et al. Serological evidence of bluetongue and a preliminary entomological study in southern Croatia. Vet Ital. 2004;40:221–5.PubMedGoogle Scholar
  24. 24.
    Djuricic B, Nedic D, Lausevic D, Pavlovic M. The epizootiological occurence of bluetongue in the central Balkans. Vet Ital. 2004;40:105–7.PubMedGoogle Scholar
  25. 25.
    Maksimovic Zoric J, Milicevic V, Veljovic L, Pavlovic I, Radosavljevic V, Valcic M, et al. Bluetongue disease-epizootiology situation in Serbia in 2015, diagnosis and differential diagnosis. Arhiv Vet Med. 2016;9:13–22.Google Scholar
  26. 26.
    Zavod za zastitu prirode Srbije. Park prirode Stara Planina. Pirot: Institute for nature conservation of Serbia; 2016. www.zzps.rs Google Scholar
  27. 27.
    Venter GJ, Meiswinkel R. The virtual absence of Culicoides imicola (Diptera: Ceratopogonidae) in a light-trap survey of the colder, high-lying area of the eastern Orange Free State, South Africa, and implications for the transmission of arboviruses. Onderstepoort J Vet Res. 1994;61:327–40.PubMedGoogle Scholar
  28. 28.
  29. 29.
    Mathieu B, Cêtre-Sossah C, Garros C, Chavernac D, Balenghien T, Carpenter S, et al. Development and validation of IIKC: an interactive identification key for Culicoides (Diptera: Ceratopogonidae) females from the Western Palaearctic region. Parasit Vectors. 2012;5:137.CrossRefGoogle Scholar
  30. 30.
    Mathieu B, Cêtre-Sossah C, Garros C, Chavernac D, Balenghien T, Vignes-Lebbe R, et al. IIKC: An Interactive Identification Key for female Culicoides (Diptera: Ceratopogonidae) from the West Palearctic region. In: Nimis PL, Vignes R, editors. Proceedings of the International congress Tools for Identifying Biodiversity: Progress and Problems. 20–22 September 2010. Paris, Trieste: EUT Edizioni Università di Trieste; 2010. p. 201–5.Google Scholar
  31. 31.
    Wenk CE, Kaufmann C, Schaffner F, Mathis A. Molecular characterization of Swiss Ceratopogonidae (Diptera) and evaluation of real-time PCR assays for the identification of Culicoides biting midges. Vet Parasitol. 2012;184:258–66.CrossRefGoogle Scholar
  32. 32.
    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.CrossRefGoogle Scholar
  33. 33.
    Ratnasingham S, Hebert PD. BOLD: The Barcode of Life Data System (http://www.barcodinglife.org). Mol Ecol Notes. 2007;7:355–64.CrossRefGoogle Scholar
  34. 34.
    Kaufmann C, Schaffner F, Ziegler D, Pfluger V, Mathis A. Identification of field-caught Culicoides biting midges using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Parasitology. 2012;139:248–58.CrossRefGoogle Scholar
  35. 35.
    Garros C, Gardes L, Allene X, Rakotoarivony I, Viennet E, Rossi S, et al. Adaptation of a species-specific multiplex PCR assay for the identification of blood meal source in Culicoides (Ceratopogonidae: Diptera): applications on Palaearctic biting midge species, vectors of Orbiviruses. Infect Genet Evol. 2011;11:1103–10.CrossRefGoogle Scholar
  36. 36.
    Townzen JS, Brower AV, Judd DD. Identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit I and cytochrome b gene sequences. Med Vet Entomol. 2008;22:386–93.CrossRefGoogle Scholar
  37. 37.
    Hofmann MA, Renzullo S, Mader M, Chaignat V, Worwa G, Thuer B. Genetic characterization of Toggenburg orbivirus, a new bluetongue virus, from goats, Switzerland. Emerg Inf Dis. 2008;14:1855–61.CrossRefGoogle Scholar
  38. 38.
    Lievaart-Peterson K, Luttikholt S, Peperkamp K, Van den Brom R. P V. Schmallenberg disease in sheep or goats: past, present and future. Vet Microbiol. 2015;181:147–53.CrossRefGoogle Scholar
  39. 39.
    Pavlovic I, Lj V, Milicevic V, Maksimovic Zoric J, Stanojevic S, Radanovic O, et al. Seasonal dynamics of the presence of Culicoides spp. in Serbia in the period 2015–2016. Arhiv Vet Med. 2017;10:3–12.Google Scholar
  40. 40.
    Bobeva A, Zehtindjiev P, Bensch S, Radrova J. A survey of biting midges of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) in NE Bulgaria, with respect to transmission of avian haemosporidians. Acta Parasitol. 2013;58:585–91.CrossRefGoogle Scholar
  41. 41.
    Ioniţă M, Mitrea IL, Buzatu MC, Dascălu L, Ionescu A. Seasonal dynamics of haematophag arthropod populations (ticks and Culicoides spp.) - vectors of pathogens in animals and humans, in different areas of Romania. Lucrări Ştiinţice Medicină Veterinară. 2009;52:629–36 (In Romanian).Google Scholar
  42. 42.
    Kaufmann C, Steinmann IC, Hegglin D, Schaffner F, Mathis A. Spatio-temporal occurrence of Culicoides biting midges in the climatic regions of Switzerland, along with large scale species identification by MALDI-TOF mass spectrometry. Parasit Vectors. 2012;5:246.CrossRefGoogle Scholar
  43. 43.
    Patakakis MJ, Papazahariadou M, Wilson A, Mellor PS, Frydas S, Papadopoulos O. Distribution of Culicoides in Greece. J Vector Ecol. 2009;34:234–51.CrossRefGoogle Scholar
  44. 44.
    Meiswinkel R, Baldet T, de Deken R, Takken W, Delecolle JC, Mellor PS. The 2006 outbreak of bluetongue in northern Europe-the entomological perspective. Prev Vet Med. 2008;87:55–63.CrossRefGoogle Scholar
  45. 45.
    Burkot TR. Non-random host selection by anopheline mosquitoes. Parasitol Today. 1988;4:156–62.CrossRefGoogle Scholar

Copyright information

© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  • Ana Vasić
    • 1
    • 2
  • Nemanja Zdravković
    • 1
    • 3
  • Dragoș Aniță
    • 4
  • Jovan Bojkovski
    • 1
  • Mihai Marinov
    • 5
  • Alexander Mathis
    • 6
  • Marius Niculaua
    • 7
  • Elena Luanda Oșlobanu
    • 4
  • Ivan Pavlović
    • 3
  • Dušan Petrić
    • 8
  • Valentin Pflüger
    • 9
  • Dubravka Pudar
    • 8
  • Gheorghe Savuţa
    • 4
  • Predrag Simeunović
    • 1
  • Eva Veronesi
    • 6
  • Cornelia Silaghi
    • 2
    • 6
    • 10
    Email author
  • the SCOPES AMSAR training group
  1. 1.Faculty of Veterinary MedicineUniversity of BelgradeBelgradeSerbia
  2. 2.Institute of Infectology, Friedrich-Loeffler-InstituteInsel RiemsGermany
  3. 3.Scientific Veterinary Institute of SerbiaBelgradeSerbia
  4. 4.Faculty of Veterinary Medicine of IaşiIaşiRomania
  5. 5.Danube Delta National Institute for Research and DevelopmentTulceaRomania
  6. 6.National Centre for Vector Entomology, Institute of Parasitology, Vetsuisse Faculty, University of ZürichZürichSwitzerland
  7. 7.Research Centre for Oenology IaşiIaşiRomania
  8. 8.Faculty for AgricultureUniversity of Novi SadNovi SadSerbia
  9. 9.Mabritec AGRiehenSwitzerland
  10. 10.Ernst-Moritz-Arndt-UniversitätGreifswaldGermany

Personalised recommendations