Study area and collection of samples
Mosquito samples
Mosquitoes were collected by UiT The Arctic University of Norway as a part of the research project Climate and Reindeer Diseases (CARD), between July and August of 2013 - 2015. Eight different geographical locations, representing reindeer summer pastures, were chosen, with four sampling sites per location (Table 1, Fig. 1). To cover a variety of different species the sampling was performed at the reindeer summer pastures twice during the summer season. Mosquitoes were caught with a mosquito trap (Mosquito Magnet Independence; Woodstream® Corporation, Pennsylvania, USA), using propane gas to produce carbon dioxide (CO2) and a biting insect attractant (Mosquito Magnet Octenol, Woodstream® Corporation, Pennsylvania, USA). The trap was set to run for two to 24 h depending on the abundance of mosquitoes and weather conditions. Mosquitoes were killed by freezing and stored at -20 °C from two days and up to two weeks after capture, until a -80 °C freezer was available. The mosquitoes from each collection site were separated into different cryotubes (Thermo Fisher Scientific, Rochester, NY, USA). The mosquitoes were stored in a 50 ml Falcon tube (Corning Science Mexico, Reynosa, Mexico) with Drierite desiccant to dehydrate and preserve the samples for visual inspection and species identification [38]. All the cryotubes were stored at -80 °C until further analysis.
Table 1 Collection of mosquitoes and semi-domesticated reindeer serum samples from eight different reindeer pastures in Norway
County
|
Site
|
UTM – Coordinates*
|
Month
|
Year
|
Troms and Finnmark
|
Tana
|
35 W 0573349 7820819
|
December
|
2013-2015
|
Troms and Finnmark
|
Lakselv
|
35 W 0441724 707695
|
December
|
2013-2015
|
Troms and Finnmark
|
Tromsø
|
34W 0429573 7738767
|
May, December, November
|
2013-2015
|
Nordland
|
Lødingen
|
33W 0540824 7589369
|
December, October
|
2013-2014
|
Nordland
|
Hattfjelldal
|
33 W 0458873 7281479
|
December, November
|
2013-2015
|
Trøndelag
|
Fosen
|
32 W 0568340 7129347
|
February, March, January
|
2013-2016
|
Trøndelag
|
Røros
|
32 V 0619596 6966550
|
February, January, November
|
2013-2015
|
Innlandet
|
Valdres
|
32 V 0490781 6794290
|
December
|
2013-2015
|
*UTM – Coordinates = Universal Transverse Mercator coordinates
Reindeer serum samples
Blood samples (n = 480) were collected from semi-domesticated reindeer (Table 1). These animals were grazing on the same summer pastures as the mosquitoes were collected. Blood was obtained from live animals by venepuncture of external jugular vein using a venoject needle (Terumo, Leuven, Belgium) and blood collecting tubes (BD Vacutainer®; BD, Plymouth, UK). From slaughtered animals, blood was collected directly in the blood collecting tubes when bled. Blood tubes were centrifuged at 3500 rpm for 10 min to collect the serum. Sera were stored at -20 °C until analysis.
Extraction of total nucleic acid from mosquitoes
Each of the 213 pools of mosquito samples were transferred to 2 ml tubes containing six steel beads (MP Biomedical Life Science, CA, USA) and 350 ml of RLT® lysis buffer with β-mercaptoethanol in a ratio of 1:10 (RNeasy Mini Kit, QIAGEN Inc., Valencia, CA, USA). They were homogenized at 4 °C by FastPrep®-24 5G instrument (MP Biomedical Life Science, CA, USA) operated at a relative velocity of 4.0 m/sec for 60 seconds. The homogenates were centrifuged at 20817 x g (5 min) to remove mosquito debris. The total nucleic acid (TNA) was extracted with MagNA Pure LC 2.0 (software v 1.1.24) instrument and MagNA Pure LC Total Nucleic Acid High-Performance Isolation kit (Roche Diagnostics GmbH, Penzburg, Germany) according to the manufacturer’s protocol. The elution volume of TNA was 60 ml.
Detection of INKV by One-step RT-PCR
To detect INKV, a RT-PCR amplifying the fragment targeting the gene encoding the non-structural protein (nsp1) of the Lövånger strain of INKV (GenBank accession number KX554935) was carried out. TNA extracted from mosquitoes were amplified by RT-PCR in a 2720 thermal cycler (Applied Biosystems, Foster City, CA, USA) using SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA polymerase kit (Invitrogen, Carlsbad, CA, USA). The amplification was carried out in a total volume of 25 µl per reaction, containing 20 µl mastermix and 5 µl extracted TNA template. The concentration of each primer and cycling conditions were modified from [39, 13, 1]. The concentrations of the INKV forward and reverse primers were 0.25 mM each. The optimized cycling conditions were: 55 °C for 30 min, 95 °C for 2 min followed by 45 cycles of 95 °C for 15 sec, 60.5 °C for 30 sec and 72 °C for 1 min, before a final extension at 72 °C for 5 min and 4 °C until the PCR products were further analysed. The PCR products were visualized on E-Gel® 48 Gels (2% agarose) with EtBr according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA) and stored at -80 °C until further analysis. A 10-fold serial endpoint dilution
(10-1 to 10-4) of INKV positive controls and RNase free water as negative control were included in each test.
Detection of SINV by One-step RT-real time PCR
Total nucleic acid from pooled mosquito samples were analysed to detect SINV RNA by probe-based real-time PCR, using qScript™ XLT One-Step RT-qPCR ToughMix® kit (Quanta Biosciences, Gaithersburg, MD, USA) in a Rotor-gene 6000 (QIAGEN, Hilden, Germany). The primers and probe used for the amplification targeted the non-structural gene (nsP1) from the Ockelbo strain of SINV (GenBank accession number M69205.1) [1, 40]. The optimized cycling conditions for the reaction were: 55 °C for 30 min, 95 °C for 2 min followed by 45 cycles of 95 °C for 15 sec, 60 °C for 30 sec and 72 °C for 1 min, before a final extension at 72 °C for 5 min. The PCR products were stored at -80 °C until further analysis.
Included on each plate was SINV RNA positive control, from the Ockelbo strain, cultured in the lab, in a 10-fold serial endpoint dilution (10-4 to 10-7) along with two RNase free water samples as negative control.
Pyrosequencing
All the PCR positive samples for INKV RNA were further analysed by Pyrosequencing according to [41], for SQA analysis with the BioTage (Pyromark Q24) system (QIAGEN, Hilden, Germany). A 10-fold serial endpoint dilution (10-1 to 10-4) of INKV positive controls and RNase free water as negative control were included in each test.
The positive controls were used as a standard to compare the sequences obtained from pyrosequencing of PCR positive samples.
Calculation of prevalence
Two hundred and thirteen pools with approximately 25 mosquitoes in each pool were analysed. The sample size was designed for other research purposes by the CARD project. Estimated pooled prevalence (EPP) of INKV was calculated by the Epitools Epidemiological calculators [42]. The estimated prevalence was assumed to be close to zero with 95% confidence interval (CI) using Method 2 [43]. This method utilizes the frequentists approach assuming a fixed pool size and perfect (100%) test sensitivity and specificity for the estimation of prevalence and confidence limits [41].
Serological screening of reindeer samples
Indirect Immunofluorescence Assay (IIFA) for detection of IgG antibody against INKV
Slides were fixed with INKV antigen, a positive fluorescence signal from 40-50% of the cells (with 50-60% of the cells negative) was required for a positive result [33]. Two INKV positive controls; anti-Tahyna mouse-ascites and INKV positive deer whole blood were included in each run.
Initially, mouse ascites (1:100), deer whole blood and reindeer serum samples (1:20), were diluted in 1X PBS (Dulbecco’s solution A, pH = 7.4, Norwegian Institute of Public Health, Oslo, Norway).
Twenty ml of the diluted reindeer sera (n = 480) and the controls were analysed on slides and incubated at 37 °C for 1 h and washed with cold PBS and water. The slides were stained with anti-mouse Alexa Fluor® 488 conjugate, (Invitrogen Life Technology, Inc., Carlsbad, CA, USA) (dilution1:30) and anti-deer IgG fluorescein isothiocyanate conjugate (FITC), (KPL, Gaithersburg, MD, USA) (dilution 1:25) for 30 min at 37 °C and washed as described previously. Each slide was air dried, coated with glycerol before examining with a fluorescence microscope (Nikon Eclipse Ci, Nikon Corporation, Tokyo, Japan). The overall seroprevalence of IgG against INKV in reindeer sera was calculated as the percentage of the total number of positives of the total number of samples tested.
Cytopathic effect neutralization test (CPE-NT) from reindeer sera
Approximately 55 of the borderline reindeer sera samples were further verified by the CPE- neutralization test at the Department of Virology, University of Helsinki as previously described [33].
Plaque Reduction Neutralization Test (PRNT)
Reindeer sera (n = 66) with strongly positive, borderline, and negative result via IIFA were confirmed with a neutralization assay to determine whether the IgG antibodies were specific to INKV, TAHV, or SSHV as previously described [32]. A 50% plaque reduction neutralization titer (PRNT50) was calculated as the reciprocal of the highest serum dilution based on 50% or greater reduction in the plaque counts. Samples with titres ≥20 were defined as INKV seropositive.
Statistical method for calculation of seroprevalence
All statistical analyses were performed by the program STATA Software v. 16 (Stata Corporation, College Station, TX, USA). The statistical difference between sites were analysed by Χ2 with Pearson correction and the interaction infection, site, and year by logistic regression. In all analyses statistical significance was indicated by p<0.05.