Virus strain

In this experiment, LSDV isolate Kubash/KAZ/16 recovered from an infected cow during an outbreak of LSD in Atyrau, Kazakhstan in 2016 were used [12]. Virus was grown on primary lamb testis (LT) and with three in vivo passages through calves. Primary LT cell cultures were derived from pre-pubertal lambs according to Plowright and Ferris [13]. Cell cultures exhibiting90% cytopathic effects (CPE), were freeze-thawed three times, and then centrifuged at 2000 g for 20 min to remove cell debris and stored at -70°C until required.

Insect colony

LSDV negative Stomoxys spp colonies used in this study were provided by the Entomology section of the Research Institute for Biological Safety Problems (RIBSP). They were maintained in an environmental chamber at 25°C and 80% humidity, and adults were fed heparinized bovine blood soaked into cotton pads twice daily, 9 a.m. and 5 p.m.

Design of experiment

As part of an ongoing study of the role of hematophagous flies in the transmission of LSDV in cattle in South Kazakhstan, we utilized three laboratory reared Stomoxys species.

Intrathoracic inoculation of LSDV in adult Stomoxys spp was conducted according to the protocol published by Rochon, Baker [14] and Perez De Leon and Tabachnick [15]. Prior to experiment commencement, approximately 200 five day old Stomoxys flies of each species (Stomoxys calcitrans, Stomoxys sitiens, Stomoxys indica) were aspirated into separate plastic cages, anaesthetized using CO₂, placed onto a light ice board, and fixed using entomological forceps. Individual flies were inoculated in the dorsal area of the thorax at the junction of the scutum and the scutellum. Virus suspension, 6μl, (Kubash/KAZ/16, 10⁶ TCID₅₀/ml) was administered using 100μl Bevel tip 1710N Series Hamilton Gastight microliter syringe (USA). A 26-gauge sterile removable needle was used and changed for each fly (Supelco Inc, Bellefonte, PA). Following virus inoculation, flies were kept in separate cages and maintained at room temperature (24°C and 70% humidity).

Stomoxys flies were tested for the presence of LSDV nucleic acid by gel-based PCR and virus isolation (VI) at 0, 6h, 12h, 24h, 2d, 3d, 4d, 5d, 6d, 7d, 8d, 9d, 10d post intrathoracic inoculation (pii). Pools of ten flies each of each Stomoxys species inoculated as described above were collected and tested by PCR and VI immediately following virus administration (0hr). Prior to running tests, pools were rinsed with PBS solution to eliminate contamination. The remaining inoculated Stomoxys flies were placed in separate cages according to species and allowed to recover for 6 h. For sampling purposes, Stomoxys spp were aspirated from the cages with a vacuum, anaesthetized in a 5% CO₂ gas chamber and transferred to a centrifuge tube and stored at -70°C until tested. Freeze–thawed flies were then washed three times in PBS and rinsed using distilled water to eliminate surface contamination, homogenized in 300μl Hanks solution, spun at 1500 g for 10 min and the supernatant was used for LSDV detection and isolation.

Additionally, three control groups of Stomoxys spp (Stomoxys calsitrans, Stomoxys sitiens, Stomoxys indica; 20 individuals of each species) were inoculated by the method previously described and tested using PCR 2 hours after inoculation to determine the total number of infected among inoculated flies as well as to determine the efficacy of the intrathoracic inoculation technique in use.

Polymerase chain reaction

Virus amplification test was conducted utilizing protocol reported by Tuppurainen, Venter [16]. For DNA extraction, a QIAamp DNA Kit (QIAGEN, USA) was used according to the manufacturer’s instructions.

For PCR assay, the forward 5’-TCC-GAGCTC-TTT-CCT-GAT-TTT-TCT-TAC-TAT-3’ and reverse 5’-TAT-GGT-ACC-TAA-ATT-ATA-TACGTA-AAT-AAC-3’ primers were utilized to produce 192 bp of amplified nucleotide reactions. [17]. For DNA amplification in a Thermal Cycler (Eppendorf Mastercycler) following parameters were adjusted: 95°C for 2 min, 95°C for 45 s, 50°C for 50 s, 72°C for 1 min (34 cycles), and 72°C for 2 min. The PCR products obtained were loaded in 1.5% agarose gel electrophoresis in the presence of Ethidium bromide, and the results visualized using Bio-Imaging Systems MiniBIS Pro (Israel).

Virus isolation

Virus isolation was performed according to standard operational procedures (SOP) of the Virology section of the RIBSP, based on OIE [18] guidance. In brief, 10 μl of supernatant were inoculated on to lamb testes cells in 25 cm² cell culture flasks and incubated at 37°C for 1 h. Following incubation, culture media was washed with Phosphate Buffered Saline (PBS) three times and overlaid with Glasgow’s Minimal Essential Medium containing 0.1% penicillin, 0.2% gentamycin and 2% foetal calf serum. The cell monolayer was monitored daily for characteristic CPE. In the case no CPE was observed, the cell culture was freeze–thawed three times and the two or three blind passages were conducted. The culture media used were stored at −70°C until needed. Cell culture flasks exhibiting CPE were tested with gel-based PCR to confirm that CPE was caused by LSDV.

Analysis

PCR and virus isolation data were analyzed with logistic ANOVA (Proc Glimmix, SAS 9.4). Time post intrathoracic inoculation (pii) was considered a continuous independent variable and species was considered a class independent variable. The interaction between species and pii was included in the model as well.

Demonstration of LSDV retention in Stomoxys calcitrans

Virus retention following intrathoracic inoculation procedures resulted in infection in 95% of inoculated flies (Table 1). Pool samples collected for PCR tested positive at time intervals 0, 6h, 12h, 1d, 2d, 3d, 4d, 5d, 6d and 7 days post intrathoracic inoculation (pii) whereas samples tested for virus isolation were LSDV positive with characteristic CPE from time interval 0 up to 24h pii (Table 2).

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Table 1. Summary obtained from individual samples of Stomoxys species tested 2 hours after inoculation.

https://doi.org/10.1371/journal.pone.0238210.t001

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Table 2. PCR and virus isolation results of Stomoxys spp at different time intervals following intrathoracic inoculation with LSDV.

https://doi.org/10.1371/journal.pone.0238210.t002

Demonstration of LSDV retention in Stomoxys sitiens

LSDV DNA retention was detected in 90% of individually infected flies (Table 1). PCR assay was positive for the presence of LSDV in pools samples taken in time intervals 0, 6h, 12h, 1d, 2d, 3d, 4d, 5d and 6 days pii (Table 2). Virus isolation was carried out in cell culture, and CPE characteristic to LSDV was observed in time intervals 0, 6h, 12h pii. Additionally, following the second blind passage virus was recovered from pool samples collected on day 1 pii (Table 2).

Demonstration of LSDV retention in Stomoxys indica

An infection rate composed 95% among intrathoracically infected individuals (Table 1). Viral nucleic acid was detected in time intervals 0, 6h, 12h, 1d, 2d, 3d, 4d, 5d, 6d and 7 days pii (Table 2). CPE similar to LSDV was seen in time intervals 0, 6h, 12h pii while on day 1 pii virus was isolated following the second blind passage in the (LT) cell culture (Table 2).