Abstract

Introduction:

Bluetongue virus (BTV) is a species of genus Orbivirus belonging to the Sedoreoviridae family. Bluetongue (BT) is endemic in India and responsible for causing significant economic losses to livestock farmers. In India, antibodies to BTV serotype 24 (BTV-24) have been reported in 2005; it was first isolated in 2010, and it caused several outbreaks in sheep during 2012–2014. The in vivo studies investigating the pathogenetic potential of various BTV serotypes in the susceptible host sheep are scarce. Furthermore, detailed investigations to elucidate the pathogenetic mechanisms of BTV-24 under experimental conditions in sheep are not available. Because of its impact on the livestock economy, the present study was undertaken for the first time to explore the infection kinetics, pathology, pathogenesis, and immune responses against the Indian isolate of BTV-24 in sheep under experimental conditions.

Methods:

Six native sheep were infected intradermally with BTV-24 at 106 TCID50/mL concentration, and six sheep were inoculated with uninfected cell culture fluid. Animals were euthanized at 4, 7, 11, 16, 45, and 60 days post-inoculation (DPI). The sequential pathology, BTV localization by immunohistochemistry, BTV quantification by quantitative PCR (qPCR), immune cell kinetics [CD4+ and CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMCs), prescapular lymph node (PSLN), and spleen] by fluorescence-activated cell sorting (FACS), and cytokine estimation by qRT-PCR were studied.

Results:

The BTV-24-infected animals showed pyrexia, conjunctival and oral mucosal congestion, cyanosis of tongue, serous to catarrhal nasal discharge, and viremia. Gross pathological lesions were observed in the lymph nodes, lungs, and kidneys, with the lymph nodes being enlarged, edematous, and hemorrhagic. Subintimal hemorrhage at the base of the pulmonary artery (pathognomonic lesion of BT) was observed at 7 DPI. Histopathological lesions were prominent in lymph nodes, spleen, heart, lungs, and cerebral endothelium. Severe hemosiderosis in spleen, and hemorrhages and hyalinization of tunica media in pulmonary artery at 7 DPI were observed. Development of clinical signs and gross and histopathological lesions in BTV-24-infected animals emphasized the moderate progression of disease and enhanced virulence of the serotype. Humoral immune response was significantly high at 5, 11, 16, 21, 45, and 60 DPI. Cell-mediated immune response-like kinetics of CD4+ and CD8+ T lymphocytes showed a sharp decline during the early stage and an increase of CD8+ T lymphocytes during later stages of infection. BTV antigen was detected consistently in tongue, thymus, trapezius muscle, heart, and pulmonary artery by immunohistochemistry and qPCR. Significant changes in the levels of cytokines [interferon-alpha (IFN-α), IFN-β, IFN-γ, interleukin-2 (IL-2), IL-12, and tumor necrosis factor-alpha (TNF-α)] and upregulated expression of apoptotic markers, B-cell lymphoma-2 (Bcl-2), and caspase-3 in the spleen and lymph nodes were correlated with peak viremia.

Conclusion:

The results of this study can be used to formulate effective preventive and control measures and to develop a suitable vaccine against BTV-24 to minimize economic losses.

1 Introduction

Bluetongue (BT) is an infectious, non-contagious vector-borne viral pathosis of ruminants caused by bluetongue virus (BTV), a prototype member of the genus Orbivirus in the family Sedoreoviridae (formerly Reoviridae). The disease is transmitted by biting midges of the genus Culicoides. The disease draws its name from the most recognizable of symptoms (BT) that an infected animal presents. The disease is also termed “malarial catarrhal fever” or “epizootic malignant catarrhal fever of sheep” (Saminathan et al., 2020). Until recently, 28 BTV serotypes have been described globally with the addition of some “atypical” serotypes, becoming a total of 36 BTV serotypes based on the differences in the genome segment-2 (Seg-2) sequence and its translated protein VP2 (Sun et al., 2016; Bumbarov et al., 2020; Caixeta et al., 2024). Because of the recent spread of BT into previously unaffected regions of the world and the incursion of few additional serotypes, BT has again risen in prominence as one of the important diseases of the 21st century (Saminathan et al., 2020). The clinical manifestations of the BT vary from asymptomatic to lethal outcome based on virus factors like serotype of BTV, passage history of the virus, and dose and route of inoculation; host factors such as age, species, breed, individual susceptibility and immune status, stress, and nutritional status of the host; and environmental factors such as solar irradiation, high temperature, and vector population (Schwartz-Cornil et al., 2008; Maclachlan et al., 2009; Saminathan et al., 2018). Virulence characteristics vary among field strains of BTV, even those of the same serotype due to the considerable genetic variability results from mutations in the different genome segments, and such changes could influence the biological properties of the virus, which might result in the variable expression of disease (Saminathan et al., 2020). Despite these facts, in vivo comparative studies focused on determining the differences in the pathogenetic potential of BTV serotypes in the susceptible host are scarce with few reports in sheep (Hamblin et al., 1998; Sanchez-Cordon et al., 2013).

Currently, BT is endemic in India. Among 28 serotypes of BTV, 23 serotypes (except 22 and 25–28) have been reported from India based on the presence of neutralizing antibodies and virus isolation (Singh et al., 2021). The virus thrives throughout tropical, subtropical, and temperate regions of the world, wherever competent vector populations predisposed to its dissemination exist. Bluetongue virus serotype 24 (BTV-24) was first reported from Africa (Nevill et al., 1992) and later isolated from America and the Mediterranean in the last decade (Brenner et al., 2010). Antibodies to BTV-24 have been reported from buffaloes in India (Chauhan et al., 2005; Saminathan et al., 2020), and the serotype was first isolated from India in 2010. BTV-24 was involved in several outbreaks in various states of India indicating the emergence and virulence of this serotype (Singh et al., 2021). Sequence analysis for Seg-2 of BTV-24 (IND2010/01) indicated that the virus is closely related to Western viruses; hence, the entry of BTV-24 to India was exotic to Australasia, which was a cause of concern (Krishnajyothi et al., 2016). The seroprevalence of BTV-24 in India was reported as 1.61% in Telangana and Andhra Pradesh during 2017–2018, which increased to 16.66% in Telangana during 2018–2019 (Reddy et al., 2010, Naresh et al., 2020; Putty et al., 2020).

The pentavalent inactivated adjuvanted vaccine containing BTV-1, -2, -10, -16, and -23 has been used for the control of BT in India (Naresh et al., 2020). Recent studies revealed that unpredictably; serotypes that are not there in the currently used pentavalent vaccine, i.e., BTV-24, -4, -5, -9, -12, and -21, have emerged with high prevalence rates in India (Naresh et al., 2020: Putty et al., 2020). Recent studies from our lab revealed that the currently used pentavalent vaccine in India did not give protection against BTV-24 and BTV-4 (data not published), which created important discussions about the revision of BTV serotypes in the current vaccine. Hence, there is a need to update or revise BTV serotypes present in the current BTV pentavalent vaccine with BTV-24 after knowing its virulence. The pathology and pathogenesis of Indian BTV-24 in the natural host sheep have not been studied yet and, thus, need to be determined for its inclusion in the existing pentavalent vaccine for control measures. Considering these critical gaps, the present study was conducted for the first time to investigate the infection kinetics, pathology, pathogenesis, and immune responses of BTV-24 infection in the natural host sheep.

2 Materials and methods2.1 Bluetongue virus serotype 24 isolate

The BTV-24 isolate used in this study was isolated from sheep, which showed symptoms of fever and hyperemia of gums and tongue with a morbidity rate of 15%–20% and a case–fatality rate of 6.91% during the BT outbreak in 2010 in Medak district, Telangana State, India (Krishnajyothi et al., 2016). The virus isolate was kindly provided by the Veterinary Biological and Research Institute, Hyderabad, Telangana, India.

2.2 Propagation of BTV-24 isolate

The BTV-24 isolate (passage 7) was propagated in the insect Culicoides sonorensis (KC) cell line maintained in Schneider’s insect medium (Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal calf serum (FCS) and antibiotic–antimycotic solution followed by cultivation in baby hamster kidney-21 (BHK-21) cells (clone-13, National Centre for Cell Science, Pune, Maharastra, India) at passage 30 maintained in modified Eagle’s medium (HiMedia Laboratories, Thane, Maharashtra, India) supplemented with 10% FCS and antibiotic–antimycotic solution containing 10,000 U of penicillin, 10 mg of streptomycin, and 25 µg of amphotericin B per milliliter (HiMedia Laboratories, Thane, Maharashtra, India) and subsequently in BHK-21 cells. The crude culture supernatant was collected and combined with the supernatant obtained after sonication of infected cells. This pooled supernatant was subjected to endpoint titration in BHK-21 cells. For long-term storage, the virus stock was maintained at −80 °C, and for short-term storage, it was maintained at 4 °C. All procedures were performed under BSL-2 containment.

2.3 Confirmation of BTV-24

The BTV-24 serotype was confirmed by PCR using self-designed serotype-specific forward: 5′-AGTGACCCACAATGGAGGAG-3′ and reverse: 5′-TGAGTGCGTCTACTATGCTACTT-3′ primers targeting the 195-bp product of VP2 gene (segment 2—gene accession number KX164150). The total RNA was extracted from the cell culture using TRIzol® reagent as per the manufacturer’s recommendations. The purity of the RNA was analyzed in a NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The reaction was carried out in Stratagene Mx3005P™ Multiplex QPCR using a One-Step RT-PCR kit (QIAGEN GmbH, Hilden, North Rhine-Westphalia, Germany). The PCR reaction mix (25 μL containing 10 pmol of forward and reverse primer) was prepared and subjected to a cycling condition of reverse transcription at 50°C for 30 min, initial PCR activation at 95°C for 15 min, template denaturation at 94°C, primer annealing at 62°C, extension at 72°C for 30 s, and final extension at 72°C for 10 min for 35 cycles. The amplicons were visualized on 1.5% agarose gel (Sigma-Aldrich, USA) prepared in 1 × TBE buffer containing ethidium bromide (0.5 μg/mL). The gel was visualized under a gel documentation system (Azure Biosystems-c300, USA) for 195-bp amplicons. The amplicon was purified using commercially available kits (QIAquick Gel Extraction Kit, Hilden, North Rhine-Westphalia, Germany). The gel-purified products were outsourced for sequencing from Eurofins Genomics India Ltd., Whitefield, Bangalore, using forward and reverse primers in a capillary sequencer.

2.4 Experimental animals

Twelve indigenous nondescript adult sheep of either sex aged between 2 and 3 years of approximately 30–40 kg were procured from livestock farmers after screening and negative for BTV antibodies using a competitive-enzyme linked immunosorbent assay (c-ELISA) kit (Bluetongue virus antibody cELISA test kit, VMRD Inc., Pullman, WA, USA). The sheep were housed in the insect-proof animal shed of the Centre for Animal Disease Research and Diagnosis (CADRAD), ICAR-Indian Veterinary Research Institute (ICAR-IVRI), Bareilly, Uttar Pradesh, India. The animals were provided feed, fodder, and water ad libitum throughout the experiment. This study was approved by IAEC (ICAR-IVRI), and all animal procedures were conducted in accordance with the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines, 2003 (Ethical Clearance Certificate’s Number 108/HRECC.FODM/VII/2017).

The animals were randomly assigned to groups. The animals of either sex were allotted to different groups irrespective of the sex. The animals were housed in the animal isolation facility of ICAR-IVRI for 28 days followed by acclimatization for 1 week at the Experimental Animal House Facility of the institute. The animals were screened for hemoparasites and antibodies against BTV prior to the study.

2.5 Experimental design

The BTV-infected and uninfected control animals were housed indoors separately. The uninfected control group with six sheep were administered 6 mL of mock infected cell culture fluid via the intradermal route at multiple sites. In the BTV-24-infected group, six sheep were inoculated with 6 mL of 1 × 106 TCID50/mL of BTV-24 via the intradermal route at multiple sites in the right pre-scapular or neck region. The infection design, including viral dose and trial structure, was based on previously published experimental BTV infection studies that have demonstrated reliable establishment of viremia and clinical response under similar conditions (Umeshappa et al., 2010, 2011; Channappanavar et al., 2012; Singh et al., 2024). The selected inoculum dose has been widely used in earlier work to ensure consistent infection dynamics, and the replication number aligns with established experimental models for BTV pathogenesis (Singh et al., 2024). The mock-control group consisted of virus-free culture medium processed identically to the infected inoculum, thereby ensuring appropriate procedural and systemic comparability.

2.6 Monitoring of clinical signs

The rectal temperature and clinical signs of each animal were recorded throughout the experimental period of 45 days daily and up to 60 days weekly before 11 a.m. to minimize any diurnal fluctuations in the estimated parameters and scored using the clinical reaction index as described by Moulin et al. (2012) and Caporale et al. (2014) with slight modifications (Supplementary Table 1).

2.7 Sample collection

Blood samples were collected at 0, 1, 3, 5, 7, 9, 11, 14, 17, 21, 28, 45, and 60 days post-inoculation (DPI) to assess the humoral immune response and viremia. Approximately 4 mL of blood was collected at each time interval for various parameters. Viremia evaluation was detailed by including blood and peripheral blood mononuclear cells (PBMCs) in the sections mentioned. One sheep from each uninfected and BTV-24-infected group was pre-euthanized by administration of a combination of xylazine (0.22 mg/kg b. wt) and ketamine (11 mg/kg b. wt) intramuscularly followed by euthanasia by administering single intravenous injection at a dose of 40 mg/kg b. wt of thiopental sodium (Thiosol sodium 1 g, Neon Laboratories Ltd., Mumbai, Maharashtra, India) via the jugular vein at 4, 7, 11, 16, 45, and 60 DPI. The required volume of thiopental was injected reasonably rapidly. The death of the animal was ensured by checking the absence of eye reflexes and heartbeat and the appearance of glazed eyes following injection, and systematic postmortem examination was carried out.

The blood samples collected before euthanasia were used for the estimation of immune cells’ (CD4+ and CD8+ T lymphocytes) kinetics. The gross pathological lesions were graded (Umeshappa et al., 2010; Sanchez-Cordon et al., 2013; Supplementary Table 2). Representative triplicate samples from lungs, heart, pulmonary artery, prescapular lymph nodes (PSLNs), spleen, trapezius muscle, skin, tonsil, and tongue were collected in 10% neutral buffered formalin for histopathology and RNAlater® (Ambion®, Austin, TX, USA) for RNA extraction. Spleen and PSLN were collected aseptically in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) in ice for the analysis of immune cells’ (CD4+ and CD8+ T lymphocytes) kinetics from the euthanized animals at 4, 7, 11, 16, 45, and 60 DPI.

2.8 Histopathology

The formalin-fixed tissues were cut into pieces of 2 to 3 mm thickness, washed with water, dehydrated in ascending grades of alcohol, and cleared in xylene. The cleared tissues were embedded in paraffin, and sections of 4–5 μm thickness were cut and stained with hematoxylin and eosin (H&E) as per the standard procedure (Luna, 1968). The histopathological lesion score was calculated semi-quantitatively by following the scoring system of Umeshappa et al. (2011) with slight modification (Supplementary Table 3).

2.9 Immunohistochemistry

The formalin-fixed tissue sections were taken on (3-aminopropyl)triethoxysilane (APES, Sigma-Aldrich, St. Louis, MO, USA)-coated slides. The sections were deparaffinized and rehydrated in graded alcohol followed by gently rinsing in distilled water. The antigen retrieval was performed by microwave in 10 mM tri-sodium citrate buffer (pH 6.0) for 15 min (three cycles of 5 min each) to unmask the antigenic sites. Then, the slides were washed three times with phosphate buffered saline (PBS, pH 7.4) for 5 min each. Endogenous peroxidase activity was blocked by incubating with freshly prepared 3% H2O2 in methanol for 30 min at room temperature (RT) in a dark chamber and washed in PBS (three times for 5 min each). Furthermore, the sections were incubated with 5% bovine serum albumin (BSA) in PBS for 1 h at RT in a humidified chamber for blocking of non-specific antigen binding sites. The primary antibody against BTV core antigen raised in rabbit at the Mukteshwar campus of the Indian Veterinary Research Institute (IVRI) was incubated on the sections at 1:20 dilution in 1% BSA in PBS overnight in a humidified chamber at 4 °C. Slides were incubated with biotinylated goat anti-rabbit IgG peroxidase conjugate (Sigma-Aldrich, St. Louis, MO, USA) at 1:200 dilution in 1% BSA (pH 7.4) for 1 h at 37 °C, followed by washing thrice in PBS for 5 min each. The slides were incubated with ImmPACT™ DAB peroxidase substrate (Vector Laboratories Inc., Burlingame, CA, USA) for 30–60 s to demonstrate immunolabeling. The slides were counterstained with Mayer’s hematoxylin. The sections were mounted with CC/Mount™ aqueous mounting medium (Sigma-Aldrich, St. Louis, MO, USA) and dried at RT. Sections prepared from known BTV-positive samples were used as positive control. In negative tissue control, tissues sections were incubated only with BSA instead of BTV primary antibody. The sections were examined under the microscope for positive signals. In the negative antibody control, tissue sections were treated identical to other slides and PBS was added instead of BTV antibody. Similarly, expression of caspase-3 was studied using rabbit anti-caspase-3 polyclonal (Santa Cruz Biotechnology, Inc. Dallas, TX, USA) as the primary antibody at 1:10 dilution and biotinylated goat anti-rabbit IgG peroxidase conjugate (Sigma-Aldrich, St. Louis, MO, USA) at 1:200 dilution as the secondary antibody.

2.10 Scoring the intensity of immunohistochemistry signals

To quantify the BTV-specific antigen semi-quantitatively, tissue sections were evaluated based on the number of positive cells for BTV antigen found in 10 high-power (40×) fields on a − to +++ scale as follows: −: absence of immunostaining; +: less than 10% cells positive for BTV antigen (weak); ++: 10%–50% cells positive (moderate); and +++: more than 50% cells positive (strong).

2.11 Estimation of humoral immunity

The development of group-specific antibodies against BTV was measured in the samples collected at different time intervals using the c-ELISA kit developed at Mukteswar Campus, ICAR-IVRI, Uttarakhand, India (Chand et al., 2017). The optical density (OD) was measured at a wavelength of 492 nm (Bio-Rad, Hercules, CA, USA). The percentage inhibition (PI) value for each sample was calculated from the OD of the test samples and the negative serum controls using the following formula. The test samples were considered positive, when the PI value of the sample is equal to or more than 50%.

2.12 Estimation of cell-mediated immunity2.12.1 Isolation of peripheral blood mononuclear cells

The pooled blood samples were used for the isolation of PBMCs using Histopaque by density gradient centrifugation as described previously by Saminathan et al. (2020). Briefly, approximately 2 mL of blood was slowly layered over an equal volume of histopaque [Histopaque® (Sigma-Aldrich, St. Louis, MO, USA) with a density of 1.077 g/mL] in a 15-mL centrifuge tube and centrifuged at 210g for 40 min, resulting in the separation of PBMCs at the plasma–histopaque interface. The PBMCs were separated carefully and washed twice with isotonic PBS (0.01 M, pH 7.3) at 375g for 10 min each. To remove the traces of red blood cells (RBCs), 1 mL of 1× RBC lysis buffer (HiMedia Laboratories, Pennsylvania, USA) was added and incubated for 10 min in ice and centrifuged at 2,000 rpm for 5 min. The PBMCs were used for the analysis of CD4+ and CD8+ T lymphocytes using fluorescence-activated cell sorting (FACS) and RNA extraction for cytokine genes expression studies.

2.12.2 Isolation of lymphocytes and splenocytes

Splenocytes were separated from spleen by mincing into small pieces as described previously (Madhu et al., 2016). Aseptically collected spleen and PSLN from BTV-infected and uninfected control animals were homogenized and passed through a cell strainer (70 µm) to make a single-cell suspension. Single-cell suspension was washed twice with PBS (0.01 M, pH 7.3) and centrifuged at 580g for 5 min at 4°C. The cell pellet was suspended in 1 mL of RBC lysis buffer, incubated at 4°C for 10 min, and centrifuged at 580g for 5 min. Separated cells were washed twice in isotonic PBS and centrifuged at 375g for 5 min each. The isolated lymphocytes and splenocytes were used for the analysis of CD4+ and CD8+ T lymphocytes using FACS and RNA extraction for cytokine gene expression studies.

2.12.3 Estimation of CD4+ and CD8+ T lymphocytes by FACS

The PBMCs from blood, lymphocytes from PSLN, and splenocytes from spleen were resuspended in 200 μL of stain buffer (FBS, BD Pharmingen™, New Jersey, USA). Cells were counted by the dye exclusion method in a Neubauer chamber after dilution at 1:10 dilution with trypan blue solution (0.01%) to adjust the cell concentration of at least 1 × 106 cells per sample. Each 10 µl of mouse anti-sheep CD4:RPE-labeled (Bio-Rad Laboratories Inc., Watford, Hertfordshire WD17 1ET, United Kingdom) and mouse anti-sheep CD8:FITC-labeled antibodies (Bio-Rad Laboratories Inc., Watford, Hertfordshire WD17 1ET, United Kingdom) was added as per the manufacturer’s instruction. Then, samples were mixed and incubated at RT in the dark for 45 min. Data were acquired using flow cytometry (BD® LSR II Flow Cytometer, BD Biosciences, Franklin Lakes, NJ, USA) and interpretation of data (10,000 events/sample) was done by comparing the data with the uninfected control animals using BD CellQuest™ Pro Software (BD Biosciences, San Jose, CA, USA).

2.12.4 RNA extraction

Total RNA was extracted from the blood and tissue samples (skin, tongue, thymus, tonsil, trapezius muscle, spleen, PSLN, lungs, heart, and pulmonary artery) using TRIzol® reagent (Invitrogen™, Thermo Fisher Scientific, Carlsbad, CA, USA) as per the manufacturer’s recommendations. The total RNA was treated with RNase-free DNase (Promega, Madison, Wisconsin, USA) followed by enzyme inactivation at 65 °C for 10 min to remove the possible traces of genomic DNA (Madhu et al., 2016). Purity of the RNA was analyzed in a NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and the integrity of the RNA was tested by electrophoresis. The RNA pellet was stored at −80 °C until further use.

2.12.5 Complementary DNA synthesis

Complementary DNA (cDNA) was synthesized from total RNA using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, Carlsbad, CA, USA) with random primers following the manufacturer’s instruction in the Thermocycler (Mastercycler Personal, Eppendorf, Hamburg, Germany). The synthesized cDNA was checked using β-actin-specific primers by polymerase chain reaction (PCR) using GoTaq® Green Master Mix (Promega, Madison, Wisconsin, USA). The synthesized cDNA was stored at −20°C until used.

2.12.6 Quantification of cytokine genes’ expression by quantitative RT-PCR

The expression of mRNA of different cytokines [interferon-alpha (IFN-α), IFN-β, IFN-γ, interleukin-2 (IL-2), IL-12, and tumor necrosis factor-alpha (TNF-α)] and apoptotic markers [B-cell lymphoma-2 (Bcl-2) and caspase-3] was quantified in the PBMCs, PSLN, and spleen of the BTV-infected and uninfected control animals at specified time intervals by comparing with GAPDH as the internal reference gene (Umeshappa et al., 2010) using QuantiFast® SYBR® Green PCR master mix (Qiagen, Maryland, USA) in a Stratagene Mx3000P™ Multiplex Quantitative PCR (QPCR) system (Agilent Technologies, Santa Clara, CA, USA). The details of the primers used for quantification of different cytokine genes in sheep are mentioned in Table 1. Reactions were performed in triplicate for each gene of interest; a dissociation curve was generated after PCR amplification and analyzed to determine the specificity of the PCR reaction. The comparative CT (2−ΔΔCt) method was used to calculate the changes in the gene expression as a relative fold change between different groups (Livak and Schmittgen, 2001).

GenePrimer sequencePrimer length (bp)Product length (bp)Annealing temperature (°C)ReferenceGAPDHF: 5′-ATCTCGCTCCTGGAAGATG-3′1922760Puech et al. (2015)R: 5′-TCGGAGTGAACGGATTCG-3′18IFN-αF: 5′-CAGACCATCTCTGTGCTCC-3′1921655Umeshappa et al. (2010)R: 5′-GTGTTTCCTCACAGCCAGG-3′19IFN-βF: 5′-CCAGATGGTTCTCCTGCTGTGT-3′2221660bin Tarif et al. (2012)R: 5′-GACCAATACGGCATCTTCCTTC-3′22IFN-γF: 5′-GATAACCAGGTCATTCAAAGG-3′2122260Umeshappa et al. (2010)R: 5′- GAGATTCTGACTTCTCTTCC-3′20IL-2F: 5′-GCTCCAAGCAAAAACCTGAA-3′2011060Wani et al. (2018)R: 5′-CAGCCTTTACTGTCGCATCA-3′20IL-12F: 5′-CGTGATGGAAGCTGTGCAC-3′1921160Umeshappa et al. (2010)R: 5′-CTTTCCCTGGACCTGAACAC-3′20TNF-αF: 5′-CTTCAACAGGCCTCTGGTTC-3′2011160Puech et al. (2015)R: 5′-GGACCTGCGAGTAGATGAGG-3′20Bcl-2F: 5′-TTCGCCGAGATGTCCAGT-3′1815158Umeshappa et al. (2010)R: 5′-ACGCTCTCCACACACATGAC-3′20Caspase-3F: 5′-TCTTCAGAGGGGACTGTTGC-3′2020658Umeshappa et al. (2010)R: 5′-ACTTTGAGTTTCGCCAGGAA-3′20

Details of oligonucleotide primers used for quantification of different cytokine genes in sheep.

2.13 Quantification of BTV-24 genome using real-time PCR

The BT viral load in blood and tissues (skin, tongue, thymus, tonsil, trapezius muscle, spleen, PSLN, lungs, heart, and pulmonary artery) at specified time points was quantified by TaqMan probe-based real-time PCR using the specific BTV NS3 (segment-10) probe [(6-FAM)-ARG CTG CAT TCG CAT CGT ACG C-(Tamra-Q)], forward primer: 5′-TGG AYA AAG CRA TGT CAA A-3′, and reverse primer: 5′-ACRTCATCACGAAACGCTTC-3′. The reaction was carried out in Stratagene Mx3005P™ Multiplex QPCR using a One-Step RT-PCR kit (QIAGEN GmbH, Hilden, North Rhine-Westphalia, Germany). The reaction mix was prepared with 2 μg of RNA as template combined with 10 pmol each of forward and reverse primers and probe with other components in the reaction mixture according to the manufacturer’s instructions. The cycling conditions were as follows: reverse transcription at 50 °C for 30 min, initial PCR activation at 95 °C for 15 min, template denaturation at 94 °C for 30 s, primer annealing at 56 °C for 30 s, extension at 72 °C for 30 s for a total of 40 cycles, and final extension at 72 °C for 10 min. Individual cycle threshold (Ct) values were determined and real-time PCR amplification was confirmed by the dissociation curve at the end of the reaction. The standard curve was used for the quantification of BTV RNA in the samples.

2.14 Statistical analysis

Data were analyzed using the statistical analysis program GraphPad Prism, Version 5.0 and IBM SPSS Statistics 20.0 software. The average cumulative clinical score was calculated for the scoring of clinical signs. The mean of the BTV-infected and uninfected control groups at specified time intervals was calculated and expressed as mean ± standard error. The clinical scores were tested for statistical significance using the Kruskal–Wallis test and Wilcoxon signed-rank test for non-parametric distribution. The differences between the baseline value (taken at day 0) and the values obtained at each time point in the BTV-infected group were analyzed using paired t-test. The differences between the uninfected control and BTV-infected groups at the same time point were analyzed using a two-way analysis of variance (ANOVA) with Bonferroni post-hoc test. For all comparisons, differences were considered significant at p < 0.05. For real-time PCR analysis, the data obtained were analyzed by using the 2–ΔΔCt method, and the relative expression (ΔCt) for each group was statistically analyzed by repeated-measures ANOVA with Bonferroni post-hoc test.

3 Results3.1 Clinical signs

In BTV-24-infected animals, a significant (p < 0.05) increase in rectal temperature was observed at 4 and 5 DPI when compared to uninfected control animals. BTV-infected animals (BT24I-6) developed hyperthermia at 2 and 3 DPI (~40°C/104°F) with a second peak at 9 and 14 DPI. BT24I-3 showed peak pyrexia at 5 (40.3°C/104.5°F) and 6 DPI (39.8°C/103.6°F). Two animals, BT24I-1 and BT24I-4, developed temperatures of 39.5°C/103.1°F and 39.7°C/103.5°F at 4 and 5 DPI, respectively (Figures 1, 2). A significant difference in the clinical sign scores was observed between BTV-24 and uninfected control animals at 4, 6, 7, and 12 DPI (Supplementary Tables 4–7). Infected animals showed moderate disease progression with congestion of conjunctival and oral mucus membranes in four out of six animals, cyanosis of the tongue in two out of six animals, and serous to catarrhal nasal discharge in three out of five animals at 5 DPI. During the mid-course of the study (11–15 DPI), clinical signs were mild in severity. Compared to uninfected controls, oral lesion score showed a significant difference at 4 DPI in infected animals. The control animals remained normal throughout the study period.

Rectal temperature in bluetongue virus serotype 24-infected animals.

Rectal temperature of BTV-24-infected and uninfected control groups. Results are presented as mean ± SE at each time point. Asterisk (*) indicates a significant difference (p < 0.05) between groups. Two-way ANOVA with Bonferroni post-hoc test was used.

3.2 Gross pathological lesions

The BTV-24-infected animals sacrificed at different time intervals showed varying degrees of gross pathological lesions. Enlarged spleen with prominently reactive splenic white pulp visible on cut sections was observed in four out of six animals. Pulmonary edema and leathery consistency of the lungs were apparent in sheep sacrificed at different time intervals (Figures 3a, c). Enlarged (4/6 animals) and hemorrhagic (3/6 animals) prescapular, mesenteric (4/6 animals), and mediastinal (3/6 animals) lymph nodes were observed (Figure 3d). Mucosal edema and vascular congestion in the small intestine and ileocecal lymph nodes were observed in three out of six animals sacrificed at 4, 7, and 16 DPI. The pathognomonic lesion of BT, subintimal hemorrhage at the base of the pulmonary artery, was observed in sheep (BT24I-2) sacrificed at 7 DPI (Figure 3b). Kidneys showed hemorrhages in medulla. The meningeal blood vessels were congested. The total gross pathological lesions score was higher in BTV-24-infected animals at 7 DPI when compared with the uninfected control animals. The uninfected control animals did not show any gross pathological lesions in the observed organs.

Gross pathological lesions in BTV-24-infected sheep. (a) Enlarged spleen at 4 DPI. (b) Subintimal hemorrhage on the base of pulmonary artery at 7 DPI. (c) Non-collapsed, heavy, and congested lungs at 7 DPI. (d) Edematous and enlarged PSLN with cortical hemorrhages at 11 DPI.

3.3 Histopathological lesions3.3.1 Four days post-inoculation

The skin of the BTV-infected animals showed moderate to severe vascular reactions in the superficial and deep dermis along with mild edema and mononuclear cell (MNC) infiltration (Figure 4a). Focal and mild to moderate infiltration of MNCs, edema, and fibrinous exudates were observed in the interstitium and around the muscle fibers of heart (Figure 4f). Lungs showed congestion of alveolar capillaries and marked interalveolar septal thickening (Figure 4k). Severe hemorrhage was observed in the thymic medulla. Mild to moderate infiltration of mononuclear inflammatory cells around the blood vessels with swollen endothelial cells were observed in the tongue. Musculature of tongue showed hyaline degeneration, loss of striations, and MNC infiltration (Figure 5a). Cortical hemorrhages were observed in PSLN (Figure 5f). In spleen, white pulp hyperactivity, thickening of tunica media of vessels, and germinal center formation was observed (Figure 5k). The uninfected control animals did not show any histopathological lesions in the examined organs.

Histopathological lesions in BTV-24-infected sheep at different DPI. (a) Dermis of skin showing moderate infiltration of mononuclear inflammatory cells at 4 DPI. H&E ×200. (b) Loose connective tissue and severe perivascular infiltration of mononuclear cells at 7 DPI. H&E ×200. (c) Fragmented and necrotic hair follicles and edematous fluid surrounding contracted and degenerated follicles at 11 DPI. H&E ×100. (d) Infiltration of mononuclear cells around the blood vessels of dermis at 16 DPI. H&E ×200. (e) Skin of control animal at 7 DPI. (f) Heart showing increased interfascicular connective tissue with edema and mild infiltration of mononuclear inflammatory cells at 4 DPI. H&E ×200. (g) Heart showing separation, thinning of muscle fibers, and interstitial infiltration of MNCs at 7 DPI. H&E ×200. (h) Heart showing interstitial and perivascular infiltration of MNCs at 11 DPI. H&E ×200. (i) Heart showing marked interfascicular infiltration of MNCs at 16 DPI. H&E ×200. (j) Heart of control animal at 7 DPI. (k) Lungs showing interalveolar septal thickening at 4 DPI. H&E ×200. (l) Lungs showing thickening of interalveolar septa and severely engorged pulmonary vessels at 7 DPI. H&E ×200. (m) Lungs showing severe interstitial proliferation and thickening of interalveolar septae at 11 DPI. H&E ×200. (n) Lungs showing mild thickening of interalveolar septae and mild hyperplasia of BALT at 16 DPI. H&E ×200. (o) Lungs of control animal at 7 DPI. H&E ×200.