Identification of bluetongue virus serotypes 1, 4, and 17 co-infections in sheep flocks during outbreaks in Brazil

Accepted Manuscript Identification of bluetongue virus serotypes 1, 4, and 17 coinfections in sheep flocks during outbreaks in Brazil

Lorena Lima Barbosa Guimarães, Júlio César Câmara Rosa, Ana Carolina Diniz Matos, Raquel Aparecida S. Cruz, Maria Isabel Maldonado Coelho Guedes, Fernanda Alves Dorella, Henrique César Pereira Figueiredo, Saulo Petinatti Pavarini, Luciana Sonne, Zélia Inês Portela Lobato, David Driemeier PII: DOI: Reference:

S0034-5288(16)30518-5 doi: 10.1016/j.rvsc.2017.09.001 YRVSC 3412

To appear in:

Research in Veterinary Science

Received date: Revised date: Accepted date:

26 October 2016 18 August 2017 3 September 2017

Please cite this article as: Lorena Lima Barbosa Guimarães, Júlio César Câmara Rosa, Ana Carolina Diniz Matos, Raquel Aparecida S. Cruz, Maria Isabel Maldonado Coelho Guedes, Fernanda Alves Dorella, Henrique César Pereira Figueiredo, Saulo Petinatti Pavarini, Luciana Sonne, Zélia Inês Portela Lobato, David Driemeier , Identification of bluetongue virus serotypes 1, 4, and 17 co-infections in sheep flocks during outbreaks in Brazil. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Yrvsc(2017), doi: 10.1016/j.rvsc.2017.09.001

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ACCEPTED MANUSCRIPT IDENTIFICATION OF BLUETONGUE VIRUS SEROTYPES 1, 4, AND 17 COINFECTIONS IN SHEEP FLOCKS DURING OUTBREAKS IN BRAZIL

Lorena Lima Barbosa Guimarãesa,# , Júlio César Câmara Rosab,c,# , Ana Carolina Diniz Matosb, Raquel Aparecida S. Cruza, Maria Isabel Maldonado Coelho Guedesb, Fernanda Alves

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Dorellac, Henrique César Pereira Figueiredoc, Saulo Petinatti Pavarinia, Luciana Sonnea, Zélia

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a

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Inês Portela Lobatob,* , David Driemeiera

Setor de Patologia Veterinária - Faculdade de Veterinária, Universidade Federal do Rio

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Grande do Sul, Av. Bento Gonçalves, 9090, Porto Alegre, Brazil. CEP 91540-000

b

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Laboratório de Pesquisa em Virologia Animal, Departamento de Medicina Veterinária

Preventiva, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Pampulha,

AQUACEN, National Reference Laboratory for Aquatic Animal Diseases, Ministry of

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c

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Belo Horizonte, Minas Gerais, Brazil. CEP 31270-901

Fisheries and Aquaculture, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627,

#

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Pampulha, Belo Horizonte, Minas Gerais, Brazil. CEP 31270-901

These authors contributed equally to this article.

Corresponding author: Z. I. P. Lobato. Universidade Federal de Minas Gerais, Escola de Medicina Veterinária, Departamento de Medicina Veterinária Preventiva, Laboratório de Pesquisa em Virologia Animal. Av. Antônio Carlos, 6627, Pampulha, Belo Horizonte, Minas Gerais, Brazil. CEP 31270-901 Phone: +55 31 3409-2129. E-mail: [email protected] 1

ACCEPTED MANUSCRIPT Abstract Bluetongue (BT) is a vector-borne viral disease caused by the Bluetongue virus (BTV), an Orbivirus from the Reoviridae family, affecting domestic and wild ruminants. BTV circulation in Brazil was first reported in 1978, and several serological surveys indicate that the virus is widespread, although with varied prevalence. In 2014, BT outbreaks affected

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sheep flocks in Rio Grande do Sul state, causing significant mortality (18.4%; 91/495) in

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BTV-infected sheep. In total, seven farms were monitored, and one or two sheep from each

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farm that died due to clinical signs of BT were necropsied. Apathy, pyrexia, anorexia, tachycardia, respiratory, and digestive disorders were noted. Additionally, an abortion was

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recorded in one of the monitored farms. The main gross lesions observed were pulmonary edema, anterior-ventral pulmonary consolidation, muscular necrosis in the esophagus and in

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the ventral serratus muscle, and hemorrhagic lesions in the heart. The blood and tissue samples were tested for BTV RNA detection by RT-qPCR targeting the segment 10. Positive

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samples were used for viral isolation. The isolated BTVs were typed by conventional RTPCR targeting the segment 2 of the 26 BTV serotypes, followed by sequencing analysis.

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BTV-1, BTV-4 and BTV-17 were identified in the analyzed samples. Double or triple BTV co-infections with these serotypes were detected. We report the occurrence of BT outbreaks

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related to BTV-1, BTV-4 and BTV-17 infections and co-infections causing clinical signs in sheep flocks in Southern Brazil, with significant mortality and lethality rates.

Key words: Bluetongue virus, Culicoides sp., outbreak, Sheep farm, Brazil

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ACCEPTED MANUSCRIPT 1. Introduction Bluetongue virus (BTV) is a non-enveloped arbovirus, a member of the Reoviridae family, and is the prototype of the genus Orbivirus (Mertens et al. 2004). The viral genome comprises ten segments of double-stranded RNA. Segment-2 (Seg-2) encodes the outercapsid protein, VP2, the most variable BTV protein that contains most of the epitopes that

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interact with neutralizing antibodies, and consequently is the main determinant of the virus

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serotype (Huismans and Erasmus, 1981; Mertens et al., 1989). Currently, 27 different BTV

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serotypes that infect ruminants have been described (Jenckel et al., 2015; Maan et al., 2012). Bluetongue (BT) is a hemorrhagic disease caused by BTV, which affects domestic and

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wild ruminants. Among domestic species, sheep are the most susceptible and the severity of clinical signs can vary according to breed, age, and immune status of the affected flock

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(Maclachlan et al. 2009). The virus replication in endothelial cells of the small vessels results in distinct disease findings associated with vascular injury, such as tissue infarction,

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hemorrhage, vascular leakage, edema, and hypovolemic shock (Maclachlan et al., 2009). BTV is a vector-borne virus, mainly transmitted by biting midges from the genus

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Culicoides. South America (SA) has ideal climatic conditions for the survival and proliferation of Culicoides spp., and as reported by indirect evidence (serological

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investigations), BTV is spread since 1978 all over the continent, with the exception of Uruguay (reviewed by Lobato et al. 2015). Most of the Brazilian territory is BT endemic. Although the BTV serotypes present in the country are poorly known, various serological surveys have shown that the virus is widespread infecting different ruminant species (reviewed by Lobato et al., 2015). Clinical signs of BT have been rarely described despite the detection of a high number of seropositive animals, and several BT cases are underreported due to the presence of very mild clinical signs, which can be mistaken for other similar endemic diseases (Lobato et al. 2015). 3

ACCEPTED MANUSCRIPT Rio Grande do Sul (RS) is the southern-most Brazilian state, bordering Argentina and Uruguay, and is one of the most important states for sheep raising in Brazil. The only BTV serological data from RS was reported in 1999, which indicated only 0.63% and 0.15% of BTV seropositivity for cattle and sheep, respectively (Costa et al. 2006). BT outbreaks affecting sheep flocks in RS state, causing significant mortality in infected sheep, were

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reported in 2014. In this study, we monitored seven BT-affected farms, described the main

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clinical and pathological aspects of the disease in these flocks, and identified the BTV

2. Material and Methods

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2.1 Flock and outbreak characterization

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serotypes involved in the outbreaks.

Outbreaks of BT affecting sheep flocks were reported in five localities in RS, Brazil

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(Taquara -29.6508°,-50.7808°; Fazenda Vilanova -29.5888°,-51.8250°; Viamão -30.0808°,51.0227°; Cachoeira do Sul -30.0388°,-51.0227° and Venâncio Aires -29.6058°,-52.1919°)

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between January and August 2014. We monitored seven farms that were named Farm A–G (Table 1; Figure 1). The epidemiological data were generated in accordance with the

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information collected from the farmers. One or two sheep that died presenting the most common clinical signs from each farm were taken as a representative of the flock and were

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numbered from one to ten (Table 2). 2.2 Sampling and tissue staining Fragments of the liver; kidney; spleen; tracheobronchial, retromandibular, and mesenteric lymph nodes; heart; pulmonary artery; lung; small and large intestine; brain; esophagus; ventral cervical serratus muscle; pro-ventricles; abomasum; tongue; ear; and skin of lip region from ten sheep and the spleen of an aborted fetus (sheep 7, farm E) were collected during necropsy.

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ACCEPTED MANUSCRIPT Tissue fragments were fixed in 10% buffered formalin, routinely processed, and stained with hematoxylin and eosin (H&E) or stored at 4°C. Blood samples were centrifuged at 2000× g for 10 minutes. The red blood cells (RBCs) were washed twice and suspended in phosphate buffered saline (PBS; pH 7.0–7.4). The washed RBCs were diluted ten-fold in sterile distilled water to lyse the cells and homogenized using a vortex. The tissue fragments

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for virus isolation were homogenized in a mortar pestle using sterile sand and diluted ten-fold

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in PBS supplemented with penicillin (200 IU/mL), streptomycin (200 µg/mL), and

2.3 RNA Extraction and BTV detection

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amphotericin B (2.5 µg/mL).

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RNA was extracted from the RBCs and homogenized tissue suspension using Trizol® reagent (Life Technologies Inc. Waltham, Massachusetts, USA) according to manufacturer’s

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instructions or with QIAamp® Viral RNA Mini Kit QIAGEN (Hilden, Germany). The BTV

Batten et al. 2015). 2.4 Virus Isolation

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nucleic acid was detected by RT-qPCR targeting Seg-10 of the virus (Hofmann et al. 2008;

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The homogenized tissue suspension or RBCs from each sheep that showed a lower Ct value were selected for virus isolation. An aliquot of 10-1 dilution of RBC or homogenized

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tissue suspension (0.1–0.2 mL) was used to infect KC cells derived from Culicoides sonorensis midges (Wechsler et al. 1989; Mertens et al. 1996). The infected KC cells were incubated at 28°C for 7 days. Alternatively, for each sample, groups of five embryonated chicken eggs (ECE) were intravenously inoculated. The ECE were candled twice a day to check the viability of embryos (Clavijo et al., 2000). Tissues from hemorrhagic embryos that died after 24 hours of incubation were homogenized and diluted ten-fold in PBS pH 7.0–7.4. The supernatant from the KC passage or the ECE tissue homogenate was inoculated on to the

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ACCEPTED MANUSCRIPT monolayer of VERO cells (ATCC, CCL-81, USA). The flasks were incubated at 37°C and monitored daily under a microscope to check for the cytopathic effects. 2.5 BTV serotyping and phylogenetic analysis The isolated BTV samples were grown in Vero cells and the dsRNA were extracted using Trizol® reagent (Attoui et al., 2000). The BTV typing assay, based on Seg-2

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amplification for each serotype, was performed using the isolated samples (Maan et al., 2012).

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The amplicons were purified using AMpure beads (Beckman Coulter, USA) and sequenced

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according to the instructions of the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, USA). Sequencing reactions were performed on an ABI 3500 Genetic Analyzer

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(Applied Biosystems, USA). Consensus sequences from each RT-PCR product were analyzed and assembled using SeqMan Software 7.1.0 (DNAStar Inc.). The nucleotide sequences were

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compared by BLAST® (https://blast.ncbi.nlm.nih.gov). The consensus sequences were aligned with the BTV Seg-2 reference strains and the sequences available on GenBank that

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presented a higher identity percentage using the ClustalW multiple alignment (Thompson et al., 1994). Phylogenetic tree was constructed using Maximum Likelihood method based on

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the Tamura 3-parameter model, and 1000 bootstrap replicates using MEGA6 software (Tamura et al. 2013).

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3. Results

3.1 Epidemiological and clinical data The disease was observed in the South American sheep breed Santa Inês, and the European sheep breeds Texel, Hampshire Down, and Corriedale. The mortality rates varied from 1.7% (4/230) to 56% (28/50), and lethality rates varied from 44.0% (11/25) to 100% (5/5; 28/28; 4/8; 13/13; 30/30) in the monitored farms (Table 1). Table 2 summarizes the outbreak date occurrence and the main clinical signs observed in affected sheep. Apathy, anorexia, pyrexia, tachycardia, digestive disorders, stiffness of the limbs, lameness and 6

ACCEPTED MANUSCRIPT respiratory symptoms, such as coughing, nasal discharge, tachypnea, and dyspnea were observed clinical signs. In the Farm E, the sheep 7 aborted (Table 2). 3.2 Macroscopic and microscopic lesions The main findings in the ten necropsied sheep were cyanosis of the oral mucosa and tongue (sheep 1–10), and mild dilation and sagging of the esophagus (sheep 4, 5, and 6)

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(Figures 2A and 2C, respectively). Hyperemia and a large amount of refractive inert plant

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fiber materials were found in the nasal cavity in most of the sheep (Figure 2B). The lungs

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were not collapsed showing elastic consistency (Figure 2D) and abundant amounts of foamy liquid were observed in the trachea and bronchi (sheep 1, 4–7, and 9). Multifocal

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consolidation areas were found predominantly in the cranioventral region of the lungs (sheep 1, 3, 4, 8, and 10) and plant fiber material in the bronchi (Figure 2E). No lesions or alterations

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were observed on the hooves of any necropsied sheep.

Necrosis of the muscles in the esophagus was observed in all the necropsied sheep that

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were markedly characterized by eosinophilic myofibers with rounded edges in cross-section

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and sometimes hyper-contracted and segmented with loss of striations (Figure 3A). Macrophage infiltration was found associated with necrotic myofibers (Figure 3B). Although these lesions were present in all the ten necropsied sheep, lesions varied in intensity and in the

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esophageal region (cranial, medial, and caudal). The intensity of the lesions in the esophageal region and in the serratus muscle were scored as absent, discreet, mild, and intense as shown in Table 3. The lip skin region of sheep 5, 6, 7, and 8 showed perivascular infiltration of lymphocytes and multifocal moderate eosinophils associated with discrete mast cell infiltration. Suppurative bronchopneumonia was observed in seven sheep (1–7) characterized by pronounced neutrophil infiltration within the bronchi and bronchioles (Figure 3C) and occasionally within the alveoli (sheep 2 and 3) and interstitial space (sheep 5 and 7). 7

ACCEPTED MANUSCRIPT Sometimes the bronchopneumonia was associated with the presence of plant fiber within bronchioles (aspiration pneumonia) (sheep 1, 3, and 4) (Figure 3D). Thrombosis (sheep 3 and 4), hemorrhage (sheep 2 and 5), fibrin exudation (sheep 4 and 5), edema, and hyperemia were present in all the cases. Tumescent and hypereosinophilic myofibers (hyaline necrosis) often with flocculation

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and fiber fragmentation (Figure 3E) were observed in the ventral cervical serratus muscle

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(sheep 1–8). Additionally, macrophage infiltration was associated with intense fibroblast

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proliferation and collagen synthesis, and multifocal mineralization areas were observed (Figure 3F).

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In the esophagus, slight vasculitis to moderate submucosal edema characterized by infiltration of lymphocytes (sheep 2, 3, 4, 6, and 8) and neutrophils (sheep 2, 4, and 5),

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multifocal hemorrhage in the adventitia, and proliferation of fibrous connective tissue (sheep 3 and 5) was observed (Figure 3G). In sheep 7 and 8, a small amount of myofibers showed

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reduced diameter and central alignment of the nuclei (muscle regeneration). Only sheep 4 had

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basophilic deposition of granular material (mineralization) in myofibers. Ulceration of the esophageal mucosal epithelium was observed in sheep 2–7. Myocardial necrosis with multifocal areas of severe bleeding associated with discrete

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mononuclear inflammatory infiltration was observed in the heart (Figure 3H) (sheep 8). In sheep 5, multifocal areas of severe hemorrhage associated with acute necrosis of cardiomyocytes were observed. Mild multifocal bleeding in the epicardium (sheep 1, 4, and 5) and myocardium, and multifocal thrombosis and severe congestion (sheep 7) were also observed. Multifocal petechiae in the epicardium of the left ventricle were observed in sheep 1, 4, 5, 6, and 7. In the pulmonary artery tunica adventitia, there was moderate bleeding and transmural focal hemorrhagic area measuring 1–3 cm in diameter (sheep 2, 6, 7, and 8).

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ACCEPTED MANUSCRIPT Macroscopic lesions were not noticed on the hooves of any necropsied sheep, and neither the fetus aborted by sheep 7 (farm E) exhibited changes. The aborted fetus did not present any notable histological lesions. 3.3 BTV detection and isolation Nine out of the ten necropsied sheep were positive for BTV by RT-qPCR analysis

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(with Ct value ≤ 35). From each animal, the sample with a lower Ct value was used for virus

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isolation (Table 2). It was possible to recover viable virus from the RBCs from sheep 5 and 6 after one passage in KC cells and from the spleen samples of sheep 2 and 3 after two passages

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in KC cells. Partial sequences of the Seg-2 of BTV-17 (774 bp – MF045074) and BTV-4 (714

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bp – MF045070) were amplified in the isolate sample from the RBCs of the sheep 5 and 6, respectively, from the same farm (Farm D) (Table 2). Partial sequences of the Seg-2 of BTV-

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1 (1502 bp – MF045068) and BTV-4 (1003 bp – MF045071) were amplified in the isolate sample from the spleen of the sheep 2 from farm B, indicating co-infection (Table 2).

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Furthermore, triple co-infection with BTV-1 (1015 bp – MF045069), BTV-4 (1082 bp – MF045072), and BTV-17 (261 bp – MF045073) was detected in the isolate sample from

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spleen of the sheep 3, also from farm B. Table 2 summarizes the C t values obtained in the RTqPCR analysis targeting the BTV Seg-10 and the BTV serotypes detected in the isolated

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samples from each sheep. The phylogenetic tree, constructed based on the partial sequence of segment 2 of BTV isolates obtained in this study, is shown in Figure 4. The analyses of the partial sequences of BTV-17 Seg-2 from the isolates of sheep 3 and 5 indicated a nucleotide similarity of 97%, and nucleotide identity of 85% and 86%, respectively, with BTV-17 isolate from Guadeloupe Island (HQ222825). BTV-17 isolated from the spleen sample of sheep 3 showed 87% nucleotide identity with the BTV-17 isolate from the USA (S72158), and the BTV-17 isolate from sheep 5 presented 88% identity with the French Guiana isolate (JQ436733). The partial Seg-2 sequences of BTV-1 isolates 9

ACCEPTED MANUSCRIPT presented 100% similarity, and shared 92% of nucleotide identity with BTV-1 isolate from the USA (KX164020) and 88% to 89% with French Guiana BTV-1 isolate (KY049854 and KY049844, respectively). The partial Seg-2 sequences from BTV-4 obtained in this study showed 100% similarity between them and with the BTV-4 vaccine strain from South Africa (KM233615). The analyses of partial Seg-2 sequences suggest that all isolates belong to

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Western topotypes (Maan et al., 2012).

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4. Discussion

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The diagnosis of BT infection in the outbreaks described herein was based on clinical symptoms, gross lesions, and microscopic findings, and was confirmed by RT-PCR and virus

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isolation. Sheep 1 presented respiratory signs and stasis of the rumen (Table 2), clinical signs that can be observed in various metabolic and infectious diseases. Although clinical

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symptoms and gross lesions were suggestive of BT, the laboratory diagnostic did not confirm BTV infection. Therefore, the clinical signs observed in sheep 1 are not a consequence of BT,

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and a BTV outbreak was not confirmed in Farm A. The low seroprevalence of BTV in RS compared to that in other regions of Brazil implies a high rate of susceptible animals in this

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area (Costa et al. 2006). Few cases of clinical diseases had been described in Brazil; most of them were in the Southern area, where BT is epidemic. The first BT outbreak was reported in

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Parana state in April 2001 and February 2002 (Clavijo et al., 2002). Sheep and goats showed clinical signs consistent with those of BT, and some of them died. Virus neutralization (VN) test was used to type the BTV isolate, and BTV-12 was identified (Clavijo et al. 2002). In March and April 2009, outbreaks of BTV affecting sheep were reported in RS in two different farms, and BTV-12 was identified as the serotype involved in these outbreaks (Antoniassi et al., 2010). The clinical and necropsy findings presented here are consistent with the acute form of the disease, due to their clinical presentation (Kusiluka & Kambarage 1996; Antoniassi et al., 10

ACCEPTED MANUSCRIPT 2010; Balaro et al., 2014). Edema and congested and cyanotic tongue are common necropsy findings in BTV-affected sheep (Brown et al. 2007). Erosions and ulcerations along the edge of the tongue, oral mucosa, esophagus, and pre-stomachs are also described in BTV-infected sheep (Antoniassi et al., 2010; Balaro et al., 2014). However, we observed only ulceration of the esophageal epithelium of two sheep, without involving the oral cavity and pre-stomachs.

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Hemorrhagic lesions in the pulmonary artery were prevalent in sheep. Generally, bleeding in

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the tunica media and the surface or intimal and adventitia up to 3.0 cm, are considered

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characteristic features of BTV infection (Maclachlan et al. 2009; Balaro et al. 2014). Pulmonary edema, a common alteration in BTV-infected sheep (Balaro et al. 2014), was

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frequently observed this study. Aspiration pneumonia, another pulmonary change, which is also reported in BTV infection, is related to aspiration of rumen contents after reflux episodes

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resulting from severe injury of the esophageal muscles (Antoniassi et al., 2010). Although suppurative bronchopneumonia was histologically observed in seven animals, only three had

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aspiration pneumonia, probably due to the sample collection site not containing vegetable fiber specimens.

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In this study, three relevant events must be outlined. First is the period of disease occurrence from January to August, including cases reported during winter (August). The

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climate of RS state is classified as subtropical. The warmest months are January and February, with average temperatures above 22°C, and the coldest is July with temperatures ranging from 18°C to −3°C. Rainfalls during the year are well distributed. However, climatic changes have been clearly noticed in recent decades. Analyses of the climate variability in RS during the period 1931–2007, indicates a decrease in amplitude between the maximum and minimum temperatures in locations situated in every geomorphological compartment in the state (Rossato, 2011). However, this fact was identified mostly in the locations in which the minimum temperature increase is greater than the maximum, particularly in the southwest, 11

ACCEPTED MANUSCRIPT central, and northeast portions, indicating a small but considerable reduction of cold cores in the state. The analysis of the total annual rain levels revealed the elevation of such levels with a trend towards concentration (Rossato, 2011). Arthropods are highly sensitive to environmental and seasonal temperature changes; the range of vector-borne diseases can be highly affected by climatic changes. An investigation on the relationships between temporal

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variability in Culicoides sp. distribution in the southeast area of RS from September 2008 to

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September 2010 indicated the presence of C. insignis throughout the sampling period; C.

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venezuelensis was associated with the period of El Niño influences, and C. caridei was associated with La Niña periods (Carrasco et al., 2014). The authors showed that variable

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humidity, temperature, precipitation, and wind speed influenced the temporal variations in the species. In South America, Central America, and the Caribbean basin, C. insignis is indicated

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as the major vector responsible for transmission of multiple BTV serotypes (Maclachlan et al. 2013), and this species is most likely related to the BTV occurrence in RS, even during

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winters.

The second interesting fact is the occurrence of BTV-1, BTV-4, and BTV-17

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circulation associated with clinical signs in Southern Brazil. In South America, BTV-1 and BTV-17 were detected and isolated from asymptomatic cattle from French Guiana in 2013

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(Viarouge et al., 2014). Phylogenetic analysis of the BTV-1 and BTV-17 isolated in the outbreaks described herein with the BTV-1 and BTV-17 isolates from South American indicated a similarity of 88%, indicating that the Brazilian isolates may form a separate cluster. BTV-4 circulation in Brazil was first identified in 1980, when this serotype was isolated and identified in asymptomatic Zebu cattle exported from Brazil to the USA (Groocock and Campbell, 1982). Then, only in 2011 and 2013, there were reports of BT clinical disease in sheep associated with BTV-4 in the Southeast region of Brazil (Balaro et al., 2014; Lima et al., 2016). In Argentina, BTV-4 was isolated from asymptomatic cattle in 12

ACCEPTED MANUSCRIPT area located at the boundary of RS (Legisa et al., 2013). The Argentinian BTV-4 strains group within a clade, and demonstrate 90% similarity with BTV-4 detected in the outbreaks in RS. The two BTV-4 strains isolated in the outbreaks in RS were closely related to the vaccine strain from South Africa (Figure 4). The use of BTV attenuated vaccines is prohibited in Brazil and the introduction of a vaccine strain in the country cannot be confirmed. A silently

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circulation of BTV-4 in cattle in Southeast region of Brazil is suspected as 86% of the herd in

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Sao Paulo state is positive by serum neutralization assay (Nogueira et al., 2016). Despite the

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previous detection of BTV-4 in Brazil, there is only two sequences of BTV-4 detected in asymptomatic bulls available in GenBank. The phylogenetic analyses of these samples

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presented only 90% similarity with the BTV-4 isolates from the outbreak described herein. The third interesting fact is the identification of double and triple co-infection with

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different serotypes in the flock. The co-circulation of different BTV strains within the same animal and flock increases the risk of re-assortment events, which are the main mechanisms

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used by segmented viruses to generate novel virus strains (Nomikou et al., 2015). Whether the viruses were recently introduced in the region by a vector or infected animal or whether they

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could only now be detected is not possible to conclude with the information obtained until now. Full genome analyses of the BTV-17 isolate from sheep 5, from the outbreak occurred in

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Cachoeira do Sul, RS, reveals that the strain has emergent through reassortment events between other BTV strains circulating in the Americas (Matos et al., 2016). BTV infection creates economic impacts related to animal deaths, loss of body condition, decreased milk production, infertility, and abortion, and determining indirect costs associated with restrictions on international trade (Legisa et al., 2013). Thus, the disease is of great importance to RS state. The virus and vector circulation combined with a high number of susceptible sheep and highly pathogenic BTV strains should trigger greater losses in small ruminant production in upcoming years. 13

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Submission declaration This research study has not been published previously elsewhere.

Conflict of interest statement

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The authors certify that there is no conflict of interest with any financial organization.

Acknowledgments

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This work was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

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(CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil (PVE, Grant No. 2392/2013). Lobato, Z. I.P. has a CNPq fellowship. We thank Peter

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Mertens and Kyriaki Nomikou for the collaboration and Jason F. Siegel for the English revision.

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5. References

Antoniassi, N.A.B., Pavarini, S.P., Henzel, A., Flores, E.F., Driemeier, D., 2010.

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Aspiration pneumonia associated with oesophageal myonecrosis in sheep due to BTV infection in Brazil. Vet. Rec. 166, 52–53. doi:10.1136/vr.b4775

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Antoniassi, N.A.B., Pavarini, S.P., Ribeiro, L.A.O., Silva, M.S., Flores, E.F., Driemeier, D., 2010. Alterações clínicas e patológicas em ovinos infectados naturalmente pelo vírus da língua azul no Rio Grande do Sul. Pesqui. Vet. Bras. 30, 1010–1016. doi:10.1590/S0100-736X2010001200002 Attoui, H., Billoir, F., Cantaloube, J.F., Biagini, P., De Micco, P., De Lamballerie, X., 2000. Strategies for the sequence determination of viral dsRNA genomes. J. Virol. Methods 89, 147–158. doi:10.1016/S0166-0934(00)00212-3 Balaro, M.F.A., Dos Santos Lima, M., Del Fava, C., de Oliveira, G.R., Pituco, E.M., 14

ACCEPTED MANUSCRIPT Brandão, F.Z., 2014. Outbreak of Bluetongue virus serotype 4 in dairy sheep in Rio de Janeiro, Brazil. J. Vet. Diagn. Invest. 26, 567–570. doi:10.1177/1040638714538020 Batten, C., Frost, L., Oura, C., 2015. Real-time reverse transcriptase PCR for the detection of bluetongue virus. Methods Mol. Biol. 1247, 125–131. doi:10.1007/978-1-4939-20044_8

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Carrasco, D., Felippe-Bauer, M.L., Dumont, L.F., D’Incao, F., 2014. Abundance of

RI

Culicoides (Diptera, Ceratopogonidae) species in salt marshes of the Patos Lagoon

SC

estuary, Rio Grande do Sul, Brazil: Influence of climatic variables. Pan-Am. J. Aquat. Sci. 9, 8–20.

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Clavijo, A., Sepulveda, L., Riva, J., Pessoa-Silva, M., Tailor-Ruthes, A., Lopez, J.W., 2002. Isolation of bluetongue virus serotype 12 from an outbreak of the disease in South

MA

America. Vet. Rec. 151, 301–302.

Clavijo, A., Heckert, R.A., Dulac, G.C., Afshar, A., 2000. Isolation and identification of

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bluetongue virus. J. Virol. Methods 87, 13–23. Costa, J.R.R., Lobato, Z.I.P., Herrmann, G.P., Leite, R.C., Haddad, J.P.A., 2006.

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Prevalência de anticorpos contra o vírus da língua azul em bovinos e ovinos do sudoeste e sudeste do Rio Grande do Sul. Arq. Bras. Med. Vet. e Zootec. 58, 273–275.

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doi:10.1590/S0102-09352006000200017 Groocock, C.M., Campbell, C.H., 1982. Isolation of an exotic serotype of bluetongue virus from imported cattle in quarantine. Can. J. Comp. Med. 46, 160–164. Hofmann, M., Griot, C., Chaignat, V., Perler, L., Thur, B., 2008. Bluetongue disease reaches Switzerland. Schweiz. Arch. Tierheilkd. 150, 49–56. doi:10.1024/00367281.150.2.49 Huismans, H., Erasmus, B.J., 1981. Identification of the serotype-specific and groupspecific antigens of bluetongue virus. Onderstepoort J. Vet. Res. 48, 51–58. 15

ACCEPTED MANUSCRIPT Jenckel, M., Breard, E., Schulz, C., Sailleau, C., Viarouge, C., Hoffmann, B., Hoper, D., Beer, M., Zientara, S., 2015. Complete coding genome sequence of putative novel bluetongue virus serotype 27. Genome Announc. 3, e00016-15. doi:10.1128/genomeA.00016-15 Legisa, D., Gonzalez, F., De Stefano, G., Pereda, a., Dus Santos, M.J., 2013. Phylogenetic

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analysis of bluetongue virus serotype 4 field isolates from Argentina. J. Gen. Virol. 94,

RI

652–662. doi:10.1099/vir.0.046896-0

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Lima, P.A., Utiumi, K.U., Yumi, K., Nakagaki, R., Biihrer, D.A., Albuquerque, A.S., Rezende, F.S., Carolina, A., Matos, D., Inês, Z., Lobato, P., Driemeier, D., Peconick,

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A.P., Varaschin, M.S., Raymundo, D.L., 2016. Diagnoses of ovine infection by the serotype-4 bluetongue virus on Minas Gerais, Brazil. Acta Sci. Vet. 44, 1–5.

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Lobato, Z.I.P., Guedes, M.I.M.C., Matos, A.C.D., 2015. Bluetongue and other orbiviruses in South America: gaps and challenges. Vet. Ital. 51, 253–262.

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doi:10.12834/VetIt.600.2892.1

Maan, N.S., Maan, S., Belaganahalli, M.N., Ostlund, E.N., Johnson, D.J., Nomikou, K.,

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Mertens, P.P.C., 2012. Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2. PLoS

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One 7, 1–9. doi:10.1371/journal.pone.0032601 Maclachlan, N.J., Drew, C.P., Darpel, K.E., Worwa, G., 2009. The pathology and pathogenesis of bluetongue. J. Comp. Pathol. 141, 1–16. doi:10.1016/j.jcpa.2009.04.003 Maclachlan, N.J., Wilson, W.C., Crossley, B.M., Mayo, C.E., Jasperson, D.C., Breitmeyer, R.E., Whiteford, A.M., 2013. Novel serotype of bluetongue virus, western North America. Emerg. Infect. Dis. 19, 665–666. doi:10.3201/eid1904.120347 Matos, A.C.D., Rosa, J.C.C., Nomikou, K., Guimarães, L.L.B., Costa, É.A., Guedes, M.I.M.C., Driemeier, D., Lobato, Z.I.P., Mertens, P.P.C., 2016. Genome sequence of 16

ACCEPTED MANUSCRIPT bluetongue virus serotype 17 isolated in Brazil in 2014. Genome Announc. 4, e01161-16. doi:10.1128/genomeA.01161-16 Mertens, P.P., Burroughs, J.N., Walton, A., Wellby, M.P., Fu, H., O’Hara, R.S., Brookes, S.M., Mellor, P.S., 1996. Enhanced infectivity of modified bluetongue virus particles for two insect cell lines and for two Culicoides vector species. Virology 217, 582–593.

PT

doi:10.1006/viro.1996.0153

RI

Mertens, P.P., Pedley, S., Cowley, J., Burroughs, J.N., Corteyn, A.H., Jeggo, M.H.,

SC

Jennings, D.M., Gorman, B.M., 1989. Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype. Virology 170, 561–565.

NU

Mertens, P.P.C., Diprose, J., Maan, S., Singh, K.P., Attoui, H., Samuel, A.R., 2004. Bluetongue virus replication, molecular and structural biology. Vet. Ital. 40, 426–437.

MA

Nogueira, A.H.C., Stefano, E., Martins, M.S.N., Okuda, L.H., Lima, M.S., Garcia, T.S., Hellwig, O.H., Lima, J.E.A., Savini, G., Pituco, E.M., 2016. Prevalence of Bluetongue

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virus serotype 4 in cattle in the State of Sao Paulo, Brazil. Vet. Ital. 52, 319-323. Nomikou, K., Hughes, J., Wash, R., Kellam, P., Breard, E., Zientara, S., Palmarini, M.,

EP T

Biek, R., Mertens, P., 2015. Widespread reassortment shapes the evolution and epidemiology of bluetongue virus following european invasion. PLoS Pathog. 11,

AC C

e1005056. doi:10.1371/journal.ppat.1005056 Rossato, M.S., 2011. The climates of Rio Grande do Sul: variability, trends and typology [Portuguese]. (Thesis). Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. 1-240. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. doi:10.1093/molbev/mst197 Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: Improving the 17

ACCEPTED MANUSCRIPT sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673– 4680. doi:10.1093/nar/22.22.4673 Viarouge, C., Lancelot, R., Rives, G., Bréard, E., Miller, M., Baudrimont, X., Doceul, V., Vitour, D., Zientara, S., Sailleau, C., 2014. Identification of bluetongue virus and

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Microbiol. 174, 78–85. doi:10.1016/j.vetmic.2014.09.006

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epizootic hemorrhagic disease virus serotypes in French Guiana in 2011 and 2012. Vet.

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Wechsler, S.J., McHolland, L.E., Tabachnick, W.J., 1989. Cell lines from Culicoides variipennis (Diptera: Ceratopogonidae) support replication of bluetongue virus. J.

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Invertebr. Pathol. 54, 385–393. doi:10.1016/0022-2011(89)90123-7

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ACCEPTED MANUSCRIPT Table 1 Localities and epidemiological aspects of the seven monitored sheep flocks. Farm

Morbidity rate (% )

Mortality rate (% )

Lethality rate (% )

Taquara

A

25.0% (5/20)

25.0% (5/20)

100.0% (5/5)

Fazenda Vilanova

B

56.0% (28/50)

56.0% (28/50)

100.0% (28/28)

Viamão

C

3.5% (8/230)

1.7% (4/230)

50.0% (4/8)

Cachoeira do Sul

Da

28.9% (13/45)

28.9% (13/45)

Cachoeira do Sul

E

31.3% (25/80)

13.8% (11/80)

Viamão

F

No data

No data

Venâncio Aires

G

42.9% (30/70)

42.9% (30/70)

No data

100.0% (30/30)

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44.0% (11/25)

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The animals died after participation in an exhibition

100.0% (13/13)

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Locality

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ACCEPTED MANUSCRIPT Table 2 Identification of sheep clinical aspects and sample isolates from the monitored sheep flocks.

2 B

Texel

3-5 3

C

Hampshire Down

7

4

D

Texel

7-10

Dyspnea, drooling, stiffness of the limbs, especially the hind limbs Apathy, dysphagia, coughing, excessive salivation, cyanosis, greenish serous nasal discharge and lameness

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5

Apathy, anorexia, depression and greenish serous nasal secretion, progressing to dyspnea

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6

Texel

5-7

7

F

Corriedale

5

8

Apathy, anorexia and dyspnea

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E

Apathy, dysphagia, drooling, coughing, greenish nasal mucosa, abortion

9 G

Texel

1-3 10

*

Mean value of duplicate RT-qPCR reaction

a

BTV isolate. Seg-2 partial sequence.

Dyspnea, pyrexia, salivation, nasal discharge and lameness

kidney (40.37), liver (No Ct), lung (No Ct)

Not identified b

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5

Serotypes

Jan/2014

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1

Tachypnea, tachycardia, pyrexia and rumen stasis, progressing to dyspnea and cyanosis

Specimen (BTV Seg10 RTqPCR Ct value*)

Date of sampling

Jan/2014

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Santa Inês

Clinical signals

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A

Breed

Necropsied sheep ID number

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Farm

Clinical course (Days)

Feb/2014

spleen a (26.43); liver (26.66) spleen a (23.36); kidney (23.82)

BTV-1 and BTV-4 BTV-1, BTV-4 and BTV-17

April/2014

spleen (24.54)

Not identified b

April/2014

RBCa (27.24)

BTV-17

April/2014

RBCa (21.14)

BTV-4

May/2014

No data

spleen (26.9); kidney (32.24); liver (32.18); placenta (32.24); liver of aborted fetus (36.68) kidney (26.48); lung (27.16)

Not identified b

Not identified b

Aug/2014

RBC (32.2)

Not identified b

Aug/2014

RBC (31.7)

Not identified b

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It was not possible to isolate the virus and identify the BTV serotype.

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ACCEPTED MANUSCRIPT Table 3 Score of muscular esophageal necrosis in the cranial, medial, caudal, and cervical ventral serratus muscle. Necropsied sheep ID number

Cranial

Medial

Caudal

1

+

+

+

+

2

+

++

+

+++

3

+

+

+

+

4

+++

+++

+++

+

5

+++

+++

+++

+++

6

0

++

+++

+++

7

+

++

++

+

8

+++

+++

+++

+++

9

+

+

+

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Cervical ventral serratus muscle

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Esophageal region

0 0

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10 0 + + 0 = absent, + = discrete, ++ = mild, +++ = intense.

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ACCEPTED MANUSCRIPT Figure 1 Map of Rio Grande do Sul state, Brazil indicating the location of the seven monitored farms.

Figure 2 Anatomical and pathological observations in BTV-infected sheep. (A) Cyanotic oral mucosa. (B) Nasal cavity with plant material. (C) Esophagus with mild dilation and

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appearance of sagging. (D) Lungs not collapsed with consolidation areas. (E) Bronchi with

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plant material.

Figure 3 Histological observations in organs from BTV-infected sheep displaying severe

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clinical disease. (A) Esophagus with moderate multifocal macrophage infiltration in the muscle layer. (B) Esophagus with macrophage infiltration between muscle fibers and

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multifocal necrosis of myofibers. (C) Lung with suppurative bronchopneumonia. (D) Bronchopneumonia associated with plant fiber within bronchioles. (E) Multifocal macrophage

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infiltration with severe multifocal necrosis of myofibers. (F) Serratus muscle macrophage infiltration with fibroblast proliferation and multifocal mineralization areas. (G) Esophagus

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with marked macrophages infiltration, necrosis, and myofiber fragmentation. (H) Heart with

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multifocal areas of bleeding associated with discrete mononuclear inflammatory infiltration.

Figure 4 Molecular phylogenetic analysis tree using Maximum Likelihood method based on the Tamura 3-parameter model, and 1000 bootstrap replicates between partial sequence of Segment-2 of the isolates from Rio Grande do Sul (black squares) and close related strains available on Genbank (https://www.ncbi.nlm.nih.gov/). The percentage of trees in which the associated taxa clustered together is shown next to the branches. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013).

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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ACCEPTED MANUSCRIPT Highlights Outbreak of Bluetongue hemorrhagic disease in seven sheep farms in Rio Grande do Sul state Brazil.



The sheep presented classical clinical signs consistent with the acute form of the disease.



Identification of two serotypes BTV-1 and BTV-17, not previously reported in the country, associated to BTV-4.



Co-circulation and co-infections of BTV serotypes 1, 4 and 17.

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