Method validation, reference values, and characterization of acute-phase protein responses to experimentally induced inflammation and bluetongue virus infection in the Iberian ibex.

BACKGROUND: Acute phase protein (APP) concentrations can change due to inflammation and be used to monitor disease in the Iberian ibex (Capra pyrenaica). OBJECTIVES: This study aimed to validate Haptoglobin (Hp) and serum amyloid A (SAA) analytes, establish reference values, and characterize Hp and SAA responses in the Iberian ibex after experimentally induced inflammation and experimental bluetongue virus (BTV) infection. METHODS: Sera from 40 free-ranging box-trapped ibexes were used to establish Hp and SAA reference values. Six healthy ibexes were subcutaneously injected with 5 mL of turpentine, then, blood samples were taken, and clinical evaluations were performed on days 0, 1, 2, 3, 4, 7, and 14 postinjection. Another seven ibexes were challenged with BTV. Serum Hp and SAA concentrations were quantified using commercial assays following the manufacturer's instructions. RESULTS: Intra-assay precision and linearity were acceptable for both Hp and SAA. Intra-assay variation for high and low concentration of Hp and SAA were 9.74% and 17.31% and 16.49% and 12.89%, respectively. Inter-assay variation was higher for the low APP concentrations. Reference values for the healthy Iberian ibexes were (median, minimum, and maximum values) 0.2 (0.12-0.64) g/L for Hp and 4.74 (0.05-29.54) mg/L for SAA. Both Hp and SAA acted as a moderate and a major APP, respectively, and each could distinguish animals with turpentine-induced inflammation from those without. Hp and SAA did not change in asymptomatic BTV-infected animals. CONCLUSION: This study validated Hp and SAA analytes and provided basal reference values for these analytes in the Iberian ibex. Both APPs were able to discriminate between healthy and diseased Iberian ibexes animals during turpentine-induced inflammatory processes.

Multiple bluetongue virus serotypes causing death in Brazilian dwarf brocket deer (Mazama nana) in Brazil, 2015-2016.

Bela Vista Biological Sanctuary (RBV) is a protected area of Itaipu Binacional, a hydroelectric power company located on the border of Brazil and Paraguay. A captive population of Brazilian dwarf brocket deer (Mazama nana, Cervidae, Artiodactyla) is maintained for conservation purposes. Despite the reproductive success of the animals, outbreaks of a fatal hemorrhagic disease have been registered over the years, compromising conservation efforts. In order to identify the etiological agents of these hemorrhagic diseases, 32 captive Brazilian dwarf brockets were sampled to investigate bluetongue virus (BTV), epizootic hemorrhagic disease (EHD), and adenovirus hemorrhagic disease (AHD), in 2015. Only one deer (1/32; 3.12%) was seropositive for BTV. After this survey, five animals died in the early autumn of 2015 and 2016, again presenting clinical signs of hemorrhagic disease. Using RT-qPCR, RT-PCR and DNA sequencing, five BTV serotypes (3, 14, 18, 19, and 22) were identified in blood and tissues collected during necropsies. These BTV serotypes had not been previously described or isolated in Brazil, either in wild or domestic ruminants. Additionally, differential diagnosis was performed for EHD and AHD, but all samples were negative for both diseases. The multiple distinct BTV serotypes identified in these outbreaks resulted in a high lethality (100%) of Brazilian dwarf brockets and indicated that various BTV serotypes are circulating in the area.

A competitive ELISA for detection of group specific antibody to bluetongue virus using anti-core antibody.

Bluetongue virus (BTV) is transmitted by biting midges, which infects domestic and wild ruminants. In present study, a competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of serogroup-specific antibodies against VP7 protein of BTV has been developed. The assay measures the competition between a group specific antibody against core protein of BTV and a test serum to an optimized concentration of BTV recombinant-VP7 (r-VP7) antigen. Serum samples (n = 895) collected from small and large ruminants were used to optimize the C-ELISA. Percent inhibition (PI) values were used for estimation of the cut-off value for the C-ELISA. On receiver operator characteristic (ROC) analysis, different cut-off values along with their diagnostic sensitivity (DSn) and diagnostic specificity (DSp) were obtained. Among these, >50% PI value was accepted as cut-off at which DSn and Dsp was achieved as 97.6% and 98.0% respectively, at >95% confidence interval. Results show the present C-ELISA assay described to be sensitive, specific and reliable and could be adopted for serological investigation of small and large ruminants.

Alterações clínicas e patológicas em ovinos infectados naturalmente pelo vírus da língua azul no Rio Grande do Sul / Clinical and pathological changes in sheep naturally infected with bluetongue virus in Rio Grande do Sul, Brazil

Língua azul (LA) é uma doença causada pelo vírus da língua azul (VLA) e transmitida por vetores do gênero Culicoides. Estudos sorológicos têm demonstrado a ampla presença do vírus no Brasil; entretanto, informações clínicas da LA na América do Sul são limitadas. Esse trabalho descreve alterações clínico-patológicas em ovinos acometidos pela LA no Sul do Brasil. Em dois surtos, em propriedades distintas, 15 ovinos apresentaram como principais sinais clínicos hipertermia, apatia, aumento de volume da face e região submandibular, dificuldade de deglutição com regurgitação, secreção nasal mucopurulenta esverdeada, alterações respiratórias, além de acentuada perda de peso e erosões na mucosa oral. Os achados de necropsia em seis ovinos afetados incluíram edema subcutâneo na face e região ventral do tórax, secreção nasal esverdeada, esôfago dilatado preenchido por grande quantidade de conteúdo alimentar, pulmões não colabados com áreas consolidadas anteroventrais, bem como luz da traquéia e brônquios preenchida por espuma misturada com conteúdo alimentar. No coração e base da artéria pulmonar, havia focos de hemorragia. Histologicamente, as principais alterações observadas ocorriam no tecido muscular cardíaco e esquelético, especialmente no esôfago e consistiam de lesões bifásicas caracterizadas por degeneração/necrose hialina e flocular de miofibras associadas com micro-calcificação e infiltrado inflamatório mononuclear. Pneumonia aspirativa associada à presença de material vegetal e bactérias na luz de brônquios também foi observada. O diagnóstico de LA foi confirmado pela detecção do genoma viral por duplex RT-PCR em amostras de sangue de animais afetados, seguido da identificação do VLA, sorotipo 12 por sequenciamento.

Bluetongue (BT) is a disease caused by bluetongue virus (BTV) and transmitted by vectors of the genus Culicoides. Serological studies have demonstrated the widespread presence of the virus in Brazil, however, clinical information of BT in South America are limited. This article describes clinical and pathological changes observed in sheep naturally infected by BTV in southern Brazil. In two outbreaks on different farms, 15 sheep showed clinical signs such as severe hyperthermia, apathy, swelling of the face and submandibular area, difficulty in swallowing with regurgitation, greenish mucopurulent nasal secretion, severe weight loss, and erosions in the oral mucosa. Necropsy findings in six sheep included subcutaneous edema of the face and ventral region of the chest, greenish nasal discharge, and dilated esophagus filled with abundant food contents, collapsed lungs with areas of anteroventral consolidation, and trachea and bronchi filled by foamy material mixed with food. In the heart and base of the pulmonary artery there were foci of hemorrhage. Histologically, the main changes were in cardiac and skeletal muscles and consisted of biphasic lesions characterized by hyaline and floccular degeneration/necrosis of myofibers associated with micro-mineralization and mononuclear cell infiltration. Pneumonia associated with the presence of organic matter and bacteria in the lumen of the bronchi was also observed. The diagnosis of BT was confirmed by detection of the viral genome by duplex RT-PCR in blood of affected animals, followed by the identification of BTV, serotype 12 by nucleotide sequencing.

A rapid assay for detecting antibody against Bluetongue virus with a latex agglutination test using recombinant VP7 antigen.

A latex agglutination test (LAT) for detecting antibody against Bluetongue virus (BTV) in ruminants was developed using latex beads coupled with recombinant VP7 protein. Compared with competitive enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of the LAT were 99.0% and 93.0%, respectively. There was excellent agreement between the results obtained by competitive ELISA and the LAT (kappa = 0.930). Because it is rapid and easy to use, the LAT could be used for BTV antibody detection, especially for screening many serum samples.

Pathological, serological, and virological findings in sheep infected simultaneously with Bluetongue, Peste-des-petits-ruminants, and Sheeppox viruses.

In this study, pathological, serological and virological examinations were performed on 15 sheep from a flock of 250 sheep and lambs that suffer from simultaneous naturally occurring BTV, PPRV and SPV outbreaks. SPV was diagnosed macroscopically and histopathologically, BTV was diagnosed by ELISA, and PPRV was diagnosed pathologically and by ELISA. Clinically fever, diarrhea, depression, polypnea, conjunctivitis, lacrimation, rhinitis, erosive stomatitis, edema of eyelids, photophobia, cutaneous eruption with erythematous areas especially noticeable in wool-free parts of the body and axilla lesions evolving into papules were observed. At necropsy, the most effected organs were lungs and gut. Subepicardial hemorrhages were also commonly seen. While typical pox lesions were observed in some lambs, usually fibrinous pleuropneumonia was more prominent lung lesion. SPV and PPRV lesions were seen at the histopathological examination of the lesioned tissues, BT lesions were mild than SPV and PPRV microscopically. Serum and leukocyte samples of 15 animals were examined for PPRV and BTV by ELISA; 5 samples were positive for PPRV and 6 BTV, 4 were positive for both PPRV and BTV simultaneously. One hundred animals died, most were lambs. Mortality rates were 100% in lambs and 80% in the herd.

The role of endothelial cell-derived inflammatory and vasoactive mediators in the pathogenesis of bluetongue.

Bluetongue is an insect-transmitted disease of sheep and wild ruminants that is caused by bluetongue virus (BTV). Cattle are asymptomatic reservoir hosts of BTV. Infection of lung microvascular endothelial cells (ECs) is central to the pathogenesis of BTV infection of both sheep and cattle, but it is uncertain as to why sheep are highly susceptible to BTV-induced microvascular injury, whereas cattle are not. Thus, to better characterize the pathogenesis of bluetongue, the transcription of genes encoding a variety of vasoactive and inflammatory mediators was quantitated in primary ovine lung microvascular ECs (OLmVECs) exposed to BTV and/or inflammatory mediators. BTV infection of OLmVECs increased the transcription of genes encoding interleukin- (IL) 1 and IL-8, but less so IL-6, cyclooxygenase-2, and inducible nitric oxide synthase. In contrast, we previously have shown that transcription of genes encoding all of these same mediators is markedly increased in BTV-infected bovine lung microvascular ECs and that BTV-infected bovine ECs produce substantially greater quantities of prostacyclin than do sheep ECs. Thus, sheep and cattle were experimentally infected with BTV to further investigate the role of EC-derived vasoactive mediators in the pathogenesis of bluetongue. The ratio of thromboxane to prostacyclin increased during BTV infection of both sheep and cattle, but was significantly greater in sheep (P = 0.001). Increases in the ratio of thromboxane to prostacyclin, indicative of enhanced coagulation, coincided with the occurrence of clinical manifestations of bluetongue in BTV-infected sheep. The data suggest that inherent species-specific differences in the production and activities of EC-derived mediators contribute to the sensitivity of sheep to BTV-induced microvascular injury.

Isolation and identification of bluetongue virus.

Bluetongue virus (BTV) is an arthropod-borne orbivirus that infects sheep, wild ruminants and occasionally cattle. Detection and specific identification of BTV is a multistep process. The first step involves the isolation of the virus from the animal's blood or other tissues, followed by inoculation of embryonating chicken eggs (ECE). After the virus has been amplified in ECE, it is passaged into BHK-21 cell culture for subsequent replication and identification. The virus is then amplified further and identified in microtiter plates by the immunoperoxidase assay using a group specific monoclonal antibody. Finally, the viral isolate is typed by a virus neutralization test.

Experimentally induced bluetongue virus infection in white-tailed deer: coagulation, clinical pathologic, and gross pathologic changes.

Ten yearling white-tailed deer (Odocoileus virginianus) were inoculated with bluetongue virus serotype 17. Two yearling white-tailed deer were inoculated with sonicated heparinized noninfected blood and served as controls. Clinical signs of bluetongue virus infection included increased rectal temperature, erythema, facial edema, coronitis, and stomatitis. By postinoculation day (PID) 8, excessive bleeding and hematoma formation at venipuncture sites, dehydration, and diarrhea developed. At necropsy, the most consistent findings were oral lesions and widespread hemorrhage, which ranged from petechia to massive hematoma formation. Bluetongue virus caused progressive prolongation of activated partial thromboplastin time and prothrombin time, and progressive reduction of Factors VIII and XII plasma activities beginning on PID 6. A progressive decrease in platelet numbers also developed on PID 6. Changes in platelet size were not detected. Mean thrombin time was shortened, but prolongation developed in 1 deer. Mean fibrinogen concentration and Factor V plasma activity initially increased and then decreased, but remained above preinoculation values. Factor V activity was low in a few deer. Results of screening tests for inhibitors of the intrinsic coagulation system were positive in 2 deer. High concentrations of fibrin(ogen) degradation products were first detected between PID 3 and 6. Hematologic changes included leukopenia, lymphopenia, neutrophilia, and low total plasma protein concentration. Differences in PCV, hemoglobin concentration, or RBC counts were not detected between infected and control deer. Serum total bilirubin concentration increased by PID 6, primarily because of increased unconjugated bilirubin concentration. Mild to severe increases in serum aspartate transaminase activity were accompanied by more marked increases in creatine kinase activity. Indirect Coombs test results were negative in all deer.(ABSTRACT TRUNCATED AT 250 WORDS)

An electron microscopic study of blood cells from calves experimentally infected with bluetongue virus.

Cellular elements from blood samples of calves experimentally inoculated with a quadrivalent mixture of bluetongue virus (BTV) types 10, 11, 13 and 17 were examined by transmission electron microscopy (TEM). Virus-like particles were observed within cytoplasmic vacuoles of infected agranular leukocytes from inoculated calves on postinoculation day (PID) 14. No virus-like particles were seen associated with other cellular blood elements nor were they observed in blood samples obtained prior to virus inoculation. The intravacuolar viral particles were 60nm in diameter, had a cockleburr appearance and lacked the outer polypeptide coats of mature virions. Increased cytoplasmic vacuolation was the most noticeable change in the infected cells. BTV infection was confirmed by viral isolation and serological testing. Pyrexia was the only consistent clinical sign seen in the viremic calves.