Antiviral Properties of Streptomyces tuirus DBZ39 Mediated Gold Nanoparticles Against Bluetongue virus.

<b>Background and Objective:</b> The proposed study involves the approach from the point of anti-viral activity of gold nanoparticles against the <i>Bluetongue virus</i>. Among viral diseases, Bluetongue is regarded as an economically scouring disease. Neither a vaccine nor an antiviral drug is available for the prevention or treatment of this disease. The antiviral activity of gold nanoparticles synthesized by a novel isolate of <i>Streptomyces tuirus</i> DBZ39 is the breakthrough of the study. <i>Streptomyces tuirus </i>DBZ39, a novel isolate obtained from alkaline soil was proved to be efficient actinomycetes, for the extracellular synthesis of gold nanoparticles. <b>Materials and Methods:</b> An upstream bioprocess was optimized and developed for the synthesis of controlled size gold nanoparticles with solitary mono dispersal pattern in aurum chloride solution. The characterization and confirmation of gold nanoparticles were illustrated by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Energy Dispersive X-ray Analysis (EDAX) and Fourier Transmission Infrared Radiation Analysis (FTIR). <b>Results:</b> Biomass size of 3 g, substrate concentration of 1 mM, pH of 8.5 and temperature of 45°C were observed as optimum conditions for the synthesis of 15-24 nm size gold nanoparticles. The <i>Bluetongue virus</i> (BTV) which belongs to the genus Orbivirus in the family Reoviridae with 26 serotypes is an etiological agent of infectious and non-contagious Bluetongue disease of main sheep and several other domestic animals. <b>Conclusion:</b> Gold nanoparticles for the 1st time, at a higher concentration of 1:64 dilutions revealed a very promising and novel antiviral property against the <i>Bluetongue virus</i>.

A low-passage insect-cell isolate of bluetongue virus uses a macropinocytosis-like entry pathway to infect natural target cells derived from the bovine host.

Bluetongue virus (BTV) causes an economically important disease in domestic and wildlife ruminants and is transmitted by Culicoides biting midges. In ruminants, BTV has a wide cell tropism that includes endothelial cells of vascular and lymphatic vessels as important cell targets for virus replication, and several cell types of the immune system including monocytes, macrophages and dendritic cells. Thus, cell-entry represents a particular challenge for BTV as it infects many different cell types in widely diverse vertebrate and invertebrate hosts. Improved understanding of BTV cell-entry could lead to novel antiviral approaches that can block virus transmission from cell to cell between its invertebrate and vertebrate hosts. Here, we have investigated BTV cell-entry using endothelial cells derived from the natural bovine host (BFA cells) and purified whole virus particles of a low-passage, insect-cell isolate of a virulent strain of BTV-1. Our results show that the main entry pathway for infection of BFA cells is dependent on actin and dynamin, and shares certain characteristics with macropinocytosis. The ability to use a macropinocytosis-like entry route could explain the diverse cell tropism of BTV and contribute to the efficiency of transmission between vertebrate and invertebrate hosts.

Syndromic surveillance of abortions in beef cattle based on the prospective analysis of spatio-temporal variations of calvings.

Our objective was to study the ability of a syndromic surveillance system to identify spatio-temporal clusters of drops in the number of calvings among beef cows during the Bluetongue epizootic of 2007 and 2008, based on calving seasons. France was partitioned into 300 iso-populated units, i.e. units with quite the same number of beef cattle. Only 1% of clusters were unlikely to be related to Bluetongue. Clusters were detected during the calving season of primary infection by Bluetongue in 28% (n = 23) of the units first infected in 2007, and in 87% (n = 184) of the units first infected in 2008. In units in which a first cluster was detected over their calving season of primary infection, Bluetongue was detected more rapidly after the start of the calving season and its prevalence was higher than in other units. We believe that this type of syndromic surveillance system could improve the surveillance of abortive events in French cattle. Besides, our approach should be used to develop syndromic surveillance systems for other diseases and purposes, and in other settings, to avoid "false" alarms due to isolated events and homogenize the ability to detect abnormal variations of indicator amongst iso-populated units.

Effect of an inactivated bluetongue serotype 8 vaccine on semen quality in rams.

The aim of this study was to determine whether a single dose of an inactivated bluetongue virus serotype 8 (BTV-8) vaccine altered semen quality in rams. Twenty sexually mature rams were assigned to three experimental groups: two groups of four animals were vaccinated and a third group of four animals was unvaccinated. The first group included rams with a history of natural BTV-8 infection in 2007 and the second and third groups included BTV-8 naïve rams. Semen was collected prior to vaccination and for 4 months post-vaccination. There were no significant differences in semen quality traits, including motility and concentration of spermatozoa, and percentages of living, normal dead and abnormal dead spermatozoa, between vaccinated and unvaccinated groups, or over time (P>0.05). The BTV-8 vaccine tested in this study did not appear to have any adverse effect on semen quality in rams.

The effect of bluetongue virus serotype 8 on milk production and somatic cell count in Dutch dairy cows in 2008.

The effect of bluetongue virus serotype 8 (BTV-8) infections was quantified on milk production and udder health. From July 2008 to December 2008, 1,074 seronegative cows in 15 herds that were not vaccinated against BTV-8 were tested every 3 wk for BTV-8 antibodies. Sampling stopped when cows seroconverted. Test-day records were provided and 3 traits were defined to evaluate the effect of BTV-8 on milk production and udder health: 1) the difference between observed and predicted fat- and protein-corrected milk production; 2) the natural logarithm of the somatic cell count (lnSCC); and 3) the occurrence of a new high SCC. In the default model, the variables were assumed influenced by BTV-8 when the test-day record of the seroconverted cow was taken within 30 d before seroconversion, thus, in the period in which the cow was infected. In sensitivity analyses, the time intervals were varied in which BTV-8 was assumed to affect milk production and udder health. During the study, 185 cows (17%) had a subclinical infection and seroconverted and 77 had a test-day result within 30 d before seroconversion. In this period, in cows that seroconverted, the fat- and protein-corrected milk production was 52 (95% confidence interval: 26 to 77) kg less than in the period before and after seroconversion and was 51 (95% CI: 26 to 76) kg less than in cows that remained seronegative. When the time interval was increased to within 42 d before seroconversion, the milk production in BTV-8-seroconverted cows decreased by 61 (95% CI: 28 to 94) kg compared with the period before and after seroconversion and decreased by 59 (95% CI: 27 to 92) kg compared with cows that remained BTV-8 seronegative. No significant effect of BTV-8 was found on SCC and odds for a high SCC. Subclinical BTV-8 infection in dairy cattle results in a decreased milk production.

Bluetongue virus infection alters the impedance of monolayers of bovine endothelial cells as a result of cell death.

Bluetongue virus (BTV) is the cause of bluetongue, an emerging, arthropod-transmitted disease of ungulates. Bluetongue is characterized by vascular injury with hemorrhage, tissue infarction and widespread edema, lesions that are consistent with those of the so-called viral hemorrhagic fevers. To further investigate the pathogenesis of vascular injury in bluetongue, we utilized an electrical impedance assay and immunofluorescence staining to compare the effects of BTV infection on cultured bovine endothelial cells (bPAEC) with those of inducers of cell death (Triton X-100) and interendothelial gap formation (tissue necrosis factor [TNF]). The data confirm that the adherens junctions of BTV-infected bPAECs remained intact until 24h post-infection, and that loss of monolayer impedance precisely coincided with onset of virus-induced cell death. In contrast, recombinant bovine TNF-alpha caused rapid loss of bPAEC monolayer impedance that was associated with interendothelial gap formation and redistribution of VE-cadherin, but without early cell death. The data from these in vitro studies are consistent with a pathogenesis of bluetongue that involves virus-induced vascular injury leading to thrombosis, hemorrhage and tissue necrosis. However, the contribution of cytokine-induced interendothelial gap formation with subsequent edema and hypovolemic shock contributes to the pathogenesis of bluetongue remains to be fully characterized.

Effects of bluetongue virus infection on sperm quality in bulls: A preliminary report.

During the recent bluetongue virus (BTV) outbreak in Germany, semen quality in bulls naturally infected with BTV-serotype 8 was evaluated. Bluetongue status was assessed by both serology and polymerase chain reaction (PCR). Six bulls became BTV-PCR positive between September and November 2007 without showing clinical signs. Between April and May 2008, all six bulls were PCR negative but remained seropositive. Semen data from non-infected test bulls recorded between 2006 and 2007 were matched for season and age and used as controls. BTV-8 infection had no effect on sperm volume and concentration, but reduced sperm motility was seen after thawing (January-August 2008: 44.1 ± 12.7% vs. 58.0 ± 7.9% in the uninfected bulls; P < 0.001). Malformed sperm in both in fresh and thawed semen from BTV-positive animals was above the 20% permitted maximal limit from December 2007 to February 2008. Infection with BTV-8 transiently impaired semen quality in bulls.

Impact of a natural bluetongue serotype 8 infection on semen quality of Belgian rams in 2007.

In 2006, bluetongue (BT) virus serotype 8 emerged in northern Europe and numerous ruminants were affected in the following year. Infertility in males is one of the consequences of BT, although its severity and duration after natural infection has not been documented. In this report, the impact of BT-8 on clinical signs and semen quality of naturally infected rams is described through a longitudinal study of two Belgian ram populations (n=12 and n=24) and a cross sectional study in a further ram population (n=43). Macroscopic semen characteristics, semen concentration, motility, percentage of living and dead spermatozoa were assessed in 167 semen samples collected on 1-6 occasions from 79 BT-8 infected rams within 5-138 days after onset of clinical disease. These were compared with healthy control animals. Significant changes in all variables were observed after natural BT-8 infection. Total recovery occurred around 85 days after clinical disease in animals undergoing a close follow-up of semen quality. Good correspondence between the results of the longitudinal and cross sectional studies suggests that semen quality of BT-8 affected rams reached normal references values 63-138 days after clinical diagnosis of BT. In addition, semen concentration seems to be a sound epidemiological indicator of ram semen quality.

Bluetongue in Belgium, 2006.

Bluetongue has emerged recently in Belgium. A bluetongue virus strain was isolated and characterized as serotype 8. Two new real-time reverse transcription-quantitative PCRs (RT-qPCRs) that amplified 2 different segments of bluetongue virus detected this exotic strain. These 2 RT-qPCRs detected infection earlier than a competitive ELISA for antibody detection.

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.

Detection by ELISA of bluetongue antigen directly in the blood of experimentally infected sheep.

An antigen-capture ELISA (Ag-ELISA) was developed to detect bluetongue virus (BTV) antigen directly from blood samples. Four blood preparations [whole blood, buffy coat, washed red blood cells (RBC) and plasma] taken pre-inoculation and on days 6 to 9 post-inoculation (PI) were used in the ELISA to study antigenaemia in forty sheep, each experimentally infected with one of 20 South African BTV serotypes. Seventeen of the 20 serotypes were detected and 27 of the 40 sheep were at some stage Ag-ELISA positive. Over the period of sampling, Ag-ELISA positive results were most frequently returned from whole blood taken on days 6 and 7 PI. However in some instances the quantity and/or duration of BTV antigenaemia was greater in buffy coat and washed RBC preparations. In a selection of samples examined, positive Ag-ELISA results were generally obtained when samples had an infectious virus titre in eggs of > 10(3.2) egg lethal doses (ELD50/ml). The appearance and duration of detectable antigenaemia was compared with the development of clinical signs and antibody responses of infected sheep. On days 6 and 7 PI the presence of fever (> 40 degrees C) was indicative to the likelihood of detectable antigenaemia. After day 5 PI antigenaemia declined and clinical signs of swollen face and inflamed feet appeared together with the first detectable antibody response. The Ag-ELISA, when used in conjunction with clinical observations and serologic data, should be useful as a rapid diagnostic procedure for bluetongue disease.

Bluetongue virus.

Bluetongue (BLU) is a noncontagious viral disease. The virus is a member of the Orbivirus genus and serves as the prototype virus of the genus. BLU is primarily a disease of domestic ruminants, some wild ruminants, and, recently, domestic dogs. The disease is caused by 1 of 24 different serotypes of virus that are distributed worldwide. This article reviews the viruses, their distribution, clinical signs, pathogenesis, and the roles they play in reproductive failure.

Aspectos epidemiológicos y entomólogicos de la enfermedad de lengua azul en ganado bovino / Epidemiologic and entomologic aspects of bluetongue in cattle in the Isthmus of Tehuantepec, Oaxaca, Mexico

Se realizó un estudio prospectivo de la enfermedad de lengua azul en 2 hatos de ganado bovino en la región del Istmo de Tehuantepec. El estudio incluyó la detección menstrual de anticuerpos grupo-específicos contra el virus de lengua azul (VLA) y la captura de moscas culicoides. En enero de 1988, el total de ganado bovino en los 2 hatos (600) fue clasificado seropositivo al VLA, utilizando un método ELISA. Con base en este resultado, 40 bovinos adultos y 35 becerros nacidos entre octubre de 1987 y enero de 1988 fueron evaluados mensualmente (enero a diciembre de 1988) para detectar la producción de anticuerpos grupo-específicos VLA. Se detectó un patrón positivo de seroconversión contra el VLA en becerros, durante la temporada de lluvia en los meses del verano (junio-septiembre). Sin embargo, en algunos becerros se determinó serológicamente actividad de VLA en la temporada seca, durante marzo y abril. Debido a la ausencia de anticuerpos maternos, la mayoría de los becerros fueron susceptibles de infección con el VLA a los 6 meses de edad. La evidencia serológica de la actividad del VLA en becerros se relacionó con la abundancia de moscas Culicoides insignis capturadas durante el estudio

The impact of bluetongue virus on reproduction.

Bluetongue virus has been recognized as an important noncontagious, arthropodborne infectious viral disease of ruminants. 24 different serotypes of virus have been recognized worldwide. The most severe clinical disease has been associated with severe clinical disease in sheep and some free ranging wild ruminants. A number of reports have implicated the viruses as causing reproductive disorders in both males and females. The bluetongue related reproductive disorders include early embryonic deaths, abortions, malformed fetal calves or lambs, transient infertility in bulls and rams, and shedding of virus in semen. Recently, bluetongue virus contamination of modified live commercial canine vaccine was associated with abortion and acute death of pregnant bitches. The pathogenesis of these various aspects of reproductive failure are discussed herein.

Bluetongue virus infection in pregnant ewes.

Inoculation of 53 ewes after 35, 45, 60, or 80 days of gestation with bluetongue virus serotypes 10, 11, 13, or 17, or with epizootic hemorrhagic disease virus serotypes 1 or 2, resulted in overt clinical disease in the 47 ewes inoculated with bluetongue virus but not in the 6 ewes inoculated with epizootic hemorrhagic disease virus. None of the lambs produced by these ewes had developmental defects or any evidence of persistence of viremia.

Experimental infection of pregnant cattle with bluetongue virus serotype 11 between postbreeding days 21 and 48.

Four bluetongue virus (BTV)-seronegative heifers and 2 BTV-seropositive heifers were inoculated with the virulent strain UC-8 of BTV-11 between postbreeding days (PBD) 21 and 30. The heifers were observed for 10-18 days after inoculation for clinical signs, and pregnancy was monitored by ultrasound examination of the uterus and by plasma progesterone levels. Blood samples were collected daily after inoculation and processed for virus isolation and titration. Heifers were euthanized between PBD 31 and PBD 48, and tissues were collected for virologic and pathologic examination. All but 1 heifer inoculated on PBD 21 remained pregnant after BTV inoculation. A cystic corpus luteum was found in the ovary of the nonpregnant heifer, but BTV was not isolated from the reproductive tract of this heifer. Three of the inoculated heifers that remained pregnant showed mild multifocal areas of perivascular lymphocytic infiltration in the ovary. BTV was reisolated from spleen and prescapular and peribronchial lymph nodes 10 days after inoculation from 3 of the 4 BTV-seronegative heifers. BTV was also reisolated from the uterus of 1 of the heifers that remained pregnant, but microscopic lesions were not found in this organ.

A cross-sectional study of bluetongue virus and Mycoplasma bovis infections in dairy cattle: I. The association between a positive antibody response and production efficiency.

In a cross-sectional study of bluetongue virus (BTV) and Mycoplasma bovis (M. bovis) infections, a sample of 572 California dairy cows was tested for the presence of antibodies to answer the question: Is it possible to identify and to assess quantitatively the associations between positive antibody test and production? Serum samples collected from these cows during December 1986 were tested for the presence of antibodies to BTV and M. bovis using the enzyme-linked immunosorbent assay (ELISA). Data on milk production were extracted from individual cow sheets of the California Dairy Herd Improvement Association (DHIA) record-keeping system and interfaced with percentage ELISA results for analysis. Univariate and multivariate statistical analyses, using the chi 2 test for categorical variables or Student's t-test for continuous variables and multiple logistic regression respectively, were carried out to evaluate for possible associations between positive antibody tests to each agent and each production variable of interest. Complete data on all variables studied were obtained for 289 (50.5%) cows for M. bovis and 423 (74%) cows for BTV. For cows with complete data on all variables, estimates of the point prevalence of antibodies to BTV and M. bovis were 70.5% and 66.1%, respectively. Results of this study indicated that Guernsey cows were more likely to have a positive BTV test than Holstein cows and that cows in higher lactations were more likely to test positive to BTV ELISA than those in lower lactations (p less than 0.05). Because all cows except those on one farm were Holstein, our confidence in the effect of breed is limited. The association between lactation number and BTV seropositive test may be an age factor identified earlier in the study. For M. bovis, the results of the analysis indicated that seropositive cows were more likely to produce less milk, on a mature equivalent basis (ORadj = 0.96, p = 0.034), and that they had less extended 305 day milk production potential (ORadj = 0.90, p less than 0.0001) than seronegative cows.

A cross-sectional study of bluetongue virus and Mycoplasma bovis infections in dairy cattle: II. The association between a positive antibody response and reproduction performance.

In a cross-sectional study to determine the possible relationship between a positive antibody test to bluetongue virus (BTV) or Mycoplasma bovis infections and reproductive performance of dairy cows, data were collected on 572 California dairy cows during December 1986 for analysis. Serum samples were tested using an enzyme-linked immunosorbent assay (ELISA). Data on reproduction variables were extracted from the individual cow sheets of the California Dairy Herd Improvement Association records and interfaced with the serological results for analysis. Similar data analyses for both BTV and M. bovis were performed to identify and quantitatively assess the association of the reproduction variables and each agent. These associations were evaluated unconditionally using the chi 2 for categorical variables and Student's t-test for continuous variables. Logistic regression analysis was used to determine if reproduction variables with significant unconditional associations remained significant when adjusted for the effects of possible confounding factors. Both the BTV and M. bovis ELISA antibody titres indicated exposure to the agents. The results of the multiple logistic regression analyses indicated that cows seropositive for BTV were significantly older at first calving (p less than 0.03). For M. bovis, seropositive cows were more likely to have longer intervals from calving to last service and longer intervals from calving to pregnancy diagnosis than seronegative cows (p less than 0.05). The other reproduction variables examined were not significantly associated with ELISA seropositivity.

Subclinical and clinical bluetongue disease in cattle: clinical, pathological and pathogenic considerations.

Calves immunized (sensitized) with alcide-inactivated bluetongue virus (BTV) developed immunoglobulin (Ig) E-specific antibody to BTV. Development of clinically apparent disease occurred after challenge with virulent BTV, and lesions correlated with peak levels of virus-specific IgE, an eosinophilic dermatitis, and high concentrations of histamine in the skin. IgE may be important in the pathogenesis of clinical bluetongue (BT) disease in cattle.