Reference Material Production and Milk Protein Concentration as Elements to Improve Bluetongue Serological Diagnosis in Bulk Tank Milk.

The serological surveillance of bluetongue in bulk tank milk is an efficient and cost-effective method for the early detection of bluetongue virus incursions in unvaccinated free areas of the disease. In addition, the availability of standardized and reliable reagents and refined diagnostic procedures with high sensitivity and specificity are essential for surveillance purposes. However, no available reference materials for bluetongue virus serological surveillance in bulk tank milk exist. This study shows the production and characterization of reference material for the implementation of a commercially available bluetongue milk ELISA test in official laboratories, as well as the evaluation of a procedure to increase the sensitivity in samples with low levels of antibodies. This procedure, based on milk protein concentration, allowed us to notably increase the ELISA test's analytical sensitivity, which is useful for milk samples from farms with low within-herd prevalence or pools of bulk tank milk samples. The standardized milk reference material produced here, together with the evaluated procedure to improve analytical sensitivity, could be applied as tools to ensure an accurate diagnosis by official laboratories in bluetongue unvaccinated free areas.

Preliminary investigation reveals novel pathological consequences of bluetongue virus-1 infection in the endocrine glands of pregnant Indian sheep.

Bluetongue virus (BTV), a major peril to the sheep industry, infects a wide range of the cells in the infected animals including mononuclear, dendritic and epithelial cells. However, little is known about its tropism for the secretory epithelial cells of endocrine glands and the pathogenesis it induces. The aim of the study was to assess the BTV load, antigen distribution in the tissue of the pituitary, thyroid as well as adrenal glands and associated histopathological consequences. BTV antigens were localized using immunohistochemistry in the thyroid's epithelial cells, zona fasciculata and zona reticularis cells and the anterior pituitary epithelial cells. The real-time PCR portrayed the high viral load in adrenals at 7th days postinoculation (DPI) and in thyroid and pituitary glands at 15th DPI. Serum examination revealed variation in the T-3 and T-4 of infected animals in comparison to the control group. Caspase-3 immunolocalization revealed BTV-1 induces apoptosis in the affected cells of endocrine gland of infected animals. Further, this study signifies the tropism of BTV in the novel sites (endocrine glands) of the host that might be one of the reasons for the poor performance of infected animals.

Clinical presentation, medical management, and outcomes in 35 hospitalized sheep diagnosed with bluetongue virus disease.

BACKGROUND: There is only limited information on the clinical presentation, medical management, and outcomes of hospitalized sheep diagnosed with bluetongue virus (BTV) disease. OBJECTIVES: To describe the signalment, history, clinical signs, clinicopathological findings, medical management, and clinical outcomes of sheep diagnosed with BTV disease. ANIMALS: Thirty-five hospitalized sheep with BTV disease. METHODS: Retrospective case series. Medical records from 1989 to 2021 were evaluated. History, signalment, clinical signs, laboratory test results, treatments, and outcomes were recorded. RESULTS: BTV disease was diagnosed from July to December, with a peak proportion (43%; 15/35) of diagnoses recorded in October. Pyrexia and anorexia, respiratory disease, vasculitis, coronitis and lameness, and ulcerative mucosal lesions were present in 71%, 71%, 66%, 49%, and 22% of sheep, respectively. BTV serotypes 10, 11, 13, and 17 were identified, with serotype 17 (75%) being the most frequent. Management of cases included administration of antimicrobials (89%), anti-inflammatories (77%), IV fluids (60%), vitamins (20%), proton-pump inhibitors (14%), diuretics (9%), and antioxidants (9%). Six ewes were pregnant on presentation, but none aborted. Six (17%) sheep died or were euthanized because of clinical deterioration, whereas 83% were discharged. CONCLUSIONS AND CLINICAL IMPORTANCE: The proportion of sheep that survived BTV disease after treatment was relatively high. Serotyping of BTV is recommended because of the mismatch between frequently identified serotypes and the serotype present in the vaccine.

Development and evaluation of gold nanoprobe based lateral flow device for rapid and sensitive serodetection of Bluetongue in sheep.

Bluetongue (BT) disease is a viral, insect borne, noncontagious illness of small ruminants caused by Orbivirus, impacting huge economic loss worldwide. The existing BT diagnostic techniques are costly, time-consuming and require both specialized equipment and also skilled personnel. So there is need to develop a rapid, sensitive, on site detection assay for diagnosis of BT. This study utilized secondary antibody derivatized Gold nanoprobes for rapid and sensitive detection of BT over lateral flow device (LFD). The detection limit for this assay was found 1.875 µg of BT IgG/ml and a comparison between LFD and indirect ELISA was performed and the sensitivity and specificity was found at 96% and 99.23%, respectively, with observed kappa value of 0.952. This developed LFD may therefore offer a quick, affordable and accurate diagnosis of BT disease at the field level.

The clinical presentation and management of a naturally occuring Bluetongue virus infection in a pregnant Rottweiler dog.

Few reports of clinical Bluetongue virus (BTV) infections have been described in dogs. Most cases were linked to inoculation with a BTV-contaminated canine modified live vaccine. In dogs, cases have only been described in pregnant females with clinical signs of fever and abortion followed by severe dyspnoea and death. A pregnant Rottweiler dog was presented with a three-day history of progressive lethargy and anorexia. The patient was a guard dog living in an enclosure where sheep were kept at night. High mortalities had been experienced in the sheep but had not been investigated. On presentation, the major clinical findings were dyspnoea and hypoxia. Clinicopathological tests showed hypoxia and systemic inflammation. Radiological findings were consistent with non-cardiogenic pulmonary oedema. The patient was treated symptomatically and recovered but did not retain the pregnancy. Bluetongue virus was identified in the patient's blood using BTV RT-PCR (Ct value 24.7). At a follow-up farm visit, an ongoing BTV outbreak in the sheep was diagnosed with affected sheep testing positive for BTV on RT-PCR. This report describes the clinical presentation, diagnostic investigations and successful treatment of a dog with BTV infection. This is the first case report of a naturally occurring clinical BTV infection in a dog. Possible routes of infection were direct contact, midgeborne, or ingestion of infected afterbirth or abortus from sheep.

Establishment of an Immunological Method for Detection of Bluetongue Virus by Fluorescence-Linked Immunosorbent Assay.

Bluetongue (BT) is a severe noncontagious infectious disease that occurs in sheep and wild ruminants but occasionally also in cattle and camels. The worldwide BT pandemic has had a significant impact on global livestock production. Rapid detection helps prevent outbreaks of bluetongue disease. Fluorescence-linked immunosorbent assay (FLISA) labeled with quantum dots (QDs) is typically used for detection due to its high sensitivity. There has been no reported detection of BT virus (BTV) using QD-based fluorescence immunoassays. In this study, monoclonal antibodies (MAbs) against BT were prepared by immunizing BALB/c mice with recombinant VP7 protein. Two MAbs with high sensitivity and specificity were selected as the detection antibody (2F11) and capture antibody (11B7). Then, the detection antibody was coupled with QDs to prepare QD-MAb fluorescence probes. Fluorescence-linked immunosorbent assay is highly specific, detecting only VP7 protein/BTV, and did not show any nonspecific reactions with other reoviruses. The detection limit of VP7 protein was 3.91 ng/mL using fluorescence-linked immunosorbent assay, with a coefficient of variation (CV) of less than 15%. The establishment of rapid, sensitive direct FLISA has potential for bluetongue virus detection and control of BT vaccine quality. IMPORTANCE Bluetongue virus causes the severe infectious disease BT. BTV has many serotypes, and there is no cross-protection among different serotypes. BT is listed as a notifiable animal infectious disease by the World Organisation for Animal Health (OIE) and occurs throughout the world, causing significant economic losses. The establishment of a fast and effective detection method is the key to controlling and preventing this disease. Current methods for detecting BTV mainly include reverse transcription-PCR (RT-PCR), enzyme-linked immunosorbent assays (ELISA), and immunochromatographic strips that are based on antigen-antibody recognition. Immunoassays are most commonly used because of their low cost, high specificity, and fast analysis, making them particularly useful for routine monitoring. These conventional detection strategies for BTV have some drawbacks. Recently, FLISA has been drawing attention due to its sensitivity, which is higher than traditional immunoassays. Fluorescence-linked immunosorbent assays (FLISA) using fluorescent materials as labels overcome ELISA's disadvantage of being time-consuming.

Multiplexed serotype-specific real-time polymerase chain reaction assays - A valuable tool to support large-scale surveillance for bluetongue virus infection.

In the last decade, real-time polymerase chain reaction (PCR) has been increasingly adopted for bluetongue diagnosis with both broadly reactive and serotype-specific assays widely used. The use of these assays and nucleic acid sequencing technologies have enhanced bluetongue virus detection, resulting in the identification of a number of new serotypes. As a result, 27 different serotypes are officially recognised, and at least three more are proposed. Rapid identification of the virus serotype is essential for matching of antigens used in vaccines and to undertake surveillance and epidemiological studies to assist risk management. However, it is not uncommon for multiple serotypes to circulate in a region either concurrently or in successive years. It is therefore necessary to have a large suite of assays available to ensure that the full spectrum of viruses is detected. Nevertheless, covering a large range of virus serotypes is demanding from both a time and resource perspective. To overcome these challenges, real-time PCR assays were optimised to match local virus strains and then combined in a panel of quadriplex assays, resulting in three assays to detect 12 serotypes directly from blood samples from cattle and sheep. These multiplex assays have been used extensively for bluetongue surveillance in both sentinel animals and opportunistically collected samples. A protocol to adapt these assays to capture variations in local strains of bluetongue virus and to expand the panel is described. Collectively, these assays provide powerful tools for surveillance and the rapid identification of bluetongue virus serotypes directly from animal blood samples.

Development of Multiwalled Carbon Nanotube-Based Immunobiosensor for the Detection of Bluetongue Virus.

Bluetongue (BT) disease is a noncontagious disease of domestic and wild ruminants (mainly sheep, cattle, deer) caused by the bluetongue virus (BTV) which is an orbivirus of the Reoviridae family and transmitted by vector Culicoides biting midges. It is a reportable disease of considerable socioeconomic concern and of major importance for the international trade of animals and animal products. Conventional diagnostic methods, such as virus propagation and isolation, immunoassays and also various molecular methods have been developed for the detection of the BTV. Here, we present a novel, rapid and pen-side test for the detection of BTV using multiwalled carbon nanotube (MWCNTs) based immunosensor. Though it is not reported yet. The MWCNTs were prepared, characterized and functionalized with carboxyl group. Viral antibodies were conjugated successfully with functionalized MWCNTs and coated on screen printed carbon electrode (SPCE). These SPCE were evaluated by using electrochemical sensor with an antigen specific to BTV antibodies, resulted in the self-assembled layer of antigen-antibody on the surface of SPCE. The approach described in the present study is a prototype for the development of simple and economic diagnostic tool which will provide the routine screening of BT disease at the door of farmers, thereby increasing the income of farmers by decreasing the cost of diagnosis.

Development of real-time RT-qPCR assays for the typing of two novel bluetongue virus genotypes derived from sheeppox vaccine.

Previously, we reported the detection of two novel bluetongue virus (BTV) strains (SPvvvv/02 and SPvvvv/03), possibly representing new BTV genotypes, in a batch of sheeppox vaccine. We developed type-specific RT-qPCR assays (targeting genome segment 2) for these two new BTV strains. The limit of detection of both assays was 10 genome copies/µl and no cross-reactivity with other BTV genotypes was observed. The performance of three other BTV group-specific diagnostic assays was also tested against the putative novel genotypes. RT-qPCR assays targeting BTV segment 9 and 10 detected both strains (SPvvvv/02 and SPvvvv/03) whereas a BTV segment 1 RT-qPCR assay was unable to detect either BTV strain. The work presented here expands upon the current repertoire of RT-qPCR assays for BTV genotype determination.

Development and Validation of an ELISA for the Detection of Bluetongue Virus Serotype 4-Specific Antibodies.

In this article, we describe the development and evaluation of a double antigen sandwich enzyme-linked immunosorbent assay (ELISA) able to detect serotype 4-specific antibodies from BTV-4 infected or vaccinated animals using a recombinant BTV-4 VP2 protein. The coding sequence of VP2 was inserted into a pVote plasmid by recombination in the Gateway® cloning system. Vaccinia virus (VacV) was used as a vector for the expression of the recombinant VP2. After production in BSR cells, recombinant VP2 was purified by immunoprecipitation using a FLAG tag and then used both as the coated ELISA antigen and as the HRP-tagged conjugate. The performance of the ELISA was evaluated with 1186 samples collected from BTV negative, infected or vaccinated animals. The specificity and sensitivity of the BTV-4 ELISA were above the expected standards for the detection of anti-BTV-4 VP2 antibodies in animals reared in Europe or in the Mediterranean basin. Cross-reactions were observed with reference sera for serotypes 10 and 20, and to a lesser extent with serotypes 12, 17 and 24, due to their genetic proximity to serotype 4. Nevertheless, these serotypes have never been detected in Europe and the Mediterranean area. This ELISA, which requires only the production of a recombinant protein, can be used to detect BTV serotype 4-specific antibodies and is therefore an attractive alternative diagnostic method to serum neutralization.

Diagnostic applications of molecular and serological assays for bluetongue and African horse sickness.

The availability of rapid, highly sensitive and specific molecular and serologic diagnostic assays, such as competitive enzyme-linked immunosorbent assay (cELISA), has expedited the diagnosis of emerging transboundary animal diseases, including bluetongue (BT) and African horse sickness (AHS), and facilitated more thorough characterisation of their epidemiology. The development of assays based on real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect and identify the numerous serotypes of BT virus (BTV) and AHS virus (AHSV) has aided in-depth studies of the epidemiology of BTV infection in California and AHSV infection in South Africa. The subsequent evaluation of pan-serotype, real-time, RT-PCR-positive samples through the use of serotype-specific RT-PCR assays allows the rapid identification of virus serotypes, reducing the need for expensive and time-consuming conventional methods, such as virus isolation and serotype-specific virus neutralisation assays. These molecular assays and cELISA platforms provide tools that have enhanced epidemiologic surveillance strategies and improved our understanding of potentially altered Culicoides midge behaviour when infected with BTV. They have also supported the detection of subclinical AHSV infection of vaccinated horses in South Africa. Moreover, in conjunction with whole genome sequence analysis, these tests have clarified that the mechanism behind recent outbreaks of AHS in the AHS-controlled area of South Africa was the result of the reversion to virulence and/or genome reassortment of live attenuated vaccine viruses. This review focuses on the use of contemporary molecular diagnostic assays in the context of recent epidemiologic studies and explores their advantages over historic virus isolation and serologic techniques.

La disponibilité d'essais diagnostiques moléculaires et sérologiques rapides, hautement sensibles et spécifiques tels que l'épreuve immuno-enzymatique de compétition (ELISAc), a accéléré le diagnostic des maladies animales transfrontalières émergentes, dont la fièvre catarrhale ovine (FCO) et la peste équine, et contribué à dresser un tableau épidémiologique plus complet de ces maladies. Grâce à la mise au point d'essais basés sur l'amplification en chaîne par polymérase en temps réel couplée à une transcription inverse (RT­PCR) qui permettent de détecter et d'identifier les nombreux sérotypes du virus de la fièvre catarrhale du mouton et du virus de la peste équine, des études approfondies ont pu être conduites sur l'épidémiologie de l'infection par le virus de la fièvre catarrhale du mouton en Californie et de l'infection par le virus de la peste équine en Afrique du Sud. L'évaluation postérieure des échantillons positifs à une RT­PCR en temps réel de groupe (détectant le virus quel que soit le sérotype) au moyen de RT­PCR spécifiques de chaque sérotype permet d'identifier rapidement le sérotype causal et de limiter le recours à des méthodes classiques onéreuses et chronophages comme l'isolement viral ou les essais de neutralisation virale spécifiques de chaque sérotype. Les outils fournis par ces essais moléculaires et par les plateformes ELISAc ont renforcé les stratégies de surveillance épidémiologique et permis de mieux connaître les altérations potentielles de comportement chez les tiques Culicoides infectées par le virus de la fièvre catarrhale du mouton. Ils ont également contribué à détecter les cas d'infection asymptomatique par le virus de la peste équine chez des chevaux vaccinés en Afrique du Sud. En outre, associés avec l'analyse de séquences du génome entier, ces tests ont révélé que le mécanisme sous-jacent aux récents foyers de peste équine dans la zone de contrôle en Afrique du Sud correspondait à une réversion vers la virulence et/ou à un réassortiment du génome des souches de vaccin à virus vivant atténué. Les auteurs passent en revue l'utilisation des essais de diagnostic moléculaire de nouvelle génération dans le contexte de récentes études épidémiologiques et cherchent à établir leurs avantages par rapport aux techniques classiques d'isolement viral et de recherche sérologique.

La existencia de ensayos moleculares y serológicos de diagnóstico rápidos y de gran sensibilidad y especificidad, como el ensayo inmunoenzimático de competición (ELISAc), ha acelerado el diagnóstico de enfermedades animales transfronterizas emergentes, como la lengua azul o la peste equina, y facilitado una caracterización más exhaustiva de su epidemiología. La creación de ensayos basados en la reacción en cadena de la polimerasa acoplada a transcripción inversa (RT?PCR) en tiempo real para detectar y caracterizar los numerosos serotipos de los virus de la lengua azul y la peste equina ha ayudado a estudiar a fondo la epidemiología de sendos episodios infecciosos causados por el virus de la lengua azul en California y por el virus de la peste equina en Sudáfrica. El subsiguiente análisis de las muestras positivas a la prueba de RT?PC en tiempo real de cualquier serotipo con empleo de ensayos RT?PCR dirigidos específicamente contra uno u otro serotipo permite identificar rápidamente los serotipos víricos, lo que hace menos necesario el uso de métodos convencionales más caros y largos, como el aislamiento del virus o técnicas de neutralización vírica adaptadas específicamente a un serotipo. Estos dispositivos de ensayo molecular o de ELISAc ponen a nuestra disposición herramientas que potencian las estrategias de vigilancia epidemiológica y ayudan a conocer mejor las eventuales alteraciones del comportamiento de los jejenes Culicoides al ser infectados por el virus de la lengua azul. Estas técnicas han ayudado también a detectar en Sudáfrica casos de infección asintomática por el virus de la peste equina en caballos vacunados. Estas pruebas, además, empleadas en combinación con el análisis de secuencias genómicas completas, han servido para aclarar que el mecanismo subyacente a los recientes brotes de peste equina surgidos en la zona de Sudáfrica donde la enfermedad estaba bajo control fue fruto de la reversión a la virulencia y/o el reordenamiento genómico de virus vacunales atenuados. Los autores, centrándose en el uso de modernos ensayos moleculares de diagnóstico como parte de recientes estudios epidemiológicos, examinan las ventajas que ofrecen en comparación con las tradicionales técnicas serológicas y de aislamiento vírico.

Critical parameters of real time reverse transcription polymerase chain reaction (RT-PCR) diagnostics: Sensitivity and specificity for bluetongue virus.

A new variant of bluetongue virus serotype 3, BTV3 ITL 2018 (here named: BTV3), was included in serial dilutions in the BT Proficiency Test 2020. Although the OIE-recommended panBTV real time RT-PCR test targeting genome segment 10 (Seg-10) detected this variant, we showed that reverse transcription (RT) at 61 °C instead of 50 °C completely abolished detection. Another Seg-10 panBTV real time RT-PCR test detected BTV3, irrespective of the temperature of RT. In silico validation showed that each of the OIE-recommended PCR primers using IVI-primers contain single mismatches at the -3 position for BTV3. In contrast, WBVR-primers of a second test completely match to the BTV3 variant. Our results suggest that single mismatches caused false negative PCR results for BTV3 at high RT temperature. Indeed, correction of both IVI-primers for BTV3 led to positive results for BTV3 but negative results for all other samples of the BT Proficiency Test 2020. Apparently, variability of the -3 position is sufficient for discriminative PCR detection, although the single mismatch in the IVI-reverse primer was the most important for this phenomenon. Extensive in silico validation showed that targets of both Seg-10 panBTV RT-PCR tests are not completely conserved, and the detailed effect of single mismatches are hard to predict. Therefore, we recommend at least two panBTV RT-PCR tests to minimize the risk of false negatives. Preferably, their PCR targets should be located at completely different and highly conserved regions of the BTV genome to guarantee adequate detection of future BTV infections.

Study of molecular diagnosis and viremia of bluetongue virus in sheep and cattle.

Bluetongue virus (BTV) is an RNA virus that infects cattle and sheep. The objective of this study was to compare two real-time PCRs for the detection of BTV and to monitor Orbivirus viremia in sheep and cattle for 6 months. The PCR results showed the occurrence of infected animals throughout the experiment without records of clinical signs. The number of positive animals reduced during the experiment, but some animals were positive for BTV RNA during the entire experiment. The performance of the two RT-qPCRs for BTV detection techniques used in this work revealed a kappa index of 0.71 for cattle and 0.75 for sheep.

Putative Novel Atypical BTV Serotype '36' Identified in Small Ruminants in Switzerland.

We identified a putative novel atypical BTV serotype '36' in Swiss goat flocks. In the initial flock clinical signs consisting of multifocal purulent dermatitis, facial oedema and fever were observed. Following BTV detection by RT-qPCR, serotyping identified BTV-25 and also a putative novel BTV serotype in several of the affected goats. We successfully propagated the so-called "BTV-36-CH2019" strain in cell culture, developed a specific RT-qPCR targeting Segment 2, and generated the full genome by high-throughput sequencing. Furthermore, we experimentally infected goats with BTV-36-CH2019. Regularly, EDTA blood, serum and diverse swab samples were collected. Throughout the experiment, neither fever nor clinical disease was observed in any of the inoculated goats. Four goats developed BTV viremia, whereas one inoculated goat and the two contact animals remained negative. No viral RNA was detected in the swab samples collected from nose, mouth, eye, and rectum, and thus the experimental infection of goats using this novel BTV serotype delivered no indications for any clinical symptoms or vector-free virus transmission pathways. The subclinical infection of the four goats is in accordance with the reports for other atypical BTVs. However, the clinical signs of the initial goat flock did most likely not result from infection with the novel BTV-36-CH0219.

Development of recombinant NS1-NS3 antigen based indirect ELISA for detection of bluetongue antibodies in sheep.

Bluetongue is an insect borne (Culicoides) viral disease of small ruminants. The virus blankets the globe with a wide serotypic variation, numbered from 1 to 28. In India 21 different serotypes have been reported to be circulating across the various agro-climatic zones of the country. Non-structural proteins (NSPs) of bluetongue virus have always remained ideal target for differentiation of infected from vaccinated animals. The current study is an extrapolation of our previous work where a novel fusion construct comprising of bluetongue viral segment NS1 and NS3 was successfully cloned, expressed, purified with an efficient strategy for its suitable implementation as a diagnostic antigen. In this study, the applicability of the fusion construct has been further evaluated and optimised for field applicability. The fusion construct used in an ELISA platform projected a relative diagnostic sensitivity and specificity of 98.1% and 95.5% respectively against a pre-established test panel. The rNS1-NS3 ELISA showed substantially good agreement with the commercial BTV antibody detection kit. Finally, the study brings together the diagnostic capability of two NSPs, which can be a handy tool for sero-surveillance of bluetongue.

An updated review on bluetongue virus: epidemiology, pathobiology, and advances in diagnosis and control with special reference to India.

Bluetongue (BT) is an economically important, non-contagious viral disease of domestic and wild ruminants. BT is caused by BT virus (BTV) and it belongs to the genus Orbivirus and family Reoviridae. BTV is transmitted by Culicoides midges and causes clinical disease in sheep, white-tailed deer, pronghorn antelope, bighorn sheep, and subclinical manifestation in cattle, goats and camelids. BT is a World Organization for Animal Health (OIE) listed multispecies disease and causes great socio-economic losses. To date, 28 serotypes of BTV have been reported worldwide and 23 serotypes have been reported from India. Transplacental transmission (TPT) and fetal abnormalities in ruminants had been reported with cell culture adopted live-attenuated vaccine strains of BTV. However, emergence of BTV-8 in Europe during 2006, confirmed TPT of wild-type/field strains of BTV. Diagnosis of BT is more important for control of disease and to ensure BTV-free trade of animals and their products. Reverse transcription polymerase chain reaction, agar gel immunodiffusion assay and competitive enzyme-linked immunosorbent assay are found to be sensitive and OIE recommended tests for diagnosis of BTV for international trade. Control measures include mass vaccination (most effective method), serological and entomological surveillance, forming restriction zones and sentinel programs. Major hindrances with control of BT in India are the presence of multiple BTV serotypes, high density of ruminant and vector populations. A pentavalent inactivated, adjuvanted vaccine is administered currently in India to control BT. Recombinant vaccines with DIVA strategies are urgently needed to combat this disease. This review is the first to summarise the seroprevalence of BTV in India for 40 years, economic impact and pathobiology.

Bluetongue in India: a systematic review and meta-analysis with emphasis on diagnosis and seroprevalence.

Bluetongue (BT) is an infectious viral disease which affects a wide range of ruminants and was first reported in India in 1964. In view of the absence of comprehensive information on the BT status in India, this study presents the seroprevalence on BT in farm animals of India based-on a systematic review and meta-analysis. A systematic review was conducted to identify the published articles (2001-2018) reporting the seroprevalence of BT in sheep, goats, cattle, buffalo, camels, and Mithun (Bos frontalis) from India. From 409 research articles, 71 fulfilled the inclusion criteria and meta-analysis for proportions was carried out targeting the eligible studies. From these, 144 strata level data were extracted with a sample size of 14048 sheep, 14696 goats, 5218 cattle, 2653 buffaloes, 2062 camels, and 222 Mithun. Overall, the analyses showed that the BT seroprevalence of 43% (95% CI: 38-49%) in goats, 39% (95% CI: 33-46%) in sheep, 38% (95% CI: 25-45%) in cattle, 34% (95% CI: 20-51%) in buffaloes, 16% (95% CI: 10-22%) in camels, and 66% (95% CI: 17-95%) in Mithun. Furthermore, the meta-regression analysis suggested that serological tests, geographical region, and sample size were the prime moderators. Meta-analytic study indicates the BT seropositivity in 25.35 million sheep (95% CI: 21.5-29.9), 58 million goats (95% CI: 51.3-66.2), 66.8 million cattle (95% CI: 47.7-86), 37.0 million buffaloes (95% CI: 21.7-55.4), 0.06 million camels (95% CI: 0.04-0.09), and 0.19 million Mithun (95% CI: 0.05-0.28). The findings highlight the variation of BT seropositivity in different geographical regions of India.

Evaluation of a commercial ELISA for detection of epizootic haemorrhagic disease antibodies in domestic and wild ruminant sera.

Bluetongue (BT) and epizootic haemorrhagic disease (EHD) are vector-borne viral diseases affecting domestic and wild ruminants. Both are notifiable under OIE rules. BT and EHD viruses (BTV and EHDV) are closely related Orbiviruses with structural, antigenic and molecular similarities. Both viruses can produce analogous clinical signs in susceptible animals. Serological tests are commonly used for BT and EHD diagnosis and surveillance. Competitive ELISA (c-ELISA) is the most widely used serological test for the specific detection of BTV or EHDV viral protein 7 (VP7) antibodies (Abs). The specificity and sensitivity of the BTV c-ELISA kits available on the market are recognized for the detection of BTV Abs. Concerning EHD, a single commercial EHDV c-ELISA kit (ELISA A kit) commonly used for diagnosis in Europe and Africa was available between 2011 and 2018 but is now no longer on the market. In this study, we evaluated a new commercial c-ELISA to detect ruminant EHDV VP7 Abs in 2,199 serum samples from cattle, sheep, goats, wild deer and zoo animals. The results showed that this ELISA kit is specific and can detect the presence of IgG anti-EHDV VP7 with a very good diagnostic specificity and a satisfactory sensitivity in domestic ruminants, zoo animals and wild deer. Therefore, the evaluated c-ELISA can detect the introduction of EHDV into an area where BTV-seropositive domestic animals are present. The performance of this kit is similar to that of the c-ELISA A kit and can thus be used for diagnosis.

Characterisation of Peste Des Petits Ruminants Disease in Pastoralist Flocks in Ngorongoro District of Northern Tanzania and Bluetongue Virus Co-Infection.

Peste des petits ruminants (PPR) disease was first confirmed in Tanzania in 2008 in sheep and goats in Ngorongoro District, northern Tanzania, and is now endemic in this area. This study aimed to characterise PPR disease in pastoralist small ruminant flocks in Ngorongoro District. During June 2015, 33 PPR-like disease reports were investigated in different parts of the district, using semi-structured interviews, clinical examinations, PPR virus rapid detection test (PPRV-RDT), and laboratory analysis. Ten flocks were confirmed as PPRV infected by PPRV-RDT and/or real-time reverse transcription-polymerase chain reaction (RT-qPCR), and two flocks were co-infected with bluetongue virus (BTV), confirmed by RT-qPCR. Phylogenetic analysis of six partial N gene sequences showed that the PPR viruses clustered with recent lineage III Tanzanian viruses, and grouped with Ugandan, Kenyan and Democratic Republic of Congo isolates. No PPR-like disease was reported in wildlife. There was considerable variation in clinical syndromes between flocks: some showed a full range of PPR signs, while others were predominantly respiratory, diarrhoea, or oro-nasal syndromes, which were associated with different local disease names (olodua-a term for rinderpest, olkipiei-lung disease, oloirobi-fever, enkorotik-diarrhoea). BTV co-infection was associated with severe oro-nasal lesions. This clinical variability makes the field diagnosis of PPR challenging, highlighting the importance of access to pen-side antigen tests and multiplex assays to support improved surveillance and targeting of control activities for PPR eradication.

Serological survey of bluetongue virus in sheep from Minas Gerais / Inquérito sorológico do vírus da língua azul em ovinos de Minas Gerais

Bluetongue is an infectious, non-contagious disease that affects domestic and wild ruminants, caused by a virus from the Orbivirus genus, Reoviridae family, transmitted by arthropod vectors of the Culicoides genus. This paper aims to be the first serological survey of bluetongue in sheep from the Meso-regions of Campo das Vertentes and South and Southeast of Minas Gerais. Samples were collected from sheep from different properties. The serum samples were submitted to Agar Gel Immunodiffusion (AGID) and competitive Enzyme-Linked Immunosorbent Assay (cELISA). 303 serum samples were submitted to AGID and cELISA. In these samples, 164 (54.13%) were positive in the AGID technique, and 171 (56.44%) positive in the cELISA technique, with an almost perfect agreement between the techniques (kappa index = 0.887). In all visited properties, positive animals have been found in the herd. Animals acquired from properties of the studied mesoregions were more likely to be positive in IDGA and cELISA tests than animals acquired from properties in other regions of Brazil (p<0.001). These results suggest that bluetongue virus (BTV) is widespread in the mesoregions of Campo das Vertentes and South and Southeast of Minas Gerais.(AU)

A língua azul (LA) é uma doença infecciosa, não contagiosa, que acomete ruminantes domésticos e silvestres, causada por um vírus do gênero Orbivirus da família Reoviridae, transmitida por vetores artrópodes do gênero Culicoides. O presente estudo representa o primeiro trabalho a realizar um inquérito sorológico da língua azul em rebanhos ovinos nas Mesorregiões de Campo das Vertentes e Sul e Sudoeste de Minas Gerais. Foram coletadas amostras de soro de ovinos de diferentes propriedades. As amostras de soro foram submetidas aos testes de imunodifusão em gel de ágar (IDGA) e ensaio de imunoadsorção enzimática por competição (cELISA). Ao todo 303 amostras de soro foram submetidas ao IDGA e cELISA. Dessas amostras, 164 (54,13%) foram positivas na técnica de IDGA e 171 (56,44%) positivas na técnica de cELISA, havendo concordância quase perfeita entre as técnicas (índice kappa = 0,887). Em todas as propriedades visitadas, foram encontrados animais positivos no rebanho. Animais adquiridos de propriedades das Mesorregiões estudadas, tiveram mais chances de serem positivos nos testes de IDGA e cELISA do que animais adquiridos de propriedades de outras Regiões do Brasil (p<0,001). Esses resultados sugerem que o vírus da língua azul encontra-se disseminado em ovinos nas Mesorregiões de Campo das Vertentes e Sul e Sudoeste de Minas Gerais.(AU)