Development of anti-Crimean-Congo hemorrhagic fever virus Gc and NP-specific ELISA for detection of antibodies in domestic animal sera.

Crimean-Congo hemorrhagic fever (CCHF) is a priority emerging disease. CCHF, caused by the CCHF virus (CCHFV), can lead to hemorrhagic fever in humans with severe cases often having fatal outcomes. CCHFV is maintained within a tick-vertebrate-tick cycle, which includes domestic animals. Domestic animals infected with CCHFV do not show clinical signs of the disease and the presence of antibodies in the serum can provide evidence of their exposure to the virus. Current serological tests are specific to either one CCHFV antigen or the whole virus antigen. Here, we present the development of two in-house ELISAs for the detection of serum IgG that is specific for two different CCHFV antigens: glycoprotein Gc (CCHFV Gc) and nucleoprotein (CCHFV NP). We demonstrate that these two assays were able to detect anti-CCHFV Gc-specific and anti-CCHFV NP-specific IgG in sheep from endemic CCHFV areas with high specificity, providing new insight into the heterogeneity of the immune response induced by natural infection with CCHFV in domestic animals.

Neethling vaccine proved highly effective in controlling lumpy skin disease epidemics in the Balkans.

Despite the wide use of the live attenuated Neethling lumpy skin disease (LSD) vaccine, only limited data existed on its efficacy and effectiveness prior to the large LSD epidemic in the Balkans, which took place during 2016-2017. In addition, analysis of risk factors for the disease was hardly performed with proper control for vaccination effects and potential differences in exposure to the virus. Data from the LSD epidemics in six Balkan countries (Bulgaria, Greece, Serbia, Montenegro, Former Yugoslav Republic of Macedonia (FYROM) and Albania) affected during 2016 were analyzed to determine vaccine effectiveness (VE) and risk factors for LSD infection at the farm level. Vaccination was performed along the occurrence of the epidemics and thus vaccination status of some of the farms changed during the epidemic. To allow for this, left truncated and right censored survival analysis was used in a mixed effects Cox proportional hazard regression model to calculate VE and risk factors for LSD. The results indicated of an average VE of 79.8% (95% CI: 73.2-84.7)) in the six countries, with the lowest VE of 62.5% documented in Albania and up to VE of more than 97% as documented in Bulgaria and Serbia. Analysis of time from vaccination to development of protective immunity showed that VE mostly developed during the first 14 days after vaccination. Data from Greece showed that the vaccination adjusted hazard ratio for LSD was 5.7 higher in grazing farms compared to non-grazing farms. However, due to a difference in geographical location of grazing and non-grazing farms and higher vaccination rate in non-grazing farms, this effect can be at least partly attributed to indirect protection due to herd immunity provided by surrounding vaccinated farms.

African Swine Fever in a Bulgarian Backyard Farm-A Case Report.

African swine fever (ASF) is one of the most threatening diseases for the pig farming sector worldwide. As an effective vaccine is lacking, strict application of control measures is the only way to fight the disease in both industrial farms and backyard holdings. With generally low biosecurity standards, the latter are at particular risk for disease introduction and offer challenging conditions for disease control. In the following case report, we describe the overall course of an ASF outbreak in a Bulgarian backyard farm and the implemented control measures. Farm facilities and available data have been investigated to estimate the possible source, spread and time point of virus introduction. Contact with contaminated fomites entering the stable via human activities was regarded to be the most likely introduction route. The slow disease spread within the farm contributes to the hypothesis of a moderate contagiosity. As no further ASF outbreaks have been detected in domestic pig farms in the region, it could be demonstrated that successful disease control in small-scale farms can be reached. Thus, the report contributes to a better understanding of ASF in the backyard sector.

A framework for reviewing livestock disease reporting systems in high-risk areas: assessing performance and perceptions towards foot and mouth disease reporting in the Thrace region of Greece, Bulgaria and Turkey.

Disease reporting is an essential frontline component of surveillance systems, particularly for detecting incursions of new and emerging diseases. It has the advantages of being comprehensive and continuous, with the potential to reduce the time of disease detection and the extent of consequent spread. A number of exotic diseases, including sheep and goat pox, lumpy skin disease, peste des petits ruminants and foot and mouth disease have historically entered into south-eastern Europe through the Thrace region, which extends across neighbouring areas of Greece, Bulgaria and Turkey. In this high-risk area, multiple factors can reduce the sensitivity of disease reporting across the diverse production systems and animal health services need robust and effective disease reporting systems. While describing a training exercise designed to provide animal health services of the three countries with the knowledge and skills for conducting comprehensive in-country assessments, we provide an initial evaluation of the sensitivity of foot and mouth disease reporting and identify gaps and constraints in the Thrace region. An expert elicitation approach was used to consult official veterinarians from central and local animal health authorities of the three countries, and scenario trees modelling was applied to analyse the collected data. The reported sensitivity of disease reporting often varied between the central and local veterinary authorities within the three countries. Awareness of clinical disease, of reporting procedures and of biosecurity measures affected the early stages of disease reporting, particularly in the production systems identified at lower reporting sensitivity such as small ruminant's herds, mixed bovine herds and backyard herds. Despite its limitations this training exercise provided an effective framework (a) to develop capacities of the veterinary services in the region and (b) to supply initial evidence for guiding further interventions targeting those sectors and stakeholders at lower reporting sensitivity to reduce risks of disease introduction.

Prediction of Pig Trade Movements in Different European Production Systems Using Exponential Random Graph Models.

In most European countries, data regarding movements of live animals are routinely collected and can greatly aid predictive epidemic modeling. However, the use of complete movements' dataset to conduct policy-relevant predictions has been so far limited by the massive amount of data that have to be processed (e.g., in intensive commercial systems) or the restricted availability of timely and updated records on animal movements (e.g., in areas where small-scale or extensive production is predominant). The aim of this study was to use exponential random graph models (ERGMs) to reproduce, understand, and predict pig trade networks in different European production systems. Three trade networks were built by aggregating movements of pig batches among premises (farms and trade operators) over 2011 in Bulgaria, Extremadura (Spain), and Côtes-d'Armor (France), where small-scale, extensive, and intensive pig production are predominant, respectively. Three ERGMs were fitted to each network with various demographic and geographic attributes of the nodes as well as six internal network configurations. Several statistical and graphical diagnostic methods were applied to assess the goodness of fit of the models. For all systems, both exogenous (attribute-based) and endogenous (network-based) processes appeared to govern the structure of pig trade network, and neither alone were capable of capturing all aspects of the network structure. Geographic mixing patterns strongly structured pig trade organization in the small-scale production system, whereas belonging to the same company or keeping pigs in the same housing system appeared to be key drivers of pig trade, in intensive and extensive production systems, respectively. Heterogeneous mixing between types of production also explained a part of network structure, whichever production system considered. Limited information is thus needed to capture most of the global structure of pig trade networks. Such findings will be useful to simplify trade networks analysis and better inform European policy makers on risk-based and more cost-effective prevention and control against swine diseases such as African swine fever, classical swine fever, or porcine reproductive and respiratory syndrome.

Spatial and Functional Organization of Pig Trade in Different European Production Systems: Implications for Disease Prevention and Control.

Understanding the complexity of live pig trade organization is a key factor to predict and control major infectious diseases, such as classical swine fever (CSF) or African swine fever (ASF). Whereas the organization of pig trade has been described in several European countries with indoor commercial production systems, little information is available on this organization in other systems, such as outdoor or small-scale systems. The objective of this study was to describe and compare the spatial and functional organization of live pig trade in different European countries and different production systems. Data on premise characteristics and pig movements between premises were collected during 2011 from Bulgaria, France, Italy, and Spain, which swine industry is representative of most of the production systems in Europe (i.e., commercial vs. small-scale and outdoor vs. indoor). Trade communities were identified in each country using the Walktrap algorithm. Several descriptive and network metrics were generated at country and community levels. Pig trade organization showed heterogeneous spatial and functional organization. Trade communities mostly composed of indoor commercial premises were identified in western France, northern Italy, northern Spain, and north-western Bulgaria. They covered large distances, overlapped in space, demonstrated both scale-free and small-world properties, with a role of trade operators and multipliers as key premises. Trade communities involving outdoor commercial premises were identified in western Spain, south-western and central France. They were more spatially clustered, demonstrated scale-free properties, with multipliers as key premises. Small-scale communities involved the majority of premises in Bulgaria and in central and Southern Italy. They were spatially clustered and had scale-free properties, with key premises usually being commercial production premises. These results indicate that a disease might spread very differently according to the production system and that key premises could be targeted to more cost-effectively control diseases. This study provides useful epidemiological information and parameters that could be used to design risk-based surveillance strategies or to more accurately model the risk of introduction or spread of devastating swine diseases, such as ASF, CSF, or foot-and-mouth disease.

Evaluation of the spatial patterns and risk factors, including backyard pigs, for classical swine fever occurrence in Bulgaria using a Bayesian model.

The spatial pattern and epidemiology of backyard pig farming and other low bio-security pig production systems and their role in the occurrence of classical swine fever (CSF) is described and evaluated. A spatial Bayesian model was used to explore the risk factors, including human demographics, socioeconomic and environmental factors. The analyses were performed for Bulgaria, which has a large number of backyard farms (96% of all pig farms in the country are classified as backyard farms), and it is one of the countries for which both backyard pig and farm counts were available. Results reveal that the high-risk areas are typically concentrated in areas with small family farms, high numbers of outgoing pig shipments and low levels of personal consumption (i.e. economically deprived areas). Identification of risk factors and high-risk areas for CSF will allow to targeting risk-based surveillance strategies leading to prevention, control and, ultimately, elimination of the disease in Bulgaria and other countries with similar socio-epidemiological conditions.

Surveillance of foot-and-mouth disease (FMD) in susceptible wildlife and domestic ungulates in Southeast of Bulgaria following a FMD case in wild boar.

Following a foot-and-mouth disease (FMD) case of serotype O in wild boar in Southeast of Bulgaria, notified in January 2011 and eleven FMD outbreaks in livestock, a control and eradication plan according to the EU legislation was implemented. Based on the epidemiological considerations a "Cordon Sanitaire" along the border to Turkey, consisting of a defined infected area (1240 km(2)) and two areas of risk (2160 km(2)) was established. Within these areas a total of 812 wild boar, 68 roe deer, 7 red deer and 2 mouflons, hunted between February 2011 and January 2012, were tested for the presence of FMD. No FMD virus could be detected. Seropositive animals were found in wild boar (6.9%) and roe deer (4.4%), most of them spatially clustered around the FMD outbreaks in livestock, limited within a radius of 20 km. The outbreaks in domestic ungulates were controlled in the framework of EU legislation, including stamping out, standstill and no use of vaccination. All villages within the Cordon Sanitaire were examined for the presence of FMD according to the control and eradication plan. Neither clinical signs nor seroconversion was detected and the region could regain its status of FMD freedom without vaccination. The relatively low seroprevalence and clustered spatial distribution of seropositive wild boar and roe deer suggest that the FMD infection in wildlife was mainly a short living event, which failed to develop into a large scale epidemic.

Evaluation of the risk of classical swine fever (CSF) spread from backyard pigs to other domestic pigs by using the spatial stochastic disease spread model Be-FAST: the example of Bulgaria.

The study presented here is one of the very first aimed at exploring the potential spread of classical swine fever (CSF) from backyard pigs to other domestic pigs. Specifically, we used a spatial stochastic spread model, called Be-FAST, to evaluate the potential spread of CSF virus (CSFV) in Bulgaria, which holds a large number of backyards (96% of the total number of pig farms) and is one of the very few countries for which backyard pigs and farm counts are available. The model revealed that, despite backyard pigs being very likely to become infected, infections from backyard pigs to other domestic pigs were rare. In general, the magnitude and duration of the CSF simulated epidemics were small, with a median [95% PI] number of infected farms per epidemic of 1 [1,4] and a median [95% PI] duration of the epidemic of 44 [17,101] days. CSFV transmission occurs primarily (81.16%) due to indirect contacts (i.e. vehicles, people and local spread) whereas detection of infected premises was mainly (69%) associated with the observation of clinical signs on farm rather than with implementation of tracing or zoning. Methods and results of this study may support the implementation of risk-based strategies more cost-effectively to prevent, control and, ultimately, eradicate CSF from Bulgaria. The model may also be easily adapted to other countries in which the backyard system is predominant. It can also be used to simulate other similar diseases such as African swine fever.

Reconstruction of the transmission history of RNA virus outbreaks using full genome sequences: foot-and-mouth disease virus in Bulgaria in 2011.

Improvements to sequencing protocols and the development of computational phylogenetics have opened up opportunities to study the rapid evolution of RNA viruses in real time. In practical terms, these results can be combined with field data in order to reconstruct spatiotemporal scenarios that describe the origin and transmission pathways of viruses during an epidemic. In the case of notifiable diseases, such as foot-and-mouth disease (FMD), these analyses provide important insights into the epidemiology of field outbreaks that can support disease control programmes. This study reconstructs the origin and transmission history of the FMD outbreaks which occurred during 2011 in Burgas Province, Bulgaria, a country that had been previously FMD-free-without-vaccination since 1996. Nineteen full genome sequences (FGS) of FMD virus (FMDV) were generated and analysed, including eight representative viruses from all of the virus-positive outbreaks of the disease in the country and 11 closely-related contemporary viruses from countries in the region where FMD is endemic (Turkey and Israel). All Bulgarian sequences shared a single putative common ancestor which was closely related to the index case identified in wild boar. The closest relative from outside of Bulgaria was a FMDV collected during 2010 in Bursa (Anatolia, Turkey). Within Bulgaria, two discrete genetic clusters were detected that corresponded to two episodes of outbreaks that occurred during January and March-April 2011. The number of nucleotide substitutions that were present between, and within, these separate clusters provided evidence that undetected FMDV infection had occurred. These conclusions are supported by laboratory data that subsequently identified three additional FMDV-infected livestock premises by serosurveillance, as well as a number of antibody positive wild boar on both sides of the border with Turkish Thrace. This study highlights how FGS analysis can be used as an effective on-the-spot tool to support and help direct epidemiological investigations of field outbreaks.

Experimental infection of wild boar and domestic pigs with a foot and mouth disease virus strain detected in the southeast of Bulgaria in December of 2010.

Foot and mouth disease (FMD) was detected in a wild boar in Southeastern Bulgaria in December 2010. The occurrence and spread of the disease in wild cloven-hoofed animals may pose an unexpected and significant threat to FMD virus (FMDV)-free areas within and outside the European Union. So far, only one well documented experimental infection with FMD in wild boar has been published. In order to obtain more epidemiologically relevant data regarding the disease in wild boar we conducted an experiment with the 2010 Bulgarian FMDV type O isolate. Two young wild boar were challenged while two domestic pigs and two additional wild boar served as contact controls. While the domestic pigs developed severe clinical signs of FMD, the wild boar showed relatively mild course of the disease. Viremia started in contact wild boar 2 days post exposure (DPE) and lasted until 6 DPE. The virus shedding lasted until 9 DPE. On 27 DPE, when the animals were slaughtered, viral RNA was detected in lymphoid tissues and oropharyngeal fluid but no virus could be isolated. Commercial ELISAs and virus neutralisation tests detected antibodies against FMDV on 8 or 6 DPE, respectively. The data of the present study will help to understand FMD in wild boar populations and can be used in models to evaluate the potential role of wild boar in FMD epidemiology.

Reemergence of human and animal brucellosis, bulgaria.

Bulgaria had been free from brucellosis since 1958, but during 2005-2007, a reemergence of human and animal disease was recorded. The reemergence of this zoonosis in the country highlights the importance of maintaining an active surveillance system for infectious diseases that will require full cooperation between public health and veterinary authorities.