Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus.

IMPORTANCE: Lumpy skin disease virus (LSDV) has a complex epidemiology involving multiple strains, recombination, and vaccination. Its DNA genome provides limited genetic variation to trace outbreaks in space and time. Sequencing of LSDV whole genomes has also been patchy at global and regional scales. Here, we provide the first fine-grained whole genome sequence sampling of a constrained LSDV outbreak (southeastern Europe, 2015-2017), which we analyze along with global publicly available genomes. We formally evaluate the past occurrence of recombination events as well as the temporal signal that is required for calibrating molecular clock models and subsequently conduct a time-calibrated spatially explicit phylogeographic reconstruction. Our study further illustrates the importance of accounting for recombination events before reconstructing global and regional dynamics of DNA viruses. More LSDV whole genomes from endemic areas are needed to obtain a comprehensive understanding of global LSDV dispersal dynamics.

Corrigendum: Overview of Cattle Diseases Listed Under Category C, D or E in the Animal Health Law for Which Control Programmes Are in Place Within Europe.

[This corrects the article DOI: 10.3389/fvets.2021.688078.].

Overview of Cattle Diseases Listed Under Category C, D or E in the Animal Health Law for Which Control Programmes Are in Place Within Europe.

The COST action "Standardising output-based surveillance to control non-regulated diseases of cattle in the European Union (SOUND control)," aims to harmonise the results of surveillance and control programmes (CPs) for non-EU regulated cattle diseases to facilitate safe trade and improve overall control of cattle infectious diseases. In this paper we aimed to provide an overview on the diversity of control for these diseases in Europe. A non-EU regulated cattle disease was defined as an infectious disease of cattle with no or limited control at EU level, which is not included in the European Union Animal health law Categories A or B under Commission Implementing Regulation (EU) 2020/2002. A CP was defined as surveillance and/or intervention strategies designed to lower the incidence, prevalence, mortality or prove freedom from a specific disease in a region or country. Passive surveillance, and active surveillance of breeding bulls under Council Directive 88/407/EEC were not considered as CPs. A questionnaire was designed to obtain country-specific information about CPs for each disease. Animal health experts from 33 European countries completed the questionnaire. Overall, there are 23 diseases for which a CP exists in one or more of the countries studied. The diseases for which CPs exist in the highest number of countries are enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis, bovine viral diarrhoea and anthrax (CPs reported by between 16 and 31 countries). Every participating country has on average, 6 CPs (min-max: 1-13) in place. Most programmes are implemented at a national level (86%) and are applied to both dairy and non-dairy cattle (75%). Approximately one-third of the CPs are voluntary, and the funding structure is divided between government and private resources. Countries that have eradicated diseases like enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis and bovine viral diarrhoea have implemented CPs for other diseases to further improve the health status of cattle in their country. The control of non-EU regulated cattle diseases is very heterogenous in Europe. Therefore, the standardising of the outputs of these programmes to enable comparison represents a challenge.

An Overview of Current Approaches and Challenges to the Control of Endemic Infectious Cattle Diseases in Albania.

Agriculture is an important production sector in Albania that makes a significant contribution to the gross domestic product (GDP) and employment. The livestock sector contributes more than half of the agricultural GDP. The Albanian cattle population represents 50% of the total livestock units and accounts for 85% of the national milk production, the rest being supplied by small ruminants. Cattle productivity, health and welfare are hindered by infectious diseases, some of which are also transmissible to humans (zoonosis). The aim of this manuscript is to provide an overview of the control of selected regulated and non-EU regulated cattle diseases in Albania and to highlight specific challenges for the Albanian cattle industry. The most important infectious cattle diseases in Albania for which national control and eradication strategies are in place are bovine brucellosis, bovine tuberculosis, and anthrax, which are all zoonotic. Additionally, lumpy skin disease recently emerged in the Balkan region and is currently subject to controls. Most of the available funds and European Union support are allocated to the control of EU regulated zoonotic diseases. For control of non-EU regulated cattle diseases, no funds are available resulting in the lack of national control programmes (CPs). Based on research, clinical investigations and laboratory results, several non-EU regulated cattle infectious diseases appear endemic in Albanian dairy farms. While no national CPs exist for any of them, regional initiatives are available on a voluntary basis to control infectious bovine rhinotracheitis and bovine viral diarrhea. In the voluntary CPs, there is no monitored requirement to prove disease freedom of purchased animals and to re-evaluate the herd's free status after the introduction of animals into a herd. Data on animal movements that are routinely collected could potentially be used to control the risk of purchase, but quality needs to be further improved to increase its usefulness in disease CPs. This overview aims to collate existing information on the CPs implemented in Albania and to evaluate these to highlight gaps and threats in disease control, as well as opportunities and strengths through a SWOT (Strengths, Weaknesses, Opportunities, and Threats) analysis, with the goal of providing a framework for the future implementation of animal disease control measures in Albania.

Existence and Quality of Data on Control Programs for EU Non-regulated Cattle Diseases: Consequences for Estimation and Comparison of the Probability of Freedom From Infection.

Some European countries have successfully implemented country-specific control programs (CPs) for infectious cattle diseases that are not regulated or are regulated only to a limited extent at the European Union (EU) level. Examples of such diseases include bovine viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR), and Johne's disease (JD). The CPs vary between countries in the design and quality of collected data as well as methods used to detect infection and estimate prevalence or probability of freedom from infection. Differences in disease status between countries and non-standardized approaches to assess freedom from infection pose a risk for countries with CPs for non-regulated diseases as infected animals may influence the progress of the disease control or eradication program. The implementation of output-based standards allows estimation and comparison of the probability of freedom for non-regulated cattle diseases in European countries. The aim of the current study was to assess the existence and quality of data that could be used for estimating freedom from infection in European countries. The online data collection tool was sent to 32 countries participating in the SOUND control COST Action and was completed by 24 countries. Data on cattle demographics and data from CPs of IBR and BVD exist in more than 50% of the response countries. However, data describing risk factors and CP of JD was reported as existing in <25% of the countries. The overall quality of data in the sections on demographics and CPs of IBR and BVD were evaluated as "good", but risk factors and JD data were mostly evaluated as "fair." Data quality was considered less good mainly due to two quality criteria: accessibility and accuracy. The results of this study show that the quantity and quality of data about cattle populations and CPs are relatively similar in many surveyed countries. The outcome of this work provides an overview of the current situation in the European countries regarding data on EU non-regulated cattle diseases and will further assist in the development and implementation of output-based standards.

Protocol optimization for simultaneous DNA and RNA co-extraction from single hard tick specimens.

Hard ticks are important vectors of DNA- and RNA-based infectious microorganisms, but they also host complex microbial communities in which pathogens and symbionts can interact among each other and with the arthropod host itself. Molecular investigations on ticks and their hosted microorganisms are important for human and animal health. These analyses often imply the use of both DNA and RNA, with prompt preservation of nucleic acids after collection, and safe handling in case of low-level containment. Several commercial kits are available for the co-extraction of DNA and RNA; however, cost can be a limiting factor for the choice of this method, which also require additional reagents for nucleic acids preservation before extraction. Additionally, extraction buffers provided in commercial kits do not guarantee the safe handling in case of hazardous biological material. With the aim of epidemiological screenings for tick-borne pathogens and gene expression analyses focused on the relationship among ticks and their microbial communities, an optimized protocol for DNA and RNA co-extraction from single adult hard tick specimens (Ixodidae) has been developed using TRIzolⓇ LS Reagent.A method for•DNA/RNA co-extraction from adult hard tick specimens;•Safe sample handling;•Obtaining DNA and RNA simultaneously for diagnostic procedures and RNA for gene expression/transcriptomic analyses.

Key Learnings During the Development of a Generic Data Collection Tool to Support Assessment of Freedom of Infection in Cattle Herds.

Various European Member States have implemented control or eradication programmes for endemic infectious diseases in cattle. The design of these programmes varies between countries and therefore comparison of the outputs of different control programmes is complex. Although output-based methods to estimate the confidence of freedom resulting from these programmes are under development, as yet there is no practical modeling framework applicable to a variety of infectious diseases. Therefore, a data collection tool was developed to evaluate data availability and quality and to collect actual input data required for such a modeling framework. The aim of the current paper is to present the key learnings from the process of the development of this data collection tool. The data collection tool was developed by experts from two international projects: STOC free (Surveillance Tool for Outcome-based Comparison of FREEdom from infection, www.stocfree.eu) and SOUND control (Standardizing OUtput-based surveillance to control Non-regulated Diseases of cattle in the EU, www.sound-control.eu). Initially a data collection tool was developed for assessment of freedom of bovine viral diarrhea virus in six Western European countries. This tool was then further generalized to enable inclusion of data for other cattle diseases i.e., infectious bovine rhinotracheitis and Johne's disease. Subsequently, the tool was pilot-tested by a Western and Eastern European country, discussed with animal health experts from 32 different European countries and further developed for use throughout Europe. The developed online data collection tool includes a wide range of variables that could reasonably influence confidence of freedom, including those relating to cattle demographics, risk factors for introduction and characteristics of disease control programmes. Our results highlight the fact that data requirements for different cattle diseases can be generalized and easily included in a data collection tool. However, there are large differences in data availability and comparability across European countries, presenting challenges to the development of a standardized data collection tool and modeling framework. These key learnings are important for development of any generic data collection tool for animal disease control purposes. Further, the results can facilitate development of output-based modeling frameworks that aim to calculate confidence of freedom from disease.

Standardizing output-based surveillance to control non-regulated cattle diseases: Aspiring for a single general regulatory framework in the European Union.

Several European countries have implemented country specific programmes to control cattle diseases with little or no regulation in the European Union (EU). These control programmes vary between member states, impairing a confident comparison of freedom from disease when cattle originate from different countries. In order to facilitate safe trade, there is a need to support the development of transparent methods that enable comparison of outputs of surveillance, control or eradication programmes. The aim of the COST Action (CA 17110), Standardizing OUtput-based surveillance to control Non-regulated Diseases in the EU (SOUND control), is the development of a generic and joint understanding of the requirements and characteristics needed for a flexible output-based framework. This framework should be able to substantiate the confidence of disease freedom and cost-effectiveness of heterogeneous surveillance, control or eradication programmes for cattle diseases in the EU. This project supports other initiatives in the development of an output-based framework which will subsequently facilitate safe trade and support the improvement of disease control measures, which is of great importance as the cattle sector contributes to one third of the total gross production value of EU agriculture.

The seroprevalence of brucellosis and molecular characterization of Brucella species circulating in the beef cattle herds in Albania.

BACKGROUND: Brucellosis is a ubiquitous zoonotic disease globally. It is endemic among bovines, sheep, and goats in Albania. The national control and eradication programs for brucellosis has been applied on sheep and goat farms as well as large dairy cattle farms, i.e., those with more than ten milking cows. The current study aims at estimating the herd and average individual animal prevalence of brucellosis in the national beef cattle herds, the missing information that was essential to propose the most appropriate control measures for this sub-population. Rose Bengal Test (RBT), Fluorescence Polarization Assay (FPA), and Enzyme-Linked Immunosorbent Assay (ELISA) were used as serological tests and classical bacteriology for isolation. Results were also used to investigate the difference in sensitivity between the assays used. METHODOLOGY: In total, 655 animals from 38 beef cattle herds from six southern districts of Albania were sampled. Sera were tested using RBT, FPA, and ELISA. Fifteen positive cows and a bull from eight high-prevalence positive herds were slaughtered, and particular tissue samples were collected for bacteriology. RESULTS: The overall herd seroprevalence in the tested beef cattle population was 55%, while the overall average within-herd prevalence (including only positive herds) was 38.3%, 42.7%, and 45.6% determined by the RBT, ELISA, and FPA, respectively. FPA was used for the first time in the diagnosis of bovine brucellosis in Albania, and its sensitivity was higher than RBT and ELISA. Three B. abortus strains were identified, two from the supra-mammary lymph node of two cows and one from the epididymis of a seropositive bull. CONCLUSION: Brucellosis was highly prevalent in beef cattle in the southern part of Albania, and B. abortus was isolated from this subpopulation. To the best of our knowledge, this is the first statistically based survey of bovine brucellosis in beef herds in Albania. Using the FPA in parallel with other serological tests improved overall diagnostic sensitivity. Test and slaughter policy is not a rational approach for the control of brucellosis in beef cattle in Albania, and vaccination is only applicable, including strict control of the movement of animals.

Assessment of an ELISA method to support surveillance of bovine tuberculosis in Albania.

BACKGROUND: Bovine tuberculosis (bTB) is an important bacterial infectious disease in Albania of concern to animal and human health; its prevalence is poorly documented. METHODS: In this longitudinal study, we tested by ELISA 2661 serum samples, from 154 herds, with the aim of establishing the suitability of this approach to screen the bovine population for bTB. In a follow-on survey of 87 animals in three villages, we assessed the usefulness of the Mycobacterium bovis IDEXX ELISA (IDEXX M. bovis Antibody (Ab) Test. IDEXX Europe B.V P.O. Box 1334, 2130 EK Hoofddorp, The Netherlands) assay by comparing IDEXX results with the results of the single intradermal cervical skin test. Skin tests were performed either after or at the time of collection of blood samples, and therefore cattle were not sensitized by tuberculin before serological testing. RESULTS: The proportion of herds in which serologically positive cattle were found was 18.2 % (95 % CI, 1.9-25.8 %) and the prevalence of seropositive cattle was 1.4 % (95 % CI, 0.8-2.1 %). In the follow-up study, two of the 87 animals reacted positively to the skin test and two produced inconclusive reactions. No overlap was found between the four animals with positive IDEXX ELISA results and the four animals with non-negative skin test results. CONCLUSION: The lack of agreement between the results of the two tests may reflect different elements of the immune response (humoral and cell-mediated immunity). In future, cattle should be sensitized by the intradermal injection of tuberculin 14 days prior to the collection of blood samples, which would then be tested by the Mycobacterium bovis IDEXX ELISA Test in order to determine more accurately the prevalence of infection.

Manganese binds to Clostridium difficile Fbp68 and is essential for fibronectin binding.

Clostridium difficile is an etiological agent of pseudomembranous colitis and antibiotic-associated diarrhea. Adhesion is the crucial first step in bacterial infection. Thus, in addition to toxins, the importance of colonization factors in C. difficile-associated disease is recognized. In this study, we identified Fbp68, one of the colonization factors that bind to fibronectin (Fn), as a manganese-binding protein (K(D) = 52.70 ± 1.97 nM). Furthermore, the conformation of Fbp68 changed dramatically upon manganese binding. Manganese binding can also stabilize the structure of Fbp68 as evidenced by the increased T(m) measured by thermodenatured circular dichroism and differential scanning calorimetry (CD, T(m) = 58-65 °C; differential scanning calorimetry, T(m) = 59-66 °C). In addition, enhanced tolerance to protease K also suggests greatly improved stability of Fbp68 through manganese binding. Fn binding activity was found to be dependent on manganese due to the lack of binding by manganese-free Fbp68 to Fn. The C-terminal 194 amino acid residues of Fbp68 (Fbp68C) were discovered to bind to the N-terminal domain of Fn (Fbp68C-NTD, K(D) = 233 ± 10 nM, obtained from isothermal titration calorimetry). Moreover, adhesion of C. difficile to Caco-2 cells can be partially blocked if cells are pretreated with Fbp68C, and the binding of Fbp68C on Fn siRNA-transfected cells was significantly reduced. These results raise the possibility that Fbp68 plays a key role in C. difficile adherence on host cells to initiate infection.