Single Nucleotide Polymorphism Genotyping and Distribution of Coxiella burnetii Strains from Field Samples in Belgium.

The genotypic characterization of Coxiella burnetii provides useful information about the strains circulating at the farm, region, or country level and may be used to identify the source of infection for animals and humans. The aim of the present study was to investigate the strains of C. burnetii circulating in caprine and bovine Belgian farms using a single nucleotide polymorphism (SNP) technique. Direct genotyping was applied to different samples (bulk tank milk, individual milk, vaginal swab, fetal product, and air sample). Besides the well-known SNP genotypes, unreported ones were found in bovine and caprine samples, increasing the variability of the strains found in the two species in Belgium. Moreover, multiple genotypes were detected contemporarily in caprine farms at different years of sampling and by using different samples. Interestingly, certain SNP genotypes were detected in both bovine and caprine samples, raising the question of interspecies transmission of the pathogen.

Diagnosis of Coxiella burnetii Cattle Abortion: A One-Year Observational Study.

Q fever is a zoonosis occurring worldwide in livestock. Often neglected in differential diagnoses, Q fever can persist in herds causing financial losses. In ruminants, well-known manifestations of Q fever are metritis, infertility, abortion, stillbirth and delivery of a weak or premature calf. In cattle, Q fever is frequently asymptomatic and/or under-reported. Few studies are available on the diagnosis of Coxiella burnetii as a cause of abortion in cattle using polymerase chain reaction (PCR) for pathogen detection while enzyme-linked immunosorbent assay (ELISA) is used to assess exposure. Moreover, existing studies include a relatively small number of abortions. The aim of this study is to assess, in the southern part of Belgium, during a year, the performance of diagnosis of C. burnetii as a cause of abortion and the putative benefit of enhanced serology using anamnesis (animal patient data, and present, past and environmental history). A one-year random selection of 1212 abortions was analysed both with the PCR method (tissues from fetuses) and two commercialised ELISAs (sera from the mothers). Relative sensitivity and specificity of the ELISA tests were assessed using PCR as the reference test. The prevalence of C. burnetii PCR positive was 8.5% (95% CI: 6.99-10.21). The diagnostic value of the ELISA tests was assessed using the area under the receiver operating characteristic curve (AUC-ROC). The sensitivity, specificity and AUC-ROC were similar for both ELISA tests. The diagnostic capacity of the ELISA was confirmed and slightly enhanced if anamnestic information was integrated with a unique scoring index system. A high negative predictive value was demonstrated and a significant reverse association between Ct values and a percentage of the ratio of the optical density between the sample and the positive control (ELISA A or ELISA B) enabling the use of ELISA as an exclusion diagnostic. This study is original by integrating the serological result and the anamnesis in a single index. It opens a new window in enhanced veterinary clinical decision-making.

First Expert Elicitation of Knowledge on Drivers of Emergence of Bovine Besnoitiosis in Europe.

Bovine besnoitiosis (BB) is a chronic and debilitating parasitic disease in cattle caused by the protozoan parasite Besnoitia besnoiti. South European countries are affected and have reported clinical cases of BB. However, BB is considered as emerging in other countries/regions of central, eastern and northern Europe. Yet, data on drivers of emergence of BB in Europe are scarce. In this study, fifty possible drivers of emergence of BB in cattle were identified. A scoring system was developed per driver. Then, the scoring was elicited from eleven recognized European experts to: (i) allocate a score to each driver, (ii) weight the score of drivers within each domain and (iii) weight the different domains among themselves. An overall weighted score was calculated per driver, and drivers were ranked in decreasing order of importance. Regression tree analysis was used to group drivers with comparable likelihoods to play a role in the emergence of BB in cattle in Europe. Finally, robustness testing of expert elicitation was performed for the seven drivers having the highest probability to play a key role in the emergence of BB: i.e., (i) legal/illegal movements of live animals from neighbouring/European Union member states or (ii) from third countries, (iii) risk of showing no clinical sign and silent spread during infection and post infection, (iv) as a consequence, difficulty to detect the emergence, (v) existence of vectors and their potential spread, (vi) European geographical proximity of the pathogen/disease to the country, and (vii) animal density of farms. Provided the limited scientific knowledge on the topic, expert elicitation of knowledge, multi-criteria decision analysis, cluster and sensitivity analyses are very important to prioritize future studies, e.g., the need for quantitative import risk assessment and estimation of the burden of BB to evidence and influence policymaking towards changing (or not) its status as a reportable disease, with prevention and control activities targeting, firstly, the top seven drivers. The present methodology could be applied to other emerging animal diseases.

Emergence of Besnoitia besnoiti in Belgium.

Bovine besnoitiosis is a cattle disease caused by a protozoan parasite called Besnoitia besnoiti. It is of serious economic concern to the cattle industry and also compromises animal welfare. For several years, it has been considered an emerging disease in some countries and regions located in the north of Europe far away from the known endemic areas in the south. This study describes the situation in the southern part of Belgium, where the parasite was recently introduced through imports of animals coming from departments of France where the disease was present. It details the detection of clinical cases as well as disease transmission features related to contacts during grazing and sales of infected cattle. A tracking and monitoring system was quickly set up and detected twelve outbreaks. Several cattle were controlled, but the lack of appropriate regulations weakens disease-management efforts. Hopefully, this predictable and silent introduction triggers the awareness of decision-makers about the need for an appropriate prevention and control policy, law enforcement, and the implementation of necessary measures to avoid bovine besnoitiosis becoming endemic in Belgium or other non-endemic countries. In addition, more proactive surveillance is required from authorities through threat analysis in the context of the risk of emergence or re-emergence of infectious animal diseases.

Environmental determinants of Anaplasma phagocytophilum infection in cattle using a kernel density function.

The study of vector-borne zoonotic diseases often relies on partial data, because of the constraints associated with observing various elements of the transmission cycle: the pathogen, the vector, the host - wild or domestic. Each angle comes with its own practical challenges, leading to data reflecting poorly either on spatial or temporal dynamics, or both. In this study, we investigated the effect of landscape on the presence of bovine ehrlichiosis infection in Walloon cattle. This disease is transmitted to cattle through the bite of a tick infected by the bacterium Anaplasma phagocytophilum. The first case of bovine ehrlichiosis in the southern region of Belgium (Wallonia) was detected in 2005 and the high seroprevalence found in herds suggests that the disease is endemic. The presence of antibodies of A. phagocytophilum in one cow selected in each of 1445 herds in 2010 and 2011 was detected using indirect immunofluorescence. Samples were geolocated at the farm. However, the precise location of infection remains uncertain. To account for the data sparsity, we elaborated a spatial index for the intensity of the presence of seropositive animals, based on a non-parametric kernel density estimation. We examined this index with the landscape surrounding the pastures, using multiple regressions. Landscape factors were selected using a conceptual framework based on the ecological resources needed for the transmission cycle of A. phagocytophilum. Results suggest that our spatial index adequately reflected infection presence in cattle in Wallonia, which was highest in central regions, corresponding to more forested and fragmented landscapes. We noticed that the presence of large hosts, wild or domestic, as well as the composition and configuration of the landscape of the pasture, influenced the capacity of the pasture to support the presence of bovine ehrlichiosis in Walloon herds. This is consistent with the ecology of A. phagocytophilum and current knowledge about risk factors of tick-borne diseases in cattle at the regional scale. The nature of the kernel density index, based on uncertainties over the location of cases positive to A. phagocytophilum, reflected the infectiousness profile at the landscape and not at the pasture level. Results also highlighted that the effects of some environmental variables remain, even when considering the different agro-geographic regions of Wallonia, which present contrasted landscapes and different levels of intensity of A. phagocytophilum infection. The kernel density index is a useful tool to help veterinary practitioner to quickly target areas where A. phagocytophilum infection is likely.

Pilot study assessing the possible benefits of a higher level of implementation of biosecurity measures on farm productivity and health status in Belgian cattle farms.

Over the last few years, the interest of decision-makers and control agencies in biosecurity (BS), aiming at preventing and controlling the introduction and spread of infectious diseases, has considerably increased. Nevertheless, previous studies highlighted a low implementation level of biosecurity measures (BSM), especially in cattle farms; different reasons were identified such as perceived costs, utility, importance, increased workload and lack of knowledge. In order to convince cattle farmers to adopt BSM, it is necessary to gather more information and evidence on their cost-effectiveness and their importance or utility in terms of disease prevention and control. The objectives of this study were to determine whether the farm or farmers' profile correlated with the implementation level of BSM and if there was a positive correlation between the BSM implementation and the farm production and health parameters. Data were collected through face-to-face interviews conducted in 100 Belgian farms as part of a stratified and randomized survey. The Regional Animal Health Services provided the farm health status and production data. A general BS score and five sub-scores related to the five BS compartments (bio-exclusion, bio-compartmentation, bio-containment, bio-prevention and bio-preservation) were calculated for each farm based on the implementation level of different BSM grouped in 16 domains. The study highlighted a significant and negative correlation between the mortality rates in adult cattle (over 24 months of age) and young calves (aged 0-7 days) and different BS compartment scores. The study also demonstrated that the farms having a higher general BS score were indeed more likely to have a BVD-free status. These evidence-based findings are encouraging as they demonstrate the benefits of implementing BSM and could promote their adoption by farmers.

Laboratory Diagnosis of Bovine Abortions Caused by Non-Maintenance Pathogenic Leptospira spp.: Necropsy, Serology and Molecular Study Out of a Belgian Experience.

: Bovine leptospirosis is a bacterial zoonotic disease caused by pathogenic Leptospira spp.. The pathology and epidemiology of this infection are influenced by the numerous existing serovars and their adaptation to specific hosts. Infections by host-maintained serovars such as Hardjo are well documented, unlike those from the incidental ones. In July 2014, an emerging phenomenon of an increased incidence of icteric abortions associated with leptospiral infection occurred in southern Belgium. First-line serological analyses targeting cattle-adapted serovars failed at initial diagnosis. This study provides a comprehensive description of laboratory findings-at the level of necropsy, serology and molecular diagnosis-regarding icteric and non-icteric abortions (n = 116) recorded during this time (years 2014-2015) and associated with incidental infection by serovars such as Grippotyphosa, Australis and Icterohaemorrhagiae. Based on these tests, a diagnostic pathway is proposed for these types of infection in cattle to establish an affordable but accurate diagnosis in the future. These investigations add insights into the understanding of the pathogenesis of bovine leptospirosis associated with serovars classically described as non-maintenance.

Characterization of three commercial ELISA kits for detection of BOHV-1 gE specific antibodies in serum and milk samples and applicability of bulk milk for determination of herd status.

Vaccination of animals with gE-deleted vaccine strains (gE- marker vaccines) and differential detection of vaccinated vs infected animals with antibody ELISA targeting the gE or the gB proteins have been proved to be useful tools in programs for control and eradication of the bovine herpesvirus 1 (BoHV-1) responsible for infectious bovine rhinotracheitis (IBR), a major pathogen of cattle. The diagnostic sensitivity (DSe) and specificity (DSp) of three commercial gE ELISA kits from IDEXX, IDVet and CIV-HIPRA were compared for serum and milk matrices. Limiting the analysis to 198 individual with concordant ELISA results in serum (91 naïve, 37 vaccinated and 70 infected) the DSe of gE kits was estimated to 0,97 for IDEXX, 0,93 for CIV-HIPRA and 0,53 for IDVet using milk samples and the DSp to 0,95 for IDEXX, 1,00 for IDVet and CIV-HIPRA. The applicability of gE ELISA for individual or bulk milk testing as an additional tool in control programs dedicated to the certification and control of vaccinated herds was evaluated. Two of the three evaluated gE ELISA kits presented substantial to good agreement individual milk and serum samples. The bulk-tank milk also proved to be suitable for the detection of BoHV-1 in vaccinated herds provided that gE prevalence is superior to 10% as false negative results are often observed at lower gE herd prevalence. This limitation could be reduced to 8% of prevalence when a prior concentration step was applied to bulk milk samples.

Complete Genome Sequence of Bovine Polyomavirus Type 1 from Aborted Cattle, Isolated in Belgium in 2014.

The complete and fully annotated genome sequence of a bovine polyomavirus type 1 (BPyV/BEL/1/2014) from aborted cattle was assembled from a metagenomics data set. The 4,697-bp circular dsDNA genome contains 6 protein-coding genes. Bovine polyomavirus is unlikely to be causally related to the abortion cases.