Jean-Baptiste Hanon

The Most Likely Time and Place of Introduction of BTV8 into Belgian Ruminants

Background In northern Europe, bluetongue (BT) caused by the BT virus (BTV), serotype 8, was first notified in August 2006 and numerous ruminant herds were affected in 2007 and 2008. However, the origin and the time and place of the original introduction have not yet been determined. Methods and Principal Findings Four retrospective epidemiological surveys have been performed to enable determination of the initial spatiotemporal occurrence of this emerging disease in southern Belgium: investigations of the first recorded outbreaks near to the disease epicenter; a large anonymous, random postal survey of cattle herds and sheep flocks; a random historical milk tank survey of samples tested with an indirect ELISA and a follow-up survey of non-specific health indicators. The original introduction of BTV into the region probably occurred during spring 2006 near to the National Park of Hautes Fagnes and Eifel when Culicoides become active. Conclusions/Significance The determination of the most likely time and place of introduction of BTV8 into a country is of paramount importance to enhance awareness and understanding and, to improve modeling of vector-borne emerging infectious diseases.

Reconstruction of the Schmallenberg virus epidemic in Belgium: Complementary use of disease surveillance approaches

Schmallenberg virus (SBV) emerged across Europe in 2011 and Belgium was among the first countries affected. In this study, published findings are combined with new data from veterinary surveillance networks and the Belgian reference laboratory for SBV at the Veterinary and Agrochemical Research centre (CODA-CERVA) to reconstruct the epidemic in Belgium. First retrospective cases of SBV were reported by veterinarians that observed decreased milk yield and fever in dairy cattle in May 2011. The number of SBV suspicions subsequently increased in adult cattle in August 2011. That month, first SBV positive pools of Culicoides were detected and extensive virus circulation occurred in Belgium during late summer and autumn 2011. As a consequence, most pregnant ruminants were infected and their fetuses exposed to the virus. This resulted in an outbreak of abortions, still-births and malformed new-borns observed between January and April 2012. The number of cases drastically diminished in 2012-2013, although multiple lines of evidence obtained from cross-sectional serological surveys, analyses on aborted foetuses, sentinel herd surveillance and surveillance of SBV in vectors prove that SBV was still circulating in Belgium at that time. Virus circulation was then probably strongly reduced in 2013-2014, while increasing evidence indicates its recirculation in 2014-2015 in Belgium. Based on the experience gathered with the closely related Akabane virus, recurrent outbreaks of congenital events can be expected for a long period. Vaccination of seronegative animals before the first mating could be used to prevent the deleterious effects of SBV. During this epidemic, different surveillance approaches including syndromic surveillance, sentinel herd surveillance, cross-sectional seroprevalence studies and pathogen surveillance in vectors have proven their utility and should be considered to continue in the future.

Seroprevalence of pertussis in Senegal: a prospective study.

Background Pertussis, also known as whooping cough, is a vaccine-preventable respiratory disease caused by Bordetella pertussis infection, against which Senegalese children are immunized with the diphtheria-tetanus-whole cell pertussis vaccine (DTwP). Seroepidemiology of pertussis has been widely described in industrialized countries, but rare are the studies referring to it in developing countries. Methods We conducted a longitudinal survey in Northern Senegal to investigate the epidemiology of B. pertussis by evaluating the IgG antibody (Ab) response against pertussis toxin (PT). A cohort of 410 children aged 1 to 9 from five villages in the Middle Senegal River Valley were followed-up for 18 months. During that period, five visits were made to assess the immunological status of the children. Principal Findings PT-specific IgG responses were significantly different according to age. Until the age of 3, there was a decrease in the Ab response, which then increased in the older groups. Assessment of IgG antibodies to PT (IgG-PT) suggested evidence of recent exposures to the pathogen. Surprisingly, in one of the five villages the average Ab response to PT was very low at all ages during the first 6 months of the study. At the third visit, IgG-PT concentrations peaked to very high levels, to slightly decline at the end of the survey. This indicates an outbreak of B. pertussis, whereas in the other villages a pertussis endemic profile could be observed. Conclusions Pertussis is endemic in Northern Senegal despite the introduction of vaccination. The circulation of the bacteria seems to differ between geographic locations and over time. A more complete understanding of the epidemiology of pertussis and its environmental determinants could provide information to adapt vaccination programs.

A trend analysis of antimicrobial resistance in commensal Escherichia coli from several livestock species in Belgium (2011-2014).

A temporal trend analysis was performed on antimicrobial resistance data collected over 4 consecutive years (2011-2014) in the official Belgian antimicrobial resistance monitoring programme. Commensal Escherichia coli strains were isolated from faecal samples of four livestock categories (veal calves, young beef cattle, broiler chickens and slaughter pigs) and the trends of resistance profiles were analysed. The resistance prevalence remained high (>50%) during the study period for ampicillin in veal calves and chickens, for ciprofloxacin and nalidixic acid in chickens, for sulfamethoxazole in veal calves, chickens and pigs and for tetracycline in veal calves. Using logistic regression and Generalized Estimating Equation and after p value adjustment for multiple testing (Linear step-up method), statistically significant decreasing temporal trends were observed for several of the 11 tested antimicrobials in several livestock categories: in veal calves (10/11), in chickens (6/11) and in pigs (5/11). A significant increasing trend was observed for the prevalence of resistance to ciprofloxacin in chickens. Multi-resistance, considered as the resistance to at least three antimicrobials of different antibiotic classes, was observed in the four livestock categories but was significantly decreasing in veal calves, chickens and pigs. Overall, the prevalence of resistance and of multi-resistance was lowest in the beef cattle livestock category and highest in broiler chickens. These decreasing temporal trends of antimicrobial resistance might be due to a decrease of the total antimicrobial consumption for veterinary use in Belgium which was reported for the period between 2010 and 2013. The methodology and statistical tools developed in this study provide outputs which can detect shifts in resistance levels or resistance trends associated with particular antimicrobial classes and livestock categories. Such outputs can be used as objective evidence to evaluate the possible efficacy of measures taken by animal health authorities and stakeholders in the livestock sector to limit antimicrobial resistance occurrence.

Effects of Malnutrition on Children's Immunity to Bacterial Antigens in Northern Senegal

To evaluate immunity to vaccine-preventable diseases according to nutritional status, a longitudinal study was conducted in Senegalese children ages 1-9 years old. A linear regression analysis predicted that weight for age was positively associated with immunoglobulin G (IgG) response to tetanus toxoid in children born during the rainy season or at the beginning of the dry season. A relationship between village, time of visits, and levels of antibodies to tetanus showed that environmental factors played a role in modulating humoral immunity to tetanus vaccine over time. Moreover, a whole-blood stimulation assay highlighted that the production of interferon-γ (IFN-γ) in response to tetanus toxoid was compromised in stunted children. However, the absence of cytokine modulation in response to Mycobacterium tuberculosis-purified protein derivatives and phytohemagglutinin suggests that the overall ability to produce IFN-γ was preserved in stunted children. Therefore, these results show that nutritional status can specifically alter the efficacy of long-lasting immunity to tetanus.

Evidence of extensive renewed Schmallenberg virus circulation in Belgium during summer of 2016 – increase in arthrogryposis‐hydranencephaly cases expected

A seroprevalence study carried out between June and September 2016 in the Belgian sheep population showed a significant increase in overall (from 25% to 62%) and between-herd (from 60% to 96%) seroprevalence against Schmallenberg virus (SBV) during this period, indicating the most extensive recirculation of SBV since its original emergence in 2011. SBV recirculation was confirmed by the detection of SBV RNA-positive Culicoides obsoletus complex midges collected in the region of Antwerp in August 2016, reaching a minimum infection rate of 3%. The recirculation of SBV in the largely unprotected ruminant population during summer 2016 will likely cause an increase in the number of arthrogryposis-hydranencephaly cases in newborn ruminants during the coming months.

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.

Evaluation of 16 commercial antibody ELISAs for the detection of bovine viral diarrhea virus–specific antibodies in serum and milk using well-characterized sample panels

We performed a thorough fit-for-purpose evaluation of commercial ELISAs for the detection of bovine viral diarrhea virus (BVDV)-specific antibodies in serum and in milk by testing 2 panels of well-characterized serum and milk samples. Sixteen ELISAs from 9 different manufacturers, available on the Belgian market at the time of our study, were assessed for their diagnostic and analytical sensitivity (DSe and ASe, respectively), diagnostic specificity (DSp), and repeatability relative to the virus neutralization (VN) test considered to be the gold standard assay. Using serum as a matrix, DSe was much lower for competitive (c)ELISAs (min. 45%, max. 65%) than for indirect (i)ELISAs (min. 85%, max. 100%), partly because of the lower detection of positive samples from vaccinated animals included in the panel. ASe was also better for iELISAs; DSp was >95% for all but 2 ELISAs. Repeatability, expressed as coefficients of variation (CV) of optical densities, was generally good, although 3 ELISAs had a mean CV >10%. With milk samples, as observed for serum, DSe was lower for cELISAs (min. 57%, max. 75%) than for iELISAs (min. 61%, max. 89%), and DSp was high for all ELISAs (min. 94%, max. 100%). Both DSe and ASe were lower when testing milk samples compared to serum samples. These results confirm that serologic monitoring of BVDV-free herds should be performed using serum samples of unvaccinated animals to avoid interference of vaccination and to maximize the chance of detecting seroconversion linked to BVDV infection. Further investigations using a larger collection of field samples are recommended.

Inter-laboratory evaluation of the performance parameters of a Lateral Flow Test device for the detection of Bluetongue virus-specific antibodies

Bluetongue (BT) is a viral vector-borne disease affecting domestic and wild ruminants worldwide. In this study, a commercial rapid immuno-chromatographic method or Lateral Flow Test (LFT) device, for the detection of BT virus-specific antibodies in animal serum, was evaluated in an international inter-laboratory proficiency test. The evaluation was done with sera samples of variable background (ruminant species, serotype, field samples, experimental infections, vaccinated animals). The diagnostic sensitivity was 100% (95% C.I. [90.5-100]) and the diagnostic specificity was 95.2% (95% C.I. [76.2-99.9]). The repeatability (accordance) and reproducibility (concordance) were 100% for seropositive samples but were lower for two of the seronegative samples (45% and 89% respectively). The analytical sensitivity, evaluated by testing positive sera at increasing dilutions was better for the BT LFT compared to some commercial ELISAs. Seroconversion of an infected sheep was detected at 4 days post infection. Analytical specificity was impaired by cross-reactions observed with some of the samples seropositive for Epizootic Haemorrhagic Disease Virus (EHDV). The agreement (Cohens kappa) between the LFT and a commercial BT competitive ELISA was 0.79 (95% CI [0.62-0.95]). Based on these results, it can be concluded that the BT LFT device is a rapid and sensitive first-line serological test that can be used in the field, especially in areas endemic for the disease where there is a lack of diagnostic facilities.

Serological monitoring on milk and serum samples in a BVD eradication program: A field study in Belgium showing antibody ELISA performances and epidemiological aspects

In a cross-sectional field study involving 51 cattle herds in Belgium, 3159 serum samples and 557 individual milk samples were collected and tested by four different commercial antibody (Ab) ELISAs on serum and two Ab ELISAs on milk. A virus neutralization test (VNT) was performed on serum samples with discording ELISA results and on all samples from non-vaccinating herds. An epidemiological survey was carried out in the same herds to collect information about herd characteristics, management practices, BVD vaccination and BVD infection status. The objective of the study was to evaluate the performances of the Ab ELISAs relatively to the VNT, to assess the possibility of using pooled samples and to give recommendations regarding serological monitoring of BVD-free herds in the context of the Belgian national BVD eradication program which started early 2015. Depending on the assays, for ELISAs on serum, the diagnostic sensitivity (DSe) was estimated to be between 93.0 and 98.7% and the diagnostic specificity (DSp) between 94.3% and 99.1%. For the two ELISAs on milk, the DSe were 91.3% and 96.7% and the DSp 94.0% and 100% respectively and the Cohens agreement coefficients between serum and milk samples were 0.75 and 0.85. Positive serum and milk samples diluted in negative samples to mimic different pool sizes were not detected by all ELISAs at dilutions above 1:5 or 1:10, leading to the conclusion that the testing of pooled samples should be used cautiously for serological monitoring and only with ELISAs with high sensitivity. The epidemiological analysis and the seroprevalence study, based on a general estimating equation model, showed that several factors had a significant influence on overall animal seroprevalence and within-herd seroprevalence such as age class, herd size, BVD herd infection status, BVD vaccination of young and/or adult cattle and the number of stables in the farm. This study showed that the best performances obtained with commercial Ab ELISAs are observed on individual serum samples, which should therefore be the preferred matrix to monitor BVD-free herds in the context of the Belgian eradication program. By regularly testing a limited number of samples from young (6-18 months) unvaccinated cattle it is possible to confirm the BVD-free herd status or to detect a recent infection.