Didier Marlier

Examination of Escherichia coli from poultry for selected adhesin genes important in disease caused by mammalian pathogenic E. coli.

A collection of 1601 extraintestinal and intestinal Escherichia coli isolated from chickens, turkeys and ducks, in Belgium, France and Spain, was hybridised with gene probes specific for fimbrial and afimbrial adhesins (F17, F18, S , Bfp, Afa, Cs31A, Intimin , Aida-1) of intestinal, urinary and invasive E. coli of mammals and with a probe specific for the P (Pap/Prs) fimbrial adhesin of urinary and invasive E. coli of mammals and birds. Three hundred and eighty-three strains (23.9%) were P-positive, 76 strains (4.8%) were Afa-positive, 75 strains (4.7%) were F17-positive, 67 strains (4.2%) were S-positive, 23 (1.4%) were Intimin-positive, and all were F18-, Cs31A-, Aida1- and Bfp-negative. The 75 F17-positive strains harboured different major subunit A-encoding gene variants, but the f17Ac variant was the most frequent (52 strains, 69.3%) and seven strains (9.3%) were not typeable. The f17G gene variant coding for the GII adhesin was the most frequent (56 strains, 75.0%), whereas the f17GI gene variant was present in four strains (5%) and 15 strains (20.0%) were not typeable. All Afa-positive strains harboured the afa-8 variant. The 23 Intimin-positive E. coli tested positive for the beta-variant (16 strains; 69.6%) or for the gamma-variant (seven strains; 30.4%) of the eae gene. Chicken and turkey E. coli were more frequently probe-positive (43.6 and 43.1%, respectively) than duck E. coli (31.5%) and extraintestinal E. coli were also more frequently probe-positive (48.4%) than intestinal strains (18.5%). Different combinations of probe positive hybridisation results were observed in 72 of the 540 probe-positive E. coli (13.3%). The most frequent combinations were between AfaE-8 and F17 probes (47 strains; 8.7%) and between P and S probes (13 strains; 2.4%). Although f17- and afa-8-related DNA sequences can be plasmid-located in mammalian E. coli, they were not in avian E. coli. Besides the P fimbrial adhesins, F17 and S fimbrial and Afa-VIII and Intimin afimbrial adhesins may thus represent colonisation factors of avian pathogenic E. coli.

Infectious agents associated with epizootic rabbit enteropathy: Isolation and attempts to reproduce the syndrome

Abstract Epizootic rabbit enteropathy (ERE), a highly lethal (30–80% mortality) disease of broiler rabbits aged 6–14 weeks, first appeared in 1997 in French intensive enclosed rabbitries and is of unknown aetiology. Bacteriological, virological and parasitical examination of the intestinal contents of rabbits that had died either in spontaneous field cases or after experimental reproduction of ERE, were undertaken in an attempt to identify infectious agents that may play a role in the disease. Two bacterial strains, Clostridium perfringens and non-enteropathogenic Escherichia coli were repeatedly isolated at high faecal counts from naturally infected animals. In field cases, a correlation between typical gross lesions of epizootic enteropathy and the presence of the alpha toxin of Cl. perfringens was observed (P <0.0001; Chi-squared test). Although attempts to reproduce the disease by inoculation with different pools of cultivable bacterial strains failed, the disease was successfully reproduced by inoculation with one French and two Belgian samples of caecal contents.

Detection of Usutu virus in a bullfinch (Pyrrhula pyrrhula) and a great spotted woodpecker (Dendrocopos major) in north-west Europe.

In October 2012, a 3-year-old bullfinch (Pyrrhula pyrrhula) held in captivity for its entire lifespan and a wild adult great spotted woodpecker (Dendrocopos major), both with neurological signs, were found 4 km from each other and 5 days apart in the Meuse Valley, Belgium. Non-suppurative encephalitis and mild degeneration and necrosis were identified in the brain and cerebellum, and Usutu virus antigen and RNA were detected by immunohistochemistry and real-time reverse transcriptase PCR, respectively. The two cases reported here represent the most western distribution of clinical disease in birds due to Usutu virus in Europe.

Cross-sectional study of the association between pathological conditions and myxoma-virus seroprevalence in intensive rabbit farms in Europe

Myxomatosis is a major viral disease of the European rabbit (Oryctolagus cuniculus). Two forms of the disease (nodular and amyxomatous) exist. The clinical diagnosis of the nodular form is easily performed on the basis of typical skin lesions whereas that of amyxomatous forms must be based on virus isolation or detection of specific antibodies to myxoma virus (MV). The seroprevalence of MV was studied between March 1998 and February 1999 in 16 farms from three European countries considered free of myxomatosis on the basis of the absence of typical clinical signs. MV antibodies were detected by enzyme-linked immunosorbent assay (ELISA) (sensitivity 100%, specificity 90%) in all 16 farms; the seroprevalences corrected for test inaccuracy (95% confidence interval) were 55+/-7.7% and 37+/-6.1% for does and broilers, respectively. The association between herd sizes, types of rabbitries, and presence of recurrent respiratory or digestive troubles and seroprevalence of MV antibodies was tested in logistic multiple regressions. In all models, the seroprevalence of MV antibodies was significantly higher in herds (does and broilers) with recurrent respiratory or digestive troubles than in herds without these problems. The seroprevalence was also higher in herds (does and broilers) where animals were housed totally or partially in outdoors rabbitries than in totally enclosed rabbitries. The effect of herd sizes on the presence of MV antibodies was the same in does and broilers; intermediate sizes were at lower risk than the smaller and larger ones.

Pyrosequencing of epizootic rabbit enteropathy inocula and rabbit caecal samples.

The aetiological agent of epizootic rabbit enteropathy (ERE) is still unknown although a bacterial infection seems the most likely hypothesis. In this study, amplification of the V5 and V6 regions of 16SrDNA from four virulent and two non-virulent caecal samples was performed using a pyrosequencing platform. The virulent samples did not group in the same cluster. The bacterial flora identified was both different and richer than the cultivable bacterial flora. These findings highlight the need for biomolecular techniques to identify the aetiological agent of ERE.

The genome of a tortoise herpesvirus (testudinid herpesvirus 3) has a novel structure and contains a large region that is not required for replication in vitro or virulence in vivo

ABSTRACT Testudinid herpesvirus 3 (TeHV-3) is the causative agent of a lethal disease affecting several tortoise species. The threat that this virus poses to endangered animals is focusing efforts on characterizing its properties, in order to enable the development of prophylactic methods. We have sequenced the genomes of the two most studied TeHV-3 strains (1976 and 4295). TeHV-3 strain 1976 has a novel genome structure and is most closely related to a turtle herpesvirus, thus supporting its classification into genus Scutavirus, subfamily Alphaherpesvirinae, family Herpesviridae. The sequence of strain 1976 also revealed viral counterparts of cellular interleukin-10 and semaphorin, which have not been described previously in members of subfamily Alphaherpesvirinae. TeHV-3 strain 4295 is a mixture of three forms (m1, m2, and M), in which, in comparison to strain 1976, the genomes exhibit large, partially overlapping deletions of 12.5 to 22.4 kb. Viral subclones representing these forms were isolated by limiting dilution assays, and each replicated in cell culture comparably to strain 1976. With the goal of testing the potential of the three forms as attenuated vaccine candidates, strain 4295 was inoculated intranasally into Hermanns tortoises (Testudo hermanni). All inoculated subjects died, and PCR analyses demonstrated the ability of the m2 and M forms to spread and invade the brain. In contrast, the m1 form was detected in none of the organs tested, suggesting its potential as the basis of an attenuated vaccine candidate. Our findings represent a major step toward characterizing TeHV-3 and developing prophylactic methods against it. IMPORTANCE Testudinid herpesvirus 3 (TeHV-3) causes a lethal disease in tortoises, several species of which are endangered. We have characterized the viral genome and used this information to take steps toward developing an attenuated vaccine. We have sequenced the genomes of two strains (1976 and 4295), compared their growth in vitro, and investigated the pathogenesis of strain 4295, which consists of three deletion mutants. The major findings are that (i) TeHV-3 has a novel genome structure, (ii) its closest relative is a turtle herpesvirus, (iii) it contains interleukin-10 and semaphorin genes (the first time these have been reported in an alphaherpesvirus), (iv) a sizeable region of the genome is not required for viral replication in vitro or virulence in vivo, and (v) one of the components of strain 4295, which has a deletion of 22.4 kb, exhibits properties indicating that it may serve as the starting point for an attenuated vaccine.

Prevalence of Salmonella serotypes in male mule ducks in Belgium

Salmonella species infections of male mule ducks were studied for 32 months in 100 flocks on nine duck farms in Belgium. The prevalence of Salmonella species infections changed significantly over time (P<0.001) with infection rates of 50, 13.4, 6.7, 2.6 and 2.9 per cent, respectively, at the time of arrival on the farm, at three, six and nine weeks of age, and when the ducks left the breeding unit to enter the force-feeding rooms (at 11 or 12 weeks of age). During the study period, 95 strains of Salmonella were isolated, belonging to 11 serotypes. S Indiana (42.1 per cent) and S Regent (36.8 per cent) were the two most common serotypes, whereas S Typhimurium and S Enteritidis were found only once (1.1 per cent). All isolated strains were resistant to at least two antimicrobials, but resistance to more than five antimicrobials was observed in 21.6 per cent of the strains.

Epizootic rabbit enteropathy: comparison of PCR-based RAPD fingerprints from virulent and non-virulent samples.

Epizootic rabbit enteropathy is a gastrointestinal disorder of unknown aetiology of farmed rabbits characterised by inanition and mortality. Genomic analyses of virulent and non-virulent samples of inocula were performed using random amplification of polymorphic DNA (RAPD). Differences in bacterial DNA composition were found between inocula, but specific sequences were not linked with field cases of epizootic rabbit enteropathy.

Improving adjuvant systems for polyclonal egg yolk antibody (IgY) production in laying hens in terms of productivity and animal welfare

The antibody production in the egg yolks of immunized laying hens is seen as a way of improving animal welfare compared with conventional production by mammals. Immunoglobulin Y (IgY) technology, however, has still to address welfare issues linked to the widespread use of an adjuvant in vaccines. Currently, Freunds adjuvants, complete (FCA) or incomplete (FIA), remain the standard. This study sought to evaluate various approaches used to enhance egg yolk antibody production in terms of both productivity and avian welfare. The outer membrane protein (OMP) of Salmonella Typhimurium was used as the prototype antigen. At 20 weeks of age, 56 ISA Brown hens, with specific-Salmonella-free status, were divided into seven groups (n=8) and received an initial intramuscular immunization. Hens in the two negative control groups received phosphate buffered saline (PBS) or FIA alone. Hens in the other groups received 80μg of Salmonella OMP emulsified with one of the following adjuvants: 200μl of FIA alone (T1); 200μl of FIA supplemented with 8μg of C-phosphate-guanosine oligodeoxynucleotides (CpG-ODN) (T2); and 280μl of Montanide ISA 70 VG (T4). Birds in the T3 group received the antigen in emulsion with FIA and were given the tested immunostimulatory component (l-carnitine) via their feed (100mg/kg). A positive control group (PC) received FCA for the first and final immunizations and FIA for the other boosters. Immunization was repeated after 20, 46, 82 and 221 days. Eggs were collected regularly until 242 days after the first immunization and the anti-Salmonella Typhimurium activities in the yolk were determined by ELISA. After 242 days, the birds were euthanized and the injection sites were evaluated for gross and microscopic lesions. Among the tested immunostimulatory approaches, supplementation of FIA with CpG-ODN led to a significant and long-lasting enhancement of the specific antibody response. This treatment was even higher than the positive benchmark using FCA in the first immunization. The study results showed that a clinical examination of injection sites is insufficient for drawing conclusions about the local tolerance of vaccines. Tissue damage was noticeable in all treatment groups. The birds receiving the Montanide adjuvant, however, had fewer and less severe lesions. Given these limited side-effects, Montanide ISA 70 VG could provide the depot effect needed to ensure the immunomodulatory efficiency of CpG-ODN. The association of these two adjuvants could prove a promising alternative to Freunds adjuvants (FA).

Corticosterone, cortisol, triglycerides, aspartate aminotransferase and uric acid plasma concentrations during foie gras production in male mule ducks (Anas platyrhynchos × Cairina moschata)

1. Corticosterone, cortisol, triglycerides, aspartate aminotransferase (AST) and uric acid (UA) plasma concentration were measured at 8 (7 days after group housing), 12 (after 7 days of force feeding) and 13 weeks of age (at slaughter after 12 days of force feeding), and 45 min after an adrenocorticotrophic hormone (ACTH) stimulation test at 8 weeks of age in 12 male mule ducks in an on-farm experiment. 2. No significant increase of corticosterone was found during the force-feeding period compared with the concentration after housing. 3. Comparison of corticosterone and cortisol values indicates that cortisol can be considered as a reliable acute stress indicator in future routine examinations. 4. Plasma concentrations of triglycerides and aspartate aminotransferase increased progressively from pre-force feeding period to slaughtering. 5. Plasma concentrations of uric acid increased from the start at 8 weeks of age to the mid-force feeding period but no difference was noticed between the mid-force feeding period and slaughtering. 6. It is concluded that acute stress induced by force-feeding is similar at the beginning and end of the commercial production of foie gras.