Véronique Duquesne

Efficiency of a phase 1 vaccine for the reduction of vaginal Coxiella burnetii shedding in a clinically affected goat herd.

The main route of human Q fever infections is inhalation of infectious aerosols. The bacterial agent, Coxiella burnetii, is frequently found throughout domestic ruminants. Females constitute potential shedders of C. burnetii through vaginal mucus, faeces and milk. Q fever is also a common cause of abortion, especially in goats. Massive load of bacteria is associated with placentas and aborted foetuses. In experimental conditions, the Coxevac inactivated phase 1 vaccine (CEVA Sante Animale, Libourne, France) was efficient, and dramatically reduced abortion and excretion of bacteria [1]. The aim of the present study was to evaluate the Coxevac vaccination impact on bacterial shedding in goats affected by Q fever in natural conditions.

Detection of Aethina tumida Murray (Coleoptera: Nitidulidae.) in Italy: outbreaks and early reaction measures

Summary The first detection of Aethina tumida Murray (the small hive beetle) in Italy occurred on 5 September 2014. Three nuclei containing honey bees (Apis mellifera) and located in a clementine (citrus) orchard near an important international harbour in the Calabria region (southern Italy) were heavily infested with adult and larval A. tumida. A. tumida infestation is a notifiable disease of honey bees in the European Union as well as an OIE listed disease. To prevent any A. tumida introduction, the importation of honey bees is regulated strictly in the European Union (Commission Regulation (EU) No. 206/2010). Early reaction measures adopted in Italy require that beekeepers must notify A. tumida discovery to the local veterinary services and cannot move their colonies. Furthermore, a protection area (20 km radius) and surveillance (100 km radius) zone should be established. The surveillance zone now includes the entire territory of Calabria region. Compulsory visits to all apiaries in the protection zone with the collection of the spatial information by means of a georeferentiation process (georeferentiation can be defined as the process to describe a location relative to the earth, in this context the process consists on the collection of the spatial coordinate of a point that represents the spatial location of the apiaries by means of a GPS device) and colony inspection according to 5% expected prevalence (95% CI) are applied. Destruction of infested apiaries is compulsory and the soil under the infested colonies must be ploughed and treated with pyrethroids. If apiaries in the protection zone are found to be negative, traps are placed. In the surveillance zone, veterinarians visit apiaries that are selected according to a risk analysis (migration in infested areas, honey bee or materials exchange) or randomly and colonies are inspected according to 2% expected prevalence (95% CI). Furthermore, in Italy as well in the rest of Europe, investigations are in progress by competent authorities to make an inventory of all bees and colonies moved from Calabria during 2014. Subsequent controls on colonies should be implemented. People from the honey bee network (beekeepers, veterinarians, beekeeping material producers and distributors) should be aware and informed of the hazard posed by A. tumida to honey bees.

Evaluation of randomly amplified polymorphic DNA (RAPD) for discrimination of Coxiella burnetii ruminant strains isolated in France

Coxiella burnetii is the causative agent of Q fever, a worldwide zoonosis. Ruminant species represent the main reservoir of the bacterium, as high levels of the bacteria are shed in birth products and other excreta. Human infection generally occurs after inhalation of contaminated aerosols [1]. The availability of methods to study the distribution and the spread of a given C. burnetii strain, from different geographical areas or hosts, would promote a better understanding of the epidemiology of this pathogen. Several genetic typing methods for C. burnetii have been evaluated and appeared to be useful tools for epidemiological and phylogenetic purposes. However, RAPD [2,3] has never been evaluated for C. burnetii typing. In this work, a RAPD protocol was used to evaluate the genetic diversity among C. burnetii ruminant strains in comparison to published MLVA data [4]. Ten French C. burnetii isolates obtained from goats, sheep, cows and the reference strain Nine Mile, were used for RAPD typing (Table 1). Whole DNA was extracted using the Qiagen Dneasy kit (Courtaboeuf, France), following the manufacturer’s protocol. To assess optimal RAPD conditions several parameters were tested, for instance using a range of MgCl2 concentrations (from 1.5 to 5.5 mM), or different brands of Taq polymerase. RAPD PCRs were performed using a set of four 10-mer primers (P4M, 5¢-AAGACGCCGT-3¢; PR5, 5¢-AGTCGTCCCC-3¢; PR10A, 5¢AGGGCCGTCT3¢; and PR12A, 5¢-CAGCTCACGA-3¢). Unless specified, the optimised PCR conditions were, for a final volume of 25 lL in dH2O: 1X Taq polymerase buffer (without MgCl2); 3 mM MgCl2; 0.2 mM each dNTPs; 1U of Taq polymerase; 0.5 lM 10-mer primer; and 10 ng of DNA (all the reagents were purchased from Invitrogen, Cergy Pontoise, France). Negative controls consisting of dH2O only were included in each run. Amplifications were performed in an Eppendorf Mastercycler (Le Pecq, France), programmed for an initial denaturation step of 5 min at 94 C, 45 cycles of 96 C for 30 s, 37 C for 30 s, 72 C for 90 s and a final extension step at 72 C for 5 min. The PCR products were separated on 0.8% agarose gel containing ethidium bromide, visualised and photographed under UV light. Gel analysis was performed using the Quantity One 1-D Analysis software from Bio-Rad (Marnes la Coquette, France). An RAPD type was defined after the combination of the patterns obtained with the four primers used in this study (Table 1). The reproducibility of the method was evaluated by repeating the same assay several times, by different manipulators, as well as by using independent DNA preparations. Briefly, RAPD profiles were stable when varying MgCl2 concentration. However, a higher yield of amplification, estimated by the intensity of the bands observed on the gels, was observed using 2.5–3.5 mM MgCl2. This yield decreased when using higher concentrations of MgCl2 (data not shown). A MgCl2 concentration of 3 mM was therefore chosen for subsequent analyses. Different RAPD patterns were observed when different brands of Taq polymerases were used (data not shown). The Hot start platinum Taq polymerase (Invitrogen) produced the best patterns in our hands and was chosen for all the experiments. After optimisation of our RAPD protocol, the use of each primer generated distinct polymorphisms allowing differentiation of the studied strains. Strains from neighbouring flocks, that were indistinguishable using MLVA analysis, could be discriminated using RAPD typing (Table 1). For instance, CbB2 and CbB5 are two strains obtained in 2001 from neighbouring flocks, but were isolated from cows displaying different clinical signs (Table 1). CbB2 was isolated from a case of metritis and CbB5 from an aborted cow. Corresponding author and reprint requests: Dr V. Duquesne, AFSSA, Sophia Antipolis, Unite de Pathologie des Petits Ruminants, 105 Route des Chappes, BP111, 06902 SophiaAntipolis, France E-mail. v.duquesne@afssa.fr

EXPERIMENTAL INFECTION OF PREGNANT PYRENEAN CHAMOIS (RUPICAPRA PYRENAICA) WITH BORDER DISEASE VIRUS SUBTYPE 4

Border disease virus (BDV) causes high mortality in Pyrenean chamois (Rupicapra pyrenaica) on the French and Spanish sides of the Pyrenees Mountains. We investigated the pathology induced by BDV in pregnant chamois via experimental infection. Three females were inoculated during the second third of pregnancy with a BDV-4 subgroup strain isolated from a wild Pyrenean chamois during an acute epizootic. A fourth pregnant chamois and one nonpregnant ewe were kept as negative controls. Animals were monitored to assess clinical signs, hematology, viremia, and serology. Postmortem examinations included necropsy, histopathology, and quantification of viral RNA in organs. Pregnancy was unsuccessful in all inoculated animals. One died 24 days postinoculation (dpi) without showing any precursory clinical signs. The second animal had profuse diarrhea from 13 dpi to its death at 51 dpi. The third aborted at 46 dpi and was euthanized at 51 dpi. All animals were viremic from 4 dpi until death. Neutralizing antibodies against BDV-4 were detected from 12 dpi. Necropsies showed generalized lymphadenomegaly, associated in one case with disseminated petechial hemorrhages in the digestive tract. Seventy-eight of 79 organs from inoculated adults and their fetuses had detectable viral RNA. The main histologic lesions in adults were mild lymphohistiocytic encephalitis associated with moderate or moderately severe lymphoid depletion. Control animals remained negative for virus (in blood and organs), antibody, and lesions upon postmortem examination. BDV infection during pregnancy in Pyrenean chamois causes severe disease leading to abortion, then death. Despite 100% fetal death following inoculation, viral RNA was recovered from all organs of infected fetuses, suggesting that persistently infected offspring could be born. Our results may help explain the reported decrease in chamois populations in several areas and suggest that great care must be taken when interpreting infection status for wildlife.

Outbreak of Q fever, Florac, Southern France, Spring 2007

INTRODUCTION In May 2007, five patients with Q fever-like symptoms were reported in an agricultural educational center in the rural southern French town of Florac. An investigation was undertaken to identify the outbreak source and risk factors for infection, and to implement control measures. MATERIALS AND METHODS We undertook active case finding. Patients were defined as individuals with an unexplained fever of ≥38.5°C who lived in, worked in, or visited Florac between April 1 and June 30, 2007. Patients were confirmed by a positive Q fever serology test. A cross-sectional survey with a seroprevalence component was carried out in the educational center and surrounding area. A standardized questionnaire on known risk factors for the infection was used and serological testing was carried out on finger prick blood specimens from participants. The veterinary services investigated local herds within a 5-mile radius using polymerase chain reaction and serological tests. RESULTS One hundred twenty-two people were included in the cross-sectional survey. Eighteen serologically confirmed acute cases were identified, of whom 12 were from the educational center. The statistical analysis showed an independent association between acute infection and living or working near an area where manure had been spread (p = 0.0.042) and male gender (p = 0.022). Frequenting the educational centers canteen was also associated with infection (p = 0.008) among staff and students. The veterinary investigations identified 11 of the 26 tested flocks of goats and sheep as seropositive for Coxiella burnetii, including 2 ovine flocks located northwest of Florac that had high shedding levels of the bacterium. DISCUSSION The observed excess of cases of Q fever in Florac, an area endemic for this infection, in spring 2007 could be explained by an aerial transmission from infectious ovine flocks situated close to the town. All local herd owners were re-educated about the risks and prevention practices for Q fever.

Evaluation of the recombinant Heat shock protein B (HspB) of Coxiella burnetii as a potential antigen for immunodiagnostic of Q fever in goats.

Coxiella burnetii is an intracellular bacterium that causes a worldwide zoonosis, the Q fever. Currently, to diagnose the infection in ruminants, whole cell antigens-based ELISAs are used. In this study a heat shock protein, the HspB, was evaluated for its ability to be recognized by the goat immune system and its capacity to sign a stage of infection. The htpB gene of C. burnetii was cloned and sequenced. A high identity (>90%) was observed among the htpB genes of four ruminant strains tested. A recombinant protein was expressed in a prokaryotic expression system. The rHspB protein was used to determine the IgG reactivity by ELISA. Sera from experimentally and naturally infected goats were tested. The rHspB is recognized early during the infection course, at 18 days post-infection. Moreover, 80-90% of the animals tested were positive at 39-60dpi. In addition, animals presenting a reactivation of the infection displayed a higher reactivity, statistically significant (p<0.05), than that of the animals in latent infection. These findings suggest that the rHspB could be a good candidate for the development of an ELISA test making possible the detection of recent C. burnetii infection in goats as well as reactivation in those with latent infection.

La fièvre Q : Problématiques et risques sanitaires

Q fever is a zoonosis caused by Coxiella burnetii. Domestic ruminants are the main reservoirs for human contamination, as infected female ruminants shed the bacteria in parturition products, milk and faeces. Several studies describe the natural evolution of bacterial shedding by domestic ruminants. AFSSA proposed recommandations on Q fever risks and on reasoned measures applicable to infected sheep, cattle, and goat herds (Rodolakis et al. 2004). Veterinary researches for the disease are designed to limit the contamination of the environment and of the population. The major challenge is to determine the best diagnostic strategies and to implement control programs. Vaccination appears to be the best way to combat Q fever. A phase I inactivated vaccine, whose preventive efficacy has been experimentally demonstrated, is authorised in France since 2004. Several research teams and professional organisations are currently evaluating the benefit of this vaccine in herds.

Introduction of Aethina tumida (Coleoptera: Nitidulidae) in the regions of Calabria and Sicily (southern Italy)

Aethina tumida (small hive beetle, SHB) was first detected in September 2014 in Calabria region, southern Italy, and in a single apiary in Sicily in November 2014. In September 2015, SHB was again recorded in Calabria, and in 2016, only sentinel honey bee nucleus colonies were found to be infested. Its phylogenetic relationship and possible origin were investigated comparing the cox1 sequences with the corresponding region available in the GenBank database. The neighbour-joining method revealed that the first Italian specimen belonged to a group also containing an African specimen from Cameroon. The Italian specimens differ from the SHBs spread worldwide and are split into two different groups: group B1 includes the AfricCam3 sequence and the first SHB identified in Calabria; group B2 includes specimens from Calabria and the only one from Sicily which share identical cox1 sequences. SHB in Italy appears to have been introduced from Africa and includes independent or contemporary incursions in the two concerned regions. The most likely scenario is that SHB was introduced into Calabria followed by man-mediated migration to Sicily.

First Description of Infection of Caprine Herpesvirus 1 (CpHV-1) in Goats in Mainland France

The purpose of this study was to investigate the epidemiological situation of the caprine herpesvirus 1 (CpHV-1) infection in nine districts in mainland France, mostly in the south, near Italy or Spain, where high seroprevalence has been observed. Two more central areas were also included in the study. The serosurvey was carried out in 9564 goats (275 herds) using bovine herpesvirus 1 (BoHV-1) glycoprotein B and E ELISAs. To confirm the presence of specific CpHV-1 antibodies, some of the samples were tested in neutralization assay. Results demonstrate, for the first time, CpHV-1 infection in goat herds on the French mainland. The analysis found cases of alphaherpesviruses infection in each district studied, with different levels of seroprevalence observed within each district (ranging from 0.2% to 31.56% at an individual level and from 9% to 46.2% for herd seroprevalence). Moreover, in the Alpes-Maritimes district, the seroprevalence seemed to be higher in older goats (79.45% of animals 6 years old or more) than in younger animals (40.99% of one-year-olds). This result suggests frequent virus re-excretion and circulation in herds. Results analysis also shows that the seroprevalence was higher when the herd size increased. In addition, the first French CpHV-1 strain was isolated from nasal swabs taken on an infected goat. The data reported herein demonstrate that CpHV-1 circulates in mainland France, which should henceforth be taken into consideration in cases of unexplained abortion in goats.

Serological and molecular characterization of AdaA: a potential marker of Q-fever abortion in goats?

Institut de Pharmacologie Mole´culaire et Cellulaire, CNRS UPR411,Universite´ de Nice Sophia Antipolis, Valbonne, FranceCoxiella burnetii is the causative agent of Q-fever, aworldwide zoonosis. C. burnetii can induce abor-tion, stillbirth or delivery of weak lambs, gener-ating non-negligible economic losses. At present,no serological tests are available to ascertain a Q-fever abortion.Western blot analysis of C. burnetii lysate indi-cated that a 28-kDa protein was recognized bysera of goats that have aborted (not shown). Asthis protein could correspond to AdaA, which haspreviously been shown to be associated withvirulence of Q-fever in human [1], the presentstudy was undertaken to determine whetherAdaA protein could be a good marker for thedetection of Q-fever abortion.The adaA gene (CBU0952) of caprine (CbC1,CbC2, CbC5), ovine (CbO1, CbO184), bovine(CbB1, CbB2, CbB3, CbB4, CbB5, CbB7) and NineMile strains of C. burnetii was amplified by PCRusing two primers containing the restrictionsite BamHI or NotI: AdaA-1 (5¢-CGCGGGATCCGCCTTCGCTGAAAATCGC-3¢) and AdaA-2(5¢-ATTTGCGGCCGCTTTATTTAAAAAAGTCGCCAC-3¢). The amplification reaction was per-formed in 25 lL containing 10 pmol of eachprimer, 0.2 mM dNTP Mix (Invitrogen, CergyPontoise, France), 3 mM MgCl