Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing

BACKGROUND: Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA). RESULTS: By applying IRS-PCR, 14 C. burnetii isolates could be divided into six groups containing up to five different isolates. Clustering as deduced from MLVA typing with 17 markers provided an increased resolution with an excellent agreement to IRS-PCR, and with the plasmid type of each strain. MLVA was then applied to 28 additional C. burnetii isolates of different origin and 36 different genotypes were identified among the 42 isolates investigated. The clustering obtained is in agreement with published Multiple Locus Sequence Typing (MLST) data. Two panels of markers are proposed, panel 1 which can be confidently typed on agarose gel at a lower cost and in any laboratory setting (10 minisatellite markers with a repeat unit larger than 9 bp), and panel 2 which comprises 7 microsatellites and provides a higher discriminatory power. CONCLUSION: Our analyses demonstrate that MLVA is a powerful and promising molecular typing tool with a high resolution and of low costs. The consistency of the results with independent methods suggests that MLVA can be applied for epidemiological studies. The resulting data can be queried on a dedicated MLVA genotyping Web service

Experimental Coxiella burnetii infection in pregnant goats: excretion routes

Q fever is a widespread zoonosis caused by Coxiella burnetii. Infected animals, shedding bacteria by different routes, constitute contamination sources for humans and the environment. To study Coxiella excretion, pregnant goats were inoculated by the subcutaneous route in a site localized just in front of the shoulder at 90 days of gestation with 3 doses of bacteria (10$^{8}$, 10$^{6}$ or 10$^{4}$ I.D.). All the goats aborted whatever the dose used. Coxiella were found by PCR and immunofluorescence tests in all placentas and in several organs of at least one fetus per goat. At abortion, all the goats excreted bacteria in vaginal discharges up to 14 days and in milk samples up to 52 days. A few goats excreted Coxiella in their feces before abortion, and all goats, excreted bacteria in their feces after abortion. Antibody titers against Coxiella increased from 21 days post inoculation to the end of the experiment. For a Q fever diagnostic, detection by PCR and immunofluorescence tests of Coxiella in parturition products and vaginal secretions at abortion should be preferred to serological tests

Identification of new antigen candidates for the early diagnosis of Mycobacterium avium subsp. paratuberculosis infection in goats

International audienceCurrently Mycobacterium avium subsp. paratuberculosis (MAP) infection is diagnosed through indirect tests based on the immune response induced by the infection. The antigens commonly used in IFN-γ release assays (IGRA) are purified protein derivative tuberculins (PPD). However, PPDs, lack both specificity (Sp) and sensitivity (Se) in the early phase of infection. This study investigated the potential of 16 MAP recombinant proteins and five lipids to elicit the release of IFN-γ in goats from herds with or without a history of paratuberculosis. Ten recombinant proteins were selected as potential candidates for the detection of MAP infection in young goats. They were found to detect 25 to 75% of infected shedder (IS) and infected non-shedder (INS) kids younger than 10 months of age. In comparison, PPD was shown to detect only 10% of INS and no IS kids. For seven antigens, Se (21–33%) and Sp (≥ 90%) of IGRA were shown to be comparable with PPD at 20 months old. Only three antigens were suitable candidates to detect IS adult goats, although Se was lower than that obtained with PPD. In paratuberculosis-free herds, IGRA results were negative in 97% of indoor goats and 86% of outdoor goats using the 10 antigens. However, 22 to 44% of one-year-old outdoor goats were positive suggesting that they may be infected. In conclusion, this study showed that ten MAP recombinant proteins are potential candidates for early detection of MAP infected goats. Combining these antigens could form a possible set of MAP antigens to optimize the Se of caprine IGRA

Isolation and characterisation of local strains of Chlamydophila abortus (Chlamydia psittaci serotype 1) from Tunisia

Chlamydiosis is one of the major diseases that can lead to abortion in ewes. Since 1997, in 5 regions of Tunisia, Chlamydia-related abortions have been reported in 15 sheep and goat flocks. One hundred and sixty-six sera and 50 vaginal swab samples were collected from adult ewes. Chlamydial antigens were detected in 29 (58%) of the vaginal swabs using Enzyme Linked Immunsorbent Assay (ELISA) while 9 (18%) were positive by cell culture. Five strains were recovered from 4 different sheep flocks. Monoclonal antibody profiles and restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA spacer region showed that these isolates were C. abortus. Using amplified fragment length polymorphism (AFLP), these Tunisian strains were shown to exhibit the same pattern as strains isolated in France.Isolement et typage de souches locales de Chlamydophila abortus (Chlamydia psittaci sérotype 1). La chlamydiose est une des principales cause d'avortements infectieux en Tunisie. Lors de la période d'agnelage de 1997, 166 prises de sang et 50 écouvillons vaginaux ont été prélevés dans 15 troupeaux répartis sur 5 gouvernorats et ayant eu des problèmes d'avortements. Des chlamydia ont été mises en évidence dans 29 (58 %) écouvillons vaginaux appartenant à 13 troupeaux différents par ELISA directement sur l'écouvillon vaginal et 9 (18 %) après multiplication sur cellules. Cinq souches tunisiennes appartenant à 4 troupeaux différents ont ainsi pu être isolées. Leur caractérisation par une panoplie d'anticorps monoclonaux et par étude du profil de restriction de l'espace intergénique 16S-23S a démontré qu'elles appartenaient toutes à l'espèce Chlamydophila abortus. Par amplification sélective de fragment de restriction les souches tunisiennes présentaient le profil caractéristique des souches françaises

Effects of a chronic stress treatment on vaccinal response in lambs

International audienceFarming systems can expose animals to chronic mild stress which is known to induce negative affective state. Affective state in animals, as in humans, can be assessed through behavioral cues. This study aimed to describe the effect of a chronic mild stress, known to induce a negative affective state, on sheep health through their response to vaccination. The study used 15 lambs subjected to a model of chronic mild stress for 15 weeks and 15 lambs reared under conventional farming as a control group. After 7 weeks of stressful treatment, the lambs were individually exposed to a judgment bias test to assess a putative stress-induced 'pessimism.' After 15 weeks of stressful treatment, antibody immune response was measured after an injection of a live vaccine challenge (Chlamydia abortus attenuated vaccine strain 1B). Stressed lambs displayed a pessimistic-like perception in the judgment bias test, revealing a negative affective state. Stressed and control animals showed different immunological reactions to vaccine challenge: stressed sheep had lower hemoglobin concentrations and higher platelet, granulocyte and acute-phase protein concentrations. Antibody response induced by the vaccine strain was not different between stressed and control sheep. Our results suggest that negative affective state induced by chronic stress treatment may induce a stronger inflammatory response to vaccine challenge in sheep. Improvement of animal health may be achieved through consideration of stressors that may affect the emotional and immunological state of sheep

A longitudinal study of the Mycobacterium avium subspecies paratuberculosis infection status in young goats and their mothers

The dynamics between Mycobacterium avium subspecies paratuberculosis (MAP) infection and the immune response of goats naturally exposed to MAP were studied in a herd where the clinical expression of paratuberculosis had been observed. Four generations of goats were observed over a 33-month period: mothers of three different generations (G1, G2, G3) and their daughters, generation 4 (G4). A MAP infection status was defined according to the combined results of an IFN-γ assay, antibody response, faecal culture and post-mortem examination. Goats were defined as non-infected (NI), infected and non-shedder (INS), infected and shedder (IS) or atypical (A). Twenty-nine percent of goats were NI, 66% were infected and either shedding (14%) or not shedding (52%) MAP, and 5% were atypical. IFN-γ responses were detected first, followed by faecal shedding and antibody responses. The results showed that in goats naturally exposed to MAP, IFN-γ responses were regularly detected earlier in non-shedders than in young infected shedder goats and were stronger in shedder than in non-shedder goats. They were also higher in the mother goats than in their daughters. Goats shedding MAP or with positive antibody response at the beginning of their pregnancy are more likely to have an infected daughter positive to an IFN-γ assay by the age of 15 months

Molecular characterization of <it>Coxiella burnetii </it>isolates by infrequent restriction site-PCR and MLVA typing

Abstract Background Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA). Results By applying IRS-PCR, 14 C. burnetii isolates could be divided into six groups containing up to five different isolates. Clustering as deduced from MLVA typing with 17 markers provided an increased resolution with an excellent agreement to IRS-PCR, and with the plasmid type of each strain. MLVA was then applied to 28 additional C. burnetii isolates of different origin and 36 different genotypes were identified among the 42 isolates investigated. The clustering obtained is in agreement with published Multiple Locus Sequence Typing (MLST) data. Two panels of markers are proposed, panel 1 which can be confidently typed on agarose gel at a lower cost and in any laboratory setting (10 minisatellite markers with a repeat unit larger than 9 bp), and panel 2 which comprises 7 microsatellites and provides a higher discriminatory power. Conclusion Our analyses demonstrate that MLVA is a powerful and promising molecular typing tool with a high resolution and of low costs. The consistency of the results with independent methods suggests that MLVA can be applied for epidemiological studies. The resulting data can be queried on a dedicated MLVA genotyping Web service.</p

Lipopentapeptide, L5P a <em>Mycobacterium avium paratuberculosis</em> specific antigen induces cell mediated immune response: potential tool in early Johne's disease

National audienc

L5P a specific antigen suitable to detect mycobacterium avium spp. paratuberculosis infection by cell mediated immune response

International audienceContext: The livestock management needs an early diagnostic test to prevent dramatic rise of Mycobacterium avium spp paratuberculosis (Map) infection in cattle. Current serology based diagnostic tests available, detect animals in later stages when they are shedding huge amount of bacillus. Cell mediated immune (CMI) responses that can be detected by Interferon Gamma Release Assays, appear before or combined with antibody responses. Therefore, diagnostic tools based on IGRA could help to identify recently infected animals in order to prevent disease transmission. The sensitivity and specificity of these tests need to define Map-specific T-cell antigens. Map produces a specific cell-wall lipopentapeptide called L5P or LP-01. L5P is suitable to detect Map-infected animals by serodiagnostic. Moreover, L5P can be synthetized chemically at high purity, large scale, and low cost. Objectives: The purpose of this work was to assess the potential of L5P to detect Map-specific cell mediated immune responses to develop an IGRA test. Methods: A panel of 36 cows were selected from two naturally infected herds where clinical paratuberculosis had occurred. We performed 1) serology with the commercial IDEXX diagnostic test and a house-made test using L5P antigen 2) IGRA with Purified Protein Derivative avian (PPD-A) or synthetic L5P antigen 3) microbiological analyses including isolation and identification of bacillus from faeces. Results: In this study 47.2 % of cows were scored positive by the commercial IDEXX diagnostic test but only 22% developed anti-L5P antibodies. PPD-A induced CMI responses in 97 % of cows while 22.2% animals were L5P responders. Map was isolated in 25.7% of animals. We provide, for the first time, evidence that Map-specific L5P is a suitable antigen for serologic and IGRA diagnosis. Perspectives: We will now carry out a longitudinal study to investigate the potential of L5P-based IGRA to predict clinical outcome of Map-infected 18-24 months cattle