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

Currently 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 10months 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 20months 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.

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.

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.

Interferon gamma response to Mycobacterium avium subsp. paratuberculosis specific lipopentapeptide antigen L5P in cattle.

After Mycobacterium avium subsp. paratuberculosis (Map) infection the cell-mediated immune (CMI) response indicative of early Th1 activation may be detected using interferon-gamma release assay (IGRA). Currently, the purified protein derivatives (PPDs), i.e., the total extract of mycobacteria antigens are used to recall CMI responses against Map. This study aimed to assess the ability of the chemically synthesized Map specific cell wall lipopentapeptide L5P to induce CMI response in cows infected by Map compared to PPD. L5P and PPD elicited an IFN-γ response in 12 and 35 animals from two Map infected herds respectively, but IFN-γ was not detected in the 13 cows recruited from a non-infected herd. Levels of IFN-γ detected were higher with PPD than with L5P. There was no correlation between the IFN-γ response and the humoral response to Map or faecal culture.

Shedding of Coxiella burnetii in ewes in two pregnancies following an episode of Coxiella abortion in a sheep flock.

Coxiella burnetii infection in pregnant sheep typically causes abortion or the birth of weak lambs. Two C. burnetii-related abortions in a group of 34 pregnant ewes were reported at their first lambing in our research institute. The seroprevalence of C. burnetii infection and bacteria shedding were investigated using an ELISA and PCR, respectively, during the course of two subsequent pregnancies. None of the ewes examined seroconverted from negative to positive at the time of the second and the third parturition and most of the ewes that were seropositive at the abortion episode remained positive throughout the investigation. The two successive pregnancies resulted in the birth of healthy lambs without PCR evidence of Coxiella infection from placenta and vaginal swabs taken postpartum. PCR assay performed on vaginal swabs taken from all animals 1, 5 or 12 days after the second lambing were also negative for Coxiella. However, one ewe that had previously experienced C. burnetii shedding at the first lambing excreted the bacteria in the genital tract after the third parturition. The bacteria could not be detected by PCR in milk and faecal samples taken up to 12 days after both parturitions.

Effect of vaccination with phase I and phase II Coxiella burnetii vaccines in pregnant goats.

Livestock is considered to be the major "source" of human Q fever. The efficacy of two currently available vaccines (Coxevac, phase I, CEVA Santé Animale and Chlamyvax FQ, phase II, MERIAL) against Coxiella excretion was investigated in terms of risks to human health. Two months before mating, 17 goats were vaccinated subcutaneously against Coxiella burnetii with an inactivated phase I vaccine and 16 goats were vaccinated with an inactivated phase II Coxiella mixed with Chlamydophila abortus vaccine. Fourteen goats were left unvaccinated. At 84 days of gestation, the goats were subcutaneously challenged with 10(4) bacteria of C. burnetii strain CbC1. Phase I vaccine was effective and dramatically reduced both abortion and excretion of bacteria in the milk, vaginal mucus and feces. In contrast, the phase II vaccine did not affect the course of the disease or excretion.

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.

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 Immunosorbent 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.