Épidémiologie de la maladie de Crohn au Québec

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Épidémiologie de la maladie de Crohn au Québec

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Risk factors for the introduction and within-herd transmission of Mycobacterium avium subspecies paratuberculosis (MAP) infection in 59 Irish dairy herds

Since 1994, Irish cattle have been exposed to greater risks of acquiring Mycobacterium avium subspecies paratuberculosis (MAP) infection as a consequence of the importation of over 70,000 animals from continental Europe. In recent years, there has been an increase in the number of reported clinical cases of paratuberculosis in Ireland. This study examines the prevalence of factors that promote the introduction and within-herd transmission of Mycobacterium avium subspecies paratuberculosis (MAP) on selected Irish dairy farms in the Cork region, and the association between these factors and the results of MAP screening tests on milk sock filter residue (MFR). A total of 59 dairy farms, selected using non-random methods but apparently free of endemic paratuberculosis, were enrolled into the study. A questionnaire was used to collect data about risk factors for MAP introduction and transmission. The MFR was assessed on six occasions over 24 months for the presence of MAP, using culture and immunomagnetic separation prior to polymerase chain reaction (IMS-PCR). Furthermore, blood samples from all entire male and female animals over one year of age in 20 herds were tested by ELISA. Eighteen (31%) farms had operated as closed herds since 1994, 28 (47%) had purchased from multiple sources and 14 (24%) had either direct or indirect (progeny) contact with imported animals. Milk and colostrum were mixed on 51% of farms, while 88% of farms fed pooled milk. Thirty (51%) herds tested negative to MFR culture and IMS-PCR, 12 (20%) were MFR culture positive, 26 (44%) were IMS-PCR positive and seven (12%) were both culture and IMS-PCR positive. The probability of a positive MFR culture was significantly associated with reduced attendance at calving, and with increased use of individual calf pens and increased (but not significantly) if multiple suckling was practised. There was poor agreement between MFR culture and MFR IMS-PCR results, but moderate agreement between MFR culture and ELISA test results. This study highlights a lack of awareness among Irish dairy farmers about the effect of inadequate biosecurity on MAP introduction. Furthermore, within-herd transmission will be facilitated by traditional calf rearing and waste management practices. The findings of viable MAP in the presence of known transmission factors in non-clinically affected herds could be a prelude to long-term problems for the Irish cattle and agri-business generally.

Mycobacterium avium subsp. paratuberculosis in foods: current evidence and potential consequences

Mycobacterium avium ssp. paratuberculosis (MAP), the cause of Johne's disease in cattle, sheep and goats, may have a role in Crohn's disease in humans. Animals with Johne's disease shed viable MAP in their milk and faeces. The organism is also widely disseminated in the blood and tissues of infected animals. Consequently, transmission to humans via consumption of animal-derived foods is a distinct possibility. Milk, other dairy products, beef and water have been identified as possible food vehicles of transmission. To date, viable MAP has been cultured from raw cows', sheep and goats' milk, retail pasteurized cows' milk, and some retail cheeses in several countries during recent studies. MAP has not been isolated from retail beef to date, although limited testing has been carried out. The public health consequences, if any, of low numbers of viable MAP being periodically consumed by susceptible individuals are uncertain. An association between MAP and Crohn's disease is not proven, but neither can it be discounted on the basis of current evidence. A precautionary approach is therefore warranted in relation to the existence of MAP in food, and action is needed to reduce the prevalence of Johne's disease in the cattle population worldwide, in order to minimize public exposure to this potential human pathogen.

Interaction between Mycobacterium avium subsp. paratuberculosis and environmental protozoa

Background: Interactions between Mycobacterium avium subsp. paratuberculosis (Map) and free-living protozoa in water are likely to occur in nature. The potential impact of ingestion of Map by two naturally occurring Acanthamoeba spp. on this pathogen's survival and chlorine resistance was investigated. Results: Between 4.6 and 9.1% of spiked populations of three Map strains (NCTC 8578, B2 and ATCC 19698), which had been added at a multiplicity of infection of 10: 1, were ingested by Acanthamoeba castellanii CCAP 1501/1B and A. polyphaga CCAP 1501/3B during co-culture for 3 h at 25 C. Map cells were observed to be present within the vacuoles of the amoebae by acid-fast staining. During extended co-culture of Map NCTC 8578 at 25 degrees C for 24 d with both A. castellanii and A. polyphaga Map numbers did not change significantly during the first 7 days of incubation, however a 1-1.5 log(10) increase in Map numbers was observed between days 7 and 24 within both Acanthamoeba spp. Ingested Map cells were shown to be more resistant to chlorine inactivation than free Map. Exposure to 2 mu g/ml chlorine for 30 min resulted in a log(10) reduction of 0.94 in ingested Map but a log(10) reduction of 1.73 in free Map (p <0.001). Conclusion: This study demonstrated that ingestion of Map by and survival and multiplication of Map within Acanthamoeba spp. is possible, and that Map cells ingested by amoebae are more resistant to inactivation by chlorine than free Map cells. These findings have implications with respect to the efficacy of chlorination applied to Map infected surface waters.

Inactivation of Mycobacterium avium subsp. paratuberculosis in milk during commercial pasteurisation

Four studies have been published relating to the inactivation of Mycobacterium avium subsp. paratuberculosis (Map) by commercial HTST pasteurization. Three of these were large surveys of commercially pasteurized milk at processing/retail level in the UK and Ontario, Canada, and the fourth a pasteurization study involving naturally infected milk and commercial-scale pasteurizing plant. Evidence that Map is capable of surviving commercial pasteurization was obtained in two of the studies: viable Map was cultured from 50 ml aliquots of commercially pasteurized milk after decontamination with 0.75% cetylpyridinium chloride for 5 h and then culture on Herrold's egg-yolk medium without antibiotics. In both studies culture did not commence until 24-72 h post-pasteurization and samples were stored at 4 degrees C in the interim period. In the other two milk surveys smaller volumes of milk were tested (1-5 ml and 15 ml) and no firm evidence of surviving Map was obtained. The three milk surveys differed in other respects - chemical decontamination, culture media used and use of antibiotics. Recent findings suggest that sub-lethally heat-injured Map in pasteurized milk have the potential to recover viability if stored at 4 degrees C for 48 h between heating and testing.

Prevalence of Mycobacterium avium subspecies paratuberculosis in Swiss raw milk cheeses collected at the retail level.

A total of 143 raw milk cheese samples (soft cheese, n = 9; semihard cheese, n = 133; hard cheese, n = 1), collected at the retail level throughout Switzerland, were tested for Mycobacterium avium ssp. paratuberculosis (MAP) by immunomagnetic capture plus culture on 7H10-PANTA medium and in supplemented BAC-TEC 12B medium, as well as by an F57-based real-time PCR system. Furthermore, pH and water activity values were determined for each sample. Although no viable MAP cells could be cultured, 4.2% of the raw milk cheese samples tested positive with the F57-based real-time PCR system, providing evidence for the presence of MAP in the raw material. As long as the link between MAP and Crohn’s disease in humans remains unclear, measures designed to minimize public exposure should also include a focus on milk products.

Mycobacterium avium ssp. paratuberculosis: its incidence, heat resistance and detection in milk and dairy products

Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease in cattle and other ruminants and has been implicated as a possible cause of Crohn's disease in humans. The organism gains access to raw milk directly through excretion into the milk within the udder and indirectly through faecal contamination during milking. MAP has been shown to survive commercial pasteurization in naturally infected milk, even at the extended holding time of 25 s. Pasteurized milk must therefore be considered a vehicle of transmission of MAP to humans. isolation methods for MAP from milk are problematical, chiefly because of the absence of a suitable selective medium. This makes food surveillance programs and research on this topic difficult. The MAP problem can be addressed in two main ways: by devising a milk-processing strategy that ensures the death of the organism: and/or strategies at farm level to prevent access of the organism into raw milk. Much of the research to date has been devoted to determining ifa problem exists and, if so, the extent of the problem. Little has been directed at possible solutions. Given the current state of information on this topic and the potential consequences for the dairy industry research is urgently needed so that a better understanding of the risks and the efficacy of possible processing solutions can be determined.

Development of a novel phage-mediated immunoassay for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis

Aims: The objective of this study was to develop a novel screening method for detection of viable Mycobacterium avium subsp. paratuberculosis (Map) in milk and faeces, as a rapid alternative to Map culture.
Methods and results: The new method couples Map-specific peptide-mediated magnetic separation technique with an optimised phage amplification assay followed by detection of released progeny phage by ELISA in a competition assay format using polyclonal antibody produced against the D29 mycobacteriophage involved in the phage assay. Sample matrices were found not to interfere with the developed method and the dynamic range of the assay was 3 X 102 – 6 X 108 phage ml-1. When low numbers of Map were present (102 CFU ml-1) the burst size of a single host Map cell was maximal (103 phage per cell) resulting in a highly sensitive screening assay.
Conclusion: A rapid, sensitive immuno-based screening method suitable for the detection of viable Map in milk and faeces was developed.
Significance and impact of study: The novel PMS-phage-ELISA permits sensitive, qualitative detection of viable Map in milk or faeces samples within 48 h, representing a substantial decrease in time to detection compared to current culture methods for Map.

An optimised milk testing protocol to ensure accurate enumeration of viable Mycobacterium avium subsp. paratuberculosis by the PMS-phage assay

There is interest in determining levels of Mycobacterium avium subsp. paratuberculosis (MAP) contamination in milk. The optimal sample preparation for raw cows' milk to ensure accurate enumeration of viable MAP by the peptide-mediated magnetic separation (PMS)-phage assay was determined. Results indicated that milk samples should be refrigerated at 4 C after collection and MAP testing should commence within 24 h, or samples can be frozen at 70 C for up to one month without loss of MAP viability. Use of Bronopol is not advised as MAP viability is affected. The vast majority (>95%) of MAP in raw milk sedimented to the pellet upon centrifugation at 2500 g for 15 min, so this milk fraction should be tested. De-clumping of MAP cells was most effectively achieved by ultrasonication of the resuspended milk pellet on ice in a sonicator bath at 37 kHz for 4 min in ‘Pulse’ mode.

Novel monoclonal antibody and peptide binders for Mycobacterium avium subsp. paratuberculosis and their application for magnetic separation

The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.

Estimation of Mycobacterium avium subsp. paratuberculosis load in raw bulk tank milk in Emilia-Romagna Region (Italy) by qPCR

Consumption of milk and dairy products is considered one of the main routes of human exposure to Mycobacterium avium subsp. paratuberculosis (MAP). Quantitative data on MAP load in raw cows’ milk are essential starting point for exposure assessment. Our study provides this information on a regional scale, estimating the load of MAP in bulk tank milk (BTM) produced in Emilia-Romagna region (Italy). The survey was carried out on 2934 BTM samples (88.6% of the farms herein present) using two different target sequences for qPCR (f57 and IS900). Data about the performances of both qPCRs are also reported, highlighting the superior sensitivity of IS900-qPCR. Seven hundred and eighty-nine samples tested MAP-positive (apparent prevalence 26.9%) by IS900 qPCR. However, only 90 of these samples were quantifiable by qPCR. The quantifiable samples contained a median load of 32.4 MAP cells mL−1 (and maximum load of 1424 MAP cells mL−1). This study has shown that a small proportion (3.1%) of BTM samples from Emilia-Romagna region contained MAP in excess of the limit of detection (1.5 × 101 MAP cells mL−1), indicating low potential exposure for consumers if the milk subsequently undergoes pasteurization or if it is destined to typical hard cheese production.

Facteurs de risque d´introduction et diagnostic de Mycobacterium avium ssp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis (MAP) est l'agent causal de la paratuberculose, maladie entérique, chronique et incurable des ruminants, avec un impact économique important. Une meilleure compréhension des facteurs de risque associés à l'introduction de la maladie dans un troupeau est essentielle pour sa prévention. L’amélioration des tests diagnostiques est aussi importante pour son contrôle. L’introduction des nouveaux animaux dans le troupeau et la présence et contact des différentes espèces sauvages et domestiques avec les vaches, semblent être des facteurs de risques d’introduction de la maladie. Nous avons réalisé une revue systématique dont l`objective était de recueillir l’information pertinente pour répondre à la question sur l’importance de ces facteurs et leur impact sur l’introduction de la maladie dans un troupeau. D`un autre côté, la détection de MAP dans les fèces par culture bactérienne demeure la méthode diagnostique de choix malgré les facteurs qui l`affectent. Une série de 3 étapes est requise afin de confirmer la présence du MAP : (1) culture (2) coloration, et (3) confirmation du MAP par PCR (si détecté à l´étape 2). Certains échantillons fécaux présentent une particularité en raison de leur forte charge de micro-organismes. Ces contaminants peuvent interférer avec la croissance et la détection de MAP. Une étude visant à : a) estimer l'impact des certain covariables sur les résultats de la culture de MAP parmi l`analyse rétrospective d`un banque des données et b) évaluer la possibilité d'optimiser le processus de diagnostic du MAP en effectuant l'analyse PCR sur les cultures déclarées comme contaminées a été réalisée.

Transmission de Mycobacterium avium ssp. paratuberculosis dans les troupeaux de bovins laitiers et dépistage de l’infection par la culture de l’environnement au Québec

Mycobacterium avium subsp. paratuberculosis (MAP) cause la maladie de Johne, une maladie chronique et incurable affectant les ruminants partout dans le monde. Plusieurs pays ont mis en place des programmes de contrôle afin de prévenir la transmission entre et au sein des troupeaux. Afin d’arriver à prévenir et contrôler cette maladie, une bonne compréhension des facteurs de risque impliqués dans la transmission est essentielle. Des tests diagnostiques performants et à coût abordable sont aussi nécessaires afin de détecter la présence du MAP et/ou les animaux infectés. L’objectif de la première étude était de réviser systématiquement la littérature scientifique concernant les facteurs de risque associés à la transmission du MAP aux génisses laitières. La présence d’une association significative entre les facteurs de risque concernant l’environnement néonatal, le colostrum, le lait, le logement des veaux et le contact des veaux avec le fumier de vaches adultes et la transmission du MAP a été compilée de 23 articles. Le contact des veaux avec le fumier de vaches adultes est le facteur de risque le plus important dans la transmission du MAP. L’objectif de la seconde étude était d’évaluer la relation entre le nombre d’échantillons de l’environnement positifs pour le MAP et la prévalence individuelle d’excrétion fécale dans les troupeaux laitiers entravés du Québec. Le nombre de cultures positives d’échantillons de l’environnement s’est avéré associé à la prévalence individuelle d’excrétion fécale du MAP. Une association significative a été trouvée entre la présence d’une forte charge bactérienne dans un échantillon de fumier individuel et la détection du MAP dans l’environnement.