J. De Buck

Medium-Chain Fatty Acids Decrease Colonization and Invasion through hilA Suppression Shortly after Infection of Chickens with Salmonella enterica Serovar Enteritidis

ABSTRACT The most common source of Salmonella infections in humans is food of poultry origin. Salmonella enterica serovar Enteritidis has a particular affinity for the contamination of the egg supply. In this study, the medium-chain fatty acids (MCFA), caproic, caprylic, and capric acid, were evaluated for the control of Salmonella serovar Enteritidis in chickens. All MCFA were growth inhibiting at low concentrations in vitro, with caproic acid being the most potent. Contact of Salmonella serovar Enteritidis with low concentrations of MCFA decreased invasion in the intestinal epithelial cell line T84. By using transcriptional fusions between the promoter of the regulatory gene of the Salmonella pathogenicity island I, hilA, and luxCDABE genes, it was shown that all MCFA decreased the expression of hilA, a key regulator related to the invasive capacity of Salmonella. The addition of caproic acid (3 g/kg of feed) to the feed of chicks led to a significant decrease in the level of colonization of ceca and internal organs by Salmonella serovar Enteritidis at 3 days after infection of 5-day-old chicks. These results suggest that MCFA have a synergistic ability to suppress the expression of the genes required for invasion and to reduce the numbers of bacteria in vivo. Thus, MCFA are potentially useful products for reducing the level of colonization of chicks and could ultimately aid in the reduction of the number of contaminated eggs in the food supply.

Colonization of the chicken reproductive tract and egg contamination by Salmonella.

1. SUMMARY The food-borne salmonellosis pandemic in humans is for a large part caused by the consumption of contaminated eggs. Infection of the reproductive organs of laying hens often is the underlying phenomenon leading to the production of contaminated eggs. To date, the pathogenesis of reproductive tract infection in hens has not received the full attention it merits in relation to its importance in transmitting Salmonella infections within the poultry population and from poultry to man. This review discusses the different possible infection routes leading to egg contamination and emphasizes on the oviduct and ovary colonization in the process of egg contamination. The role of known bacterial virulence factors in the pathogenesis of reproductive tract infection is discussed. Immune responses in the oviduct, related to Salmonella infection, are described. Finally, different possible approaches to protect laying hens against reproductive tract infection by Salmonella are reviewed.

Invasion of Salmonella enteritidis in avian intestinal epithelial cells in vitro is influenced by short-chain fatty acids.

Fermentation reactions in the caeca of chickens, the predominant place for Salmonella colonization, result in high concentrations of short-chain fatty acids (SCFA). Thus Salmonella bacteria are in close contact with SCFA during their life cycle. A study was carried out to analyse the effects of SCFA on invasion of Salmonella enteritidis in an avian intestinal epithelial cell line. Preincubation of S. enteritidis for 4 h in growth media supplemented with various concentrations of propionate or butyrate resulted in decreased invasion compared to bacteria, preincubated in nonsupplemented media, and to bacteria, preincubated in media supplemented with formate or acetate. Incubation of the S. enteritidis bacteria in media supplemented with mixtures of SCFA mimicking the in vivo caecal concentrations resulted in increased invasion compared with butyrate-exposed bacteria, but equal invasion compared with nonexposed bacteria. Increasing the butyrate concentration in these mixtures did not modify invasion compared with the original mixtures.

Identification of lactobacilli isolated from the cloaca and vagina of laying hens and characterization for potential use as probiotics to control Salmonella Enteritidis.

Aims:  To select Lactobacillus strains from laying hens for potential use as probiotic to control Salmonella Enteritidis infection.

Effect of type 1 fimbriae of Salmonella enterica serotype Enteritidis on bacteraemia and reproductive tract infection in laying hens

Research on the role of type 1 fimbriae of Salmonella enterica serotype Enteritidis in poultry to date has been focused on the intestinal phase of the infection. This study aimed to investigate the role of type 1 fimbriae in a systemic infection by intravenously inoculating chickens with a fimD mutant or its parent strain. The fimD mutant was present in the blood for 3 weeks after infection, while the wild type parent strain was cleared within the first 3 days. The fimD mutant was isolated at least as much as the parent strain from the liver and spleen for up to 3 weeks after inoculation. The wild type strain was cleared from the caeca in the second week, while the fimD mutant was isolated from the caeca for up to 3 weeks after infection. The ovaries were more heavily infected by the fimD mutant than by the wild-type strain. In the first and second week after inoculation, the oviducts were more frequently infected by the mutant strain. The eggs of birds infected with the fimD mutant were less frequently contaminated with Salmonella. The shells of the eggs were more frequently contaminated by the wild type strain than with the mutant strain. Thus, the absence of type 1 fimbriae prolongs bacteraemia, modifies reproductive tract infection and reduces egg shell contamination by Salmonella enterica serovar Enteritidis.

Bacteria-host interactions of Salmonella Paratyphi B dT(+) in poultry

In recent years, a dramatic increase in incidence of the dextro-rotatory tartrate-positive variant (dT+) of Salmonella enterica subspecies enterica serovar Paratyphi B has been observed in poultry and poultry products. In the present study the interactions of this bacterium with the host were studied in vivo and in vitro in an attempt to explain the preferential association of this serotype with poultry. The ability of this organism to invade and multiply in chicken intestinal epithelial cells and the intracellular behaviour in chicken macrophages was studied in vitro using chicken cell lines. In vivo challenge experiments in specific pathogen-free chickens were carried out to determine the level of colonization of caeca and internal organs early after experimental infection. An in vivo trial with commercial broiler chickens, using a seeder model, was performed to determine whether S. Paratyphi B dT+ could persist and spread in broilers until slaughter. S. Paratyphi B dT+ invaded and multiplied in the chicken epithelial cell line and survived in a chicken macrophage cell line. The strain used colonized caeca and internal organs of chickens to a high extent 1 week after infection with a low-dose inoculum. Moreover, the strain was efficiently transmitted within a group of broilers and persisted until slaughter. It was concluded that S. Paratyphi B dT+ was well adapted to poultry and therefore it is suggested that specific control measures against this serotype should be considered.

High herd-level prevalence of Mycobacterium avium subspecies paratuberculosis in Western Canadian dairy farms, based on environmental sampling

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive enteritis in ruminants. The pathogen is present in most countries with modern dairy production, causing substantial economic losses for the industry. The objectives of this study were to estimate dairy herd prevalence of MAP in the Western Canadian provinces of Alberta and Saskatchewan, and to determine whether herd size and housing system (tie-stall versus freestall or loose housing) affected the risk of a herd testing positive for MAP. Six environmental samples were collected on 360 Alberta farms (60% of registered producers) and on 166 Saskatchewan dairy farms (99%). In total, 47% of the sampled farms in Alberta and 53% of the sampled farms in Saskatchewan had at least one environmental sample that was MAP culture positive and were, therefore, defined as infected. Sensitivity of environmental sampling was estimated using 3 subsequent annual tests performed on 82 farms. Because laboratory protocols were continuously improved throughout the project, the sensitivity increased over time. Therefore, a mean of the sensitivity estimates weighted on sampling year was constructed; this resulted in sensitivities of 68 and 69% for Alberta and Saskatchewan, respectively. Implementing those estimates in an approximate Bayesian computation model resulted in a true herd prevalence of 68% (95% probability interval: 60-80%) for Alberta and 76% (95% probability interval: 70-85%) for Saskatchewan. Herds with >200 cows had 3.54 times higher odds of being environmental sample positive and had more positive samples than herds with <50 cows (neither province nor housing system affected those results). In conclusion, the majority of Alberta and Saskatchewan dairy farms were infected with MAP and larger herds were more often MAP positive than smaller herds.

Identification of bovine-associated coagulase-negative staphylococci by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a direct transfer protocol

This study evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) for the identification of bovine-associated coagulase-negative staphylococci (CNS), a heterogeneous group of different species. Additionally, we aimed to expand the MALDI-ToF MS database with new reference spectra as required to fill the gaps within the existing commercial spectral library. A total of 258 isolates of CNS were used in the study, covering 16 different CNS species. The majority of the isolates were previously identified by rpoB gene sequencing (n = 219), and the remainder were identified by sequencing of 16S rRNA, hsp60, or both rpoB and hsp60. The genotypic identification was considered the gold standard identification. All MALDI-ToF MS identifications were carried out using the direct transfer method. In a preliminary evaluation (n = 32 isolates; 2 of each species) with the existing commercial database, MALDI-ToF MS showed a typeability of 81% (26/32) and an accuracy of 96% (25/26). In the main evaluation (n = 226 isolates), MALDI-ToF MS with the existing commercial Biotyper (Bruker Daltonics Inc., Billerica, MA) database achieved a typeability of 92.0% (208/226) and an accuracy of 99.5% (207/208). Based on the assessment of the existing commercial database and prior knowledge of the species, a total of 13 custom reference spectra, covering 8 species, were created and added to the commercial database. Using the custom reference spectra expanded database, isolates were identified by MALDI-ToF MS with 100% typeability and 100% accuracy. Whereas the MALDI-ToF MS manufacturers cutoff for species-level identification is 2.000, the reduction of the species level cutpoint to ≥1.700 improved the species-level identification rates (from 64 to 92% for the existing commercial database) when classifying CNS isolates. Overall, MALDI-ToF MS using the direct transfer method was shown to be a highly reliable tool for the identification of bovine-associated CNS.

Sampling location, herd size, and season influence Mycobacterium avium ssp. paratuberculosis environmental culture results.

Mycobacterium avium subspecies paratuberculosis (MAP), the etiologic agent of Johnes disease, a chronic progressive enteritis, is a common pathogen on dairy farms. Environmental sampling is frequently used to detect MAP-infected herds, because it does not require sample collection from individual animals. The objectives were to determine (1) location-specific odds of MAP-positive environmental sampling results and whether season or herd size affect results; (2) whether season and herd size affect the odds of collection of samples from certain locations; and (3) whether sample-set composition affects the odds of a positive set. Herd veterinarians, producer organization staff, and University of Calgary staff collected 5,588 samples on dairy farms in Alberta and Saskatchewan. Samples from sick-cow and calving pens and samples from dry-cow housing had lower odds of testing MAP-positive than lactating cow-pen samples (odds ratio=0.3 and 0.4, respectively). Samples collected from bedding packs and manure piles were less frequently MAP-positive than those collected from alleyways and lagoons, whereas samples collected in spring and summer more often tested MAP-positive than those collected in winter. Sample sets collected in summer more often included samples from all locations than samples collected in winter; therefore, we inferred that collectors had difficulties accessing certain areas in winter. Substitution of sample locations with others had minor effect on the sensitivity of sample sets containing 6 samples. However, set composition had an effect on the sensitivity of sample sets containing only 2 samples. In that regard, whereas sets with 2 manure-storage-area samples detected 81% of farms with at least one positive environmental sample, sets with only dry, sick, or calving-pen samples detected only 59%. Environmental samples should be collected from areas where manure from numerous cows accumulates and can be well mixed (e.g., alleyways and manure lagoons). Collection of samples should be performed in spring or summer when locations are easier to access and samples have higher odds for testing MAP-positive.

PCR amplification and high‐resolution melting curve analysis as a rapid diagnostic method for genotyping members of the Mycobacterium avium–intracellulare complex

Some of the members of the Mycobacterium avium–intracellulare (MAI) complex are recognized as human pathogens in both immunocompromised and immunocompetent patients. The current molecular methods that are available for genotyping the MAI complex members can be both expensive and technically demanding. In this report, we describe for the first time the application of a real-time PCR and high-resolution melt approach to differentiate between the complex members by targeting a member of the Pro- Pro-Glu gene family, MACPPE24. To this end, reference strains of the M. avium subspecies and Mycobacterium intracellulare were used to optimize the technique. Then, this real-time PCR–high-resolution melt approach was used to distinguish ten M. avium ssp. hominissuis field isolates from the M. intracellulare reference strain.