The Validation of the VereBeef™ Detection Kit.

VereBeef™ Detection Kit, incorporating both multiplex PCR and microarray technologies on a lab-on-chip platform, is intended for qualitative detection and differentiation of Escherichia coli O157:H7, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121, E. coli O145, Shiga toxin-producing E. coli (STEC) virulence factors (stx1A, stx2A, eae), and Salmonella species in one test using raw beef trim samples. This product underwent extensive evaluations, including inclusivity-exclusivity, method comparison, robustness, lot-to-lot variability, and stability studies. The inclusivity/exclusivity study demonstrated that VereBeef Detection Kit specifically detects and identifies target analytes without occurrence of false-positive and false-negative detection. In the method comparison study, the performance of the VereBeef Detection Kit was compared with U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook's methods for target organism detection in raw beef trim using E. coli O157:H7 single inoculation and Salmonella and non-O157 STEC dual inoculation. Data demonstrated equivalence in both methods. The robustness study showed that changes in the test parameters do not impact assay performance. Collectively, VereBeef Detection Kit is able to detect target pathogens in raw beef trim with a minimum enrichment time of 8 h for E. coli O157:H7 detection and 10 h for Salmonella and non-O157 STEC detection.

Pathogen Characterization of Fresh and Stored Mesophilic Anaerobically Digested Biosolids.

Culturable bacterial pathogens (Campylobacter, Salmonella, Listeria, Yersinia) and indicators (E. coli, enterococci, Clostridium perfringens) were quantified at six water resource recovery facilities that land apply anaerobically digested biosolids in Ontario, Canada. Cryptosporidium parvum and Giardia lamblia were also quantified by polymerase chain reaction (PCR). Salmonella and Listeria were frequently detected in sludge and liquid biosolids (70-100% of samples) but less often in fresh dewatered cake biosolids (50-60%); with low levels in fresh cake (<100 cells/g dw). Yersinia were in 20 to 30% of samples, typically at very low levels (<10 cell/g dw). Giardia and Cryptosporidium were detected in 80 and 20% of cake biosolids at geometric means of 270 cysts/g dw and 70 oocysts/g dw, respectively. E. coli reduction was typically >2-log10 while pathogen reduction was variable. "Sudden increase" of pathogens was not observed, however, Salmonella and E. coli showed regrowth (at 1 to 3 orders of magnitude) after 2- to 3-day storage at 30 °C.

Validation of the ANSR® for Campylobacter Method for Detection of Thermophilic Campylobacter spp. in Chicken Carcass Rinse and Turkey Sponge Samples.

A performance validation of the ANSR® for Campylobacter method was conducted in selected matrixes. This assay used selective nicking enzyme amplification technology to amplify target genes. Samples were enriched for 20 to 24 h and then lysed. The assay was completed within 50 min using real-time detection in a combination incubator/fluorescence detector and software. When 50 distinct strains of Campylobacter jejuni, C. lari, or C. coli were tested for inclusivity, all 50 strains produced positive results. In exclusivity testing, 31 strains of related organisms, including seven nontarget Campylobacter strains and other common species, were evaluated. All 31 species generated negative ANSR assay results, including the nontarget Campylobacter strains. The ANSR for Campylobacter method was compared to the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference method using naturally contaminated chicken carcass rinse or turkey carcass sponge samples. ANSR method performance was not statistically different from the reference method using two different enrichment options. Equivalent results were observed at both time points (20 and 24 h) and in both atmospheres (microaerobic and aerobic) to reference methods. Method performance with chicken carcass rinse was confirmed in an independent laboratory study. Additionally, in robustness testing, small, deliberate changes to the assay parameters minimally affected ANSR method performance. Finally, accelerated stability results from three independently manufactured lots supported a shelf life of 6 months when stored at 4°C. The ANSR assay offered greater efficiency and flexibility when compared to the reference method with a 20-24 h single-step enrichment in a microaerobic or an aerobic atmosphere.

Yersinia enterocolitica-Specific Infection by Bacteriophages TG1 and ϕR1-RT Is Dependent on Temperature-Regulated Expression of the Phage Host Receptor OmpF.

UNLABELLED: Bacteriophages present huge potential both as a resource for developing novel tools for bacterial diagnostics and for use in phage therapy. This potential is also valid for bacteriophages specific for Yersinia enterocolitica To increase our knowledge of Y. enterocolitica-specific phages, we characterized two novel yersiniophages. The genomes of the bacteriophages vB_YenM_TG1 (TG1) and vB_YenM_ϕR1-RT (ϕR1-RT), isolated from pig manure in Canada and from sewage in Finland, consist of linear double-stranded DNA of 162,101 and 168,809 bp, respectively. Their genomes comprise 262 putative coding sequences and 4 tRNA genes and share 91% overall nucleotide identity. Based on phylogenetic analyses of their whole-genome sequences and large terminase subunit protein sequences, a genus named Tg1virus within the family Myoviridae is proposed, with TG1 and ϕR1-RT (R1RT in the ICTV database) as member species. These bacteriophages exhibit a host range restricted to Y. enterocolitica and display lytic activity against the epidemiologically significant serotypes O:3, O:5,27, and O:9 at and below 25°C. Adsorption analyses of lipopolysaccharide (LPS) and OmpF mutants demonstrate that these phages use both the LPS inner core heptosyl residues and the outer membrane protein OmpF as phage receptors. Based on RNA sequencing and quantitative proteomics, we also demonstrate that temperature-dependent infection is due to strong repression of OmpF at 37°C. In addition, ϕR1-RT was shown to be able to enter into a pseudolysogenic state. Together, this work provides further insight into phage-host cell interactions by highlighting the importance of understanding underlying factors which may affect the abundance of phage host receptors on the cell surface. IMPORTANCE: Only a small number of bacteriophages infecting Y. enterocolitica, the predominant causative agent of yersiniosis, have been previously described. Here, two newly isolated Y. enterocolitica phages were studied in detail, with the aim of elucidating the host cell receptors required for infection. Our research further expands the repertoire of phages available for consideration as potential antimicrobial agents or as diagnostic tools for this important bacterial pathogen.

Validation of Modifications to the ANSR(®) Listeria Method for Improved Ease of Use and Performance.

A study was conducted to validate minor reagent formulation, enrichment, and procedural changes to the ANSR(®) Listeria method, Performance-Tested Method(SM) 101202. In order to improve ease of use and diminish risk of amplicon contamination, the lyophilized reagent components were reformulated for increased solubility, thus eliminating the need to mix by pipetting. In the alternative procedure, an aliquot of the lysate is added to lyophilized ANSR reagents, immediately capped, and briefly mixed by vortexing. When three foods (hot dogs, Mexican-style cheese, and cantaloupe) and sponge samples taken from a stainless steel surface were tested, significant differences in performance between the ANSR and U.S. Food and Drug Administration Bacteriological Analytical Manual or U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedures were seen with hot dogs and Mexican-style cheese after 16 h enrichment, with the reference methods producing more positive results. After 24 h enrichment, however, there were no significant differences in method performance for any of the four matrixes tested. Robustness testing was also conducted, with variations to lysis buffer volume, lysis time, and sample volume having no demonstrable effect on assay results. Accelerated stability testing was carried out over a 10-week period and showed no diminishment in assay performance. A second phase of the study examined performance of the ANSR assay following enrichment in a new medium, LESS Plus broth, designed for use with all food and environmental sample types. With the alternative LESS Plus broth, there were no significant differences in performance between the ANSR method and the reference culture procedures for any of the matrixes tested after either 16 or 24 h enrichment, although 24 h enrichment is recommended for hot dogs due to higher sensitivity. Results of inclusivity and exclusivity testing using LESS Plus broth showed that the ANSR assay is highly specific, with 100% expected results for target and nontarget bacteria.

Validation of the ANSR(®) Listeria monocytogenes Method for Detection of Listeria monocytogenes in Selected Food and Environmental Samples.

Work was conducted to validate performance of the ANSR(®) for Listeria monocytogenes method in selected food and environmental matrixes. This DNA-based assay involves amplification of nucleic acid via an isothermal reaction based on nicking enzyme amplification technology. Following single-step sample enrichment for 16-24 h for most matrixes, the assay is completed in 40 min using only simple instrumentation. When 50 distinct strains of L. monocytogenes were tested for inclusivity, 48 produced positive results, the exceptions being two strains confirmed by PCR to lack the assay target gene. Forty-seven nontarget strains (30 species), including multiple non-monocytogenes Listeria species as well as non-Listeria, Gram-positive bacteria, were tested, and all generated negative ANSR assay results. Performance of the ANSR method was compared with that of the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedure for detection of L. monocytogenes in hot dogs, pasteurized liquid egg, and sponge samples taken from an inoculated stainless steel surface. In addition, ANSR performance was measured against the U.S. Food and Drug Administration Bacteriological Analytical Manual reference method for detection of L. monocytogenes in Mexican-style cheese, cantaloupe, sprout irrigation water, and guacamole. With the single exception of pasteurized liquid egg at 16 h, ANSR method performance as quantified by the number of positives obtained was not statistically different from that of the reference methods. Robustness trials demonstrated that deliberate introduction of small deviations to the normal assay parameters did not affect ANSR method performance. Results of accelerated stability testing conducted using two manufactured lots of reagents predicts stability at the specified storage temperature of 4°C of more than 1 year.

Complete genome sequence of bacteriophage vB_YenP_AP5 which infects Yersinia enterocolitica of serotype O:3.

BACKGROUND: Bacteriophage vB_YenP_AP5 is a lytic bacteriophage capable of infecting Yersinia enterocolitica strains of serotype O:3, an epidemiologically significant serotype within this bacterial species that causes yersiniosis in humans. This work describes the complete genome sequence of this phage. RESULTS: The genome consists of linear double-stranded DNA of 38,646 bp, with direct terminal repeats of 235 bp in length, and a GC content of 50.7%. There are 45 open reading frames which occupy 89.9% of the genome. Most of the proteins encoded by this virus exhibit sequence similarity to Yersinia phage φYeO3-12 and Salmonella phage φSG-JL2 proteins. CONCLUSIONS: Genomic and morphological analyses place the bacteriophage vB_YenP_AP5 in the T7likevirus genus of the subfamily Autographivirinae within the family Podoviridae.

Campylobacter, Salmonella, Listeria monocytogenes, verotoxigenic Escherichia coli, and Escherichia coli prevalence, enumeration, and subtypes on retail chicken breasts with and without skin.

This study examined the prevalence, counts, and subtypes of Campylobacter, Salmonella, Listeria monocytogenes, verotoxigenic Escherichia coli (VTEC), and E. coli on raw retail chicken breast with the skin on versus the skin off. From January to December 2007, 187 raw skin-on chicken breasts and 131 skin-off chicken breasts were collected from randomly selected retail grocery stores in the Region of Waterloo, Ontario, Canada. Campylobacter isolates were recovered from a higher proportion of the skin-off chicken breasts, 55 (42%) of 131, than of the skin-on chicken breasts tested, 55 (29%) of 187 (P = 0.023). There was no difference in the proportion of Salmonella isolates recovered from the two meat types (P = 0.715): 40 (31%) of 131 skin-off chicken breasts versus 61 (33%) of 187 skin-on chicken breasts. L. monocytogenes isolates were recovered from a statistically lower proportion of the skin-off chicken breasts, 15 (15%) of 99, than of the skin-on chicken breasts, 64 (34%) of 187 (P = 0.001). There was no difference in the proportion of E. coli isolates recovered from the skin-off chicken breasts, 33 (33%) of 99, than from the skin-on chicken breasts, 77 (41%) of 187 (P = 0.204). VTEC was detected on a single skin-off chicken breast. Campylobacter jejuni was the most frequent species isolated on both types of chicken meat: skin-on, 48 (87%) of 55, and skin-off, 51 (94%) of 54. Salmonella serotypes Kentucky and Heidelberg and L. monocytogenes serotype 1/2a were the most frequently detected serotypes from both skin-off and skin-on chicken breasts. Although there appeared to be a trend toward higher enumeration values of these pathogens and E. coli on the skin-on chicken, the differences did not exceed 1 log. This study suggested that skin-off chicken breast may represent a higher risk of consumer exposure to Campylobacter, a similar risk for Salmonella, VTEC, and E. coli, and a lower risk for L. monocytogenes than skin-on chicken breast.

Multiple-locus variable number of tandem repeat analysis (MLVA) of Listeria monocytogenes directly in food samples.

Listeria monocytogenes is the etiologic agent of listeriosis responsible for severe and fatal infections in humans. Listeria contamination occurs quite often in a wide range of foods due to its ubiquitous nature. Isolates need to be characterized to a strain level for accurate diagnosis of Listeria infection, epidemiological studies, investigation of outbreaks and effective prevention and control of food-borne listeriosis. The purpose of this research was to evaluate the multiple-locus variable number of tandem repeat analysis (MLVA) for sub-typing L. monocytogenes isolates in pure cultures and in food matrices. Two multiplex PCR assays were formulated to amplify six specific loci using fluorescently-labeled primers; and the amplicons were analyzed by capillary electrophoresis. The MLVA method resulted in 34 unique DNA fingerprint patterns from 46 L. monocytogenes isolates of 10 serotypes which had 29 or 30 PFGE patterns with a single restriction enzyme and 34 AFLP patterns. The MLVA patterns of the 46 isolates remained unchanged in the presence of pre-enriched food matrices including sausage, ham, chicken, milk and lettuce. The MLVA method successfully typed L. monocytogenes strains spiked in cheese, roast beef, egg salad and vegetable samples after 48 h enrichment at the initial inoculation levels of 1-5 CFU per 25 g of food or higher. The limits of detection (typing) of the MLVA method were 10(3)-10(4)CFU/mL of pre-enriched food broth when evaluated using post-spiked sausage, ham, chicken, milk and lettuce samples. The MLVA method was simple, highly discriminatory, and easy to perform with portable (numerical) results. To our knowledge, this is the first report that describes the application of the MLVA method directly to food samples and demonstrates the possibility to obtain rapid and accurate subtyping results before an isolate is obtained.

Evaluation of immunomagnetic separation in combination with ALOA Listeria chromogenic agar for the isolation and identification of Listeria monocytogenes in ready-to-eat foods.

A study was conducted to evaluate the performance of the ALOA (chromogenic media) in combination with immunomagnetic separation (IMS) for the detection of Listeria monocytogenes in ready-to-eat food products. IMS-ALOA method was found to be equivalent to Health Canada's reference culture method as well as comparable to BAX-PCR method in terms of the sensitivity of the methods for the detection of L. monocytogenes in ready-to-eat foods such as turkey roast, beef roast, mixed vegetable salads, potato and egg salad, soft cheese and smoked salmon. The IMS-ALOA method gave 100% sensitivity in the inclusivity tests with 42 pure L. monocytogenes strains. Exclusivity testing with five other species of Listeria genus and 29 pure non-L. monocytogenes strains from 21 different genera showed 97% specificity. The method was able to detect L. monocytogenes at levels near or below 1cfu/25g regulatory limit in ready-to-eat food matrices after 24h enrichment, with a turnaround time of 3days compared to 7-8days for culture method. IMS-ALOA method is a valuable alternate test method for the screening of L. monocytogenes in a variety of foods especially ready-to-eat foods.

Application of an automated immunomagnetic separation-enzyme immunoassay for the detection of Salmonella enterica subspecies enterica from poultry environmental swabs.

An automated immunomagnetic separation (IMS) and enzyme immunoassay (EIA) was applied to the detection of Salmonella enterica subspecies enterica serotypes from poultry environmental samples. The analytical sensitivity and specificity of the IMS-EIA for 46 S. enterica serotypes and 33 non-salmonellae isolates belonging to 21 different genera were 91.3% and 90.9%, respectively. From post enrichment S. enterica cultures, the limit of detection of the assay was estimated at 10(4)-10(6) CFU/mL. Application of IMS-EIA on 850 naturally contaminated poultry environmental samples achieved 98.4% sensitivity and 96.8% specificity, as compared with a standard culture reference method performed concurrently on the same set of samples. The IMS-EIA described, allows for the identification of suspect positive samples within 48 h of testing versus 4-6 days required by standard culture methods while significantly reducing the materials and labour required for the detection of S. enterica serotypes in poultry environmental samples.

Comparison of milk culture, direct and nested polymerase chain reaction (PCR) with fecal culture based on samples from dairy herds infected with Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in cattle and other farm ruminants, and is also a suspected pathogen of Crohn's disease in humans. Development of diagnostic methods for MAP infection has been a challenge over the last few decades. The objective of this study was to investigate the relationship between different methods for detection of MAP in milk and fecal samples. A total of 134 milk samples and 110 feces samples were collected from 146 individual cows in 14 MAP-infected herds in southwestern Ontario. Culture, IS900 polymerase chain reaction (PCR) and nested PCR methods were used for detecting MAP in milk; results were compared with those of fecal culture. A significant relationship was found between milk culture, direct PCR, and nested PCR (P < 0.05). The fecal culture results were not related to any of the 3 assay methods used for the milk samples (P > 0.10). Although fecal culture showed a higher sensitivity than the milk culture method, the difference was not significant (P = 0.2473). The number of MAP colony-forming units (CFU) isolated by culture from fecal samples was, on average, higher than that isolated from milk samples (P = 0.0083). There was no significant correlation between the number of CFU cultured from milk and from feces (Pearson correlation coefficient = 0.1957, N = 63, P = 0.1243). The animals with high numbers of CFU in milk culture may not be detected by fecal culture at all, and vise versa. A significant proportion (29% to 41%) of the positive animals would be missed if only 1 culture method, instead of both milk and feces, were to be used for diagnosis. This suggests that the shedding of MAP in feces and milk is not synchronized. Most of the infected cows were low-level shedders. The proportion of low-level shedders may even be underestimated because MAP is killed during decontamination, thus reducing the chance of detection. Therefore, to identify suspected Johne's-infected animals using the tests in this study, both milk and feces samples should be collected in duplicate to enhance the diagnostic rate. The high MAP kill rate identified in the culture methods during decontamination may be compensated for by using the nested PCR method, which had a higher sensitivity than the IS900 PCR method used.

Microbial survey of selected Ontario-grown fresh fruits and vegetables.

Recent produce-related outbreaks have been receiving heightened media coverage, which has increased public concern toward the safety of fresh fruits and vegetables. In response, the microbial contamination of Ontario-grown fresh fruits and vegetables was evaluated by the Ontario Ministry of Agriculture, Food and Rural Affairs during the summer of 2004. Prior to this survey, information specific to the microbial contamination of Ontario-produced fruits and vegetables was limited. This nonregulatory survey had two objectives: (i) to obtain a general microbiological profile of selected fruits and vegetables produced in Ontario and (ii) to use the information and knowledge gained from this survey to direct and support future on-farm food safety research and food safety programs to manage potential risks. In all, 1,183 samples, including muskmelon (151), scallions and green onions (173), leaf lettuce (263), organic leaf lettuce (112), head lettuce (155), parsley (127), cilantro (61), and fresh market tomatoes (141), were collected and analyzed. Samples were analyzed for Salmonella, Shigella, and generic E. coli. Enrichment cultures positive for E. coli were further assessed for verotoxigenicity. One sample each of Roma tomato and organic leaf lettuce were positive for Salmonella, with no samples yielding Shigella or verotoxigenic E. coli. The E. coli prevalence was highest in parsley (13.4%), followed by organic leaf lettuce (11.6%), leaf lettuce (6.5%), scallions (6.4%), cilantro (4.9%), muskmelon (1.3%), head lettuce (0%), and fresh market tomatoes (0%). These findings, in combination with foodborne illness data, will help target those commodities that require more focused risk mitigation efforts.

Improved template DNA preparation procedure for detection of Mycobacterium avium subsp. paratuberculosis in milk by PCR.

Factors affecting the detection of Mycobacterium avium subsp. paratuberculosis (MAP) by PCR in raw milk and their interactions were investigated. Three day old bulk tank raw milk (50 ml) samples were seeded with MAP at a level of an estimated 30 CFU/ml. Heat-treatment of raw milk before centrifugation significantly affected the partitioning of MAP in the cream, whey and pellet fractions. Based on the IS900 PCR results, MAP preferentially partitioned into the cream fraction in unheated raw milk, and into the pellet fraction in the heat-treated milk. Treatment with 0.75% hexadecylpyridinium chloride (HPC) helped collect MAP in cream fraction. Heat treatment, use of pooled cream and pellet fractions and treatment with HPC improved the detection by PCR significantly, while washing of pellets prior to DNA extraction did not. The limit of detection using our optimized procedure was an estimated 15-50 CFU in 50 ml, or

Evaluation of BBL CHROMagar Listeria agar for the isolation and identification of Listeria monocytogenes from food and environmental samples.

The performance of BBL CHROMagar Listeria chromogenic agar for the detection of Listeria monocytogenes was evaluated for its ability to isolate and identify L. monocytogenes from food and environmental samples. The medium was compared to non-chromogenic selective agars commonly used for Listeria isolation: Oxford, Modified Oxford, and PALCAM. BBL CHROMagar Listeria had a sensitivity of 99% and 100% for the detection of L. monocytogenes from 200 natural and artificially inoculated food samples, respectively, with a colony confirmation rate of 100%. The sensitivity of non-chromogenic selective media for the detection of L. monocytogenes from these same samples was 97-99% with colony confirmation rates of 65-67.5%. From 93 environmental samples, BBL CHROMagar Listeria agar results correlated 100% with a Listeria spp. visual immunoassay (TECRA) performed on these same samples and the USDA-FSIS standard culture method for the isolation of L. monocytogenes. From environmental samples, the L. monocytogenes confirmation rate was 100% for BBL CHROMagar Listeria as compared to 50% for conventional agars tested. On BBL CHROMagar Listeria, L. monocytogenes forms a translucent white precipitation zone (halo) surrounding blue-pigmented colonies of 2-3 mm in diameter, with an entire border. BBL CHROMagar Listeria offers a high degree of specificity for the confirmation of suspect L. monocytogenes colonies, whereas non-chromogenic selective agars evaluated were not differential for L. monocytogenes from other Listeria species.

Effect of environmental stresses on the mean and distribution of individual cell lag times of Escherichia coli O157:H7.

The effect of starvation, heat or acid stress on duration of individual cell lag time (tau) and standard deviation (SD) of tau was investigated using Escherichia coli O157:H7. Cells were stressed by exposure to acid (pH 3.5), heat (50 degrees C), or starvation in either glucose-free mineral medium (MOPS), tryptic soy broth (TSB) or Luria broth (LB). Stressed cells were then diluted into wells of a Bioscreen plate to obtain single cells per well. Replicate time to detection (td) values were obtained using the Bioscreen and used to calculate the tau and SD. Significant (P< or =0.05) increases in tau over untreated controls were found for the following treatments: 14 days in acid; 2 h of heating; 3 days starvation in MOPS; and 2 days starvation in either TSB or LB. The largest increase in tau was >2-fold from 2.5 to 5.6 h observed with the heat treatment. MOPS starvation was more detrimental to the cells than was acid treatment over the same time period. A significant increase in SD was found with 21 days acid treatment, and 2 days starvation in either TSB or LB. No significant increase in SD was found for MOPS starvation or heat treatment. Lognormal, Gamma, ExtremeValue and Weibull distributions were fitted to the tau data using BestFit. The results suggest that the Lognormal distribution is suitable for fitting tau data from either stressed or unstressed cells.

Microbial assessment of irrigation water used for production of fruit and vegetables in Ontario, Canada.

Five hundred one irrigation water samples were collected from 27 irrigation water sources on 17 farms in southern Ontario, Canada, over a single irrigation season in 2002. The water samples were tested for the presence of the following bacterial water quality indicators: total coliform bacteria, fecal coliforms, Escherichia coli, and fecal streptococci. The median values per 100 ml of these indicators in the irrigation water samples were 3,000, 33, 15, and 1, respectively. Between 70.6 and 98.2% of irrigation water samples contained acceptable levels of fecal coliforms or E. coli, according to published irrigation water quality guidelines. Significant correlations (P < 0.05) were observed between the concentrations of different bacterial indicators and the degree of recent precipitation and concentrations of total coliforms and fecal streptococci. With the exception of fecal streptococci, which increased in number toward the end of the study, none of the indicators displayed a significant trend over the course of the season, as determined by linear regression analysis of indicator concentrations over time (P > 0.05).

Development of improved method for isolation of Mycobacterium avium subsp. paratuberculosis from bulk tank milk: effect of age of milk, centrifugation, and decontamination.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease in cattle and it has been suggested that this organism may be associated with Crohn's disease in humans. Cows at the advanced stage of the disease shed this organism into both their milk and feces. The objective of this study was to develop a more efficient procedure for isolating MAP from bulk tank raw milk. Bulk tank raw milk (50 mL) samples 3 to 13 d old after collection without spiking were investigated to evaluate the effects of milk age on the efficacy of decontamination. Milk samples, 2 to 3 d old, were seeded with MAP at levels of 50 to 200 colony forming units/mL in experiments involving factorial design to evaluate 1) the effects of different decontaminating reagents and decontamination procedures on recovery of MAP, and 2) partition MAP in milk fractions after centrifugation in raw milk. Decontamination in 20 mL of 0.75% hexadecylpyridinium chloride (HPC) at room temperature (22 degrees C) for 2 to 5 h, with shaking, at intervals was found to be the most effective procedure for decontaminating milk 2 to 3 d old. Prolonged exposure to decontaminants, additional incubations in antibiotics, or at higher temperature (37 degrees C) significantly reduced recovery of live MAP. Enhanced growth of microbial contaminants was noticed in samples decontaminated overnight at room temperature compared to those decontaminated for 2 to 5 h. Decontamination of 6 d old milk samples required extra incubation in antibiotic brew. Decontamination of milk samples that are 8 d and older was not effective in removing microbial contaminants. The MAP cells preferentially partitioned into the cream fraction after centrifugation, and combining the milk cream and pellet fractions enhanced recovery of MAP. A recovery rate of 16.6% was estimated with the use of our improved protocol.

Evaluation of an automated immunomagnetic separation method for the rapid detection of Salmonella species in poultry environmental samples.

Automated immunomagnetic separation (AIMS), using Dynabeads anti-Salmonella (Dynal, Oslo), was evaluated for its ability to detect Salmonella spp. in poultry environmental samples in comparison with standard, culture-based method (Health Canada, Health Protection Branch, MFHPB-20). AIMS was found to be more reliable in detecting Salmonella from artificially inoculated enrichment broths at low levels and exhibited a 15.5% higher sensitivity value than the culture method.

Evaluation of methods for the identification of Salmonella enterica serotype Typhimurium DT104 from poultry environmental samples.

An increase in the prevalence of Salmonella enterica serotype Typhimurium DT104 has been reported worldwide. This study examined the prevalence of this microorganism in poultry environmental samples from commercial layer flocks and pullet environments as well as the sensitivity and specificity of a PCR-based method, and multiple antibiotic resistance profile of Salmonella serogroup B isolates in relation to the serotype and phagetype reference method for the identification of Salmonella Typhimurium DT104. A total of 435 Salmonella isolates were obtained from poultry house environmental samples tested during a 20-month period representing a prevalence of 5.5%. Of these, 313 (72%) isolates were identified as Salmonella serogroup B isolates. These isolates were tested by a PCR-based assay, and for resistance to five antibiotics: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT) for the rapid identification of Salmonella Typhimurium DT104. Upon comparing the antibiotic resistance and PCR results with serotype and phage type data, the sensitivity and specificity for the identification of Salmonella Typhimurium DT104 of both methods were found to be 100%, and 99.6%, respectively. Both methods can be completed within 24 h after obtaining an isolate, while serotyping and phagetyping required more than 5 days to complete.