Food Safety Research Forum - Poster Session

Ontario Food Safety Research Forum

Poster Session

Wednesday, April 23, 2008
Hosted by the Research and Innovation Branch, OMAFRA
Victoria Park East Golf Club, 1096 Victoria Road, S., Guelph

1. The Application of Immunomagnetic Separation in Combination with ALOA Listeria Chromogenic Agar for the Isolation and Identification of Listeria monocytogenes in a Variety of Foods

   Carlos G. Leon-Velarde¹, Nathan Larson¹, Joseph A. Odumeru¹

   ¹Laboratory Services Division, University of Guelph.
   95 Stone Rd. West, Zone 2, Guelph, Ontario N1H 8J7, Canada

The use of an immunomagnetic separation method (IMS) in combination with ALOA Listeria chromogenic agar was investigated for the isolation and identification of Listeria monocytogenes from a variety of foods. Raw meat, processed meat, fish and seafood, cultured and non cultured dairy, egg and egg products, and produce, were inoculated at a level of 1, 10, and 100 cfu/25g with L. monocytogenes ATCC 19115 and stressed at 4ºC for 24 hours prior to testing. IMS was performed as described by Dynal Biotech (Oslo, Norway) using a BeadRetriever Automated IMS system (Dynal Biotech) followed by spread plating of 25 uL each onto ALOA Listeria chromogenic agar (AES Chemunex) and Modified Oxford agar, a conventional selective agar. As a comparison method, the same sample enrichments were tested as described by the BAX Q7 Listeria monocytogenes PCR Assay (Dupont). Results showed that at the 10 and 100 cfu/25 g inoculum level, both methods showed a 100% correlation. However, at the 1 cfu/25g inoculum level were fractional recovery was observed, 70 positive samples of 110 were reported by the BAX L. monocytogenes PCR Assay compared to 75 positive samples by the IMS-ALOA Listeria chromogenic agar combination. Analysis of the BAX secondary enrichment by standard culture methods confirmed the presence of L. monocytogenes indicating that these BAX PCR results were false negatives. The IMS-ALOA Listeria chromogenic agar combination was used successfully in isolating and identifying L. monocytogenes from seeded samples in the same time frame (48 h) and in some cases faster (in selected food matrices) that a screening result is obtained by use of the BAX PCR Listeria Assay. Further work including the selection of a single enrichment medium, optimization of incubation conditions, alternate IMS protocols, further evaluation of chromogenic agars, as well testing of naturally contaminated samples are being pursued.

2. Development of a Miniaturized Tube Array for Salmonella Serotyping

   Kristyn Franklin¹, Adam Crossley¹, Stewart Loker¹, Andre Villegas¹, Clifford Clark², Andrew Kropinski¹, Kris Rahn¹

   ¹Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph, ON., ² National Laboratory for Enteric Pathogens, Winnipeg, MB, Canada

Foodborne microbial pathogens are a significant cause of morbidity and mortality in Canada. Non-typhoidal Salmonella infections cause an estimated 1.3 million cases of gastroenteritis in Canada each year, resulting in approximately 15,000 hospitalizations and 550 deaths. An important early step in the characterization of clinical Salmonella is the determination of serotype. Determination of the O (somatic) and H (flagellar) antigenic epitopes, based on the Kauffman White scheme, is performed using rabbit polyclonal antiserum. Serotyping with this method is labour-intensive, time-consuming and expensive. In order to improve outbreak prevention and control a rapid and inexpensive microarray based serotyping assay is being developed to detect the 100 most frequently isolated Salmonella serotypes in Canada and the United States.

The Clondiag ArrayTube^TM based miniarray consists of 60 oligonucleotide probes printed in duplicate. Controls include gapA, an enteric specific control, and invA, a Salmonella specific gene target. Targets are PCR amplified with biotin-labelled primers before hybridization to the miniarray. Probes specific for antigens within the rfb cluster were designed based on publicly available sequence data and newly acquired sequence data from the National Microbiology Laboratory. To date, somatic probes representing 98% of isolates in Canada and the US have been printed on the miniarray of which 97% can be positively identified. The remaining rfb clusters are being cloned, sequenced and annotated using long range PCR and pyrosequencing from which antigen specific probes will be designed. The remaining probes on the miniarray are antigen-specific target sequences within the flagellin antigen phase 1 (fliC) and phase 2 (fljB) genes. Currently 70% of isolates containing phase 1 flagellar antigens can be positively identified. However, due to the genetic similarity between phase 2 antigens present in the 1-complex (1,2; 1,2,7; 1,5; 1,5,7; 1,6; and 1,7), as well as the genetic diversity within target antigen sequences, only 13% of isolates containing phase 2 antigens can be positively identified. To make sequence specific probes all of the 1-complex antigens present in the top 100 Salmonella have been sequenced. This miniarray technology offers a user-friendly platform coupled with reliable and economical array processing. These characteristics make technology transfer to diagnostic labs feasible with the added possibility of commercialization.

Please click here for a PDF of the poster (PDF - 984KB)

3. The control of Salmonella in broilers by defined and non-defined probiotics and fructooligosaccharides

   J.R. Chambers¹, J. Gong¹, C.L. Gyles², M.A. Hayes², B. Sanei³ and S. Sharif<sup>2
   
   1</sup>Guelph Food Research Centre, AAFC, 93 Stone Road West, Guelph, Ontario, N1G 5C9, ²Department of Pathobiology, OVC, University of Guelph, Guelph, Ontario, N1G 2W1, ³Ontario Ministry of Agriculture, Food and Rural Affairs, Department of Pathobiology, OVC, University of Guelph, Guelph, Ontario, N1G 2W1

Broiler chicks were challenged orally with Nal^R Salmonella, either typhimurium or heidelberg, in a set of two trials to determine their ability to reduce caecal Salmonella counts. In each trial, hatched chicks were treated with no probiotics (control), a defined probiotic (<6 known organisms) and a non-defined probiotic (numerous organisms not all known) with half of each group fed a fructooligosaccharide product (92.5% FOS, 0.5% product in ration by wt). One day later, caeca from one chick per treatment group, six in total, were tested for existing Salmonella (none detected) before the remaining 132 chicks were challenged with 10⁴ cfu of Salmonella in 0.5 ml suspension by gavage. Chicks were provided a crumbled, broiler starter ration and distilled H₂O throughout the trial. They were confined to 6 small pens with litter floors and solid walls. This plan was repeated with a second pair of probiotics in an additional set of two trials. At 1, 2 and 4 weeks post-challenge, caecal contents of 7 chicks per treatment group were sampled and cultured on BGS agar plates with nalidixic acid. For samples with no detectable colonies we cultured material previously preenriched in Selenite Cystine broth to distinguish between low level positive and negative status. Salmonella counts were analysed using the GLM procedure of SAS with probiotics, prebiotic, age and trial (Salmonella serotype) and their interactions in the model. The two sets (trial pairs) were analysed separately because probiotics in each set differed. The probiotics reduced Salmonella, both typhimurium and heidelberg, by about 0.5 logs (defined) and 2.5 to 4.5 logs (non-defined) at 1 and 2 weeks post infection. Effects of FOS were modest, 1.0 log at one week post infection if used with non-defined probiotics. Decline in Salmonella excretion over time in the control chickens prevented demonstration of probiotic effects at 4 weeks. Modern technology for identification of probiotic organisms, poultry pathogens and antibiotic resistance genes is required to enable developers to define products and approval bodies to permit use of currently non-defined probiotics for improved control of food-borne pathogens in poultry.

4. Virulence gene expression in Escherichia coli O157:H7 present on lettuce

   C. M. Carey¹, M. Kostrzynska¹, S. Thompson¹

   ¹Agriculture and Agri-Food Canada, Guelph Food Research Centre, 93 Stone Road West, Guelph, Ontario, N1G 5C9

In recent years the number of E. coli O157:H7 outbreaks linked to the consumption of leafy-green vegetables has increased dramatically. In this study, the effect of various storage conditions on virulence gene expression in E. coli O157:H7 present on contaminated Romaine lettuce was investigated. A two-step reverse-transcription comparative quantitative real-time PCR assay was employed to evaluate the expression of genes coding for intimin (eaeA), flagellin (fliC), and genes encoding the A subunit of Shiga-toxin 1 and 2 (stx1A and stx2A) in E. coli O157:H7. Up-regulation of stx1A and stx2A was observed during 9-day storage of contaminated lettuce at 4°C and 15°C, however stx2A was up-regulated to a greater degree when stored at 4°C. In addition, fliC was up-regulated during storage at 15°C, while transcription of fliC gene changed only slightly when stored at 4°C. Expression of gene encoding intimin (eaeA) was variable at 15°C with a tendency towards down-regulation. However this gene was slightly up-regulated when stored at 4°C. In conclusion, our results suggest that E. coli O157:H7 may become more virulent with prolonged storage of contaminated lettuce.

5. The diagnostic potential of miniaturised DNA microarray

   Muriel Mafura*¹, Miranda Batchelor*¹, Katie Hopkins², Guanghui Wu¹, Sarah North¹, John Threlfall², Martin J. Woodward¹ and Muna F. Anjum¹.

   ¹Department of Food and Enviornmental Safety, Veterinary Laboratories Agency-Weybridge, New Haw, Addlestone, Surrey KT15 3NB, U.K. ²Laboratory of the Enteric Pathogens, Health Protection Agency Centre for Infections, London NW9 5EQ.

   * these authors have contributed equally.

The DNA microarray chip offers a new way for biologists to understand the complexities of a living organism. In our laboratory we have developed miniaturized microarray chips suitable for high throughput use for detection of virulence and antimicrobial resistance (AMR) genes. The virulence gene chip contains 60 oligonucleotide probes representing 7 different Escherichia coli pathotypes, whilst the AMR gene chip contains 58 probes representing 46 different AMR groups present in enteric bacteria. The virulence pathotype of 63 E. coli clinical isolates of human and animal source was established using the virulence chip. Strains of STEC and EPEC pathotypes showed similar virulence characteristics with presence of the stx toxin genes being the main differential determinant. In contrast, isolates characterised as ExPEC, ETEC, EIEC or EAEC tended to show distinct virulence profiles. Others harboured novel combinations of virulence determinants, the significance of which remains to be determined. For the AMR chip presence of resistance genes in a panel of 50 E. coli and 43 Salmonella clinical isolates of human and animal origin was tested. The mean number of resistant genes present in E. coli isolates was found to be 8 and in Salmonella, 5. The most common gene detected in both E. coli and Salmonella isolates was the extended-spectrum b-lactamase TEM gene, which was present in 90 and 56% of the isolates tested, respectively. The results demonstrate that these arrays provide an effective, economic and simple method for detection of genes in clinical isolates, which will help to understand the epidemiology of pathogens, and to detect gene linkage to infection.

Please click here for a PDF of the poster (PDF - 148KB)

6. Dump tank water sanitation technologies

   Arthur, Lindsay¹, Knight, Kelley², Zhou, Ting²

   ¹Ontario Ministry of Agriculture, Food and Rural Affairs, 1 Stone Rd., W., Guelph, ON N1G 4Y2. ²Agriculture Agri-Food Canada. 90 Stone Rd. W., Guelph, ON N1G 5C9.
   

Many horticultural producers use water as a method to remove field heat and/or to clean produce. Water is a well-known vector for microbial contamination, and a number of trace back investigations have suggested that poor dump tank water quality contributed to outbreaks in fresh fruit and vegetables. To avoid this, producers typically treat their dump tank water with sodium or calcium hypochlorite; however, these treatments are corrosive, have worker safety issues, and are not in accordance with organic production standards. In addition, the efficacy of these treatments is reduced by the presence of organic matter (OM) which can be present in high concentrations depending on the type of produce. As a result, seven water treatment systems: calcium hypochlorite, sodium hypochlorite, chlorine dioxide, ultra violet radiation, ozone and two different peroxyacetic acid treatments, were evaluated for efficacy, ease of use and disposal, worker safety, cost and changes in produce quality as a result of their use. The research, conducted in a research pilot plant, was designed as 2 x 2 factorial experiment with high and low levels of OM and Escherichia coli for each of the seven treatments in a randomized complete block design with four replications.. Asparagus, lettuce and peaches were assessed for colour and textural changes against all seven treatments. All treatments exhibited at least a 94% reduction in E. coli. Each treatment varies significantly in terms of cost and ease of use. Colour and textural changes due to the varying treatments were determined. This research provides producers practical information enabling them to make informed decisions implementing water treatment technology, which enhances food safety while meeting their production needs.

7. A longitudinal study of the Salmonella status on Ontario swine farms within the time period 2001-2006

   Vahab Farzan¹, Robert Friendship¹,Catherine Dewey¹, Cornelis Poppe², Julie Funk³, Anne Muckle⁴

   ¹Department of Population Medicine, University of Guelph, Guelph, Ontario N1G 2W1, ² Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph, ON N1G 3W4, ³ National Food Safety and Toxicology Center, Michigan State University, 165 Food Safety and Toxicology Building, East Lansing, MI 48824-1302 USA, ⁴Diagnostic Services, Atlantic Veterinary College, University Prince Edward Island, 550 University Avenue, Charlottetown PE C1A 4P3
   

Salmonella enterica subspecies enterica are important foodborne pathogens associated with pork products. In order to limit and control Salmonella occurrence in swine herds, it may be useful to conduct epidemiological studies to determine the prevalence of Salmonella and provide knowledge on the distribution of Salmonella serovars on swine farms. In order to describe the farm-level of Salmonella status, 113 Ontario swine farms were tested annually for Salmonella 1 to 5 times within the time period 2001-2006. During 422 visits, 6844 fecal samples were collected and cultured for Salmonella. Salmonella was recovered from 437 (6.38%) of the fecal samples and 69 (61%) of the farms had at least one positive sample over the entire period of the study. Salmonella was not recovered on 11 farms of the 54 farms visited five times, nor from 7 of the 17 farms visited four times. On 7 farms Salmonella was not recovered over the first 4 visits but were cultured on the fifth visit. The isolates belonged to 30 different serovars and serogroup B and C1 were the most common serogroups. Salmonella Typhimurium var. Copenhagen was the most common serovar followed by S. Typhimurium, rough Salmonella isolates (Rough-O), S. Derby, S. Infantis, S. Brandenburg, and S. Mbandaka. The most frequent phage type was DT104 (including DT104a and DT104b) followed by DT12, DT193, and U302. Significant trends were detected in apparent farm-level prevalence of Salmonella during 5 years of this study. Although the observed trends may be partly attributed to the different culturing methods, different types of samples, and sampling strategies used in each year, it may also denote the dynamics of Salmonella as a bacterial population on swine farms. These findings indicate that monitoring over time may be useful to detect changes in Salmonella on swine farms.

8. Molecular epidemiology of Salmonella Typhimurium DT104 on Ontario swine farms
   

   Vahab Farzan¹, Robert Friendship¹, Catherine Dewey¹,Cornelis Poppe², Laura Martin², Julie Funk³

   ¹Department of Population Medicine, University of Guelph, Guelph, Ontario N1G 2W1, ² Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph, ON N1G 3W4, ³ National Food Safety and Toxicology Center, Michigan State University, 165 Food Safety and Toxicology Building, East Lansing, MI 48824-1302 USA.

Multi-drug resistant Salmonella Typhimurium DT104 has become a worldwide public health concern. It has been also the first or second most common Salmonella serovar reported from human and food-producing animals in Canada. However, S. Typhimurium DT 104 strains isolated from different sources might not be distinguished based on phenotypic characteristics. This study was conducted to examine antimicrobial resistances, plasmid profiles and pulsed-field gel electrophoresis patterns of 80 S. Typhimurium (including var. Copenhagen) DT104 strains (including DT104a and DT104b) recovered from pig and environmental fecal samples on 17 swine farms in Ontario. No resistance was observed to amoxicillin/clavulanic acid, apramycin, carbadox, cephalothin, ceftriaxone, ceftiofur, cefoxitin, ciprofloxacin, nalidixic acid, trimethoprim, and tobramycin. However, the isolates exhibited resistance against 4 to 10 antimicrobials with most frequent resistance to sulfonamides, ampicillin, streptomycin, spectinomycin, chloramphenicol, tetracycline, and florfenicol. Thirteen distinct resistance patterns were determined but 88% of isolates shared the typical resistance pattern "ACSpSSuT". Twelve different plasmid profiles were observed; the 62 MDa virulence-associated plasmid was detected in 92.6% of the isolates. The 2.1 MDa plasmid was the second most frequent one, which was harbored by 65% isolates. The isolates were classified into 23 distinct genotypes by PFGE-SpeI+BlnI when difference in at least one fragment was defined as a distinct genotype. In total, 39 distinct "types" were observed when defining a "type" based on the combination of antimicrobial resistance, plasmid pattern, and PFGE-SpeI+BlnI for each isolate. The highest diversity was 0.96 (95% CI: 0.92, 0.96) for the "type" described above followed by 0.92 (95% CI: 0.88, 0.93) for PFGE-SpeI+BlnI. The diversity of DT104 isolates indicates there might be multiple sources for this microorganism on swine farms. This knowledge might be used to track these sources, as well as to study the extent of human salmonellosis attributed to pork compared to food products derived from other food-producing animals.

9. The prevalence of Salmonella spp based on fecal culture and serum antibody from pigs on farms using liquid or dry feeding

   Vahab Farzan¹, Robert Friendship¹, Catherine Dewey¹,Keith Warriner², Anne Muckle³, Kim Klotins⁴

   ¹Department of Population Medicine, University of Guelph, Guelph, Ontario N1G 2W1, ² Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, ³ Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph, Ontario N1G 3W4, ⁴ Ontario Ministry of Agriculture and Food, 1 Stone Road West Guelph, Ontario N1G 4Y2.

Salmonella enterica serovars are important foodborne pathogens of public health concern and pork products play an important role in the transmission of Salmonella to humans. Feed is an important component in a Salmonella control program and among risk factors the type of feed appears to be strongly associated with the presence of Salmonella. The use of liquid-feeding has been recently established in Ontario in more than 100 swine farms and it is estimated that liquid-feeding accounts for about 20% of pigs sent to market. The objective of this study was to determine whether liquid-feeding is associated with a lower shedding and antibody titre to Salmonella than dry-feeding in grower-finisher pig operations in Ontario. Twenty liquid-feeding farms and 61 dry-feeding farms were selected. Blood samples were collected from 15 finisher pigs on each. Rectal feces were obtained from these same 15 pigs. Fecal samples were cultured for Salmonella and sera were analyzed by ELISA for the presence of antibodies against Salmonella. Salmonella were isolated from 25 of 420 fecal samples (6%) from dry- feeding farms compared to three of 400 samples (0.8%) from liquid-feeding farms. Furthermore, eight farms (38%) using dry-feeding had at least one positive sample compared to only three liquid-feeding farms (15%). The Salmonella Typhimurium isolated from the liquid-feeding farm was susceptible to all antimicrobials tested. In contrast, the S. Typhimurium isolates from dry-feeding farms were resistant to four or more antimicrobial agents. At the cut-off of 10, 98% of dry feeding-farms and 84% of liquid-feeding farms tested positive (P = 0.01). Dry-feeding, antimicrobial daily usage, and additional pigs were predicted to increase the value of OD. Liquid-feeding tended to be associated with a decrease in both culture of Salmonella and serum Salmonella antibody and may be considered as intervention to control Salmonella in swine. However, liquid-feeding is a new technology for the swine industry in Canada and it tends to be found in newer facilities compared to dry-feeding.

10. Antimicrobial resistance in Salmonella on Ontario swine farms, 2001-2006
    

    Vahab Farzan¹, Robert Friendship¹, Cornelis Poppe², Catherine Dewey¹, Julie Funk³

    ¹Department of Population Medicine, University of Guelph, Guelph, Ontario N1G 2W1, ² Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph, Ontario N1G 3W4, ³ National Food Safety and Toxicology Center, Michigan State University, 165 Food Safety and Toxicology Building, East Lansing, MI 48824-1302 USA.

Salmonella serovars demonstrating multiple antimicrobial resistance are important worldwide public health concern. Antimicrobial resistance associated to Salmonella might be transferred to other swine pathogens causing serious problems in treatment and control of infectious diseases. Therefore, epidemiological studies need to be conducted in order to gain a better understanding of antimicrobial resistance in Salmonella on swine farms. The point estimate studies cannot represent the true status of Salmonella and antimicrobial resistance in swine and it is very important to test swine farms for Salmonella over a period of time. The objective of this study was to describe the farm-level prevalence of antimicrobial resistance in Salmonella isolated on Ontario swine farms during the years 2001-2006. Salmonella isolates recovered on 113 swine farms within the time period 2001-2006 were tested for antimicrobial susceptibility by the agar dilution method. No resistance was determined to amikacin and ciprofloxacin and only one nalidixic acid resistant isolate was recovered in 2001. Only 1% of the isolates were resistance against ceftiofur, ceftriaxone, apramycin, apramycin, cephalothin, amoxicillin/clavulanic acid, cefoxitin, and gentamicin. Resistance to ceftiofur, ceftriaxone, and apramycin was observed only in the last year of the study. The most frequent resistance was seen against sulfisoxazole (45.1%), tetracycline (43.4%), streptomycin (42.5%), spectinomycin (41.6%), chloramphenicol (36.3%), and ampicillin (35.4%) followed by neomycin (23%), kanamycin (23%), and nitrofurantoin (18.6%). Resistance against sulfamethoxazole/ trimethoprim (10.6%), trimethoprim (8.8%), cephalosporins (3.5%) was observed only in 2002 and 2006. Resistance to the cephalosporins and carbadox was found on 2 farms each in the last year of the study. Resistance to carbadox deserves a serious consideration and follow up because the use of this drug in swine industry has been banned in Canada since 2001. Three groups of farms were defined based on Salmonella status for each year of the study. A farm was classified either as Group 1 if no Salmonella were recovered during the entire study period, as Group 2 if Salmonella without antimicrobial resistance were isolated, or classified as Group 3 if Salmonella with antimicrobial resistance were cultured. When defining these 3 groups for the entire study period, 44 (39%), 30 (27%), and 39 (35%) farms were categorized into Group 1, Group 2, and Group 3, respectively. Further studies need to be conducted to compare the risk factors that distinguish these three groups of farms. Significant trends were detected in farm-level prevalence of antimicrobial resistance during 5 years of this study. These trends need to be interpreted with caution since we used different culturing methods and different types of sample and sampling strategies in each year. However, these findings indicate that monitoring over time may be useful to detect changes in antimicrobial resistance patterns on swine farms.

11. Enteric Infectious Disease Pathogens in Ontario Commercial Rabbitries: Findings of a Prevalence Study

    Marina Brash¹, Patricia V. Turner², Ashley Whiteman², Emily Martin¹, Durda Slavic¹, Scott Weese², Robert Wright³, Brian Tapscott³, Lindsay Arthur⁴

    ¹Animal Health Laboratory, Laboratory Services Division, University of Guelph, Guelph, ON, N1H 6R8, ² Department of Pathobiology, OVC, University of Guelph, Guelph, ON, N1G 2W1, ³ Ontario Ministry of Agriculture, Food and Rural Affairs, Wellington Place, R.R. # 1, Fergus, ON, N1M 2W3, ⁴Ontario Ministry of Agriculture, Food and Rural Affairs, 1 Stone Road West, Guelph, ON, N1G 4Y2
    

A collaborative study was undertaken in early 2007 that involved surveying the Ontario commercial rabbit industry for prevalence of infectious disease agents from clinically affected rabbits with diarrhea. Both fryers and does with enterocolitis were necropsied in the summer and winter of 2007 with subsequent histopathologic and microbiologic evaluations. In addition, the Ontario industry was surveyed by questionnaire regarding on-farm hygiene and disposal practices. E coli, including enteropathogenic serotypes, was isolated from both age groups from all farms surveyed, with a higher prevalence in the winter; however, prevalence of coinfection with other pathogens, such as Lawsonia intracellularis and Clostridium perfringens differed between fryers and does and was low overall. No C. difficile or EHEC or VTEC strains of E. coli were isolated from any operation. Salmonella agona was isolated from a nonclinical doe in one operation. Based on the survey findings, few biosecurity practices were in place on the operations surveyed. As multiple species are frequently managed on the same farm, opportunities may exist for enhancing biosecurity awareness and personal hygiene practices to prevent cross-species transmission of infectious disease agents.

12. A monoclonal antibody-based antigen capture Enzyme Linked Immunosorbent Assay (ELISA) for the detection of serogroup D Salmonella serovar Enteritidis from farm environmental samples: a comparison with routine culture methods

    B.W. Brooks¹, J. Devenish¹,C.L. Lutze-Wallace¹, M. Elmufti¹, T. Burke¹, S. Duff¹, K. Habib¹.

    ¹Canadian Food Inspection Agency, Ottawa Laboratory (Fallowfield), PO Box 11300, Station H, Ottawa, ON K2H 8P9

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for use as a presumptive screening test for detection of Salmonella Enteritidis and other serogroup D Salmonella. A mixture of two monoclonal antibodies, that recognize two different forms of the serogroup D lipopolysaccharide O-polysaccharide, was used in the ELISA for specific detection of serogroup D Salmonella. The performance of the ELISA was evaluated in comparison to standard culture procedures for Salmonella detection. A total of 444 environmental samples collected from poultry hatcheries were analysed by both culture and ELISA. Culture included nonselective enrichment with buffered peptone water, and direct and delayed secondary enrichment with tetrathionate and Rappaport-Vassiliadis broths, five enrichment broths per sample (total of 2220 broths). Highest recovery of S. Enteritidis was obtained with delayed secondary enrichment and Rappaport-Vassiliadis broth. A high correlation was obtained between ELISA results and culture results. The data indicate that this ELISA is a useful test for screening field samples for the presence of S. Enteritidis. Furthermore the ELISA format is rapid and cost effective for screening large numbers of samples.

13. Canadian Integrated Program for Antimicrobial Resistance Surveillance: Retail Food Program

Brent Avery^1,2, Carolee Carson^1,2,Danielle Daignault¹, Anne Deckert^1,2, Lucie Dutil1³, Sheryl Gow1⁴, David Léger^1,2, Jane Parmely^1,5, Richard Reid-Smith^1,2, Rebecca Irwin¹.

¹Antimicrobial Resistance Unit, Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph, Ontario / St-Hyacinthe, Québec / Saskatoon, Saskatchewan; ²Department of Population Medicine, University of Guelph, Guelph, Ontario; ³Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec; ⁴Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan; ⁵Centre for Coastal Health, Nanaimo, British Columbia

The Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) was initiated in 2002 to monitor trends in antimicrobial use and antimicrobial resistance in selected bacterial organisms. Retail meat represents a logical sampling node for surveillance of antimicrobial resistance in the food chain as it is the endpoint of the food pathway (i.e. the point of consumer exposure prior to the kitchen). For this reason, a retail food surveillance component was added to CIPARS in 2003. The objective of CIPARS-Retail Surveillance is to investigate antimicrobial resistance among bacteria found in food at retail. This surveillance framework can easily be modified (e.g. food commodities, bacteria, regions) as necessary to function as a research platform to investigate specific questions regarding antimicrobial resistance in the agri-food sector. The unit of concern is the bacterial isolate cultured from one of the commodities of interest and tested for antimicrobial susceptibility to a standard panel of antimicrobials. Commodities of interest are raw meat and poultry products commonly consumed by Canadians and mirror those commodities sampled in the CIPARS Abattoir and On-Farm Surveillance components including: chicken (skin-on chicken legs or wings), pork (shoulder chops) and beef (ground beef). The bacteria of interest in chicken meat are Campylobacter spp., Salmonella spp, Enterococcus spp., and generic E. coli. In pork and beef, only generic E. coli is routinely cultured, given the low prevalence of Campylobacter spp. and Salmonella spp. in these commodities as determined in the early phases of the retail program. The target population is consumers of retail meat in Canada. The majority of retail sampling involves continuous, weekly sample submissions from randomly selected census divisions, weighted by population, in each of the participating provinces. Currently, CIPARS-Retail surveillance is underway in Québec, Ontario, Saskatchewan and British Columbia with regular surveillance activities scheduled to commence in Atlantic Canada in the near future. Selected results from this program will be presented from 2003 through 2006.

14. Canadian Integrated Program for Antimicrobial Resistance Surveillance: ON-FARM SWINE PROGRAM

    Anne Deckert^1,3, David Léger^1,3, Brent Avery^1,3, Sheryl Gow^1,4, Danielle Daignault¹, Lucie Dutil1^,2, Richard Reid-Smith^1,3, Rebecca Irwin¹.

    ¹Antimicrobial Resistance Unit, Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph, Ontario / St-Hyacinthe, Québec / Saskatoon, Saskatchewan; ²Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec; ³Department of Population Medicine, University of Guelph, Guelph, Ontario; ⁴Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan.

In 2006, the CIPARS On-farm surveillance program was implemented in swine herds across the five major pork producing provinces in Canada. The swine industry was selected as the pilot commodity for surveillance infrastructure development because there is extensive implementation of the Canadian Quality Assurance (CQA®) program by the industry, there was the absence of a recent foreign animal disease outbreak and there is a similar initiative in swine in the United States (Collaboration in Animal Health and Food Safety Epidemiology). Upon completion of the programs first year and after consultation with collaborators program refinements were implemented in 2007.

The objectives of the CIPARS On-farm surveillance program are to:

• Establish infrastructure to support a national surveillance program.
• Provide data on antimicrobial use and resistance.
• Investigate associations between antimicrobial use and resistance.
• Provide data for human health risk assessments.

The surveillance program focuses on grower-finisher hogs. Nationally, 108 sentinel grower-finisher sites are enrolled. In each of the 5 participating provinces, the number of CIPARS sentinel sites is proportional to the national total of grower-finisher units. To provide producer anonymity, herd veterinarians conduct the sample and data collection. These veterinarians were purposively selected from provincial sampling frames. Using specified inclusion/exclusion criteria, each veterinarian selected a set number of sentinel farm sites. Antimicrobial use data are collected using sampling day questionnaires and CQA® forms. Pooled fecal samples are collected from pens of close to market weight finisher hogs three times annually. In a subset of herds, specific cohorts of pigs are followed. Cohort pens have pooled fecal samples collected at arrival and again when close to market. All fecal samples are cultured for generic E. coli, Enterococcus and Salmonella and quantitative antimicrobial susceptibility testing is performed using the Sensititre® Microbiology System (Trek Diagnostic Systems, Cleveland, OH, USA). Preliminary analysis of herd demographic data and antimicrobial resistance data will be presented.

15. Simple method for the speciation analysis of bio-accessible arsenic in seafood using on-line continuous leaching and ion exchange chromatography coupled to inductively coupled plasma mass spectrometry

    Vincent Dufailly¹, Thierry Guérin¹, Laurent Noël¹, Jean-Marc Frémy², and Diane Beauchemin³.

    ¹Unité des Contaminants Inorganiques et Minéraux de l'Environnement, AFSSA-LERQAP, 23 Avenue du Général de Gaulle, F-94706 Maisons-Alfort Cedex, France; ²Unité d'Evaluation des Risques Physico-Chimiques, AFSSA-DERNS, 27-31, Avenue du Général Leclerc, F-94701 Maisons-Alfort, France; ³Department of Chemistry, Queen's University, 90 Bader Lane, Kingston, Ontario, K7L 3N6, Canada.

A quick and simple on-line leaching method was used to assess the maximum bio-accessibility of As in seafood samples. Artificial saliva, gastric juice and intestinal juice were successively pumped through a mini-column of sample (maintained at 37°C in a thermostated water bath), which is connected to the nebulizer of an inductively coupled plasma mass spectrometry (ICP-MS) instrument. In contrast to the usual batch method, this approach allows the continuous monitoring of the progressive release of As by the 3 reagents. So, the dissolution equilibrium is driven to the right, thereby enabling the determination of the maximum amount of analyte that can be dissolved (i.e. the worst-case scenario). Results for 4 certified reference materials (CRMs) show that saliva alone was sufficient to release in less than 5 min all the bio-accessible total As that was mobilised by saliva and gastric juices in the batch mode. The As speciation in each leachate was then determined by ion-exchange chromatography (IEC) coupled to ICP-MS, after adjustment to the gradient program previously optimised by experimental design, along with a 5-fold sample dilution and increased stabilisation time. Under these optimised conditions, 7 different arsenic species in saliva could be separated within 18 min in a single chromatographic run. In all cases, the sum of bio-accessible and residue As concentrations agreed with the certified value.

16. Evaluation of the Alaska FastrAK* Assay System for the Detection of Salmonella in Foods

    Parmjot S Swatch^1,2, Carlos G. Leon-Velarde¹, Nathan Larson¹, Joseph A. Odumeru^1,2.

    ¹Laboratory Services Division, Guelph, Ontario, Canada; ¹Department of Food Science, University of Guelph, Guelph, Ontario, Canada.

A newly developed phage-based Alaska FastrAK* System was evaluated for the rapid detection of Salmonella spp. in artificially contaminated foods commonly associated with Salmonellosis. The sensitivity of the novel method was determined by comparing the performance of the method with that of a reference culture method for Salmonella as described in the Health Canada Compendium of Analytical Methods (HFHPB-20).The system gave 100% accuracy in terms of inclusivity and exclusivity with 28 pure Salmonella strains and 22 pure non-Salmonella strains respectively. The detection limit of the assay was established between 10 to 10³ CFU/mL using 4 different Salmonella serovars. The phage-based method was able to detect Salmonella spp. in animal foods such as raw chicken, raw beef, and processed chicken and liquid pasteurized egg at levels near or below the 1CFU /25 g (or mL) regulatory limit after 16 h enrichment. The system gave high number of false negative results when tested on raw turkey meat because of competitive micro flora and could not detect Salmonella in cheddar cheese because of high fat content. The phage based detection system is a viable alternative to immunological and DNA-based rapid methods for detection of Salmonella in selected foods, especially low fat and processed foods. A thorough evaluation of system is required for the raw and processed chicken meat including naturally contaminated samples before adopting the phage-based Alaska System for routine testing this food matrix.

17. Evaluating the Effectiveness of Search Strategies for Systematic Reviews in Zoonotic Public Health

    Vi Nguyen¹, Lisa Waddell^1,2, Janet Harris¹, and Andrijana Rajic^1,2,

    ¹Laboratory for Foodborne Zoonoses, Guelph, PHAC; ²Department of Population Medicine, University of Guelph.

The study objective was to compare the effectiveness of modified search strategies with the original SR searches that were utilised in three previously completed systematic reviews (SRs) addressing specific zoonotic public health topics. Reducing the number of major databases included in the electronic search to at least three, in combination with comprehensive search terms, yielded high sensitivity in capturing relevant citations for two out of three SRs. The effect of reducing both the number of databases and search terms resulted in decreased sensitivity. Results reveal that in order to achieve efficient and effective searching, a balance between comprehensive and brief searches is required

18. Challenges and Opportunities for Utilizing Systematic Reviews in Zoonotic Public Health

    Lisa Waddell^1,2, Andrijana Raji?^1,2, Jan Sargeant^2,3, Wendy Wilkins⁴, and Scott McEwen².

    ¹Policy Advice and Effectiveness Program, Laboratory for Foodborne Zoonoses, PHAC, Guelph, Ontario; ²Department of Population Medicine, Ontario Veterinary College, Guelph, Ontario; ³Centre for Zoonoses, Ontario Veterinary College, Guelph, Ontario; ⁴Western College of Veterinary Medicine, Saskatoon, Saskatchewan.

Zoonotic public health (ZPH) spans multiple scientific disciplines and a variety of stakeholders. Examples include avian influenza, antimicrobial resistance, and transmissible spongiform encephalopathy. These issues are often inherently complex with primary research resulting in contradictory findings and recommendations to policy makers. Systematic reviews (SR) have been utilized to a limited extent in ZPH. The Policy Advice and Effectiveness Program, Public Health Agency of Canada (established in 2004), together with academic collaborators, developed a guide1 for conducting SRs in this area and published several SRs and meta-analyses (MA) addressing ZPH questions. Our objective is to highlight the major challenges and opportunities experienced with the implementation of SRs on ZPH, using three already conducted SRs as examples.

19. The Methodological Soundness of Literature Reviews in Zoonotic Public Health

    Lisa Waddell^1,2, Andrijana Raji?^1,2, Jan Sargeant^2,3, Sarah Parker ⁴, Anne Deckert¹ and Scott McEwen².

    ¹Policy Advice and Effectiveness Program, Laboratory for Foodborne Zoonoses, PHAC, Guelph, Ontario; ²Department of Population Medicine; ³Centre for Public Health and Zoonoses, Ontario Veterinary College, Guelph, Ontario; ⁴Western College of Veterinary Medicine, Saskatoon, Saskatchewan.

The study objective was to evaluate the methodological soundness of literature reviews in zoonotic public health (ZPH). Review articles (n=132), published over the last five years and addressing three known zoonotic or debatable zoonotic issues, were evaluated for methodological soundness using 13 criteria and two independent reviewers. None of the reviews met more than eight criteria and two met only one criterion. Literature reviews in ZPH should adhere to structured and transparent methods that are employed in systematic reviews. These would allow their users to assess the review validity and the appropriateness of its utilization in a decision making process.

20. Linking Research Synthesis and Risk-Based Tools: Addressing Policy Makers' Needs in Zoonotic Public Health

    Andrijana Rajic^1,2, Aamir Fazil¹, Javier Sanchez³, and Scott McEwen²,

    ¹Laboratory for Foodborne Zoonoses - Guelph, PHAC; ²Department of Population Medicine, University of Guelph; ³Population Health Research Group, University of Prince Edward Island.

Our objective was to evaluate the opportunities and challenges for linking research synthesis and risk-based tools using 'Salmonella issue in pork' as a model. Global knowledge-base on the effectiveness of interventions against Salmonella in the 'farm-to-processing' pork continuum was mapped out, appraised and summarized, utilizing a systematic review (SR). No single on-farm intervention was universally beneficial for Salmonella reduction in pork. SR-meta-analysis (MA) approach was applied to investigate factors affecting reported prevalence of Salmonella. A meta-regression revealed that prevalence estimates based on cultured feces or tissues were 12-27% lower than estimates obtained from serological tests; estimates based on convenience sampling were 10% higher than from random sampling. Evidence-based intervention and prevalence summaries, generated through SR-MA approach, were refined using expert-panel opinions and incorporated into a quantitative risk assessment (QRA) to investigate the effect of selected interventions. A package of interventions produced the largest (93%) overall prevalence reduction of Salmonella at the end of processing. SR-MA should be considered as a routine tool for generating evidence-based inputs for QRA.

21. Bacteriophage Control of Salmonella in Pig Production

    M. Bassit¹, R. Friendship², R. Johnson³, M. Kostrzynska⁴, and K. Warriner¹

    ¹Department of Food Science, University of Guelph, Guelph, ON N1G 2W1; ²Population Medicine, University of Guelph; ³Laboratory for Foodborne Zoonoses Health Canada 110 Stone Road West Guelph, ON N1G 3W4; ⁴Agriculture and Agri-Food Canada, Guelph Food Research Centre, 93 Stone Road West, Guelph, Ontario, N1G 5C9.

Over 50% of pigs within Ontario are thought to carry Salmonella and hence considered a significant source of the enteric pathogen. Several strategies have been introduced to control the prevalence of Salmonella within pig herds. These have included increased surveillance, pest control, all-in-all-out production and sanitation. Despite such efforts the prevalence of Salmonella remains high.

In the following, a bacteriophage based biocontrol method has been developed and evaluated for controlling Salmonella in pigs. Baseline studies isolated lytic bacteriophage from fecal and effluent samples derived from Ontario pig farms. From over 300 isolates recovered, 5 were selected for further study by using a selection criteria based on host range, lytic strength and ability to replicate under sub-optimal temperature conditions. The five phages were combined to form a cocktail that was subsequently applied in pig trials to assess the efficacy of the preparation to control Salmonella. Within the trials pigs were administered bacteriophage through introducing the cocktail via inoculated milk, in addition to daily spraying the bacteriophage in the pig pen. Salmonella Typhimurium DT104 was introduced into the pigs 3 days following phage treatment. The weight and diarrhea score, in addition to microbial sampling (Salmonella and bacteriophage) was performed throughout the 28 day trial. From the results obtained it was evident that the application of bacteriophage was insufficient to eliminate Salmonella associated with pigs and their environment. However, levels of the enteric pathogen were only low and could only be detected by enrichment. The majority of Salmonella recovered from pigs were identified as serotype Typhimurium although not the ST DT104 strain introduced into pigs. Significantly all of the Salmonella isolates recovered were sensitive to phage indicating that generation of resistant mutants did not occur.

In conclusion the study demonstrated the utility of bacteriophage to reduce the levels of Salmonella associated with pigs and their environment. However, it is likely that addition interventions are required in combination with bacteriophage to completely eliminate the enteric pathogen from pigs.

22. Suppression of Salmonella on Sprouting Mung Beans by Using a Combination of Antagonistic bacteria and Lytic Bacteriophage

    J. Ye¹, M. Kostrzynska², K. Dunfield³ and K. Warriner¹

    ¹Department of Food Science, University of Guelph, Guelph, ON N1G 2W1; ²Agriculture and Agri-Food Canada, Guelph Food Research Centre, 93 Stone Road West, Guelph, Ontario, N1G 5C9; ³Landscape Sciences, University of Guelph.

Sprouted seeds, such as mung bean sprouts, have been implicated in several high profile foodborne illness outbreaks. One of the largest ever recorded occurred within Ontario in 2005 when bean sprouts contaminated with Salmonella resulted in over 600 cases of foodborne illness. In the majority of outbreaks the pathogens could be traced to the seed used to produce sprouts. To reduce the risk of producing contaminated sprouts it has been recommended to sanitize the seeds with hypochlorite prior to sprouting. However, it is well established that hypochlorite has only limited efficacy in decontaminating seeds. In addition, a high proportion of sprouts are produced for the organic market where the use of chemical sanitizers is restricted.

The following study reports on the efficacy of a biocontrol method to suppress the growth of Salmonella on sprouting mung beans. The biocontrol preparation consisted of an Enterobacter asburiae strain recovered from mung bean sprouts and a cocktail of five Salmonella infecting bacteriophage isolated from pig farms. When applied individually Ent asburiae and bacteriophage could reduce the growth of Salmonella in vitro by 3 log cfu/mL or 1-2 log cfu/mL respectively. However, when Ent asburiae and bacteriophage were used in combination total suppression of Salmonella growth was observed although residual populations (0.1 log cfu/mL) persisted. The growth suppressing effect was independent on the Multiplicity of Infection (MOI) applied in the range of 0.1 - 100. The same results were obtained with trials using inoculated mung beans. Here, mung beans were inoculated with Salmonella followed by Ent asburiae and/or bacteriophage. When applied individually both Ent asburiae and bacteriophage could only reduce Salmonella populations. However, when used in combination Salmonella could only be detected by using enrichment. The research provides a promising approach to control Salmonella in sprouting mung beans and other sprout types.

23. Development of Microencapsulated Bacteriophage against Salmonella

    Yongsheng Ma^1,2, Jennifer C. Pacan¹, Qi Wang¹*, Xiaoqing Huang¹, Parviz M. Sabour¹*

    ¹Guelph Food Research Center, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada; ²Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China.

    *Corresponding author: Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, N1G 5C9, Canada.

Bacteriophages can eliminate pathogenic bacteria without disturbing the normal or beneficial microflora in the guts. However, viability of some bacteriophages may decrease in upper gastrointestinal tract, thus their protection is necessary. In our previous study, we have shown that free phage Felix O1 is extremely sensitive to acidic environment of stomach and to a lesser degree to bile salts. In a simulated In vitro tests, using gastric fluid (SGF) and bile salts solutions we showed that encapsulation of phage Felix O1 in alginate-CaCl₂ microspheres, coated by chitosan, detrimental effects were minimized. In addition, phages were completely released from the microspheres upon exposure to simulated intestinal fluids at pH 6.8, within six hours. Here we report further technical improvement of the procedure for encapsulating phage Felix O1. Incorporation of CaCO3 into the alginate-CaCl₂ formulation, significantly improved the resistance of encapsulated phages to acidic conditions. The encapsulated phages retained full viability when stored wet at 4ºC during the testing period (6 weeks). However, drying process and further storage of dried product severely reduced phage viability. A number of stabilizing agents including trehalose, sucrose and skim milk were tested and found to increase the viability of encapsulated phages during drying and storage. The current encapsulation technique enables a larger proportion of Felix O1 bacteriophage to remain bioactive in simulated gastrointestinal tract condition, providing a more effective delivery of therapeutic phages.

24. Acidification of raw cow milk and effects on the culturability of Mycobacterium avium subsp. paratuberculosis

    Lucy M Mutharia¹, Melinda Raymond¹

    ¹Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1.

A source of Mycobacterium avium subs. paratuberculosis (MAP)-free milk for calf feeding is needed to control MAP transmission to calves, and is a key component in the success of the national Johne's disease (JD) control programs. JD affects up to 30% of Ontario Dairy herds and similar rates are reported for dairy and beef herds across Canada. Calves are most susceptible to infection when they ingest the bacterium in colostrums and milk contaminated either in the mammary glands or post harvest with feces of cows with JD. At the present, installing pasteurization systems, or purchasing pasteurized and commercial milk replacers to feed calves are the only options available to producers. For producers, these options present added economic costs, since they buy commercial products while discarding readily available colostrums or waste (non-saleable) milk. In this study, raw milk cow milk and colostrum seeded with cultured clinical MAP strains was employed to investigate the following, (i) the pH necessary and the minimum contact time required to achieve log-reduction in MAP viability, (ii) whether MAP bacteria subjected to acidic treatments were actually 'killed' or could resuscitate once milk is stored, neutralized or diluted, (iii) whether acidification destroyed milk immunoglobulins. Here we discuss our results on the effect of acidification on MAP culturability. Our results show that acidification affectrd the recovery of MAP from milk and decreased by up to 70% the culturability of the bacterium. HPC decontamination and antibiotic treatment further decreased the culturability of MAP bacteria. In our hands, acidification significantly reduced the cfu of MAP in milk. Neutralization of the milk prior to recovery of the bacterium did not counteract the effect of acidification. It remains to be determined whether acidification affects the nutritional quality of the milk and the functions of the colostrum immunoglobulins.

(* Poster was presented at the 9th ICP Tokyo Japan)

 

**As titles and abstracts are submitted, they will be posted here.

Contact: Stacy Favrin
E-mail: stacy.favrin@ontario.ca
Tel: 519-826-3976

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