Genetic Diversity of Culicoides stellifer (Diptera: Ceratopogonidae) in the Southeastern United States Compared With Sequences From Ontario, Canada.

Much of the bluetongue (BT) and epizootic hemorrhagic disease (EHD) research in North America focuses on white-tail deer and Culicoides sonorensis (Wirth & Jones) (Diptera: Ceratopogonidae), though several other biting midge species have been suggested as vectors. Culicoides stellifer (Coquillett) has been associated with hosts susceptible to hemorrhagic disease (HD), and more recently, specimens from Florida have tested positive for EHD and BT viral RNA. If C. stellifer is acting as a vector, this could have an impact on the distribution of HD in North America. To determine if gene flow is occurring across the range of C. stellifer within the southeast United States, a mitochondrial haplotype analysis was performed using the COI gene. Our haplotype network showed no population structure in C. stellifer from Florida, Texas, and South Carolina, as the overall genetic divergence between these sites was equal to the genetic divergence within each. We also compared these haplotypes to published sequences of C. stellifer collected in Ontario, Canada. Surprisingly, the genetic diversity of the flies from Ontario was two times greater than what was observed between the southeast U.S. collection sites. This considerable divergence could be evidence of a cryptic species. A better understanding of the connectivity between C. stellifer populations across all of North America will give insight into the distribution of HD. Our results show that gene flow is occurring between sites in the southeastern United States and potentially throughout the eastern distribution of the species.

Foodborne Illness Outbreak Investigation Training Needs: A Survey Among State Public Health Staff in the Northeast and Mid-Atlantic United States.

CONTEXT: In 2011, the Food Safety and Modernization Act established Integrated Food Safety Centers of Excellence across the United States to train, educate, and enhance the skill of foodborne illness outbreak investigation teams. To target regional training efforts, the New York Integrated Food Safety Center of Excellence (NYCoE) identified training needs in food safety and foodborne illness investigations among public heath staff in 11 states and 1 large metropolitan area in the Northeast. OBJECTIVE: To identify topics so as to develop training materials relevant to food safety and foodborne disease outbreaks in order to improve and impact foodborne outbreak investigations regionally and nationally. DESIGN: Cross-sectional, paper-based survey conducted in January-February 2016. SETTING: Eleven Northeast and Mid-Atlantic states, and 1 large metropolitan area. PARTICIPANTS: Foodborne illness outbreak investigators in the NYCoE region. MAIN OUTCOME MEASURES: Identification of training needs, as self-reported by participants, regarding general foodborne outbreak investigation needs and those specific to epidemiologists, environmental health specialists, and laboratorians. Topics included basic food safety/processing knowledge, communication and metrics, and training formats. Information regarding demographics, utility of the NYCoE, and certificate programs was also collected. RESULTS: Surveys returned from 33 respondents (from 10 states and 1 metropolitan area) identified metrics (100%), increasing use of culture-independent diagnostic tests (85%), and guidance on implementation of the Council to Improve Foodborne Outbreak Response (guidelines (89%) as the top training needs. By field, epidemiologists cited training in applying whole genome sequencing (100%), environmental health specialists cited training on the National Outbreak Reporting System (67%), and laboratorians cited training on whole genome sequencing (91%) as important. Short, online, or in-person, 1- to 2-day trainings were the preferred training formats (≥91%). Respondents wanted certificate programs in food safety (73%). CONCLUSION: New diagnostic and molecular techniques are highly desirable topics for trainings, as is understanding national guidelines and outcome measures. Shorter and hands-on training formats, as well as certificate programs, are desirable. The NYCoE used the results of this survey to (i) select training topics for a Food Safety symposium conducted in July 2016 and to (ii) drive other activities.

Distribution of internalin gene profiles of Listeria monocytogenes isolates from different sources associated with phylogenetic lineages.

Listeria monocytogenes is a human foodborne pathogen with a broad range of hosts. While the L. monocytogenes genome encodes a number of internalins, which are leucine-rich repeat bacterial surface proteins with putative or confirmed roles in host cell attachment and invasion, the specific function of many internalins remains unclear. The distribution of 7 internalin genes (inlC, inlC2, inlD, inlE, inlF, inlG, and inlH) in 120 L. monocytogenes isolates from humans, foods, and animals was investigated to determine if the distribution of these proteins differed with respect to source or phylogenetic lineage. Isolates were classified into 6 different profiles based on internalin gene presence or absence, and a phylogeny based on one stress response (sigB) and two housekeeping (gap and prs) genes was used to correlate these profiles with L. monocytogenes phylogenetic lineages. All 69 isolates classified into L. monocytogenes lineage I, which is overrepresented among human disease cases, had the same internalin profile (presence of inlC and the inlC2DE operon). Lineage II (48 isolates), which is common among food and environmental sources, represented 4 internalin gene profiles, with most isolates carrying inlG and inlF in addition to inlC and inlC2DE. Our data indicate that L. monocytogenes isolates show diverse and distinct patterns of internalin gene presence/absence and L. monocytogenes internalin profiles are associated with phylogenetic lineages.

Markov chain approach to analyze the dynamics of pathogen fecal shedding--example of Listeria monocytogenes shedding in a herd of dairy cattle.

Fecal shedding is an important mechanism of spreading of a number of human and animal pathogens. Understanding of the dynamics of pathogen fecal shedding is critical to be able to control or prevent the spread of diseases caused by these pathogens. The objective of this study was to develop a model for analysis of the dynamics of pathogen fecal shedding. Fecal shedding of Listeria monocytogenes in dairy cattle was used as a model system. A Markov chain model (MCM) with two states, shedding and non-shedding, has been developed for overall L. monocytogenes fecal shedding (all L. monocytogenes subtypes) and fecal shedding of three L. monocytogenes subtypes (ribotypes 1058A, 1039E and 1042B) using data from one study farm. The matrices of conditional probabilities of transition between shedding and non-shedding states for different sets of covariates have been estimated by application of logistic regression. The covariate-specific matrices of conditional probabilities, describing the presence of different risk factors, were used to estimate (i) the stationary prevalence of dairy cows that shed any L. monocytogenes subtype or ribotypes 1058A, 1039E, and 1042B, (ii) the duration of overall and subtype specific fecal shedding, and (iii) the duration of periods without shedding. A non-homogeneous MCM was constructed to study how the prevalence of fecal shedders changes over time. The model was validated with data from the study farm and published literature. The results of our modeling work indicated that (i) the prevalence of L. monocytogenes fecal shedders varies over time and can be higher than 90%, (ii) L. monocytogenes subtypes exhibit different dynamics of fecal shedding, (iii) the dynamics of L. monocytogenes fecal shedding are highly associated with contamination of silage (fermented feed) and cows' exposure to stress, and (iv) the developed approach can be readily used to study the dynamics of fecal shedding in other pathogen-host-environment systems.

Daily variability of Listeria contamination patterns in a cold-smoked salmon processing operation.

An understanding of Listeria transmission and contamination patterns in processing environments of ready-to-eat foods is critical for improving control of Listeria monocytogenes. A cold-smoked fish processing operation was the site used to study variability in Listeria contamination in a processing environment associated with a ready-to-eat food product throughout one production week (five consecutive days). Intensive testing was conducted on finished products and environmental samples collected at the beginning, middle, and end of each working day. A total of 20 finished products and 22 to 36 environmental samples were collected at each sampling time, and an additional 12 environmental samples were collected on days 4 and 5. Overall, a total of 782 samples, 300 finished products and 482 environmental samples, were tested. All samples were collected from processing steps after smoking, including skinning, trimming, slicing, staging, and packing. A total of 28 finished and 57 environmental samples (9.3 and 11.8%, respectively) were positive for Listeria spp. (including 1 and 5 samples positive for L. monocytogenes, respectively). DNA sequencing of the sigB gene allowed differentiation of eight Listeria subtypes. Listeria prevalence varied significantly between days, and a high prevalence in both environmental samples and finished products on day 3 was likely associated with a point source contamination event by a single Listeria welshimeri subtype. There were no consistent differences in Listeria prevalence among samples collected from the beginning, middle, and end of the production day, but subtype data often revealed unique contamination patterns for samples collected at different times of a given day. Listeria contamination patterns and prevalences were highly variable between days and within a given day. These findings indicate that chance events play an important role in the contamination of finished products, thus complicating efforts to define Listeria transmission patterns in processing environments associated with ready-to-eat foods.

Evaluation of farm management practices as risk factors for clinical listeriosis and fecal shedding of Listeria monocytogenes in ruminants.

OBJECTIVE: To assess seasonal variation in prevalence of Listeria monocytogenes on ruminant farms and identify management practices associated with ruminant listeriosis and fecal shedding of L. monocytogenes. STUDY DESIGN: Case-control study. SAMPLE POPULATION: 2056 samples of feces, feed, soil, and water from 24 case farms with listeriosis and 28 control farms without listeriosis. PROCEDURE: Samples were collected and evaluated via bacterial culture for L. monocytogenes. Univariate associations between farm management practices and listeriosis and fecal shedding of L. monocytogenes were assessed. Multivariate models were developed to identify farm management practices associated with listeriosis and fecal shedding of L. monocytogenes. RESULTS: The prevalence of L. monocytogenes on cattle, goat, and sheep farms was seasonal, especially in fecal samples, with peak prevalence in winter. Although the prevalence of L. monocytogenes in feedstuffs from small-ruminant farms also peaked during winter, the bacterium was detected at a constant rate in cattle farm feedstuffs throughout the year. Farm management practices, animal health and hygiene, and feedstuff quality and storage were associated with ruminant listeriosis and fecal shedding of L. monocytogenes. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the prevalence of L. monocytogenes on ruminant farms is seasonal, management practices are associated with ruminant listeriosis and fecal shedding of L. monocytogenes, and the epidemiologic features of listeriosis differ in cattle versus small ruminants. Awareness of risk factors may be used to develop control measures to reduce animal disease and introduction of L. monocytogenes into the human food chain.

Prevalence and growth of Listeria on naturally contaminated smoked salmon over 28 days of storage at 4 degrees C.

Only limited data are available on the growth characteristics of Listeria in naturally contaminated ready-to-eat foods. To evaluate Listeria contamination patterns and growth in smoked salmon, 72 smoked salmon product samples from two processing plants were tested for Listeria spp. and L. monocytogenes. Samples were divided into four approximately equal portions: one portion was tested on receipt, and the other three were vacuum sealed and stored at 4 degrees C for 7, 14, and 28 days. Listeria testing was performed using both an enrichment procedure and direct plating to enumerate Listeria in samples that contained >2 to 10 CFU/g. Five samples were positive for Listeria spp., including one sample that was positive for L. monocytogenes. Most samples yielded only sporadic positive results among the portions tested on days 0, 7, 14, and 28. Only one sample contained Listeria spp. in numbers above the detection limit for enumeration. For this sample, the portions tested on days 7 and 28 contained 46 and 52 CFU/g, respectively, whereas the portion tested on day 14 was negative. Overall, our data indicate that there is considerable heterogeneity in Listeria spp. distribution within a single positive smoked fish sample. Even with refrigerated storage for 28 days, none of the naturally contaminated samples reached Listeria spp. numbers >100 CFU/g, which indicates that Listeria growth was limited within a 4-week storage period. However, because of the apparent heterogeneity of Listeria distribution within samples, the interpretation of growth data collected on naturally contaminated samples is difficult.