Genomic-based identification of environmental and clinical Listeria monocytogenes strains associated with an abortion outbreak in beef heifers

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Bono, Michael L. Clawson, John D. Loy, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.13582/v3 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Feb, 2020 Read the published version in BMC Veterinary Research → Version 3 posted 2 You are reading this latest preprint version Show more versions Abstract Background In a beef cattle facility an outbreak of abortions occurred over a 36-day period and included two aborted fetuses and 21 post-abortion clinical cases sampled. There are numerous etiologies, including clinical listeriosis. At the species level, Listeria monocytogenes is ubiquitous in cattle production environments, including soil, feed, and occasionally water sources, and is a common enteric resident of cattle and other mammals. There are four genetically distinct lineages of L. monocytogenes (I-IV), with most lineage III and IV isolates obtained from ruminants. Definitive diagnosis of L. monocytogenes as a causative agent in disease outbreaks relies upon case identification, appropriate sample collection, and laboratory confirmation. Furthermore, clearly establishing a relationship between a pathogen source and clinical disease is difficult. Results Of the two fetal and 21 clinical case submissions, 19 were positive for L. monocytogenes. Subsequent culture for L. monocytogenes from water and silage sources identified both as potential origins of infection. Using whole-genome sequencing and phylogenetic analyses, clinical, water and silage L. monocytogenes strains grouped into two of four lineages. All water and silage strains, plus 11 clinical strains placed in lineage III, with identical or nearly identical genomic sequences. The remaining eight clinical strains placed in lineage I, with seven having nearly identical sequences and one distinctly different. Conclusion Three genetically distinct strains within two lineages of L. monocytogenes caused the abortion outbreak. The etiology of abortion in 11 cases was directly linked to water and silage contamination from a lineage III L. monocytogenes strain. The source of infection for the remaining abortion cases with two different strains from lineage I is unknown. This is the first report of L. monocytogenes genomics being used as part of an outbreak investigation of cattle abortion. Large Animal Medicine Cattle abortion listeriosis silage whole-genome sequencing outbreak Figures Figure 1 Figure 2 Background Listeria monocytogenes is a well-known saprophytic bacterial pathogen that is ubiquitous in the cattle production environment. Cattle and many other mammals including humans, can be asymptomatic shedders. However, L. monocytogenes can cause a number of diseases across species, including visceral listeriosis, neurologic listeriosis, and importantly, reproductive listeriosis, which is implicated in late-term abortion in cattle. Certain populations of cattle, particularly those who are immunocompromised, pregnant, young, old, or stressed are more affected by environmental risk factors associated with L. monocytogenes infection [1-3]. The potential routes of infection leading to clinical listeriosis in cattle is either ingestion, inhalation, direct contact or oral mucosal lesions [4-7]. The primary route in cattle is suspected to be the consumption of contaminated feed or water and subsequent passage through the gastrointestinal tract, a pathway that has been demonstrated in goats and sheep [8, 9]. Once in the gastrointestinal tract, L. monocytogenes is able to bind, penetrate and move within and between the epithelial cells [10]. Translocation of L. monocytogenes occurs from the intestines to the liver and hepatocytes via macrophages, leading to a bacteremia that results in either an effective cell-mediated immune response, neurological disease, or reproductive infection [11]. Across mammalian species, the incubation period from infection to clinical signs of listeriosis is variable, generally 2-6 weeks [12]. Consequently, the delayed onset of clinical signs following infection makes identification of a potentially contaminated feed source challenging, in that the feed may have been entirely consumed by the time clinical listeriosis is observed. Even if L. monocytogenes is detected in available feed, the many available molecular diagnostic techniques [13] may not be specific enough to directly link the source strain of L. monocytogenes to the clinical strain due to its pervasiveness in the environment. L. monocytogenes diversity has been assessed using various methods. Historically, serotyping [14] and pulsed-field gel electrophoresis (PFGE) [15, 16] have been implemented in source origin identification of strains in human and cattle listeriosis outbreaks. However, these diagnostics do not definitively establish genetic relationships between strains. Genomic sequencing has advantages over traditional diagnostics in that it interrogates the complete genome and can establish a more definitive relationship between isolated strains. In situations where L. monocytogenes can be established as an etiology for clinical disease as well as an environmental contaminant, utilizing genomic sequencing can verify that a specific strain found in the environment is responsible for clinical disease. There are four genetically distinct lineages of L. monocytogenes (I-IV) that have been found across mammalian species and in the environment [17, 18]. L. monocytogenes strains can be classified into one of four lineages based on a number of techniques including: pulse-field gel electrophoresis, ribotyping [14, 19], multi-locus enzyme electrophoresis, multilocus sequencing typing and more recently whole genome sequencing. Of the four lineages, strains from lineage III are often isolated from cattle, and lineage IV is currently exclusive to ruminants. Lineages I through III have been documented in clinical listeriosis in cattle and other mammals [17]. In this report, an outbreak investigation of L. monocytogenes abortion in a beef cattle operation is described. Aborted bovine fetuses, cervico-vaginal (CV) swabs, and retained fetal membranes (RFM), if present, from aborting females, as well as their water and feed sources were sampled and cultured for L. monocytogenes . To establish a definitive source of the abortion outbreak, all L. monocytogenes culture positive strains were subjected to whole-genome sequencing, phylogenetic analyses, and lineage classification. Results Cattle An abortion outbreak occurred at the United States Meat Animal Research Center (USMARC) in late winter/early spring of 2014. A total of 28 heifers aborted over a 36-day period, with 24 abortions occurring in a 12-day period. By comparison, four mature cows aborted during the calving season, none within the acute abortion outbreak timespan and none were positive for L. monocytogenes . Three heifer management sites (A-C) were affected, and all were managed similarly, fed from the same silage source, and from the same feed truck (Figure 1). Two- and three-year old females were never grouped with mature cows, but rather rotated through pastures, occupying sites where cows had resided approximately seven months previous. Females were aborting late term (3 rd trimester) fetuses, with no other observed clinical signs, except some with blood on the perineum or RFM, and some groups had refused feed as recently as the day prior to the first abortion. Descriptive statistics of abortions during the calving season are listed in Table 1. The mature cow known abortion percentage was 0.1%, but was at 3% for the heifer population, much higher than spring calving herd reports by the National Animal Health Monitoring System (NAHMS) [20]. The majority of abortions occurred within a 12-day time period, prompting the outbreak investigation. During the outbreak, two aborted bovine fetuses were submitted to the University of Nebraska Veterinary Diagnostic Center, Lincoln, NE (UNLVDC), for aerobic culture, C. fetus culture, Leptospira spp. , Bovine herpesvirus 1 (BHV1), bovine viral diarrhea virus (BVDV) PCR, and histopathology. Cervico-vaginal swabs or RFM from 21 of the 28 aborting heifers were also collected on follow-up sampling and submitted to the UNLVDC for aerobic culture, followed by L. monocytogenes culture and/or L. monocytogenes PCR, if needed. Both fetuses and 17/21 heifer samples were positive for L. monocytogenes, specifically 11/11 RFM and 6/10 CV swabs. Fetal tissues were examined by board certified pathologists as part of the abortion diagnostic investigation. Fetus 1 had liver lesions consistent with listeriosis and included scattered foci of acute necrosis with moderate neutrophilic inflammatory response. Fetus 2 had marked autolysis within the liver with presence of few mononuclear cells in the hepatic capsule. Both pathologists diagnosed listeria as the cause of abortion. Fetuses were negative to all other diagnostics, including PCR for Leptospira spp., BHV1 and BVDV, and culture for Campylobacter sp. No other diagnostics were performed on the RFM or CV samples. Descriptive statistics of the sample submissions and results are summarized in Table 2. Feed and Water Samples Nine water sources were tested, and four were culture positive for L. monocytogenes (Table 3). Site A had two water tanks positive for L. monocytogenes while site B contained one positive water tank and one positive water hole by the feed bunk. Water tanks associated with site C were sampled, but no L. monocytogenes was isolated. Of the 15 silage samples tested, Listeria spp. were detected in 14 samples, of which four samples were culture positive for L. monocytogenes . Of those positive for L. monocytogenes , three strains were derived from feeds that had a pH less than 5.0. Feed sample timing, type, location, pH, and test results are summarized in Table 4. Sample locations of the pile face correspond to Additional file 1. Genomics Thirty one strains were subjected to short read Illumina sequencing in this study, of which 27 were part of the USMARC outbreak and four were available from the UNLVDC (see Additional file 2 for strain information). All 31 strains passed post-sequencing processing for low quality reads and adapter sequence. The lowest read coverage was 12.8X while the highest coverage was 132X with the average being 58X. Parsnp was used to create an initial phylogenetic tree based on Strategic Kmer Extension for Scrupulous Assemblies (SKESA) assembled chromosomes of the 31 sequenced strains. The tree was divided into three major lineages with six minor clusters. One strain from each cluster of the initial tree was also sequenced using long-read PacBio sequencing to generate reference complete closed genomes for each cluster. The four major L. monocytogenes lineages were reproduced in a maximum-likelihood tree calibrated with 20 L. monocytogenes chromosomes of known lineage and serotype affiliations that were available from the National Center for Biotechnology Information (NCBI, Additional file 2, Figure 2). The tree also contained 25 SKESA assembled L. monocytogenes chromosomes and six closed PacBio generated chromosomes from this project that were assigned to lineages based on their placement in the tree. Lineage I contained seven NCBI genomes representing serotypes 1/2b, 3b, 4b, 4d, 4e, and 7, and eight clinical strains from cattle located in two different USMARC management sites (A and C) and one UNLVDC strain. Seven of the lineage I clinical L. monocytogenes strains molecularly serotyped as 1/2b based on their in silico multi-locus sequence typing (MLST) patterns, and one molecularly serotyped as 4b. Lineage II contained seven genomes from NCBI that represented serotypes 1/2a, 1/2c, 3a and 3c plus one UNLVDC strain that molecularly serotyped as 1/2a (Additional file 2, Figure 2). No lineage II strains were isolated from any of the three cattle management sites, feed, or water. Lineage III contained two NCBI genomes that represented serotypes 4a and 4c, and two UNLVDC strains that could not be molecularly serotyped because there were no matching MLST allele patterns in the Listeria Pasteur MLST or Center for Genomic Epidemiology databases [21, 22]. Lineage III also contained a monophyletic cluster of eight environmental strains and eleven clinical strains that collectively originated from feed and water or cattle at all three sites, respectively (Additional file 2, Figure 2). These strains also could not be molecularly serotyped based on their MLST allele patterns. While all clinical strains from the USMARC abortion outbreak placed in either lineages I or III, the feed and water strains only placed in lineage III. Of the eight USMARC clinical strains that placed in lineage I seven grouped with a UNLVDC strain and were either identical or differed from each other by no more than six single nucleotide polymorphisms (SNPs). In contrast, the UNLVDC strain differed from the seven clinical strains by more than 60 SNPs. These seven clinical strains were all isolated from cattle at site A and molecularly serotyped as 1/2b. The other clinical strain in lineage I was isolated from a heifer at site C and was more closely related to NCBI genome NC_018642, a 4b serotype isolated from cheese [23]. Consequently, lineage I contained two genetically distinct L. monocytogenes strains that originated from clinical cases in two different management sites with no apparent connection to strains isolated from feed and water samples. All 11 clinical strains that placed in lineage III were either identical or no more than eight SNPs different from the lineage III strains recovered from the silage pile. Additionally, two clinical, two water and two corn silage strains were identical across their core genome, which represented 99% of a closed, circular, PacBio-generated reference genome for isolate 52330 (Additional file 3). Consequently, all lineage III strains from the outbreak were either completely clonal, or only differed by a few SNP alleles. These results indicate that there were three genetically distinct L. monocytogenes strains responsible for the cattle abortion outbreak, with two that placed in lineage I and one that placed in lineage III. Discussion Although an important diagnostic sample, only two aborted fetuses were recovered by cattle managers for L. monocytogenes testing in this outbreak. Consequently, alternative sampling was needed to confirm the etiology of the abortions and to quickly and efficiently implement appropriate management changes. To that end, CV swabs or RFM from females that aborted proved to be valuable diagnostic samples in this outbreak when fetuses were unavailable. An etiologic diagnosis was achieved in 81% (17/21) of all examined clinical cases, with 100% (11/11) of the retained placental samples and 60% (6/10) of the CV swabs positive for L. monocytogenes (Refer to Table 2). This high rate of diagnostic success was achieved despite sampling being delayed up to seven days from the time of abortion. Additionally, 17/19 of the overall positive cultures (including two fetal tissue submissions) were isolated from primary aerobic culture and did not require additional enrichment. This is important as cold enrichment of L. monocytogenes can take weeks, further delaying the time to a diagnosis. Diagnostic investigation indicated the causative agent for the abortion outbreak was L. monocytogenes , however the source of the organism(s) was not immediately clear. A positive L. monocytogenes result from a day one fetal tissue sample submission was not reported until day eight of the outbreak, with 19 abortions occurring within that week. Mitigating the abortion outbreak was critical for the remaining pregnant females in the herd. Previous literature indicated that poorly ensiled feed was associated with L. monocytogenes contamination [24-28]. Because of this, focus was placed on the supplemental silage as a potential source, and feeding was discontinued on day eight until results from the remaining case samples and environmental testing were completed. Abortions cases ceased four days after removing suspected silage from the ration, with the exception of one occurring 19 days later. The reason for the abrupt halt to the abortions following silage removal is unclear. The incubation period for L. monocytogenes would suggest that abortions should have continued for a longer time if exposure occurred and the dose was sufficient. Immediately after day one fetal tissue sample submission results were confirmed positive for L. monocytogenes , a novel and organized approach to feed and water sampling was employed, resulting in more effective and efficient collection of environmental samples and subsequent source identification. Both silage and water samples were culture positive for L. monocytogenes . Of the four positive water samples, three required enrichment to facilitate growth, indicating a smaller bacterial load in these sources. Therefore, we suspected early on in the investigation that the source of L. monocytogenes in the water was likely from heifers transferring feed into the water soon after consuming affected silage. Interestingly, some feed samples positive for L. monocytogenes were at a pH of less than 5.0, which should have been lethal to the bacteria. This finding is supportive of other research indicating that L. monocytogenes can survive at a pH of less than 5.0 [26, 29], bringing the traditional acceptable pH for appropriately ensiled feeds into question. While clinical and laboratory findings were supportive of L. monocytogenes in the environment as the likely etiology for the abortions, we wanted to determine if a genetic relationship between the isolates from the environment and clinical samples existed and if the feed was the source of L. monocytogenes . Disposing of several hundred tons of silage had significant economic and resource implications, but herd health and welfare related to feeding contaminated silage was a major concern, and the cattle could be exposed to other risk factors associated with L. monocytogenes infection, such as oral-fecal transmission and the ubiquitous nature of the bacteria. Proving that the silage was the source of the outbreak, and not another cause would further strengthen the decision to dispose of all contaminated silage. To our knowledge, full genomic sequencing of L. monocytogenes has not been previously used in a bovine abortion outbreak investigation. Using this technique would give the highest resolution for determining the source of the outbreak. Two silage, two water, and two clinical L. monocytogenes strains were identical to each other, and nearly identical to all remaining silage (n=2) and water (n=2) strains, and clinical strains (n=9). Thus, the same lineage III genetic strain type was found in silage, water, and clinical case samples. The direction of the feed delivery pathway (Figure 1) would not have allowed for water or cattle to contact the original silage pile. This implicated the silage as the most likely source of the pathogen and the cause of abortion in at least 11 heifers. The remaining eight L. monocytogenes positive clinical strains consisted of two genetically distinct lineage I strain types. Females infected with either of the two lineage I strain types were kept with other heifers that were infected with the distinct lineage III strain type that traced back to the silage. Of note, when silage feeding was discontinued, all abortions but one ceased four days later. Thus, it is possible that all three genetically distinct strains implicated in the outbreak could have been in the feed, with two undetected. Importantly, identification of seven lineage I clinical L. monocytogenes strains as sertotype 1/2b provided risk information for cattle handlers. Serotypes 1/2b and 4b, along with serotype 1/2a, are the main serotypes that cause of human disease and represents 90-95% of cases [30-32]. Consequently, personnel working with cattle from the outbreak were at an increased risk to become infected with L. monocytogenes . Because L. monocytogenes was isolated from the silage at different sampling times and at different depths, the integrity of the entire silage pile was called into question, and therefore it was decided that the feed was unsuitable for further use. The remaining affected silage pile was spread onto pastures that would not be populated for several months, so that UV light and desiccation could synergistically eradicate the remaining L. monocytogenes [33-35]. In addition to routine cleaning of the water tanks, recommendations for future silage management were made including: appropriate packing of newly harvested silage to ensure an anaerobic environment within the pile, vermin mitigation, surface protection and routine silage pile sampling throughout the subsequent feeding season to monitor pH and L. monocytogenes presence. The silage sampling scheme would follow the protocol used in the investigation, with suggested 2-4 week sampling intervals. Management strategies to prevent another abortion outbreak due to L. monocytogenes appeared to be effective in subsequent production years. Following the acute outbreak, all additional aborted fetal submissions screened for L. monocytogenes in the 2014 calving season and following seasons did not detect or isolate L. monocytogenes . Additionally, no clinical listeriosis cases of any kind in the cowherd were observed during that time. No negative sequelae to reproductive performance was observed in the recovered animals. Of the 17 L. monocytogenes positive females, five were culled prior to the 2014 breeding season, and the remaining 12 were diagnosed pregnant (100%) by ultrasound in the fall of the same year. In the 2015 calving season, those heifers all carried a term calf. Eleven of 12 calves were born alive, with dystocia as the documented cause of the only calf death loss, resulting in a 91.6% calving percentage for those females exposed. Conclusion This case investigation resulted in a very complete epidemiologic picture of an L. monocytogenes abortion outbreak in beef cattle. L. monocytogenes was quickly identified as the cause through strategic sampling of affected cattle and their environment, with contaminated silage initially implicated as the probable source. The outbreak was brought under control through the elimination of the contaminated silage. Subsequent whole genome sequencing showed that three strains were involved in the outbreak and confirmed that silage was the primary source of at least one of them. Abortions in beef cattle can be a major problem in herds, particularly if a larger than normal percentage of the population is affected, or if outbreaks occur over a short period of time. During L. monocytogenes abortion outbreaks, challenges in timely disease recognition and diagnosis can create limitations for treatment of affected cattle and management decisions. Thus, quickly establishing a clear connection between the host, pathogen, and environment through strategic animal and environmental sampling, followed by strain identification using whole genome sequencing of L. monocytogenes isolates allows for appropriate management of feed and environmental risks, as well as strategies for risk management of future outbreaks. Using whole-genome sequencing of pathogens in outbreak investigations will give veterinarians and epidemiologists greater flexibility and stronger evidence for confirming the strains and sources involved. Methods Cattle population and management All sampled cattle in this report resided at the USMARC in Clay Center, Nebraska, and sampling protocols were approved by the Institutional Animal Care and Use Committee at the University of Nebraska, Lincoln (IACUC, #1383). Veterinary intervention and sampling were requested by the owner soon after the abortions began. The entire cattle population was considered closed; bovine semen was the only source of new genetic material and biosecurity measures were in place to prevent direct contact with outside cattle. Staff and equipment were shared between cattle locations within USMARC. As a part of routine management practices, all females were previously vaccinated for Brucella abortus and given an initial Campylobacter fetus and Leptospira canicola-grippotyphosa-hardjo-icterohaemorrhagiae-pomona bacterin (Spirovac VL5, Zoetis, Kalamazoo, MI), followed by a modified live IBR, BVD types 1 and 2, and PI 3 vaccine in combination with C. fetus , and Leptospira spp. bacterin (PregGuard Gold FP 10, Zoetis, Kalamazoo, MI) 30 days before the 2013 breeding season. Pregnancy was diagnosed via rectal ultrasound between 45-100 days gestation. A total of 28 females (27 two-year old heifers and 1 three-year old female, herein referred to as “heifers”) aborted in late winter/early spring of 2014. Twenty-one of these cases were sampled (described below) and diagnostics performed at the UNLVDC in Lincoln, Nebraska. All pregnant females were on pasture prior to, during, and after all sampling. During the winter months, all cattle were supplemented primarily with corn silage stored and fed from the same feed storage site. Silage was analyzed for nutritional content and combined with mineral supplementation to meet nutritional requirements. Rations were delivered by the same truck and dispensed in mobile bunk or tire feeders. These feeders were moved periodically when conditions around the bunks were deemed unsanitary. Heifers that aborted were from one of three management sites (A-C) that were managed in a similar fashion. A site map of the operation with feed delivery pathway is identified in Figure 1. Clinical case sampling Twenty-eight heifers and four mature cows (four years or greater) aborted by the end of the 2014 calving season, with 24 heifer abortions occurring over 12 days. All collected tissues and sampling of females that had aborted were performed by or under the direct supervision of a licensed veterinarian (lead author). Aborted fetal tissues, including fresh and fixed heart, lung, liver, spleen and kidney, as well as stomach contents and ear notches, were submitted to the UNLVDC on day one and day eight of the investigation,, the days that they were discovered and recovered by cattle managers. All fetal tissues were placed in insulated cooler with icepacks and shipped overnight to the UNLVDC. Of the 28 heifers that were identified by cattle managers and suspected of aborting, 21 were humanely restrained in a commercial cattle chute and were subjected to individual examination and sample collection (day 8, 14, or 27). The perineum of each female was cleaned with soap and water, rinsed and dried, and the tail held away from the region. If RFM were present, a clean, gloved hand was inserted into the vagina and the membranes were extracted caudally until the hand and membranes were outside the vulva, then a section of membrane was aseptically removed and placed in a sterile collection bag. In heifers that did not have RFM, a uterine culture double-guarded swab (Jorgensen Laboratories, Loveland, CO) was used to sample near and within the cervical opening (CV swab). To facilitate the entry of the swab, a disposable lubricated vaginal speculum was used to visualize the cervical opening and the area just proximal to the opening. Approximately 1-2 cm of the distal cervix and the area just proximal to the opening were swabbed. This method prevented undue contamination from the vagina, which in many cases contained discharge and fluid as a result of metritis or pyometra. Once swabs were collected, samples were contained within the protective unit of the swab mechanism. All case samples were placed in insulated cooler with icepacks and shipped overnight to the UNLVDC. Heifers were released after sampling into communal pens for health observation, then subsequently released back onto pasture. Feed and water sampling for L. monocytogenes and pH Supplemental feed consisted of corn silage, earlage, and haylage separated into open concrete bunkers. Corn silage and earlage were sampled by visually dividing the piles into three stratified layers (top, middle and base) and crosswise sections (left, middle, and right) to provide targeted and documented sampling locations (Additional file 1). Samples were obtained by first brushing away loose materials on the surface that were potentially transferred or contaminated by feeding equipment. Exposed feed was then collected at a depth of approximately 10 cm. Additional samples included loose surface material from the center face and apron of the piles as well as loose material present in the drainage tube of each pile. Samples were collected from haylage in a similar fashion as silage piles but from only three of the quadrants: center-top, mid-left and bottom-right. All samples were collected with freshly gloved hands, placed in sterile bags, and transported to the laboratory where they were held at 4 °C until processing. Silage and earlage pile samples were collected a second time, ten days after the initial sampling, and were processed in the same manner. The second sample was taken after removal of at least one meter of the silage face to determine if contamination existed deeper within the silage piles, or if contamination was only present in isolated areas. Thirty-six feed samples were collected over this period. Accessible water either from a tank or standing water in close proximity to the tank was collected where clinical cases of listeriosis had been identified. Nine water samples (three from each management site) were obtained by placing a 50 mL screw cap conical test tube into the water source. A scooping motion was used, such that the bottom of the tank or water hole was contacted by the tube to capture any sediment present. Tubes were capped and transported immediately to the laboratory for processing. Water sources were only sampled once. After sampling, water tanks were drained, disinfected, allowed to dry, and refilled. To measure the pH of the feed, collected samples were placed in a 100 mm petri dish then wetted with approximately 10 mL sterile distilled deionized water. A surface pH meter (ExTech Instrument Corp., Nashua, NH) was used to measure the pH of the moistened surface. For each dish of silage, average pH was determined by measuring pH at three separate areas corresponding to 12, 4 and 8 o’clock positions in the petri dish. L. monocytogenes detection and isolation from clinical samples All diagnostic specimens collected during the case investigation were shipped overnight in insulated coolers with icepacks to the UNLVDC. Submitted samples included fresh and fixed fetal tissues (collected and submitted on day 1 and 8) and CV swabs or RFM from the 21 sampled heifers (day 8, 14, and 27). Requested diagnostics for fetal tissues included: aerobic culture and sensitivity, C. fetus culture, Leptospira spp. , BHV1, and BVDV PCR, and histopathology. Fetuses and fetal tissues, including spleen, heart, lung, kidney, and liver were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin, examined histopathologically by board certified pathologists with other ancillary testing at their discretion to determine a diagnosis. Testing at UNLVDC is by standard operating procedures accredited under the American Association of Veterinary Laboratory Diagnosticians (AAVLD). Fetal tissues (lung and liver) and stomach contents were subjected to culture testing. Requested diagnostics on the CV and RFM samples collected included aerobic culture and bacterial identification. Fetal tissue samples were flame sterilized and directly plated on to TSA with 5% sheep’s blood, chocolate agar, Colombia CNA agar with naldixic acid and 5% sheep’s blood (CNA), Campy CVA agar and MacConkey’s agar (Thermo Fisher Scientific, Waltham, MA). Cervico-vaginal swabs, RFM, and fetal stomach contents were directly plated onto the same agar media as the tissues. Remaining samples were macerated (tissues) or agitated (swabs) into Fraser’s Broth (Thermo Fisher Scientific, Waltham, MA). All media except Campy CVA were incubated for 18-24 hours at 37º C with 5% CO 2 supplementation, then examined by trained laboratory technicians following the UNLVDC Standard Operating Procedures. Media without pathogenic bacterial growth were re-incubated and observed following an additional 18-24 hour incubation. Campy CVA agar was incubated 48 hours at 37º C in a GasPak EZ Campy microaerophilic environment container (BD Diagnostics, Sparks, MD). Bacterial colonies with morphology consistent with members of the genus Listeria were sub-cultured onto TSA with 5% sheep’s blood for purity. Sub-cultured, suspect Listeria colonies were subjected to gram staining, catalase testing, and were phenotypically tested using a commercial identification platform using manufacturer’s instructions for Protocol A (Biolog, Omnilog, Hayward, CA). Samples that did not have growth consistent with L. monocytogenes or L. ivanovii on primary isolation media were subjected to PCR testing specific for Listeria hemolysin ( hly ). Nucleic acid was extracted from culture samples in Fraser’s media using a commercial DNA extraction kit (Qiagen, DNA mini kit and QIACube) per manufacturer’s instructions for bacteria. Samples that were negative following PCR testing were placed into cold enrichment (4 ºC) for six weeks. Cultures with a lack of esculin hydrolysis in Fraser media after six weeks of cold enrichment were considered negative for L. monocytogenes . No additional diagnostics were performed on L. monocytogenes negative submissions. L. monocytogenes detection and isolation from feed and water Feed and water samples were screened for the presence of Listeria spp and L. monocytogenes using BAX System Real-Time PCR Assays (Dupont Qualicon, Wilmington, DE) and Atlas Detection assays (Roka Bioscience, Lake Forest Park, WA). Water samples were screened directly and after culture enrichment, while feed samples were only screened after enrichment. For direct screening, 1 mL of each water sample was placed in an Atlas G2 Sample Tube (Roka Bioscience) and subjected to testing. Feed samples were enriched for rapid screening by mixing 50 g feed into 200 mL of Listeria enrichment broth (LEB; Dupont Qualicon, Wilmington, DE) and water samples were enriched by diluting 10 mL of each water sample into 90 mL of LEB. Samples were incubated at 30 °C for 24 h. After incubation, a 1mL portion was placed into an Atlas G2 Sample Tube (Roka Bioscience) and a 20 mL portion was used to prepare a BAX template lysis. All Atlas G2 sample tubes were processed through the RokaBioscience Atlas instrument using the Atlas Listeria LSP Assay and the Atlas LmG2 Assay for Listeria spp and L. monocytogenes respectively. The BAX lysis preparations from each sample were processed through the BAX Q7 instrument using a BAX System Real-Time PCR Assay for L. monocytogenes according to the manufacturer’s instructions. To isolate and confirm L. monocytogenes from the feed and water samples the 24 h LEB enrichments were streaked for isolation onto a Difco Oxford agar plate (Beckton Dickinson and Co., Franklin Lakes, NJ) and a CHROMagar Listeria plate (DRG International, Inc., Springfield, NJ) using a sterile cotton swab and inoculating loop. Plates were incubated at 37 °C overnight then viewed for suspect colony phenotypes; black colonies on Oxford agar for Listeria spp. and blue colonies without and with halos on chromogenic agar for Listeria spp. and L. monocytogenes respectively, were targeted. Suspect colonies were selected and placed into a 96-well block containing 1 mL per well of Fraser media containing 5% ferric ammonium citrate, and then incubated at 37 °C overnight. The 24 h LEB enrichments of feed and water were further incubated for another 24 h (48 h total) and the above streaking for isolation onto chromogenic Listeria and Oxford agar was repeated. A secondary 48 h enrichment in Fraser media was incubated an additional 48 h at 30 °C and was streaked for isolation, incubated and viewed for suspect colonies as described above. Suspect colonies were confirmed to the species level using a Listeria spp. specific PCR and biochemical tests. Listeria species monocytogenes, innocua, grayi, ivanovii, seeligeri and welshimeri were identified through Listeria spp. multiplex PCR [36]. The isolates that were identified as L. monocytogenes were further characterized using the serovar multiplex PCR [21]. Suspect isolates that were found to possess the phosphoribosyl pyrophosphate synthetase ( prs ) gene, indicative of all Listeria spp., but which could not be identified through PCR were further identified using biochemical tests. Each suspect Listeria was streaked for isolation on tryptic soy agar containing 0.6% yeast extract, incubated at 37 °C overnight and then processed using a Remel Micro-ID Listeria Kit (Thermo Fisher Scientific, Lenexa, KS) according to the manufacturer’s protocol. L. monocytogenes strains selected for sequencing A total of 31 L. monocytogenes strains were selected for whole-genome DNA sequencing on a MiSeq instrument (Illumina, San Diego, CA). Of those, 19 clinical strains were isolated from USMARC cattle aborted fetuses, placentas, or uterine swabs. Another eight strains were isolated from four different corn silage samples, three water tanks and one standing water source. Additionally, four other strains from bovine abortions were obtained from the UNLVDC that originated from other regions in Nebraska for use as controls and references DNA preparation and Illumina MiSeq whole-genome sequencing All 31 L. monocytogenes strains were passaged twice from -80 °C frozen stocks on chocolate agar plates (Hardy Diagnostics, Santa Maria, CA) at 37 °C. A single colony of each isolate was then inoculated in 1.5 mL of Brain-Heart Infusion (BHI) broth and grown overnight without shaking at 37 °C. Genomic DNA was extracted from the cultures using MO BIO microbial DNA isolation kits (MO BIO Laboratories, Carlsbad, CA) according to the manufacturer’s instructions. The extracted DNAs were quantified and checked for purity using 260/280 absorbance readings on a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). Individual libraries were constructed for each of the strain DNA preparations using Illumina Nextera XT DNA sample preparation kits with appropriate indices tags according to the manufacturer’s instructions (Illumina Inc., San Diego, CA). The libraries were pooled together and run on an Illumina MiSeq DNA sequencer (Illumina Inc., San Diego, CA). The genome of each strain was sequenced to a minimal depth of 10X coverage. Assembly of L. monocytogenes chromosomes and phylogenetic trees Adapter sequence and low-quality bases were trimmed using Trimmomatic [37]. Trimmed fastq reads were assembled using SKESA. The L. monocytogenes assembled chromosomes from 27 unique USMARC strains and 4 clinical strains from the UNLVDC were imported into parsnp [38] for genome alignments and subsequent identification of core-genome SNPs and construction of a preliminary phylogenetic tree. The strains grouped into six initial clusters. One strain from each cluster was selected for PacBio sequencing to obtain a complete closed chromosome representative of each cluster (described below). Illumina reads from strains not also sequenced with PacBio were assembled using SKESA. The SKESA, PacBio, and 20 closed L. monocytogenes chromosomes from GenBank [39] were then used in parsnp to create a new phylogenetic tree. The 20 GenBank chromosomes represented all four known L. monocytogenes lineages and were utilized to calibrate the final phylogenetic tree. Evolview was used to populate the tree with phenotypic metadata [40]. DNA preparation and PacBio whole-genome sequencing library construction High molecular weight DNA was extracted from L. monocytogenes cultures using Qiagen Genomic-tip 100/G columns and a modified manufacturer’s protocol as previously described [41] with the addition of mutanolysin with the proteinase K step followed by incubation at 50 °C for 1 h. Ten micrograms of DNA were sheared to a targeted size of 20 kb using a g-TUBE (Corvaris, Woburn, MA) and concentrated using 0.45X volume of AMPure PB magnetic beads (Pacific Biosciences, Menlo Park, CA) following the manufacturer’s protocol. Sequencing libraries were created using 5 µg of sheared, concentrated DNA and the PacBio DNA Template Prep Kit 2.0 (3Kb - 10Kb) according to the manufacturer’s protocol. The library was bound with polymerase P5 followed by sequencing on a Pacific BioSciences (PacBio) RS II sequencing platform with chemistry C3 and the 120 min data collection protocol. PacBio Sequence Assembly into Closed Circularized Genomes PacBio reads were assembled using HGAP3 (SMRTanalysis Version 2.1) and the resulting contigs were imported into Geneious. Within Geneious, overlapping sequence on the ends of the contigs were removed from the 5’ and 3’ ends to generate a circularized chromosome. The chromosome was reoriented to start with a putative origin of replication with Ori-Finder [42]. The chromosome was initially polished for accuracy using the Resequencing 1.0 protocol in SMRTanalysis by mapping corrected PacBio reads to the chromosome. To correct PacBio sequencing errors (homopolymers and SNPs), Illumina reads were mapped to the initially polished chromosome using Pilon. Then, both PacBio and Illumina reads were mapped to the Pilon-generated chromosome using Geneious Mapper. Additional sequencing errors were identified and corrected by manual editing in Geneious, resulting in a finished closed circularized chromosome. Chromosome sequences were deposited into NCBI (Additional file 2) and annotated using the Prokaryotic Genome Annotation Pipeline version 4.5. MLST 2.0 [43] was used to determine the MLST allelic profiles for the L. monocytogenes strains from USMARC and UNLVDC. MLST allelic profiles were then used with the Listeria Pasteur MLST database to determine serotype, lineage and clonal complex [21, 22]. Abbreviations PFGE: Pulse-field gel electrophoresis CV: cervico-vaginal RFM: retained fetal membranes USMARC: United States Meat Animal Research Center NAHMS: National Animal Health Monitoring System UNLVDC: University of Nebraska, Lincoln, Veterinary Diagnostic Center SKESA: Strategic Kmer Extension for Scrupulous Assemblies NCBI: National Center for Biotechnology Information MLST: multi-locus sequence typing SNPs: single nucleotide polymorphisms Declarations Ethics approval and consent to participate This study was approved by the author’s institution (University of Nebraska IACUC, #1383) and owner consent was verbally confirmed as part of routine veterinary diagnostic sampling for an outbreak investigation. Consent for publication Not applicable Availability of data and material The Illumina sequencing reads for the 31 isolates sequenced on that platform have all been placed in the NCBI sequence read archive (SRA) for public availability. Additionally, the complete genomes of all six isolates sequenced and assembled with PacBio and Illumina sequences are publicly available in GenBank (CP032668-CP-32673). Competing interests The authors declare that they have no competing interests. Funding The authors would like to thank the US Meat Animal Research Center for providing financial support for sample submission to the veterinary diagnostic laboratory and for machine use and material costs associated with genomic sequencing. The authors would also like to thank the University of Nebraska Great Plains Veterinary Educational Center for financial support of material costs during sample collection, labor for case management, and cost of manuscript submission. Funding sources did not have any involvement in the study design, data analysis and interpretation, or writing of the manuscript. Author’s contributions Clinical sample collection and submission were performed by KJW and JDO. KJW was responsible for clinical case management, writing, and revision of the manuscript. JDL was responsible for clinical case Listeria diagnostics and providing Listeria controls. Feed and water sample collection and Listeria diagnostics on those samples was completed by JMB and TMA. Genomic analysis and manuscript revisions were provided by JLB and MLC. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank Sandy Fryda-Bradley, Gennie Schuller, and Karen Schuck for excellent technical assistance. The authors also thank the USMARC core sequencing facility, USMARC cattle operations, the UNL GPVEC for technical support and the UNL Veterinary Diagnostic Center. The mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply approval to the exclusion of other products that might be suitable. The USDA is an equal opportunity employer and provider. 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Isolation and identification of Listeria monocytogenes in vegetable byproduct silages containing preservative additives and destined for animal feeding. Anim Feed Sci Tech. 1990;31:285-291. Doumith M, Buchrieser C, Glaser P, Jacquet C, Martin P. Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. J Clin Microbiol. 2004;42:3819-3822. Graves LM, Hunter SB, Ong AR, Schoonmaker-Bopp D, Hise K, Kornstein L, DeWitt WE, Hayes PS, Dunne E, Mead P, Swaminathan B. Microbiological aspects of the investigation that traced the 1998 outbreak of listeriosis in the United States to contaminated hot dogs and establishment of molecular subtyping-based surveillance for Listeria monocytogenes in the PulseNet network. J Clin Microbiol. 2005;43:2350-2355. Farber JM, Peterkin PI. Listeria monocytogenes , a food-borne pathogen. Microbiol Rev.1991;55:476-511. Rowan NJ, MacGregor SJ, Anderson JG, Fouracre RA, McIlvaney L, Farish O. Pulsed-light inactivation of food-related microorganisms. Appl Environ Microbiol. 1999;65:1312-1315. Oloketuyi SF, Khan F. Inhibition strategies of Listeria monocytogenes biofilms-current knowledge and future outlooks. J Basic Microbiol. 2017;57:728-743. Hingston P, Chen J, Dhillon BK, Laing C, Bertelli C, Gannon V, Tasara T, Allen K, Brinkman FS, Truelstrup Hansen L, Wang S. Genotypes associated with Listeria monocytogenes isolates displaying impaired or enhanced tolerances to cold, salt, acid, or desiccation stress. Front Microbiol. 2017;8:369. Bubert A, Hein I, Rauch M, Lehner A, Yoon B, Goebel W, Wagner M. Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR. Appl Environ Microbiol. 1990;65:4688-4692. Bolger AM, Lohse M, Usadel B. Trimmomatic: flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114-2120. Treangen TJ, Ondov BD, Koren S, Phillippy AM. The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes. Genome Biol. 2014;15:524. Kuenne C, Billion A, Mraheil MA, Strittmatter A, Daniel R, Goesmann A, Barbuddhe S, Hain T, Chakraborty T. Reassessment of the Listeria monocytogenes pan-genome reveals dynamic integration hotspots and mobile genetic elements as major components of the accessory genome. BMC Genomics. 2013;14:47. He Z, Zhang H, Gao S, Lercher MJ, Chen WH, Hu S. Evolview v2: an online visualization and management tool for customized and annotated phylogenetic trees. Nucleic Acids Res. 2016;44:W236-241. Clawson ML, Keen JE, Smith TP, Durso LM, McDaneld TG, Mandrell RE, Davis MA, Bono JL. Phylogenetic classification of Escherichia coli O157:H7 strains of human and bovine origin using a novel set of nucleotide polymorphisms. Genome Biol. 2009;10:R56. Gao F, Zhang CT. Ori-Finder: a web-based system for finding oriC s in unannotated bacterial genomes. BMC bioinformatics. 2008;9:79. Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, Jelsbak L, Sicheritz-Ponten T, Ussery DW, Aarestrup FM, Lund O. Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol. 2012;50:1355-1361. Tables Table 1 (Page 6, Line 114) Descriptive statistics relative to the female population over the calving season. Category Total Population Season Abortion Count Season Abortion % Day 1-12 Abortion Count % Total Abortions Occurring Day 1-12 Mature Cows 3633 4 0.1% 0 0 Heifers 936 28 3.0% 24 89% Total 4569 31 0.7% 24 77% Site A 369 17 4.6% 16 94% Site B 283 3 1.1% 3 100% Site C 284 8 2.8% 5 71% Table 2 (Page 7, Line 124) Descriptive statistics of clinical submissions. Sample ID Sample Type Site Location Aerobic Culture LM PCR LM Culture 1 F A + 2 F B + 3 S A - - - 4 P A + 5 S A + 6 S A - - - 7 S A + 8 S A + 9 P A - + 10 S A - - - 11 P C + 12 P C + 13 P A + 14 P A + 15 P A + 16 P A + 17 P A + 18 S A - - - 19 S C - + 20 S A + 21 S C + 22 P C + 23 P A + LM= L. monocytogenes , F=fetal tissues, P=retained fetal membranes, S=cervico-vaginal (CV) swabs Table 3 (Page 7, Line 127) Summary of L. monocytogenes water samples. Sample Site Direct LM PCR Enriched LM PCR Enriched LM Culture Tank 1 A + + + Tank 2 A - + + Tank 3 B - + + Tank 4 C - - - Hole 5* A - + - Hole 6 B - + + Hole 7 B - - - Hole 8 C - - - Hole 9 C - - - *Culture positive for L. innocua -not sequenced. Table 4 (Page 7, Line 133) Summary of L. monocytogenes positive feed samples. Sample timing Pile location pH LM culture LM PCR 1st 1 4.54 + + 1st 10 8.09 + + 2nd 1 7.09 + + 2nd 5 4.34 + + Sample timing: 2 nd sample collected 10 days after 1 st sample. All positive samples were from corn silage. Pile location is in reference to the position on the silage face (Additional file 1). Additional Files Additional file 1 .docx Sampling scheme of corn silage and earlage piles Image of silage face with grid overlay. Eight samples (ovals; 1-8) were collected from each pile according the gridded lines. Loose surface materials (9-10) and the drainage pipe (11) were also collected. Additional file 2 .xlsx L. monocytogenes strains used in the project with corresponding genotypes, sequencing information and sample type. Additional file 3 .pptx Gingr visualization of the genomes of lineage III L. monocytogenes strains from cattle, water or feed associated with the outbreak. The genomes were aligned with parsnp. The 19 strains in this figure comprised the monophyletic clade in Figure 2. The outer taxonomic units in the phylogenetic tree are aligned with their corresponding row in the alignment. Each row represents the entire genome for a strain. Vertical purple lines in the alignment indicate base differences. Asterisks are to the right of the six strains whose core genomes are identical with their names highlighted according to sample type; cattle (green), water (blue) or feed (red). Supplementary Files Additionalfile1Silagesamplingschematic.pdf Additionalfile3LM..pptx Additionalfile2Tableisolatesv1edits.xlsx Cite Share Download PDF Status: Published Journal Publication published 22 Feb, 2020 Read the published version in BMC Veterinary Research → Version 3 posted Submission checks completed at journal 05 Feb, 2020 Editorial decision: Accept 05 Feb, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4291","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":333422,"identity":"1f19db33-3f98-4bf4-9523-b7e8fdbac44e","order_by":1,"name":"Katherine Whitman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYDACCcYGEGXA2A6iDSyI0tIIVsvYcwBESRCjhQFsjQGDRAKESxAY3G5uf3Sj5o4x88znVzf8KJBg4G/vTsCv5c7BxuacY8/MGGfnlN3sATpM4szZDXi1mN1IBGphO2wD1JJ2gweoxUAilxgt/4BaZp5Ju/mHaC25bYfNGGewH7tNlC32QC2zc/sOGzP25LDdljGQ4CHoF8kZ6Q8+53w7bLix/fizm2/+2Mjxt/fi1wIHhg08BiCahzjlICDPwP6AeNWjYBSMglEwogAAq31PKatRC1AAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-5156-5288","institution":"University of Nebraska","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Katherine","middleName":"","lastName":"Whitman","suffix":""},{"id":333423,"identity":"598c082e-07a4-47b5-b6bf-cf462786afe0","order_by":2,"name":"James L. 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Ondrak","email":"","orcid":"","institution":"USDA-ARS Roman L Hruska US Meat Animal Research Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jeff","middleName":"D.","lastName":"Ondrak","suffix":""}],"badges":[],"createdAt":"2019-08-22 13:46:22","currentVersionCode":3,"declarations":"","doi":"10.21203/rs.2.13582/v3","doiUrl":"https://doi.org/10.21203/rs.2.13582/v3","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-020-2276-z","type":"published","date":"2020-02-22T20:36:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":454962,"identity":"77a7ebea-55ab-4fef-bcf8-752e59c75928","added_by":"auto","created_at":"2020-02-05 22:44:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25633,"visible":true,"origin":"","legend":"Layout of operation and feed pathway from shared feed location.","description":"","filename":"1.PNG","url":"https://assets-eu.researchsquare.com/files/81790fec-d07c-4260-9284-304050e92725/v3/1.PNG"},{"id":454964,"identity":"86b86275-5ed9-43c8-9f04-a501ed791d5a","added_by":"auto","created_at":"2020-02-05 22:44:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":69549,"visible":true,"origin":"","legend":"Parsnp generated tree of complete closed or draft genomes of L. monocytogenes. \n(See attached .pptx file: “Figure2_LM_manuscript_tree_final2”)","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/81790fec-d07c-4260-9284-304050e92725/v3/Figure 2.png"},{"id":15666083,"identity":"a314ada2-0c5d-4504-8b88-e56b49000087","added_by":"auto","created_at":"2021-11-18 13:35:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":570212,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4291/v3/661daad0-d70b-4802-8528-1653b69e67e6.pdf"},{"id":454966,"identity":"31a153ff-dda4-48d2-adc3-48014cc862c4","added_by":"auto","created_at":"2020-02-05 22:44:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":279109,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1Silagesamplingschematic.pdf","url":"https://assets-eu.researchsquare.com/files/81790fec-d07c-4260-9284-304050e92725/v3/Additional file 1-Silage sampling schematic.pdf"},{"id":454965,"identity":"ddf8221a-2550-4957-9642-3acf6ede79af","added_by":"auto","created_at":"2020-02-05 22:44:25","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":76887,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3LM..pptx","url":"https://assets-eu.researchsquare.com/files/81790fec-d07c-4260-9284-304050e92725/v3/Additional_file_3_LM..pptx"},{"id":454963,"identity":"e8dbbeef-eb50-4b55-a44b-169188f24191","added_by":"auto","created_at":"2020-02-05 22:44:24","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13947,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2Tableisolatesv1edits.xlsx","url":"https://assets-eu.researchsquare.com/files/81790fec-d07c-4260-9284-304050e92725/v3/Additional file 2_Table_isolatesv1-edits.xlsx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eGenomic-based identification of environmental and clinical \u003cem\u003eListeria monocytogenes\u003c/em\u003e strains associated with an abortion outbreak in beef heifers\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cem\u003eListeria monocytogenes\u003c/em\u003e is a well-known saprophytic bacterial pathogen that is ubiquitous in the cattle production environment. Cattle and many other mammals including humans, can be asymptomatic shedders. However, \u003cem\u003eL. monocytogenes\u003c/em\u003e can cause a number of diseases across species, including visceral listeriosis, neurologic listeriosis, and importantly, reproductive listeriosis, which is implicated in late-term abortion in cattle.\u0026nbsp; Certain populations of cattle, particularly those who are immunocompromised, pregnant, young, old, or stressed are more affected by environmental risk factors associated with \u003cem\u003eL. monocytogenes\u003c/em\u003e infection [1-3].\u003c/p\u003e\n\u003cp\u003eThe potential routes of infection leading to clinical listeriosis in cattle is either ingestion, inhalation, direct contact or oral mucosal lesions [4-7].\u0026nbsp; The primary route in cattle is suspected to be the consumption of contaminated feed or water and subsequent passage through the gastrointestinal tract, a pathway that has been demonstrated in goats and sheep [8, 9]. Once in the gastrointestinal tract, \u003cem\u003eL. monocytogenes \u003c/em\u003eis able to bind, penetrate and move within and between the epithelial cells [10]. Translocation of \u003cem\u003eL. monocytogenes\u003c/em\u003e occurs from the intestines to the liver and hepatocytes via macrophages, leading to a bacteremia that results in either an effective cell-mediated immune response, neurological disease, or reproductive infection [11].\u003c/p\u003e\n\u003cp\u003eAcross mammalian species, the incubation period from infection to clinical signs of listeriosis is variable, generally 2-6 weeks [12]. Consequently, the delayed onset of clinical signs following infection makes identification of a potentially contaminated feed source challenging, in that the feed may have been entirely consumed by the time clinical listeriosis is observed. Even if \u003cem\u003eL. monocytogenes\u003c/em\u003e is detected in available feed, the many available molecular diagnostic techniques [13] may not be specific enough to directly link the source strain of \u003cem\u003eL. monocytogenes\u003c/em\u003e to the clinical strain due to its pervasiveness in the environment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e L. monocytogenes\u003c/em\u003e diversity has been assessed using various methods. Historically, serotyping [14] and pulsed-field gel electrophoresis (PFGE) [15, 16] have been implemented in source origin identification of strains in human and cattle listeriosis outbreaks. However, these diagnostics do not definitively establish genetic relationships between strains. Genomic sequencing has advantages over traditional diagnostics in that it interrogates the complete genome and can establish a more definitive relationship between isolated strains. In situations where \u003cem\u003eL. monocytogenes\u003c/em\u003e can be established as an etiology for clinical disease as well as an environmental contaminant, utilizing genomic sequencing can verify that a specific strain found in the environment is responsible for clinical disease.\u003c/p\u003e\n\u003cp\u003eThere are four genetically distinct lineages of \u003cem\u003eL. monocytogenes \u003c/em\u003e(I-IV) that have been found across mammalian species and in the environment [17, 18]. \u003cem\u003eL. monocytogenes\u003c/em\u003e strains can be classified into one of four lineages based on a number of techniques including: pulse-field gel electrophoresis, ribotyping [14, 19], multi-locus enzyme electrophoresis, multilocus sequencing typing and more recently whole genome sequencing. Of the four lineages, strains from lineage III are often isolated from cattle, and lineage IV is currently exclusive to ruminants. Lineages I through III have been documented in clinical listeriosis in cattle and other mammals [17].\u003c/p\u003e\n\u003cp\u003eIn this report, an outbreak investigation of \u003cem\u003eL. monocytogenes \u003c/em\u003eabortion in a beef cattle operation is described. Aborted bovine fetuses, cervico-vaginal (CV) swabs, and retained fetal membranes (RFM), if present, from aborting females, as well as their water and feed sources were sampled and cultured for \u003cem\u003eL. monocytogenes\u003c/em\u003e. To establish a definitive source of the abortion outbreak, all \u003cem\u003eL. monocytogenes\u003c/em\u003e culture positive strains were subjected to whole-genome sequencing, phylogenetic analyses, and lineage classification.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCattle \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn abortion outbreak occurred at the United States Meat Animal Research Center (USMARC) in late winter/early spring of 2014. A total of 28 heifers aborted over a 36-day period, with 24 abortions occurring in a 12-day period. By comparison, four mature cows aborted during the calving season, none within the acute abortion outbreak timespan and none were positive for \u003cem\u003eL. monocytogenes\u003c/em\u003e. Three heifer management sites (A-C) were affected, and all were managed similarly, fed from the same silage source, and from the same feed truck (Figure 1).\u0026nbsp; Two- and three-year old females were never grouped with mature cows, but rather rotated through pastures, occupying sites where cows had resided approximately seven months previous. Females were aborting late term (3\u003csup\u003erd\u003c/sup\u003e trimester) fetuses, with no other observed clinical signs, except some with blood on the perineum or RFM, and some groups had refused feed as recently as the day prior to the first abortion.\u0026nbsp; Descriptive statistics of abortions during the calving season are listed in Table 1. The mature cow known abortion percentage was 0.1%, but was at 3% for the heifer population, much higher than spring calving herd reports by the National Animal Health Monitoring System (NAHMS) [20]. The majority of abortions occurred within a 12-day time period, prompting the outbreak investigation.\u003c/p\u003e\n\u003cp\u003eDuring the outbreak, two aborted bovine fetuses were submitted to the University of Nebraska Veterinary Diagnostic Center, Lincoln, NE (UNLVDC), for aerobic culture, \u003cem\u003eC. fetus\u003c/em\u003e culture, \u003cem\u003eLeptospira spp.\u003c/em\u003e, Bovine herpesvirus 1 (BHV1), bovine viral diarrhea virus (BVDV) PCR, and histopathology. Cervico-vaginal swabs or RFM from 21 of the 28 aborting heifers were also collected on follow-up sampling and submitted to the UNLVDC for aerobic culture, followed by \u003cem\u003eL. monocytogenes\u003c/em\u003e culture and/or \u003cem\u003eL. monocytogenes\u003c/em\u003e PCR, if needed. Both fetuses and 17/21 heifer samples were positive for \u003cem\u003eL. monocytogenes, \u003c/em\u003especifically 11/11 RFM and 6/10 CV swabs. Fetal tissues were examined by board certified pathologists as part of the abortion diagnostic investigation.\u0026nbsp; Fetus 1 had liver lesions consistent with listeriosis and included scattered foci of acute necrosis with moderate neutrophilic inflammatory response.\u0026nbsp; Fetus 2 had marked autolysis within the liver with presence of few mononuclear cells in the hepatic capsule.\u0026nbsp; Both pathologists diagnosed listeria as the cause of abortion. Fetuses were negative to all other diagnostics, including PCR for \u003cem\u003eLeptospira\u003c/em\u003e spp., BHV1 and BVDV, and culture for \u003cem\u003eCampylobacter\u003c/em\u003e sp. No other diagnostics were performed on the RFM or CV samples. Descriptive statistics of the sample submissions and results are summarized in Table 2.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFeed and Water Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNine water sources were tested, and four were culture positive for \u003cem\u003eL. monocytogenes\u003c/em\u003e (Table 3). Site A had two water tanks positive for \u003cem\u003eL. monocytogenes\u003c/em\u003e while site B contained one positive water tank and one positive water hole by the feed bunk. Water tanks associated with site C were sampled, but no \u003cem\u003eL. monocytogenes\u003c/em\u003e was isolated.\u0026nbsp; Of the 15 silage samples tested, \u003cem\u003eListeria\u003c/em\u003e spp. were detected in 14 samples, of which four samples were culture positive for \u003cem\u003eL. monocytogenes\u003c/em\u003e. Of those positive for \u003cem\u003eL. monocytogenes\u003c/em\u003e, three strains were derived from feeds that had a pH less than 5.0. Feed sample timing, type, location, pH, and test results are summarized in Table 4. Sample locations of the pile face correspond to Additional file 1.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty one strains were subjected to short read Illumina sequencing in this study, of which 27 were part of the USMARC outbreak and four were available from the\u0026nbsp; UNLVDC (see Additional file 2 for strain information). All 31 strains passed post-sequencing processing for low quality reads and adapter sequence. The lowest read coverage was 12.8X while the highest coverage was 132X with the average being 58X.\u0026nbsp; Parsnp was used to create an initial phylogenetic tree based on Strategic Kmer Extension for Scrupulous Assemblies (SKESA) assembled chromosomes of the 31 sequenced strains. The tree was divided into three major lineages with six minor clusters. One strain from each cluster of the initial tree was also sequenced using long-read PacBio sequencing to generate reference complete closed genomes for each cluster.\u003c/p\u003e\n\u003cp\u003eThe four major \u003cem\u003eL. monocytogenes\u003c/em\u003e lineages were reproduced in a maximum-likelihood tree calibrated with 20 \u003cem\u003eL. monocytogenes\u003c/em\u003e chromosomes of known lineage and serotype affiliations that were available from the National Center for Biotechnology Information (NCBI, Additional file 2, Figure 2). The tree also contained 25 SKESA assembled \u003cem\u003eL. monocytogenes\u003c/em\u003e chromosomes and six closed PacBio generated chromosomes from this project that were assigned to lineages based on their placement in the tree.\u0026nbsp; Lineage I contained seven NCBI genomes representing serotypes 1/2b, 3b, 4b, 4d, 4e, and 7, and eight clinical strains from cattle located in two different USMARC management sites (A and C) and one UNLVDC strain.\u0026nbsp; Seven of the lineage I clinical \u003cem\u003eL. monocytogenes\u003c/em\u003e strains molecularly serotyped as 1/2b based on their \u003cem\u003ein silico\u003c/em\u003e multi-locus sequence typing (MLST) patterns, and one molecularly serotyped as 4b.\u003c/p\u003e\n\u003cp\u003eLineage II contained seven genomes from NCBI that represented serotypes 1/2a, 1/2c, 3a and 3c plus one UNLVDC strain that molecularly serotyped as 1/2a (Additional file 2, Figure 2). No lineage II strains were isolated from any of the three cattle management sites, feed, or water. Lineage III contained two NCBI genomes that represented serotypes 4a and 4c, and two UNLVDC strains that could not be molecularly serotyped because there were no matching MLST allele patterns in the Listeria Pasteur MLST or Center for Genomic Epidemiology databases [21, 22].\u0026nbsp; Lineage III also contained a monophyletic cluster of eight environmental strains and eleven clinical strains that collectively originated from feed and water or cattle at all three sites, respectively (Additional file 2, Figure 2).\u0026nbsp; These strains also could not be molecularly serotyped based on their MLST allele patterns.\u003c/p\u003e\n\u003cp\u003eWhile all clinical strains from the USMARC abortion outbreak placed in either lineages I or III, the feed and water strains only placed in lineage III. Of the eight USMARC clinical strains that placed in lineage I seven grouped with a UNLVDC strain and were either identical or differed from each other by no more than six single nucleotide polymorphisms (SNPs). In contrast, the UNLVDC strain differed from the seven clinical strains by more than 60 SNPs. These seven clinical strains were all isolated from cattle at site A and molecularly serotyped as 1/2b. The other clinical strain in lineage I was isolated from a heifer at site C and was more closely related to NCBI genome NC_018642, a 4b serotype isolated from cheese [23]. Consequently, lineage I contained two genetically distinct \u003cem\u003eL. monocytogenes\u003c/em\u003e strains that originated from clinical cases in two different management sites with no apparent connection to strains isolated from feed and water samples.\u003c/p\u003e\n\u003cp\u003eAll 11 clinical strains that placed in lineage III were either identical or no more than eight SNPs different from the lineage III strains recovered from the silage pile. Additionally, two clinical, two water and two corn silage strains were identical across their core genome, which represented 99% of a closed, circular, PacBio-generated reference genome for isolate 52330 (Additional file 3). Consequently, all lineage III strains from the outbreak were either completely clonal, or only differed by a few SNP alleles.\u0026nbsp; These results indicate that there were three genetically distinct \u003cem\u003eL. monocytogenes\u003c/em\u003e strains responsible for the cattle abortion outbreak, with two that placed in lineage I and one that placed in lineage III.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough an important diagnostic sample, only two aborted fetuses were recovered by cattle managers for \u003cem\u003eL. monocytogenes\u003c/em\u003e testing in this outbreak. Consequently, alternative sampling was needed to confirm the etiology of the abortions and to quickly and efficiently implement appropriate management changes. To that end, CV swabs or RFM from females that aborted proved to be valuable diagnostic samples in this outbreak when fetuses were unavailable. An etiologic diagnosis was achieved in 81% (17/21) of all examined clinical cases, with 100% (11/11) of the retained placental samples and 60% (6/10) of the CV swabs positive for \u003cem\u003eL. monocytogenes \u003c/em\u003e(Refer to Table 2). This high rate of diagnostic success was achieved despite sampling being delayed up to seven days from the time of abortion.\u0026nbsp; Additionally, 17/19 of the overall positive cultures (including two fetal tissue submissions) were isolated from primary aerobic culture and did not require additional enrichment. This is important as cold enrichment of \u003cem\u003eL. monocytogenes \u003c/em\u003ecan take weeks, further delaying the time to a diagnosis. \u003c/p\u003e\n\u003cp\u003eDiagnostic investigation indicated the causative agent for the abortion outbreak was \u003cem\u003eL. monocytogenes\u003c/em\u003e, however the source of the organism(s) was not immediately clear. A positive \u003cem\u003eL. monocytogenes\u003c/em\u003e result from a day one fetal tissue sample submission was not reported until day eight of the outbreak, with 19 abortions occurring within that week. Mitigating the abortion outbreak was critical for the remaining pregnant females in the herd. Previous literature indicated that poorly ensiled feed was associated with \u003cem\u003eL. monocytogenes \u003c/em\u003econtamination [24-28]. Because of this, focus was placed on the supplemental silage as a potential source, and feeding was discontinued on day eight until results from the remaining case samples and environmental testing were completed. Abortions cases ceased four days after removing suspected silage from the ration, with the exception of one occurring 19 days later. The reason for the abrupt halt to the abortions following silage removal is unclear. The incubation period for \u003cem\u003eL. monocytogenes\u003c/em\u003e would suggest that abortions should have continued for a longer time if exposure occurred and the dose was sufficient. Immediately after day one fetal tissue sample submission results were confirmed positive for \u003cem\u003eL. monocytogenes\u003c/em\u003e, a novel and organized approach to feed and water sampling was employed, resulting in more effective and efficient collection of environmental samples and subsequent source identification.\u003c/p\u003e\n\u003cp\u003eBoth silage and water samples were culture positive for \u003cem\u003eL. monocytogenes\u003c/em\u003e. Of the four positive water samples, three required enrichment to facilitate growth, indicating a smaller bacterial load in these sources. Therefore, we suspected early on in the investigation that the source of \u003cem\u003eL. monocytogenes\u003c/em\u003e in the water was likely from heifers transferring feed into the water soon after consuming affected silage. Interestingly, some feed samples positive for \u003cem\u003eL. monocytogenes\u003c/em\u003e were at a pH of less than 5.0, which should have been lethal to the bacteria. This finding is supportive of other research indicating that \u003cem\u003eL. monocytogenes\u003c/em\u003e can survive at a pH of less than 5.0 [26, 29], bringing the traditional acceptable pH for appropriately ensiled feeds into question.\u003c/p\u003e\n\u003cp\u003eWhile clinical and laboratory findings were supportive of \u003cem\u003eL. monocytogenes\u003c/em\u003e in the environment as the likely etiology for the abortions, we wanted to determine if a genetic relationship between the isolates from the environment and clinical samples existed and if the feed was the source of \u003cem\u003eL. monocytogenes\u003c/em\u003e. Disposing of several hundred tons of silage had significant economic and resource implications, but herd health and welfare related to feeding contaminated silage was a major concern, and the cattle could be exposed to other risk factors associated with \u003cem\u003eL. monocytogenes\u003c/em\u003e infection, such as oral-fecal transmission and the ubiquitous nature of the bacteria. Proving that the silage was the source of the outbreak, and not another cause would further strengthen the decision to dispose of all contaminated silage.\u003c/p\u003e\n\u003cp\u003eTo our knowledge, full genomic sequencing of \u003cem\u003eL. monocytogenes\u003c/em\u003e has not been previously used in a bovine abortion outbreak investigation. Using this technique would give the highest resolution for determining the source of the outbreak. Two silage, two water, and two clinical \u003cem\u003eL. monocytogenes\u003c/em\u003e strains were identical to each other, and nearly identical to all remaining silage (n=2) and water (n=2) strains, and clinical strains (n=9). Thus, the same lineage III genetic strain type was found in silage, water, and clinical case samples. The direction of the feed delivery pathway (Figure 1) would not have allowed for water or cattle to contact the original silage pile. This implicated the silage as the most likely source of the pathogen and the cause of abortion in at least 11 heifers.\u003c/p\u003e\n\u003cp\u003eThe remaining eight \u003cem\u003eL. monocytogenes\u003c/em\u003e positive clinical strains consisted of two genetically distinct lineage I strain types.\u0026nbsp; Females infected with either of the two lineage I strain types were kept with other heifers that were infected with the distinct lineage III strain type that traced back to the silage. Of note, when silage feeding was discontinued, all abortions but one ceased four days later. Thus, it is possible that all three genetically distinct strains implicated in the outbreak could have been in the feed, with two undetected. Importantly, identification of seven lineage I clinical \u003cem\u003eL. monocytogenes\u003c/em\u003e strains as sertotype 1/2b provided risk information for cattle handlers. Serotypes 1/2b and 4b, along with serotype 1/2a, are the main serotypes that cause of human disease and represents 90-95% of cases [30-32].\u0026nbsp; Consequently, personnel working with cattle from the outbreak were at an increased risk to become infected with \u003cem\u003eL.\u003c/em\u003e \u003cem\u003emonocytogenes\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eBecause \u003cem\u003eL. monocytogenes\u003c/em\u003e was isolated from the silage at different sampling times and at different depths, the integrity of the entire silage pile was called into question, and therefore it was decided that the feed was unsuitable for further use. The remaining affected silage pile was spread onto pastures that would not be populated for several months, so that UV light and desiccation could synergistically eradicate the remaining \u003cem\u003eL. monocytogenes \u003c/em\u003e[33-35]. In addition to routine cleaning of the water tanks, recommendations for future silage management were made including:\u0026nbsp; appropriate packing of newly harvested silage to ensure an anaerobic environment within the pile, vermin mitigation, surface protection and routine silage pile sampling throughout the subsequent feeding season to monitor pH and \u003cem\u003eL. monocytogenes\u003c/em\u003e presence. The silage sampling scheme would follow the protocol used in the investigation, with suggested 2-4 week sampling intervals.\u003c/p\u003e\n\u003cp\u003eManagement strategies to prevent another abortion outbreak due to \u003cem\u003eL. monocytogenes\u003c/em\u003e appeared to be effective in subsequent production years. Following the acute outbreak, all additional aborted fetal submissions screened for \u003cem\u003eL. monocytogenes\u003c/em\u003e in the 2014 calving season and following seasons did not detect or isolate \u003cem\u003eL. monocytogenes\u003c/em\u003e. Additionally, no clinical listeriosis cases of any kind in the cowherd were observed during that time. No negative sequelae to reproductive performance was observed in the recovered animals.\u0026nbsp; Of the 17 \u003cem\u003eL. monocytogenes\u003c/em\u003e positive females, five were culled prior to the 2014 breeding season, and the remaining 12 were diagnosed pregnant (100%) by ultrasound in the fall of the same year. In the 2015 calving season, those heifers all carried a term calf. Eleven of 12 calves were born alive, with dystocia as the documented cause of the only calf death loss, resulting in a 91.6% calving percentage for those females exposed.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis case investigation resulted in a very complete epidemiologic picture of an \u003cem\u003eL. monocytogenes\u003c/em\u003e abortion outbreak in beef cattle.\u0026nbsp; \u003cem\u003eL. monocytogenes \u003c/em\u003ewas quickly identified as the cause through strategic sampling of affected cattle and their environment, with contaminated silage initially implicated as the probable source.\u0026nbsp; The outbreak was brought under control through the elimination of the contaminated silage.\u0026nbsp; Subsequent whole genome sequencing showed that three strains were involved in the outbreak and confirmed that silage was the primary source of at least one of them. \u003c/p\u003e\n\u003cp\u003eAbortions in beef cattle can be a major problem in herds, particularly if a larger than normal percentage of the population is affected, or if outbreaks occur over a short period of time. During \u003cem\u003eL. monocytogenes\u003c/em\u003e abortion outbreaks, challenges in timely disease recognition and diagnosis can create limitations for treatment of affected cattle and management decisions. Thus, quickly establishing a clear connection between the host, pathogen, and environment through strategic animal and environmental sampling, followed by strain identification using whole genome sequencing of \u003cem\u003eL. monocytogenes\u003c/em\u003e isolates allows for appropriate management of feed and environmental risks, as well as strategies for risk management of future outbreaks. Using whole-genome sequencing of pathogens in outbreak investigations will give veterinarians and epidemiologists greater flexibility and stronger evidence for confirming the strains and sources involved.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCattle population and management\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll sampled cattle in this report resided at the USMARC in Clay Center, Nebraska, and sampling protocols were approved by the Institutional Animal Care and Use Committee at the University of Nebraska, Lincoln (IACUC, #1383). Veterinary intervention and sampling were requested by the owner soon after the abortions began. The entire cattle population was considered closed; bovine semen was the only source of new genetic material and biosecurity measures were in place to prevent direct contact with outside cattle. Staff and equipment were shared between cattle locations within USMARC.\u003c/p\u003e\n\u003cp\u003eAs a part of routine management practices, all females were previously vaccinated for \u003cem\u003eBrucella abortus\u003c/em\u003e and given an initial \u003cem\u003eCampylobacter fetus\u003c/em\u003e and \u003cem\u003eLeptospira canicola-grippotyphosa-hardjo-icterohaemorrhagiae-pomona \u003c/em\u003ebacterin (Spirovac VL5, Zoetis, Kalamazoo, MI), followed by a modified live IBR, BVD types 1 and 2, and PI\u003csub\u003e3\u003c/sub\u003e vaccine in combination with \u003cem\u003eC. fetus\u003c/em\u003e, and \u003cem\u003eLeptospira spp. \u003c/em\u003ebacterin (PregGuard Gold FP 10, Zoetis, Kalamazoo, MI) 30 days before the 2013 breeding season. Pregnancy was diagnosed via rectal ultrasound between 45-100 days gestation. A total of 28 females (27 two-year old heifers and 1 three-year old female, herein referred to as \u0026ldquo;heifers\u0026rdquo;) aborted in late winter/early spring of 2014. Twenty-one of these cases were sampled (described below) and diagnostics performed at the UNLVDC in Lincoln, Nebraska.\u003c/p\u003e\n\u003cp\u003eAll pregnant females were on pasture prior to, during, and after all sampling. During the winter months, all cattle were supplemented primarily with corn silage stored and fed from the same feed storage site. Silage was analyzed for nutritional content and combined with mineral supplementation to meet nutritional requirements. Rations were delivered by the same truck and dispensed in mobile bunk or tire feeders. These feeders were moved periodically when conditions around the bunks were deemed unsanitary. Heifers that aborted were from one of three management sites (A-C) that were managed in a similar fashion. A site map of the operation with feed delivery pathway is identified in Figure 1.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical case sampling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-eight heifers and four mature cows (four years or greater) aborted by the end of the 2014 calving season, with 24 heifer abortions occurring over 12 days. All collected tissues and sampling of females that had aborted were performed by or under the direct supervision of a licensed veterinarian (lead author). Aborted fetal tissues, including fresh and fixed heart, lung, liver, spleen and kidney, as well as stomach contents and ear notches, were submitted to the UNLVDC on day one and day eight of the investigation,, the days that they were discovered and recovered by cattle managers. All fetal tissues were placed in insulated cooler with icepacks and shipped overnight to the UNLVDC.\u003c/p\u003e\n\u003cp\u003eOf the 28 heifers that were identified by cattle managers and suspected of aborting, 21 were humanely restrained in a commercial cattle chute and were subjected to individual examination and sample collection (day 8, 14, or 27). The perineum of each female was cleaned with soap and water, rinsed and dried, and the tail held away from the region.\u0026nbsp; If RFM were present, a clean, gloved hand was inserted into the vagina and the membranes were extracted caudally until the hand and membranes were outside the vulva, then a section of membrane was aseptically removed and placed in a sterile collection bag. In heifers that did not have RFM, a uterine culture double-guarded swab (Jorgensen Laboratories, Loveland, CO) was used to sample near and within the cervical opening (CV swab).\u0026nbsp; To facilitate the entry of the swab, a disposable lubricated vaginal speculum was used to visualize the cervical opening and the area just proximal to the opening. Approximately 1-2 cm of the distal cervix and the area just proximal to the opening were swabbed.\u0026nbsp; This method prevented undue contamination from the vagina, which in many cases contained discharge and fluid as a result of metritis or pyometra.\u0026nbsp; Once swabs were collected, samples were contained within the protective unit of the swab mechanism. All case samples were placed in insulated cooler with icepacks and shipped overnight to the UNLVDC. Heifers were released after sampling into communal pens for health observation, then subsequently released back onto pasture.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFeed and water sampling for \u003cem\u003eL. monocytogenes\u003c/em\u003e and pH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplemental feed consisted of corn silage, earlage, and haylage separated into open concrete bunkers. Corn silage and earlage were sampled by visually dividing the piles into three stratified layers (top, middle and base) and crosswise sections (left, middle, and right) to provide targeted and documented sampling locations (Additional file 1).\u0026nbsp; Samples were obtained by first brushing away loose materials on the surface that were potentially transferred or contaminated by feeding equipment. Exposed feed was then collected at a depth of approximately 10 cm.\u0026nbsp; Additional samples included loose surface material from the center face and apron of the piles as well as loose material present in the drainage tube of each pile. Samples were collected from haylage in a similar fashion as silage piles but from only three of the quadrants: center-top, mid-left and bottom-right. All samples were collected with freshly gloved hands, placed in sterile bags, and transported to the laboratory where they were held at 4 \u0026deg;C until processing. Silage and earlage pile samples were collected a second time, ten days after the initial sampling, and were processed in the same manner.\u0026nbsp; The second sample was taken after removal of at least one meter of the silage face to determine if contamination existed deeper within the silage piles, or if contamination was only present in isolated areas. Thirty-six feed samples were collected over this period.\u003c/p\u003e\n\u003cp\u003eAccessible water either from a tank or standing water in close proximity to the tank was collected where clinical cases of listeriosis had been identified. Nine water samples (three from each management site) were obtained by placing a 50 mL screw cap conical test tube into the water source.\u0026nbsp; A scooping motion was used, such that the bottom of the tank or water hole was contacted by the tube to capture any sediment present.\u0026nbsp; Tubes were capped and transported immediately to the laboratory for processing. Water sources were only sampled once. After sampling, water tanks were drained, disinfected, allowed to dry, and refilled.\u003c/p\u003e\n\u003cp\u003eTo measure the pH of the feed, collected samples were placed in a 100 mm petri dish then wetted with approximately 10 mL sterile distilled deionized water.\u0026nbsp; A surface pH meter (ExTech Instrument Corp., Nashua, NH) was used to measure the pH of the moistened surface.\u0026nbsp; For each dish of silage, average pH was determined by measuring pH at three separate areas corresponding to 12, 4 and 8 o\u0026rsquo;clock positions in the petri dish.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e L. monocytogenes \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003edetection and isolation from clinical samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll diagnostic specimens collected during the case investigation were shipped overnight in insulated coolers with icepacks to the UNLVDC. Submitted samples included fresh and fixed fetal tissues (collected and submitted on day 1 and 8) and CV swabs or RFM from the 21 sampled heifers (day 8, 14, and 27). Requested diagnostics for fetal tissues included: aerobic culture and sensitivity, \u003cem\u003eC. fetus\u003c/em\u003e culture, \u003cem\u003eLeptospira spp.\u003c/em\u003e, BHV1, and BVDV PCR, and histopathology. Fetuses and fetal tissues, including spleen, heart, lung, kidney, and liver were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin, examined histopathologically by board certified pathologists with other ancillary testing at their discretion to determine a diagnosis.\u0026nbsp; Testing at UNLVDC is by standard operating procedures accredited under the American Association of Veterinary Laboratory Diagnosticians (AAVLD). Fetal tissues (lung and liver) and stomach contents were subjected to culture testing. Requested diagnostics on the CV and RFM samples collected included aerobic culture and bacterial identification.\u003c/p\u003e\n\u003cp\u003eFetal tissue samples were flame sterilized and directly plated on to TSA with 5% sheep\u0026rsquo;s blood, chocolate agar, Colombia CNA agar with naldixic acid and 5% sheep\u0026rsquo;s blood (CNA), Campy CVA agar and MacConkey\u0026rsquo;s agar (Thermo Fisher Scientific, Waltham, MA).\u0026nbsp; Cervico-vaginal swabs, RFM, and fetal stomach contents were directly plated onto the same agar media as the tissues.\u0026nbsp; Remaining samples were macerated (tissues) or agitated (swabs) into Fraser\u0026rsquo;s Broth (Thermo Fisher Scientific, Waltham, MA). All media except Campy CVA were incubated for 18-24 hours at 37\u0026ordm; C with 5% CO\u003csub\u003e2\u003c/sub\u003e supplementation, then examined by trained laboratory technicians following the UNLVDC Standard Operating Procedures.\u0026nbsp; Media without pathogenic bacterial growth were re-incubated and observed following an additional 18-24 hour incubation. Campy CVA agar was incubated 48 hours at 37\u0026ordm; C in a GasPak EZ Campy microaerophilic environment container (BD Diagnostics, Sparks, MD).\u0026nbsp; Bacterial colonies with morphology consistent with members of the genus\u003cem\u003e Listeria\u003c/em\u003e were sub-cultured onto TSA with 5% sheep\u0026rsquo;s blood for purity. \u003c/p\u003e\n\u003cp\u003eSub-cultured, suspect \u003cem\u003eListeria\u003c/em\u003e colonies were subjected to gram staining, catalase testing, and were phenotypically tested using a commercial identification platform using manufacturer\u0026rsquo;s instructions for Protocol A (Biolog, Omnilog, Hayward, CA). Samples that did not have growth consistent with \u003cem\u003eL. monocytogenes\u003c/em\u003e or \u003cem\u003eL. ivanovii\u003c/em\u003e on primary isolation media were subjected to PCR testing specific for Listeria hemolysin (\u003cem\u003ehly\u003c/em\u003e).\u0026nbsp; Nucleic acid was extracted from culture samples in Fraser\u0026rsquo;s media using a commercial DNA extraction kit (Qiagen, DNA mini kit and QIACube) per manufacturer\u0026rsquo;s instructions for bacteria. Samples that were negative following PCR testing were placed into cold enrichment (4 \u0026ordm;C) for six weeks.\u0026nbsp; Cultures with a lack of esculin hydrolysis in Fraser media after six weeks of cold enrichment were considered negative for \u003cem\u003eL. monocytogenes\u003c/em\u003e. No additional diagnostics were performed on \u003cem\u003eL. monocytogenes \u003c/em\u003enegative submissions.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e L. monocytogenes\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e detection and isolation from feed and water\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFeed and water samples were screened for the presence of \u003cem\u003eListeria\u003c/em\u003e spp and \u003cem\u003eL. monocytogenes\u003c/em\u003e using BAX System Real-Time PCR Assays (Dupont Qualicon, Wilmington, DE) and Atlas Detection assays (Roka Bioscience, Lake Forest Park, WA).\u0026nbsp; Water samples were screened directly and after culture enrichment, while feed samples were only screened after enrichment.\u0026nbsp; For direct screening, 1 mL of each water sample was placed in an Atlas G2 Sample Tube (Roka Bioscience) and subjected to testing. Feed samples were enriched for rapid screening by mixing 50 g feed into 200 mL of \u003cem\u003eListeria\u003c/em\u003e enrichment broth (LEB; Dupont Qualicon, Wilmington, DE) and water samples were enriched by diluting 10 mL of each water sample into 90 mL of LEB. Samples were incubated at 30 \u0026deg;C for 24 h. After incubation, a 1mL portion was placed into an Atlas G2 Sample Tube (Roka Bioscience) and a 20 mL portion was used to prepare a BAX template lysis.\u0026nbsp; All Atlas G2 sample tubes were processed through the RokaBioscience Atlas instrument using the Atlas Listeria LSP Assay and the Atlas LmG2 Assay for \u003cem\u003eListeria\u003c/em\u003e spp and \u003cem\u003eL. monocytogenes \u003c/em\u003erespectively. The BAX lysis preparations from each sample were processed through the BAX Q7 instrument using a BAX System Real-Time PCR Assay for \u003cem\u003eL. monocytogenes\u003c/em\u003e according to the manufacturer\u0026rsquo;s instructions. \u003c/p\u003e\n\u003cp\u003eTo isolate and confirm \u003cem\u003eL. monocytogenes\u003c/em\u003e from the feed and water samples the 24 h LEB enrichments were streaked for isolation onto a Difco Oxford agar plate (Beckton Dickinson and Co., Franklin Lakes, NJ) and a CHROMagar \u003cem\u003eListeria\u003c/em\u003e plate (DRG International, Inc., Springfield, NJ) using a sterile cotton swab and inoculating loop.\u0026nbsp; Plates were incubated at 37 \u0026deg;C overnight then viewed for suspect colony phenotypes; black colonies on Oxford agar for \u003cem\u003eListeria\u003c/em\u003e spp. and blue colonies without and with halos on chromogenic agar for \u003cem\u003eListeria\u003c/em\u003e spp. and \u003cem\u003eL. monocytogenes\u003c/em\u003e respectively, were targeted.\u0026nbsp; Suspect colonies were selected and placed into a 96-well block containing 1 mL per well of Fraser media containing 5% ferric ammonium citrate, and then incubated at 37 \u0026deg;C overnight.\u003c/p\u003e\n\u003cp\u003eThe 24 h LEB enrichments of feed and water were further incubated for another 24 h (48 h total) and the above streaking for isolation onto chromogenic \u003cem\u003eListeria\u003c/em\u003e and Oxford agar was repeated.\u0026nbsp; A secondary 48 h enrichment in Fraser media was incubated an additional 48 h at 30 \u0026deg;C and was streaked for isolation, incubated and viewed for suspect colonies as described above.\u003c/p\u003e\n\u003cp\u003eSuspect colonies were confirmed to the species level using a \u003cem\u003eListeria\u003c/em\u003e spp. specific PCR and biochemical tests. \u003cem\u003eListeria\u003c/em\u003e species \u003cem\u003emonocytogenes, innocua, grayi, ivanovii, seeligeri \u003c/em\u003eand\u003cem\u003e welshimeri \u003c/em\u003ewere identified through \u003cem\u003eListeria\u003c/em\u003e spp. multiplex PCR [36]. The isolates that were identified as \u003cem\u003eL. monocytogenes\u003c/em\u003e were further characterized using the serovar multiplex PCR [21]. Suspect isolates that were found to possess the phosphoribosyl pyrophosphate synthetase (\u003cem\u003eprs\u003c/em\u003e) gene, indicative of all \u003cem\u003eListeria\u003c/em\u003e spp., but which could not be identified through PCR were further identified using biochemical tests.\u0026nbsp; Each suspect \u003cem\u003eListeria\u003c/em\u003e was streaked for isolation on tryptic soy agar containing 0.6% yeast extract, incubated at 37 \u0026deg;C overnight and then processed using a Remel Micro-ID Listeria Kit (Thermo Fisher Scientific, Lenexa, KS) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e L. monocytogenes\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e strains selected for sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 31 \u003cem\u003eL. monocytogenes\u003c/em\u003e strains were selected for whole-genome DNA sequencing on a MiSeq instrument (Illumina, San Diego, CA).\u0026nbsp; Of those, 19 clinical strains were isolated from USMARC cattle aborted fetuses, placentas, or uterine swabs.\u0026nbsp;\u0026nbsp; Another eight strains were isolated from four different corn silage samples, three water tanks and one standing water source.\u0026nbsp; Additionally, four other strains from bovine abortions were obtained from the UNLVDC that originated from other regions in Nebraska for use as controls and references\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA preparation and Illumina MiSeq whole-genome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 31 \u003cem\u003eL. monocytogenes \u003c/em\u003estrains were passaged twice from -80 \u0026deg;C frozen stocks on chocolate agar plates (Hardy Diagnostics, Santa Maria, CA) at 37 \u0026deg;C.\u0026nbsp; A single colony of each isolate was then inoculated in 1.5 mL of Brain-Heart Infusion (BHI) broth and grown overnight without shaking at 37 \u0026deg;C. Genomic DNA was extracted from the cultures using MO BIO microbial DNA isolation kits (MO BIO Laboratories, Carlsbad, CA) according to the manufacturer\u0026rsquo;s instructions.\u0026nbsp; The extracted DNAs were quantified and checked for purity using 260/280 absorbance readings on a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE).\u0026nbsp; Individual libraries were constructed for each of the strain DNA preparations using Illumina Nextera XT DNA sample preparation kits with appropriate indices tags according to the manufacturer\u0026rsquo;s instructions (Illumina Inc., San Diego, CA).\u0026nbsp; The libraries were pooled together and run on an Illumina MiSeq DNA sequencer (Illumina Inc., San Diego, CA). The genome of each strain was sequenced to a minimal depth of 10X coverage.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssembly of \u003cem\u003eL. monocytogenes\u003c/em\u003e chromosomes and phylogenetic trees\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdapter sequence and low-quality bases were trimmed using Trimmomatic [37]. Trimmed fastq reads were assembled using SKESA. The \u003cem\u003eL. monocytogenes\u003c/em\u003e assembled chromosomes from 27 unique USMARC strains and 4 clinical strains from the UNLVDC were imported into parsnp [38] for genome alignments and subsequent identification of core-genome SNPs and construction of a preliminary phylogenetic tree.\u0026nbsp; The strains grouped into six initial clusters.\u0026nbsp; One strain from each cluster was selected for PacBio sequencing to obtain a complete closed chromosome representative of each cluster (described below). Illumina reads from strains not also sequenced with PacBio were assembled using SKESA. \u0026nbsp;The SKESA, PacBio, and 20 closed \u003cem\u003eL. monocytogenes\u003c/em\u003e chromosomes from GenBank [39] were then used in parsnp to create a new phylogenetic tree.\u0026nbsp; The 20 GenBank chromosomes represented all four known \u003cem\u003eL. monocytogenes\u003c/em\u003e lineages and were utilized to calibrate the final phylogenetic tree.\u0026nbsp; Evolview was used to populate the tree with phenotypic metadata [40].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA preparation and PacBio whole-genome sequencing library construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh molecular weight DNA was extracted from \u003cem\u003eL. monocytogenes\u003c/em\u003e cultures using Qiagen Genomic-tip 100/G columns and a modified manufacturer\u0026rsquo;s protocol as previously described [41] with the addition of mutanolysin with the proteinase K step followed by incubation at 50 \u0026deg;C for 1 h. Ten micrograms of DNA were sheared to a targeted size of 20 kb using a g-TUBE (Corvaris, Woburn, MA) and concentrated using 0.45X volume of AMPure PB magnetic beads (Pacific Biosciences, Menlo Park, CA) following the manufacturer\u0026rsquo;s protocol.\u0026nbsp; Sequencing libraries were created using 5 \u0026micro;g of sheared, concentrated DNA and the PacBio DNA Template Prep Kit 2.0 (3Kb - 10Kb) according to the manufacturer\u0026rsquo;s protocol.\u0026nbsp; The library was bound with polymerase P5 followed by sequencing on a Pacific BioSciences (PacBio) RS II sequencing platform with chemistry C3 and the 120 min data collection protocol.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePacBio Sequence Assembly into Closed Circularized Genomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePacBio reads were assembled using HGAP3 (SMRTanalysis Version 2.1) and the resulting contigs were imported into Geneious. Within Geneious, overlapping sequence on the ends of the contigs were removed from the 5\u0026rsquo; and 3\u0026rsquo; ends to generate a circularized chromosome.\u0026nbsp; The chromosome was reoriented to start with a putative origin of replication with Ori-Finder [42]. The chromosome was initially polished for accuracy using the Resequencing 1.0 protocol in SMRTanalysis by mapping corrected PacBio reads to the chromosome. To correct PacBio sequencing errors (homopolymers and SNPs), Illumina reads were mapped to the initially polished chromosome using Pilon. Then, both PacBio and Illumina reads were mapped to the Pilon-generated chromosome using Geneious Mapper. Additional sequencing errors were identified and corrected by manual editing in Geneious, resulting in a finished closed circularized chromosome. Chromosome sequences were deposited into NCBI (Additional file 2) and annotated using the Prokaryotic Genome Annotation Pipeline version 4.5.\u0026nbsp; MLST 2.0 [43] was used to determine the MLST allelic profiles for the \u003cem\u003eL. monocytogenes\u003c/em\u003e strains from USMARC and UNLVDC. MLST allelic profiles were then used with the Listeria Pasteur MLST database to determine serotype, lineage and clonal complex [21, 22].\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePFGE:\u0026nbsp; Pulse-field gel electrophoresis\u003c/p\u003e\n\u003cp\u003eCV:\u0026nbsp; cervico-vaginal\u003c/p\u003e\n\u003cp\u003eRFM:\u0026nbsp; retained fetal membranes\u003c/p\u003e\n\u003cp\u003eUSMARC:\u0026nbsp; United States Meat Animal Research Center\u003c/p\u003e\n\u003cp\u003eNAHMS:\u0026nbsp; National Animal Health Monitoring System\u003c/p\u003e\n\u003cp\u003eUNLVDC:\u0026nbsp; University of Nebraska, Lincoln, Veterinary Diagnostic Center\u003c/p\u003e\n\u003cp\u003eSKESA:\u0026nbsp; Strategic Kmer Extension for Scrupulous Assemblies\u003c/p\u003e\n\u003cp\u003eNCBI:\u0026nbsp; National Center for Biotechnology Information\u003c/p\u003e\n\u003cp\u003eMLST:\u0026nbsp; multi-locus sequence typing\u003c/p\u003e\n\u003cp\u003eSNPs:\u0026nbsp; single nucleotide polymorphisms\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the author\u0026rsquo;s institution (University of Nebraska IACUC, #1383) and owner consent was verbally confirmed as part of routine veterinary diagnostic sampling for an outbreak investigation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and material\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe Illumina sequencing reads for the 31 isolates sequenced on that platform have all been placed in the NCBI sequence read archive (SRA) for public availability.\u0026nbsp; Additionally, the complete genomes of all six isolates sequenced and assembled with PacBio and Illumina sequences are publicly available in GenBank (CP032668-CP-32673).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the US Meat Animal Research Center for providing financial support for sample submission to the veterinary diagnostic laboratory and for machine use and material costs associated with genomic sequencing. \u0026nbsp;The authors would also like to thank the University of Nebraska Great Plains Veterinary Educational Center for financial support of material costs during sample collection, labor for case management, and cost of manuscript submission. Funding sources did not have any involvement in the study design, data analysis and interpretation, or writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthor\u0026rsquo;s contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eClinical sample collection and submission were performed by KJW and JDO. KJW was responsible for clinical case management, writing, and revision of the manuscript. JDL was responsible for clinical case \u003cem\u003eListeria\u003c/em\u003e diagnostics and providing \u003cem\u003eListeria\u003c/em\u003e controls. Feed and water sample collection and \u003cem\u003eListeria\u003c/em\u003e diagnostics on those samples was completed by JMB and TMA.\u003c/p\u003e\n\u003cp\u003eGenomic analysis and manuscript revisions were provided by JLB and MLC. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Sandy Fryda-Bradley, Gennie Schuller, and Karen Schuck for excellent technical assistance. The authors also thank the USMARC core sequencing facility, USMARC cattle operations, the UNL GPVEC for technical support and the UNL Veterinary Diagnostic Center. The mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply approval to the exclusion of other products that might be suitable. The USDA is an equal opportunity employer and provider.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNightingale KK, Fortes ED, Ho AJ, Schukken YH, Grohn YT, Wiedmann M. 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Microbiological aspects of the investigation that traced the 1998 outbreak of listeriosis in the United States to contaminated hot dogs and establishment of molecular subtyping-based surveillance for \u003cem\u003eListeria monocytogenes\u003c/em\u003e in the PulseNet network. J Clin Microbiol. 2005;43:2350-2355.\u003c/li\u003e\n\u003cli\u003eFarber JM, Peterkin PI. \u003cem\u003eListeria monocytogenes\u003c/em\u003e, a food-borne pathogen. Microbiol Rev.1991;55:476-511.\u003c/li\u003e\n\u003cli\u003eRowan NJ, MacGregor SJ, Anderson JG, Fouracre RA, McIlvaney L, Farish O. Pulsed-light inactivation of food-related microorganisms. Appl Environ Microbiol. 1999;65:1312-1315.\u003c/li\u003e\n\u003cli\u003eOloketuyi SF, Khan F. Inhibition strategies of \u003cem\u003eListeria monocytogenes\u003c/em\u003e biofilms-current knowledge and future outlooks. 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J Clin Microbiol. 2012;50:1355-1361.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 115%; font-family: 'Times New Roman',serif;\"\u003eTable 1 \u003c/span\u003e\u003c/strong\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 115%; font-family: 'Times New Roman',serif;\"\u003e(Page 6, Line 114)\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 115%; font-family: 'Times New Roman',serif;\"\u003eDescriptive statistics relative to the female population over the calving season.\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse: collapse; border: none;\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 80.1pt; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"107\"\u003e\n\u003cp\u003e\u003cstrong\u003eCategory\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 82.9pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp\u003e\u003cstrong\u003eTotal Population\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp\u003e\u003cstrong\u003eSeason Abortion Count\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp\u003e\u003cstrong\u003eSeason Abortion %\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp\u003e\u003cstrong\u003eDay 1-12 Abortion Count\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 64.95pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"87\"\u003e\n\u003cp\u003e\u003cstrong\u003e% Total Abortions Occurring Day 1-12\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 80.1pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"107\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eMature Cows\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 82.9pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e3633\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e4\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; 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border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e0.7%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e24\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 64.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"87\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e77%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 80.1pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"107\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eSite A\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 82.9pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e369\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e17\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e4.6%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e16\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 64.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"87\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e94%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 80.1pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"107\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eSite B\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 82.9pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e283\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e1.1%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 64.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"87\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e100%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 80.1pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"107\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eSite C\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 82.9pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e284\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e8\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e2.8%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"106\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 64.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"87\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e71%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size: 12.0pt; 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border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"90\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 58.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; 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border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eS\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; 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border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003eA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 67.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"90\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 58.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eS\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eA\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 67.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"90\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 58.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; 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border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003eC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 67.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"90\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 58.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003eA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 67.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"90\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 58.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; 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border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003eA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 67.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"90\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 58.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003eA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 67.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"90\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 58.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; 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border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eS\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eA\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 67.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"90\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 58.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; 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border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eS\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003eA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 67.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"90\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 58.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eS\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003eC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 67.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"90\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 58.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003eC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 67.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"90\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 58.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 61.95pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"83\"\u003e\n\u003cp style=\"text-align: center;\"\u003e23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 73.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"98\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003eA\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.85pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"color: black;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 67.4pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"90\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 58.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"78\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"line-height: 200%; tab-stops: 4.5pt;\"\u003eLM=\u003cem\u003eL. monocytogenes\u003c/em\u003e, F=fetal tissues, P=retained fetal membranes, S=cervico-vaginal (CV) swabs\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: 4.5pt;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"line-height: 200%; tab-stops: 4.5pt;\"\u003e\u003cstrong\u003e\u003cspan style=\"line-height: 200%;\"\u003eTable 3 \u003c/span\u003e\u003c/strong\u003e\u003cem\u003e\u003cspan style=\"line-height: 200%;\"\u003e(Page 7, Line 127)\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 115%; font-family: 'Times New Roman',serif;\"\u003eSummary of \u003cem\u003eL. monocytogenes\u003c/em\u003e water samples.\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"width: 463.25pt; border-collapse: collapse; border: none;\" width=\"618\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 53.75pt; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.5pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eSite\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eDirect LM PCR\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eEnriched LM PCR\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eEnriched LM Culture\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 53.75pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eTank 1\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.5pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center;\"\u003eA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 53.75pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eTank 2\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.5pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 53.75pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eTank 3\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.5pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 53.75pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eTank 4\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.5pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center;\"\u003eC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 53.75pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eHole 5*\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.5pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 53.75pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eHole 6\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.5pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center;\"\u003eB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 53.75pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eHole 7\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.5pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center;\"\u003eB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 53.75pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eHole 8\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.5pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center;\"\u003eC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 53.75pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eHole 9\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.5pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center;\"\u003eC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 120.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"160\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Culture positive for \u003cem\u003eL. innocua\u003c/em\u003e-not sequenced.\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 115%; font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 115%; font-family: 'Times New Roman',serif;\"\u003eTable 4 \u003c/span\u003e\u003c/strong\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 115%; font-family: 'Times New Roman',serif;\"\u003e(Page 7, Line 133)\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 115%; font-family: 'Times New Roman',serif;\"\u003eSummary of \u003cem\u003eL. monocytogenes\u003c/em\u003e positive feed samples.\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse: collapse; border: none;\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 77.2pt; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003eSample timing\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003ePile location\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003epH\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003eLM culture\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.25pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003eLM PCR\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 77.2pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e1st\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e4.54\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.25pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 77.2pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e1st\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e10\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e8.09\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.25pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 77.2pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e2nd\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e7.09\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.25pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 77.2pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e2nd\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e4.34\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.25pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"103\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 12.0pt; font-family: 'Times New Roman',serif;\"\u003e+\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 115%; font-family: 'Calibri',sans-serif;\"\u003eSample timing:\u0026nbsp; 2\u003csup\u003end\u003c/sup\u003e sample collected 10 days after 1\u003csup\u003est\u003c/sup\u003e sample. All positive samples were from corn silage. Pile location is in reference to the position on the silage face (Additional file 1).\u003c/span\u003e\u003c/p\u003e"},{"header":"Additional Files","content":"\u003cp\u003eAdditional file 1 .docx\u003c/p\u003e\n\u003cp\u003eSampling scheme of corn silage and earlage piles\u003c/p\u003e\n\u003cp\u003eImage of silage face with grid overlay. Eight samples (ovals; 1-8) were collected from each pile according the gridded lines. Loose surface materials (9-10) and the drainage pipe (11) were also collected.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditional file 2 .xlsx\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e L. monocytogenes\u003c/em\u003e strains used in the project with corresponding genotypes, sequencing information and sample type.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditional file 3 .pptx\u003c/p\u003e\n\u003cp\u003eGingr visualization of the genomes of lineage III \u003cem\u003eL. monocytogenes\u003c/em\u003e strains from cattle, water or feed associated with the outbreak. \u0026nbsp;The genomes were aligned with parsnp.\u0026nbsp; The 19 strains in this figure comprised the monophyletic clade in Figure 2.\u0026nbsp; The outer taxonomic units in the phylogenetic tree are aligned with their corresponding row in the alignment.\u0026nbsp; Each row represents the entire genome for a strain.\u0026nbsp; Vertical purple lines in the alignment indicate base differences.\u0026nbsp; Asterisks are to the right of the six strains whose core genomes are identical with their names highlighted according to sample type; cattle (green), water (blue) or feed (red).\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cattle abortion, listeriosis, silage, whole-genome sequencing, outbreak","lastPublishedDoi":"10.21203/rs.2.13582/v3","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.13582/v3","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground In a beef cattle facility an outbreak of abortions occurred over a 36-day period and included two aborted fetuses and 21 post-abortion clinical cases sampled. There are numerous etiologies, including clinical listeriosis. At the species level, Listeria monocytogenes is ubiquitous in cattle production environments, including soil, feed, and occasionally water sources, and is a common enteric resident of cattle and other mammals. There are four genetically distinct lineages of L. monocytogenes (I-IV), with most lineage III and IV isolates obtained from ruminants. Definitive diagnosis of L. monocytogenes as a causative agent in disease outbreaks relies upon case identification, appropriate sample collection, and laboratory confirmation. Furthermore, clearly establishing a relationship between a pathogen source and clinical disease is difficult. \u003c/p\u003e\u003cp\u003eResults Of the two fetal and 21 clinical case submissions, 19 were positive for L. monocytogenes. Subsequent culture for L. monocytogenes from water and silage sources identified both as potential origins of infection. Using whole-genome sequencing and phylogenetic analyses, clinical, water and silage L. monocytogenes strains grouped into two of four lineages. All water and silage strains, plus 11 clinical strains placed in lineage III, with identical or nearly identical genomic sequences. The remaining eight clinical strains placed in lineage I, with seven having nearly identical sequences and one distinctly different. \u003c/p\u003e\u003cp\u003eConclusion Three genetically distinct strains within two lineages of L. monocytogenes caused the abortion outbreak. The etiology of abortion in 11 cases was directly linked to water and silage contamination from a lineage III L. monocytogenes strain. The source of infection for the remaining abortion cases with two different strains from lineage I is unknown. This is the first report of L. monocytogenes genomics being used as part of an outbreak investigation of cattle abortion.\u003c/p\u003e","manuscriptTitle":"Genomic-based identification of environmental and clinical Listeria monocytogenes strains associated with an abortion outbreak in beef heifers","msid":"","msnumber":"","nonDraftVersions":[{"code":3,"date":"2020-02-05 22:44:23","doi":"10.21203/rs.2.13582/v3","editorialEvents":[{"type":"communityComments","content":0},{"type":"checksComplete","content":"","date":"2020-02-05T12:00:00+00:00","index":"","fulltext":""},{"type":"decision","content":"Accept","date":"2020-02-05T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary 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policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-01-20T12:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-01-16T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2019-12-27T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2019-12-26T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2019-12-26T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary 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policy.\n"},{"type":"reviewerAgreed","content":"","date":"2019-09-13T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2019-09-10T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2019-09-10T12:00:00+00:00","index":1,"fulltext":""},{"type":"checksComplete","content":"","date":"2019-08-21T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2019-08-12T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2019-08-11T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2019-08-07T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary 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