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Abstract Background Management of inferior ramus of the pubis-ischium ramus remains controversial, and related research is sparse. The main intention of this study is to describe the biomechanical and clinical outcomes of pubis-ischium ramus fractures in Tile B pelvic injuries and to identify the feasibility and necessity of fixation of the inferior ramus of the pubis-ischium ramus. Methods This study comprised two parts: a biomechanical test and a retrospective clinical study. For the biomechanical tests, Tile B-type pelvic injuries were modeled in six cadaver specimens by performing pubis-ischium osteotomies and disruption of the anterior and interosseous sacroiliac ligaments. The superior and/or inferior rami of the pubis-ischium ramus were repaired with reconstruction plates and separated into three groups (A, B, and C). Specimens were placed in the standing position and were loaded axially with two-leg support for three cycles at 500 N. The displacements of sacroiliac joints at osteotomy were measured with Vernier calipers and compared using statistical software. To investigate the clinical outcomes of this technique, 26 patients were retrospectively analyzed and divided into a superior ramus fixation group (Group D) and a combined superior and inferior ramus of the pubis-ischium ramus fixation group (Group E). The main outcome measures were time of operation, blood loss, postoperative radiographic reduction grading, and functional outcomes. Results In the vertical loading test, Group E showed better pelvic ring stability than Group D (P

Abstract Purpose Chronic kidney disease (CKD) has been one of the most common complications in type 2 diabetes mellitus (T2DM) patients. This retrospective study aimed to investigate the regional differences in the prevalence and management of CKD in T2DM inpatients from two grassroots hospitals in Beijing and Taiyuan. Methods The sociodemographic status, health history, lifestyle information, biochemical parameters and drug choices of the patients were collected from the Diabetes Care Information System using a retrospective cross-sectional analysis. The presence of CKD was defined as albuminuria (urine albumin-to-creatinine ratio of ≥ 30 mg/g) and/or as a reduced estimated glomerular filtration rate (

Antimicrobial resistance (AMR) in bacteria is a major public health concern in the US and around the world. Campylobacter is an important foodborne pathogen that resides in the gut of pigs and is shed in feces, with the potential to be transmitted to humans. In pigs, the oral route, either in-feed or in-water, is by far the most common route of administration of antimicrobials. Because the distribution of the antibiotic in the gut and the dosages are different, the impact of in-feed vs. in-water administration of antibiotics on the development of AMR is likely to be different. Therefore, a study was conducted to compare in-feed vs. in-water administrations of chlortetracycline (CTC) and/or tiamulin on fecal prevalence and AMR profiles of Campylobacter among weaned nursery piglets. A total of 1,296 weaned piglets, allocated into 48 pens (27 piglets per pen), were assigned randomly to six treatment groups: Control (no antibiotic), in-feed CTC, in-water CTC, in-feed tiamulin, in-water tiamulin, or in-feed CTC and tiamulin. Fecal samples were collected randomly from 5 piglets from each pen during the pre-treatment (days 0, 7), treatment (days 14, 21), and post-treatment (days 28, 35) phases. Bacterial isolations and species identifications were conducted by culture and PCR, respectively. The microbroth dilution method with SensititreTM plates was used to determine the antimicrobial susceptibility and resistance of Campylobacter isolates. The results on resistance were interpreted based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cutoff values for Campylobacter. The overall prevalence of Campylobacter was 18.2% (262/1440). Speciation of Campylobacter isolates by PCR indicated the prevalence of only two species: Campylobacter hyointestinalis (17.9%; 258/1440) and C. coli (0.3%; 4/1440). Campylobacter isolates were resistant to tetracycline (98.5%), ciprofloxacin (89.3%), and nalidixic acid (60.3%). Neither the antibiotic nor the route of administration had an effect (p > 0.05) on the prevalence of AMR Campylobacter in the feces of piglets.

Growing resistant rice cultivars is the most effective strategy to control bacterial blight (BB), a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Screening resistant germplasm and identifying resistance (R) genes are prerequisites for breeding resistant rice cultivars. We conducted a genome-wide association study (GWAS) to detect quantitative trait loci (QTL) associated with BB resistance using 359 East Asian temperate Japonica accessions inoculated with two Chinese Xoo strains (KS6-6 and GV) and one Philippine Xoo strain (PXO99A). Based on the 55K SNPs Array dataset of the 359 Japonica accessions, eight QTL were identified on rice chromosomes 1, 2, 4, 10, and 11. Four of the QTL coincided with previously reported QTL, and four were novel loci. Six R genes were localized in the qBBV-11.1, qBBV-11.2, and qBBV-11.3 loci on chromosome 11 in this Japonica collection. Haplotype analysis revealed candidate genes associated with BB resistance in each QTL. Notably, LOC_Os11g47290 in qBBV-11.3, encoding a leucine-rich repeat receptor-like kinase, was a candidate gene associated with resistance to the virulent strain GV. Knockout mutants of Nipponbare with the susceptible haplotype of LOC_Os11g47290 exhibited significantly improved BB resistance. These results will be useful for cloning BB resistance genes and breeding resistant rice cultivars.

Abstract Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause illnesses in humans ranging from mild to hemorrhagic enteritis with complications of hemolytic uremic syndrome and even death. Cattle are a major reservoir of STEC, which reside in the hindgut and are shed in the feces, a major source of food and water contaminations. Seven serogroups, O26, O45, O103, O111, O121, O145 and O157, called ‘top-7’, are responsible for the majority of human STEC infections in North America. Additionally, 151 serogroups of E. coli are known to carry Shiga toxin genes (stx). Not much is known about fecal shedding and prevalence and virulence potential of STEC other than the top-7. Our primary objectives were to identify serogroups of STEC strains, other than the top-7, isolated from cattle feces and subtype stx genes to assess their virulence potential. Additional objective was to develop and validate a novel multiplex PCR assay to detect and determine prevalence of six serogroups, O2, O74, O109, O131, O168, and O171, in cattle feces. A total of 351 strains, positive for stx gene and negative for the top-7 serogroups, isolated from feedlot cattle feces were used in the study. Of the 351 strains, 291 belonged to 16 serogroups and 60 could not be serogrouped. Among the 351 strains, 63 (17.9%) carried stx1 gene and 300 (82.1%) carried stx2, including 12 strains positive for both. The majority of the stx1 and stx2 were of stx1a (47/63; 74.6%) and stx2a subtypes (234/300; 78%), respectively, which are often associated with human infections. A novel multiplex PCR assay developed and validated to detect six serogroups, O2, O74, O109, O131, O168, and O171, which accounted for 86.9% of the STEC strains identified, was utilized to determine their prevalence in fecal samples (n = 576) collected from a commercial feedlot. Four serogroups, O2, O109, O168, and O171 were identified as the dominant serogroups prevalent in cattle feces. In conclusion, cattle shed in the feces a number of STEC serogroups, other than the top-7, and the majority of the strains isolated possessed stx2, particularly of the subtype 2a, suggesting their potential risk to cause human infections.

Abstract Background Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most devastating bacterial diseases of rice in temperate and tropical regions. Breeding and deployment of resistant cultivars carrying major resistance (R) genes has been the most effective approach for BB management. However, because of specific interaction of each R gene with the product of the corresponding pathogen avirulence or effector gene, new pathogen strains that can overcome the deployed resistance often emerge rapidly. To deal with ever-evolving Xoo, it is necessary to identify novel R genes and resistance quantitative trait loci (QTL). Results BB resistance of a diverse panel of 340 accessions from the 3000 Rice Genomes Project (3 K RGP) was evaluated by artificial inoculation with four representative Xoo strains, namely Z173 (C4), GD1358 (C5), V from China and PXO339 (P9a) from Philippines. Using the 3 K RG 4.8mio filtered SNP Dataset, a total of 11 QTL associated with BB resistance on chromosomes 4, 5, 11 and 12 were identified through a genome-wide association study (GWAS). Among them, eight resistance loci, which were narrowed down to relatively small genomic intervals, coincided with previously reported QTL or R genes, e.g. xa5, xa25, xa44(t). The other three QTL were putative novel loci associated with BB resistance. Linear regression analysis showed a dependence of BB lesion length on the number of favorable alleles, suggesting that pyramiding QTL using marker-assisted selection would be an effective approach for improving resistance. In addition, the Hap2 allele of LOC_Os11g46250 underlying qC5–11.1 was validated as positively regulating resistance against strain C5. Conclusions Our findings provide valuable information for the genetic improvement of BB resistance and application of germplasm resources in rice breeding programs.

To improve the oil recovery of a block in the Wutonggou Formation of the Changji Oilfield, viscosity reducing and foaming agent was optimized to improve the development effect of the water flooding reservoir. The core flooding experiment and microscopic visual experiment were conducted to investigate the production characteristics and EOR mechanism of nitrogen foam flooding. The results show that the 0.5 wt% viscosity reducing and foaming agent DXY-03 was optimized. In the process of microscopic oil displacement by nitrogen foam, nitrogen foam continuously expands and spreads, improves oil displacement efficiency, and greatly improves oil recovery through emulsification and viscosity reduction, squeezing action, dragging action, and Jamin effect. The core flooding experiment shows that on the basis of the water flooding recovery rate of 20.3%, the nitrogen foam huff and puff is increased by 9.2%. The viscosity reducing and foaming agent flooding is increased by 7.8%, and the nitrogen foam flooding is increased by 12.9%. The main EOR mechanism of the viscosity reducing and foaming agent is that it can reduce the interfacial tension between oil and water and can promote heavy oil emulsification and dispersion, thereby forming an oil/water- (O/W-) type emulsion. The reduction in the viscosity of heavy oil makes crude oil easier to extract, realizing the synergistic viscosity reducing and efficiency enhancing effect of nitrogen and viscosity reducing and foaming agents. This study is helpful to provide reference for the development of low-permeability and high-viscosity medium-deep heavy oil reservoirs by chemical agents combined with cold production.

Escherichia coli carrying prophage with genes that encode for Shiga toxins are categorized as Shiga toxin-producing E. coli (STEC) pathotype. Illnesses caused by STEC in humans, which are often foodborne, range from mild to bloody diarrhea with life-threatening complications of renal failure and hemolytic uremic syndrome and even death, particularly in children. As many as 158 of the total 187 serogroups of E. coli are known to carry Shiga toxin genes, which makes STEC a major pathotype of E. coli. Seven STEC serogroups, called top-7, which include O26, O45, O103, O111, O121, O145, and O157, are responsible for the majority of the STEC-associated human illnesses. The STEC serogroups, other than the top-7, called “non-top-7” have also been associated with human illnesses, more often as sporadic infections. Ruminants, particularly cattle, are principal reservoirs of STEC and harbor the organisms in the hindgut and shed in the feces, which serves as a major source of food and water contaminations. A number of studies have reported on the fecal prevalence of top-7 STEC in cattle feces. However, there is paucity of data on the prevalence of non-top-7 STEC serogroups in cattle feces, generally because of lack of validated detection methods. The objective of our study was to develop and validate 14 sets of multiplex PCR (mPCR) assays targeting serogroup-specific genes to detect 137 non-top-7 STEC serogroups previously reported to be present in cattle feces. Each assay included 7–12 serogroups and primers were designed to amplify the target genes with distinct amplicon sizes for each serogroup that can be readily identified within each assay. The assays were validated with 460 strains of known serogroups. The multiplex PCR assays designed in our study can be readily adapted by most laboratories for rapid identification of strains belonging to the non-top-7 STEC serogroups associated with cattle.

As the vertical-well fire flooding technology is industrially applied in the steam-injection old heavy oil areas of Xinjiang and Liaohe oilfields, its enhanced oil recovery potential is gradually clear. According to laboratory experiment, field test and reservoir engineering, the displacement characteristics of vertical-well fire flooding in the steam-injection old heavy oil areas are systematically investigated. Laboratory experiments and core data show that the vertical-well fire flooding has significantly high flooding efficiency, no residual oil are remained in the firing front sweeping zone, and the lateral sweep efficiency and ultimate recovery can be achieved. The vertical-well fire flooding is a strategic replacement technology to enhance the recovery greatly. Development characteristics and advantages of areal and linear fire flooding are well investigated, and research results are applied in the design of industrial test plan of fire flooding in the Hongqian block, Xinjiang. The research shows that the linear well pattern has advantages of easy construction of ground facilities and management, less well of stage management, simple matching technology and easy achievement of purposeful control for fire front; the areal well pattern has advantages in reducing the air/oil ratio during the fire flooding period, increasing the total recovery rate of reservoirs and reducing the geological and reservoir management risks. To select well patterns of fire flooding, some factors such as geology, reservoir, fluid properties, oil price, and reservoir development degree should be mainly considered. In the Hongqian block, an improved linear well pattern with a combination of new wells and old wells is applied, this not only absorb experiences of linear well pattern pilot test, but also use advantages of areal well pattern. Keywords: Old heavy oil area, Fire flooding, Residual oil, Oil displacement efficiency, Sweep efficiency, Linear and areal well patterns

A real-time PCR (qPCR) assay targeting on invA and pagC genes was developed and validated for the detection and quantification of Salmonella enterica strains (Bai et al., 2018) [1]. A host gene, normally an endogenous housekeeping gene (Beer-Davidson et al., 2018; Poon et al., 2004) [2,3], or an irrelevant exogenous gene (Cheng et al., 2015; Sedlak et al., 2014) [4,5] has been widely used as an internal control to monitor nucleic acid extraction efficiencies and potential PCR inhibitions in PCR-based detection assays. An endogenous internal control designed based on the 18S rRNA gene was used in the above-mentioned qPCR assay. This 18S rRNA internal control amplifies the target gene in multiple species including bovine, swine, ovine, caprine and cervine. Data was generated by the duplex qPCR assay on 138 enriched cattle lymph node samples without the internal control, and compared with data on the same samples tested by the triplex qPCR assay that has the 18S rRNA gene as internal control. Threshold cycle (Ct) data for the duplex and the triplex qPCR on the 138 samples were similar, and are presented in this brief report. Keywords: Real-time PCR, Threshold cycle, Internal control, Salmonella, Foodborne pathogen, Lymph node

Escherichia coli O145 serogroup is one of the big six non-O157 Shiga toxin producing E. coli (STEC) that causes foodborne illnesses in the United States and other countries. Cattle are a major reservoir of STEC, which harbor them in their hindgut and shed in the feces. Cattle feces is the main source of hide and subsequent carcass contaminations during harvest leading to foodborne illnesses in humans. The objective of our study was to determine the virulence potential of STEC O145 strains isolated from cattle feces and hide samples. A total of 71 STEC O145 strains isolated from cattle feces (n = 16), hide (n = 53), and human clinical samples (n = 2) were used in the study. The strains were subjected to whole genome sequencing using Illumina MiSeq platform. The average draft genome size of the fecal, hide, and human clinical strains were 5.41, 5.28, and 5.29 Mb, respectively. The average number of genes associated with mobile genetic elements was 260, 238, and 259, in cattle fecal, hide, and human clinical strains, respectively. All strains belonged to O145:H28 serotype and carried eae subtype γ. Shiga toxin 1a was the most common Shiga toxin gene subtype among the strains, followed by stx2a and stx2c. The strains also carried genes encoding type III secretory system proteins, nle, and plasmid-encoded virulence genes. Phylogenetic analysis revealed clustering of cattle fecal strains separately from hide strains, and the human clinical strains were more closely related to the hide strains. All the strains belonged to sequence type (ST)-32. The virulence gene profile of STEC O145 strains isolated from cattle sources was similar to that of human clinical strains and were phylogenetically closely related to human clinical strains. The genetic analysis suggests the potential of cattle STEC O145 strains to cause human illnesses. Inclusion of more strains from cattle and their environment in the analysis will help in further elucidation of the genetic diversity and virulence potential of cattle O145 strains.

Escherichia coli O104:H4, a Shiga toxin-producing hybrid pathotype that was implicated in a major foodborne outbreak in Germany in 2011, has not been detected in cattle. However, serotypes of O104, other than O104:H4, have been isolated from cattle feces, with O104:H7 being the most predominant. In this study, we investigated, based on whole genome sequence analyses, the virulence potential of E. coli O104 strains isolated from cattle feces, since cattle are asymptomatic carriers of E. coli O104. The genomes of ten bovine E. coli O104 strains (six O104:H7, one O104:H8, one O104:H12, and two O104:H23) and five O104:H7 isolated from human clinical cases were sequenced. Of all the bovine O104 serotypes (H7, H8, H12, and H23) that were included in the study, only E. coli O104:H7 serotype possessed Shiga toxins. Four of the six bovine O104:H7 strains and one of the five human strains carried stx1c. Three human O104 strains carried stx2, two were of subtype 2a, and one was 2d. Genomes of stx carrying bovine O104:H7 strains were larger than the stx-negative strains of O104:H7 or other serotypes. The genome sizes were proportional to the number of genes carried on the mobile genetic elements (phages, prophages, transposable elements and plasmids). Both bovine and human strains were negative for intimin and other genes associated with the type III secretory system and non-LEE encoded effectors. Plasmid-encoded virulence genes (ehxA, epeA, espP, katP) were also present in bovine and human strains. All O104 strains were negative for antimicrobial resistance genes, except one human strain. Phylogenetic analysis indicated that bovine E. coli O104 strains carrying the same flagellar antigen clustered together and STEC strains clustered separately from non-STEC strains. One of the human O104:H7 strains was phylogenetically closely related to and belonged to the same sequence type (ST-1817) as the bovine O104:H7 STEC strains. This suggests that the bovine feces could be a source of human illness caused by E. coli O104:H7 serotype. Because bovine O104:H7 strains carried virulence genes similar to human clinical strains and one of the human clinical strains was phylogenetically related to bovine strains, the serotype has the potential to be a diarrheagenic pathogen in humans.

Escherichia coli O104:H4, a hybrid pathotype reported in a large 2011 foodborne outbreak in Germany, has not been detected in cattle feces. However, cattle harbor and shed in the feces other O104 serotypes, particularly O104:H7, which has been associated with sporadic cases of diarrhea in humans. The objective of our study was to assess the virulence potential of Shiga toxin-producing E. coli (STEC) O104:H7 isolated from feces of feedlot cattle using DNA microarray. Six strains of STEC O104:H7 isolated from cattle feces were analyzed using FDA-E. coli Identification (ECID) DNA microarray to determine their virulence profiles and compare them to the human strains (clinical) of O104:H7, STEC O104:H4 (German outbreak strain), and O104:H21 (milk-associated Montana outbreak strain). Scatter plots were generated from the array data to visualize the gene-level differences between bovine and human O104 strains, and Pearson correlation coefficients (r) were determined. Splits tree was generated to analyze relatedness between the strains. All O104:H7 strains, both bovine and human, similar to O104:H4 and O104:H21 outbreak strains were negative for intimin (eae). The bovine strains were positive for Shiga toxin 1 subtype c (stx1c), enterohemolysin (ehxA), tellurite resistance gene (terD), IrgA homolog protein (iha), type 1 fimbriae (fimH), and negative for genes that code for effector proteins of type III secretory system. The six cattle O104 strains were closely related (r = 0.86-0.98) to each other, except for a few differences in phage related and non-annotated genes. One of the human clinical O104:H7 strains (2011C-3665) was more closely related to the bovine O104:H7 strains (r = 0.81-0.85) than the other four human clinical O104:H7 strains (r = 0.75-0.79). Montana outbreak strain (O104:H21) was more closely related to four of the human clinical O104:H7 strains than the bovine O104:H7 strains. None of the bovine E. coli O104 strains carried genes characteristic of E. coli O104:H4 German outbreak strain and unlike other human strains were also negative for Shiga toxin 2. Because cattle E. coli O104:H7 strains possess stx1c and genes that code for enterohemolysin and a variety of adhesins, the serotype has the potential to be a diarrheagenic foodborne pathogen in humans.

Escherichia coli serotype O157:H7 continues to pose a serious health threat to human beings. Cattle, a major reservoir of the pathogen, harbor E. coli O157:H7 in their gastrointestinal tract and shed variable concentrations of E. coli O157:H7 into the environment. Genetic characterization of cattle-shed E. coli O157 strains is of interest to the livestock industry, food business, and public health community. The present study applied whole genome shotgun sequencing (WGS) and single nucleotide variant (SNV) calling to characterize 279 cattle-shed E. coli O157:H7 strains isolated from a single feedlot located in southwestern region of the US. More than 4,000 SNVs were identified among the strains and the resultant phylogenomic tree revealed three major groups. Using the Sakai strain genome as reference, more than 2,000 SNVs were annotated and a detailed SNV map generated. Results clearly revealed highly polymorphic loci along the E. coli O157:H7 genome that aligned with the prophage regions and highly variant genes involved in processing bacterial genetic information. The WGS data were further profiled against a comprehensive virulence factor database (VFDB) for virulence gene identification. Among the total 285 virulence genes identified, only 132 were present in all the strains. There were six virulence genes unique to single isolates. Our findings suggested that the genome variations of the E. coli O157:H7 were mainly attributable to dynamics of certain phages, and the bacterial strains have variable virulence gene profiles, even though they came from a single cattle population, which may explain the differences in pathogenicity, host prevalence, and transmissibility by E. coli O157:H7.

Escherichia coli O103, harbored in the hindgut and shed in the feces of cattle, can be enterohemorrhagic (EHEC), enteropathogenic (EPEC), or putative non-pathotype. The genetic diversity particularly that of virulence gene profiles within O103 serogroup is likely to be broad, considering the wide range in severity of illness. However, virulence descriptions of the E. coli O103 strains isolated from cattle feces have been primarily limited to major genes, such as Shiga toxin and intimin genes. Less is known about the frequency at which other virulence genes exist or about genes associated with the mobile genetic elements of E. coli O103 pathotypes. Our objective was to utilize whole genome sequencing (WGS) to identify and compare major and putative virulence genes of EHEC O103 (positive for Shiga toxin gene, stx1, and intimin gene, eae; n = 43), EPEC O103 (negative for stx1 and positive for eae; n = 13) and putative non-pathotype O103 strains (negative for stx and eae; n = 13) isolated from cattle feces. Six strains of EHEC O103 from human clinical cases were also included. All bovine EHEC strains (43/43) and a majority of EPEC (12/13) and putative non-pathotype strains (12/13) were O103:H2 serotype. Both bovine and human EHEC strains had significantly larger average genome sizes (P < 0.0001) and were positive for a higher number of adherence and toxin-based virulence genes and genes on mobile elements (prophages, transposable elements, and plasmids) than EPEC or putative non-pathotype strains. The genome size of the three pathotypes positively correlated (R2 = 0.7) with the number of genes carried on mobile genetic elements. Bovine strains clustered phylogenetically by pathotypes, which differed in several key virulence genes. The diversity of E. coli O103 pathotypes shed in cattle feces is likely reflective of the acquisition or loss of virulence genes carried on mobile genetic elements.

Liver abscesses (LA) continue to be an economic challenge for the beef industry. Chronic ruminal acidosis and ruminitis contribute to LA development. Fusobacterium necrophorum subsp. necrophorum, a ruminal bacterium, is the primary causative agent. Damaged ruminal, and possibly other gut regions, become susceptible to invasion and colonization by subsp. necrophorum, which reaches the liver via portal blood to cause abscesses. We hypothesize that the LA occurrence is related to the presence and concentrations of subsp. necrophorum in gut contents and epithelial tissues. Our primary objective was to detect, isolate and quantify subsp. necrophorum in gut contents and tissues in steers with experimentally induced LA. An experimental model, which included ruminal acidosis followed by intraruminal inoculation of subsp. necrophorum alone or with Salmonella enterica serotype Lubbock, was used to assess the relationship. Steers (n = 61) from three independent studies with three treatment groups were used. Treatments included: 1) forage-based diet with intraruminal inoculation of subsp. necrophorum; 2) ruminal acidosis with intraruminal inoculation of subsp. necrophorum; and 3) ruminal acidosis with intraruminal inoculation of subsp. necrophorum and S. Lubbock. Steers were euthanized 3 wk after bacterial inoculation and liver, gut contents, and tissue samples (ruminal, ileal and colonic) from steers with LA (n = 27) and with healthy livers (n = 34) were collected and subjected to culture method and quantitative PCR to isolate, detect, and quantify subsp. necrophorum. Data were analyzed by PROC GLIMMIX of SAS after summarizing frequency of isolation in various sample types within each study for steers with and without LA and converted to a binomial proportion. Concentrations were analyzed using steer as the experimental unit. The model included the fixed effect of liver status (abscessed or not) and study as the random effect. The frequency of isolation and mean concentration of subsp. necrophorum in purulent materials of LA were 100% and 3.5 x 107 Log10 CFU/g, respectively. The frequency of isolation of subsp. necrophorum from ruminal tissues of steers with LA was greater than steers with healthy livers (35.4 vs 14.5%; P = 0.04). Likewise, the concentration of subsp. necrophorum in ruminal tissues was greater in steers with LA than with healthy livers (4.2 vs 3.6 Log10 CFU/g; P < 0.001). None of the ileal and colonic tissues, except one ileal tissue in each group, yielded subsp. necrophorum. The concentrations of subsp. necrophorum in ruminal, ileal, and colonic contents did not differ (P = 0.9) between steers with LA or with healthy livers. Our data suggest that the occurrence of LA is positively related to prevalence and concentration of subsp. necrophorum in ruminal tissue, and the colonization likely serves as a source of continuous flow of bacterial emboli into the portal blood to initiate LA development. [ABSTRACT FROM AUTHOR]

Fusobacteria necrophorum is an important opportunistic bacterial pathogen associated with liver abscesses, foot rot, necrotic laryngitis and endometritis in both dairy and beef cattle. Recently, we identified F. necrophorum as one of the taxa in the uterine microbiota that are positively associated with pregnancy outcome in beef cows, and that Fusobacterium was identified as the most dominant genus in the bovine seminal microbiota. These findings indicate that Fusobacterium spp. may have a role in reproductive health and cattle fertility. Our objectives were to detect and isolate the F. necrophorum subsp necrophorum (FNN), F. necrophorum subsp. funduliforme (FNF) and F. varium (FV) in seminal, vaginal and uterine microbial ecosystems as well as fetal samples by culture and real-time PCR (qPCR) methods. The qPCR was performed on the DNA (n = 215) extracted from bull semen (n = 37), ram semen (n = 100), and vaginal (n = 25) and uterine swabs (n = 32), caruncles (n = 9), allantoic fluid (n = 6), and amniotic fluid (n = 6) from pregnant beef cows. The qPCR targeted the hgdA gene, and leukotoxin promoter regions (lktA-n and lktA-f) to detect and quantify FV, FNN and FNF, respectively. Cryopreserved samples (n = 174; 57 bull semen, 10 ram semen, 38 vaginal, and 47 uterine swabs) were cultured to detect and isolate FNN, FBF and FV by direct plating and after culturing in anaerobic peptoneyeast extract broth containing sodium lactate broth with josamycin, vancomycin and norfloxacin for selective enrichment of FN and FV. Also, 28 samples of 6-mo-old bovine fetuses caruncles, allantoic and amniotic fluids, and intestinal tissue) were screened for Fusobacterium species. By qPCR, FNF was detected in 75.7% of bull semen, and 8.0% of vaginal swabs as well as 33.3% of both allantoic fluid and caruncles, but was undetected in ram semen, uterine swabs, and amniotic fluids. Subspecies necrophorum was identified in 37.8% of bull semen, 3% of ram semen, 8.0% of vaginal and 15.6% of uterine 55.6 % of caruncles, 50% of allantoic fluid samples, but was not detected in amniotic fluid samples. Meanwhile FV was only detected in vaginal, uterine swabs and caruncles at 4%, 6.3%, and 11.1%, respectively. The culture method detected FNF in 63.2% of bull semen and 5.3% of vaginal samples, while FNN was identified in 1.8% of bull semen and 2.1% of uterine samples. F. varium was not isolated from any of the samples. Overall, FNF was the most prevalent subspecies present in the seminal microbiome of bulls. Identification of FNN subspecies and FV in seminal, vaginal, and uterine microbiota of healthy cattle, as well as in healthy calf fetus-associated samples suggests potential transfer from bulls to cows and then on to offspring, and potential involvement of the Fusobacterium spp. in cattle fertility. [ABSTRACT FROM AUTHOR]

Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the public health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx1, stx2) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and was meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system.

Escherichia coli O104:H4, an hybrid pathotype of Shiga toxigenic and enteroaggregative E. coli, involved in a major foodborne outbreak in Germany in 2011, has not been detected in cattle feces. Serogroup O104 with H type other than H4 has been reported to cause human illnesses, but their prevalence and characteristics in cattle have not been reported. Our objectives were to determine the prevalence of E. coli O104 in feces of feedlot cattle, by culture and PCR detection methods, and characterize the isolated strains. Rectal fecal samples from a total of 757 cattle originating from 29 feedlots were collected at a Midwest commercial slaughter plant. Fecal samples, enriched in E. coli broth, were subjected to culture and PCR methods of detection. The culture method involved immunomagnetic separation with O104-specific beads and plating on a selective chromogenic medium, followed by serogroup confirmation of pooled colonies by PCR. If pooled colonies were positive for the wzxO104 gene, then colonies were tested individually to identify wzxO104-positive serogroup and associated genes of the hybrid strains. Extracted DNA from feces were also tested by a multiplex PCR to detect wzxO104-positive serogroup and associated major genes of the O104 hybrid pathotype. Because wzxO104 has been shown to be present in E. coli O8/O9/O9a, wzxO104-positive isolates and extracted DNA from fecal samples were also tested by a PCR targeting wbdDO8/O9/O9a, a gene specific for E. coli O8/O9/O9a serogroups. Model-adjusted prevalence estimates of E. coli O104 (positive for wzxO104 and negative for wbdDO8/O9/O9a) at the feedlot level were 5.7% and 21.2%, and at the sample level were 0.5% and 25.9% by culture and PCR, respectively. The McNemar's test indicated that there was a significant difference (P < 0.01) between the proportions of samples that tested positive for wzxO104 and samples that were positive for wzxO104, but negative for wbdDO8/O9/O9a by PCR and culture methods. A total of 143 isolates, positive for the wzxO104, were obtained in pure culture from 146 positive fecal samples. Ninety-two of the 143 isolates (64.3%) also tested positive for the wbdDO8/O9/O9a, indicating that only 51 (35.7%) isolates truly belonged to the O104 serogroup (positive for wzxO104 and negative for wbdDO8/O9/O9a). All 51 isolates tested negative for eae, and 16 tested positive for stx1 gene of the subtype 1c. Thirteen of the 16 stx1-positive O104 isolates were from one feedlot. The predominant serotype was O104:H7. Pulsed-field gel electrophoresis analysis indicated that stx1-positive O104:H7 isolates had 62.4% homology to the German outbreak strain and 67.9% to 77.5% homology to human diarrheagenic O104:H7 strains. The 13 isolates obtained from the same feedlot were of the same PFGE subtype with 100% Dice similarity. Although cattle do not harbor the O104:H4 pathotype, they do harbor and shed Shiga toxigenic O104 in the feces and the predominant serotype was O104:H7.

Culture-based methods to detect the six major non-O157 (O26, O45, O103, O111, O121 and O145) Shiga toxin-producing E. coli (STEC) are not well established. Our objectives of this study were to develop a culture-based method to detect the six non-O157 serogroups in cattle feces and compare the detection with a PCR method. Fecal samples (n = 576) were collected in a feedlot from 24 pens during a 12-week period and enriched in E. coli broth at 40° C for 6 h. Enriched samples were subjected to immunomagnetic separation, spread-plated onto a selective chromogenic medium, and initially pooled colonies, and subsequently, single colonies were tested by a multiplex PCR targeting six serogroups and four virulence genes, stx1, stx2, eae, and ehxA (culture method). Fecal suspensions, before and after enrichment, were also tested by a multiplex PCR targeting six serogroups and four virulence genes (PCR method). There was no difference in the proportions of fecal samples that tested positive (74.3 vs. 77.4%) for one or more of the six serogroups by either culture or the PCR method. However, each method detected one or more of the six serogroups in samples that were negative by the other method. Both culture method and PCR indicated that O26, O45, and O103 were the dominant serogroups. Higher proportions (P < 0.05) of fecal samples were positive for O26 (44.4 vs. 22.7%) and O121 (22.9 vs. 2.3%) serogroups by PCR than by the culture method. None of the fecal samples contained more than four serogroups. Only a small proportion of the six serogroups (23/640; 3.6%) isolated carried Shiga toxin genes. The culture method and the PCR method detected all six serogroups in samples negative by the other method, highlighting the importance of subjecting fecal samples to both methods for accurate detection of the six non-O157 STEC in cattle feces.

We evaluated the association of diet type, ruminal acidosis bout frequency, and intraruminal bacterial inoculum concentration on liver abscess (LA) development in steers. Beef × dairy steers [n = 40, initial body weight (BW) = 107 ± 11 kg] were assigned randomly to 1 of 5 treatments that included either a highdose (HD; 1 × 109 and 1 × 109 CFU/mL) or a low-dose (LD; 1 × 106 and 1 × 106 CFU/mL) of Fusobacterium necrophorum subsp. necrophorum and Salmonella enterica serovar Lubbock, respectively. Treatments included: NCON = high-forage diet with no intraruminal inoculation; CON+HD = high-forage diet with a single, HD inoculation; 2AD+HD = 2 acidotic diet cycles with a single, HD inoculation; 4AD+3LD = 4 acidotic diet cycles with a series of 3 LD inoculations; and 4AD+HD = 4 acidotic diet cycles with a single, HD inoculation. Individual animal was the experimental unit. Continuous data were analyzed using mixed models, and categorical data were analyzed as binomial proportions with treatment, time, and their interactions as the fixed effects. Hematology was evaluated before harvest on d 21 to evaluate indications of systemic disease. Although no difference was observed in rumenitis among treatments (P = 0.79), steers with LA presented with 8 percentage points greater rumenitis than steers without LA. Steers consuming the high-forage diet also presented with rumenitis. Prevalence of LA was 0, 37.5, 37.5, 50, and 12.5% for NCON, CON+HD, 2AD+HD, 4AD+3LD, and 4AD+HD, respectively (P = 0.68). A key observation of this study was that steers fed a highforage diet presented with LA when dosed with of F. necrophorum and S. enterica, even in the absence of the acidotic diet. [ABSTRACT FROM AUTHOR]