Identification of Aspergillus and Mucorales in formalin-fixed, paraffin-embedded tissue samples: Comparison of specific and broad-range fungal qPCR assays.

Establishing the etiology of invasive fungal infections is important to guide therapeutic options and for epidemiologic purposes. Formalin-fixed, paraffin-embedded (FFPE) tissue specimens from patients with proven invasive fungal infections are valuable to determine the etiology of systemic fungal infections. We compared different polymerase chain reaction (PCR) amplification strategies from FFPE tissue blocks to identify agents of invasive fungal infections. We found that specific PCR assays show superior sensitivity in the identification of DNA of Mucorales and Aspergillus and mixed infections caused by both as compared to broad-range PCR assays. Shorter amplicon lengths and less detection of contaminating fungal DNA are potential factors involved. However, detection of fungal DNA by highly sensitive specific PCR assays in the absence of demonstration of fungal elements in tissue suggests that PCR results should be interpreted in the context of the histopathology and clinical findings.

Outcomes after prophylactic gastrectomy for hereditary diffuse gastric cancer.

BACKGROUND: Patients with hereditary diffuse gastric cancer and a CDH1 mutation have a 60-80 per cent lifetime risk of developing diffuse gastric cancer. Total prophylactic gastrectomy eliminates this risk, but is associated with considerable morbidity. The effectiveness (removal of all gastric mucosa) and outcomes of this procedure were evaluated retrospectively. METHODS: All consecutive individuals undergoing a prophylactic gastrectomy for a CDH1 mutation or gastric signet ring cell foci at the authors' institute between 2005 and 2017 were included. RESULTS: In 25 of 26 patients, intraoperative frozen-section examination (proximal resection margin) was used to verify complete removal of gastric mucosa. All definitive resection margins were free of gastric mucosa, but only after the proximal margin had been reresected in nine patients. In the first year after surgery, five of the 26 patients underwent a relaparotomy for adhesiolysis (2 patients) or jejunostomy-related complications (3 patients). Six patients were readmitted to the hospital within 1 year for nutritional and/or psychosocial support (4 patients) or surgical reintervention (2 patients). Mean weight loss after 1 year was 15 (95 per cent c.i. 12 to 18) per cent. For the 25 patients with a follow-up at 1 year or more, functional complaints were reported more frequently at 1 year than at 3 months after the operation: bile reflux (15 versus 11 patients respectively) and dumping (11 versus 7 patients). The majority of patients who worked or studied before surgery (15 of 19) had returned fully to these activities within 1 year. CONCLUSION: The considerable morbidity and functional consequences of gastrectomy should be considered when counselling individuals with an inherited predisposition to diffuse gastric cancer. Intraoperative frozen-section examination is recommended to remove all risk-bearing gastric mucosa.

The effects of progressing and nonprogressing Mycobacterium avium ssp. paratuberculosis infection on milk production in dairy cows.

Longitudinal data from 3 commercial dairy herds in the northeast United States, collected from 2004 to 2011, were analyzed to determine the effect of Mycobacterium avium ssp. paratuberculosis (MAP) infection status and progression path on milk production. Disease status, as indicated by MAP test results, was determined through quarterly ELISA serum testing, biannual fecal culture, and culture of tissues and feces at slaughter. Milk production data were collected from the Dairy Herd Information Association. Animals with positive MAP test results were categorized, based on test results over the full course of the study, as high path (at least one high-positive culture) or low path (at least one positive culture or ELISA). The cumulative numbers of positive ELISA and culture results were recorded. The effects of both MAP infection path, status, and number of positive tests on milk production were analyzed using a mixed linear model with an autocorrelation random effect structure. Low- and high-path animals produced more milk before their first positive test than always-negative animals, especially high-path animals. Although mean production decreased after a first positive test, low-path animals were shown to recover some productivity. High-path animals continued to exhibit a decrease in milk production, especially after their first high-positive fecal culture. These results show that not all animals that test positive for MAP will have long-term production losses. Milk production decreased significantly with each additional positive test. Ultimately, production loss appeared to be a function of MAP infection progression.

Molecular detection of the index case of a subclinical Salmonella Kentucky epidemic on a dairy farm.

Salmonella enterica commonly colonizes the intestinal tract of cattle and is a leading cause of foodborne illness. A previously described investigation into the prevalence of S. enterica on a dairy farm revealed an 8-year-long asymptomatic S. enterica epidemic caused by serotypes Cerro and Kentucky in the lactating herd. To investigate the source of the S. Kentucky strains, the genomes of two S. Kentucky isolates were sequenced; one collected prior to the epidemic (2004) and one collected during the epidemic (2010). Comparative genomic analysis demonstrated significant polymorphisms between the two strains. PCR primers targeting unique and strain-specific regions were developed, and screening of the archived isolates identified the index case of the asymptomatic S. Kentucky epidemic as a heifer that was raised off-site and transported onto the study farm in 2005. Analysis of isolates collected from all heifers brought onto the farm demonstrated frequent re-introduction of clones of the epidemic strain suggesting transmission of pathogens between farms might occur repeatedly.

Antimicrobial Activity of Ceftolozane-Tazobactam, Ceftazidime-Avibactam, and Cefiderocol against Multidrug-Resistant Pseudomonas aeruginosa Recovered at a German University Hospital.

Multidrug-resistant (MDR) Pseudomonas aeruginosa increasingly causes health care-associated infections. In this study, we determined the activity of ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol against 223 MDR P. aeruginosa clinical isolates recovered from 2013 to 2017 at the University Hospital Frankfurt by using MIC test strips. Furthermore, we evaluated the presence of genes encoding major ß-lactamases, such as VIM, IMP, NDM, GIM, SPM, and KPC; the extended spectrum ß-lactamase (ESBL)-carbapenemase GES; and the virulence-associated traits ExoS and ExoU, as in particular ExoU is thought to be associated with poor clinical outcome. For MDR P. aeruginosa isolates, the MIC50/MIC90 values of ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol were 8/>256 mg/L, 16/>256 mg/L, and 0.25/1 mg/L, respectively. Cefiderocol showed the highest susceptibility rate (97.3%) followed by ceftazidime-avibactam (48.4%) and ceftolozane-tazobactam (46.6%). In 81 (36.3%) isolates, carbapenemase gene blaVIM was detected, and in 5 (2.2%) isolates, blaGES was detected (with a positive association of exoU and blaVIM). More than half of the isolates belong to the so-called international P. aeruginosa "high-risk" clones, with sequence type 235 (ST235) (24.7%) being the most prevalent. This study underlines that ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol are important options for the treatment of infections due to MDR P. aeruginosa, with cefiderocol currently being the most active available antipseudomonal ß-lactam agent. According to our clinical experience, the outcome of cefiderocol therapy (8 patients) was favorable especially in cases of MDR P. aeruginosa-associated complicated urinary tract infections. IMPORTANCE After testing ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol against a collection of 233 multidrug-resistant (MDR) Pseudomonas aeruginosa, we showed that cefiderocol is the most active antipseudomonal ß-lactam agent (susceptibility rates were 46.6%, 48.4%, and 97.4%, respectively). The most prevalent one was sequence type 235 (ST235) (24.7%), followed by ST244, ST175, and ST233, with all belonging to the top 10 P. aeruginosa high-risk clones with worldwide distribution. Our data indicate that during surveillance studies special attention should be paid to the MDR and highly virulent VIM- and ExoU-producing variant of ST235. Furthermore, in the case of infections caused by carbapenemase-producing MDR P. aeruginosa, cefiderocol is the preferred treatment option, while outcomes of complicated urinary tract infections and hospital-acquired pneumonia with cefiderocol were favorable.

Identification of loci associated with tolerance to Johne's disease in Holstein cattle.

Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (Map), is a fatal disease in cattle. The objective of this study was to identify loci associated with tolerance in cows infected with Map. Tolerance was defined as a cow's fitness at a given level of Map infection intensity. Fitness was measured by Map faecal cultures, and Map infection intensity was measured by culturing four gut tissues. The quantitative phenotype of tolerance was defined by numerical indexes of cultures of peak (peak tolerance, PT) and average (average tolerance, AT) faecal and tissue Map from 245 Holstein cows. The categorical phenotype was defined as: ≥ 100 cfu Map tissue infection, and faecal shedding ≥ 75 cfu (intolerant) or <10 cfu (tolerant cows). In 94 cows, Map was identified in ≥ 1 tissue, including 44 cows with ≥ 100 Map tissue cfu and 36 with ≥ 1 faecal cfu. A genome-wide association analysis was performed after filtering, leaving genotypes for 45,789 SNPs in 90 animals for the quantitative phenotype and 16 cases and 25 controls for the categorical analysis of tolerance. rs41748405:A>C (BTA15) was associated with PT (P = 1.12 × 10(-7)) and AT (P = 2.17 × 10(-6)). Associations were identified with PT and adjacent SNPs ss61512613:A>G and ss61530518:A>G (BTA6) (P < 3.0 × 10(-5)), and with AT for ss61469568:A>G (BTA 2) (P = 3.3 × 10(-5)) and ss86284768:A>G (BTA1) (P = 3.31 × 10(-5)). For the categorical phenotype, an association was found with ss8632653:A>G (BTA6) (P < 5.0 × 10(-5)). This is the first study to identify loci associated with tolerance to Johne's disease.

Effect of somatic cell count in goat milk on yield, sensory quality, and fatty acid profile of semisoft cheese.

This study investigated the effect of somatic cell count (SCC) in goat milk on yield, free fatty acid (FFA) profile, and sensory quality of semisoft cheese. Sixty Alpine goats without evidence of clinical mastitis were assigned to 3 groups with milk SCC level of <500,000 (low), 500,000 to 1,000,000 (medium), and 1,000,000 to 1,500,000 (high) cells/mL. Thirty kilograms of goat milk with mean SCC levels of 410,000 (low), 770,000 (medium), and 1,250,000 (high) cells/mL was obtained for the manufacture of semisoft cheese for 2 consecutive weeks in 3 lactation stages. The composition of milk was analyzed and cheese yield was recorded on d 1. Cheese samples on d 1, 60, and 120 were analyzed for total sensory scores, flavor, and body and texture by a panel of 3 expert judges and were also analyzed for FFA. Results indicated that milk composition did not change when milk SCC varied from 214,000 to 1,450,000 cells/mL. Milk with higher SCC had a lower standard plate count, whereas coliform count and psychrotrophic bacteria count were not affected. However, milk components (fat, protein, lactose, casein, and total solids) among the 3 groups were similar. As a result, no significant differences in the yield of semisoft goat cheeses were detected. However, total sensory scores and body and texture scores for cheeses made from the high SCC milk were lower than those for cheeses made from the low and medium SCC milks. The difference in milk SCC levels also resulted in diverse changes in cheese texture (hardness, springiness, and so on) and FFA profiles. Individual and total FFA increased significantly during ripening, regardless the SCC levels. It is concluded that SCC in goat milk did not affect the yield of semisoft cheese but did result in inferior sensory quality of aged cheeses.

Biofilm in milking equipment on a dairy farm as a potential source of bulk tank milk contamination with Listeria monocytogenes.

The objective of this study was to assess the presence of a Listeria monocytogenes-containing biofilm in milking equipment as a potential source of bulk tank milk contamination on a dairy farm where milk contamination had been previously documented. Samples were collected from milking equipment and milking parlor premises on 4 occasions and analyzed for the presence of L. monocytogenes. Pulsed-field gel electrophoresis (PFGE) typing was conducted on L. monocytogenes isolates from the milking equipment, parlor and storage room floors, bulk tank milk, and in-line milk filters. Pieces from milk meters and rubber liners were obtained to visually assess the presence of a biofilm using scanning electron microscopy. A total of 6 (15%), 4 (25%), and 1 (6%) samples were culture-positive for L. monocytogenes in the first, second, and third sample collection, respectively. Two samples were L. monocytogenes hly PCR-positive but were culture-negative in the fourth sample collection. Combined AscI and ApaI restriction analysis yielded 6 PFGE types for 15 L. monocytogenes isolates obtained from milking equipment, parlor, bulk tank milk, and milk filters. A predominant and persistent PFGE type (PFGE type T) was observed among these L. monocytogenes isolates (9/15 isolates). Scanning electron microscopy of samples from the bottom cover of 2 milk meters showed the presence of individual and clusters of bacteria, mainly associated with surface scratches. The presence of a bacterial biofilm was observed on the bottom covers of the 2 milk meters. Prevention of the establishment of biofilms in milking equipment is a crucial step in fulfilling the requirement of safe, high-quality milk.

Effect of Johne's disease status on reproduction and culling in dairy cattle.

Among the costs attributed to Mycobacterium avium ssp. paratuberculosis (MAP) infection in dairy cattle, the effects on reproduction and culling are the least documented. To estimate the cost of MAP infections and Johne's disease in a dairy herd, the rates of calving and culling were calculated for cows in each stage of MAP infection relative to uninfected cows. Data from 6 commercial dairy herds, consisting of 2,818 cows with 2,754 calvings and 1,483 cullings, were used for analysis. Every cow in each study herd was tested regularly for MAP, and herds were followed for between 4 and 7 yr. An ordinal categorical variable for Johne's disease status [test-negative, low-positive (low-shedding or ELISA-positive only), or high-shedding] was defined as a time-dependent variable for all cows with at least 1 positive test result or 2 negative test results. A Cox regression model, stratified on herd and controlling for the time-dependent infection variable, was used to analyze time to culling. Nonshedding animals were significantly less likely to be culled in comparison with animals in the low-shedding or ELISA-positive category, and high-shedding animals had nonsignificantly higher culling rates than low-shedding or ELISA-positive animals. Time to calving was analyzed using a proportional rates model, an analog to the Andersen-Gill regression model suitable for recurrent event data, stratifying on herd and weighted to adjust for the dependent censoring caused by the culling effects described above. High-shedding animals had lower calving rates in comparison with low-shedding or ELISA-positive animals, which tended to have higher calving rates than test-negative animals.

A whole genome association analysis identifies loci associated with Mycobacterium avium subsp. paratuberculosis infection status in US holstein cattle.

The purpose of this study was to identify loci associated with Mycobacterium avium subspecies paratuberculosis (Map) infection status in US Holsteins using the Illumina BovineSNP50 BeadChip whole genome single nucleotide polymorphism (SNP) assay. Two hundred forty-five cows from dairies in New York, Pennsylvania and Vermont enrolled in longitudinal herd studies between January 1999 and November 2007 were assessed for the presence of Map in both faecal and tissue samples. An animal was considered tissue infected if any sample contained at least one colony forming unit of Map per gram of tissue (CFU/g) and the same definition was employed for faecal samples. Each animal was genotyped with the Illumina BovineSNP50 BeadChip and after quality assurance filtering, 218 animals and 45 683 SNPs remained. We sought to identify loci associated with four different case/control classifications: presence of Map in the tissue, presence of Map in faeces, presence of Map in both tissue and faeces and presence of Map in tissue but not faeces. A case-control genome wide association study was conducted to test the four different classifications of Map infection status (cases) when compared with a Map-negative control group (control). Regions on chromosomes 1, 5, 7, 8, 16, 21 and 23 were identified with moderate significance (P < 5 x 10(-5)). Two regions, one on chromosome 3 (near EDN2) and another on chromosome 9 (no positional gene candidates), were identified with a high level of association to the presence of Map in tissue and both tissue and faeces respectively (P < 5 x 10(-7), genome-wide Bonferonni P < 0.05).

A longitudinal study on the impact of Johne's disease status on milk production in individual cows.

Longitudinal data from 3 commercial dairy herds in the northeast United States were collected from 2004 to 2007. Johne's disease status, as indicated by Mycobacterium avium ssp. paratuberculosis infection levels, was determined through quarterly ELISA serum testing, biannual fecal culture, and culture of tissues at slaughter. Milk production data were collected from the Dairy Herd Improvement Association. The effect of Johne's disease status on milk production was analyzed using a mixed linear model with an autocorrelation random effect structure. Infected animals produced more milk than uninfected cows before they began shedding M. avium ssp. paratuberculosis. Cows infected with M. avium ssp. paratuberculosis had monthly decreases of 0.05 to 1 kg in daily milk production relative to uninfected animals, with greater decreases in progressive disease categories. Animals with fecal culture results of >30 cfu/g produced approximately 4 kg less milk per day compared with uninfected cows. These results will be valuable in calculating the economic effect of Johne's disease.

Dynamics of endemic infectious diseases of animal and human importance on three dairy herds in the northeastern United States.

Endemic infectious diseases in dairy cattle are of significant concern to the industry as well as for public health because of their potential impact on animal and human health, milk and meat production, food safety, and economics. We sought to provide insight into the dynamics of important endemic infectious diseases in 3 northeastern US dairy herds. Fecal samples from individual cows and various environmental samples from these farms were tested for the presence of major zoonotic pathogens (i.e., Salmonella, Campylobacter, and Listeria) as well as commensal bacteria Escherichia coli and enterococci. Additionally, the presence of Mycobacterium avium ssp. paratuberculosis was tested in fecal and serum samples from individual cows. Test results and health and reproductive records were maintained in a database, and fecal, plasma, DNA, and tissue samples were kept in a biobank. All bacteria of interest were detected on these farms and their presence was variable both within and between farms. The prevalence of Listeria spp. and L. monocytogenes in individual fecal samples within farm A ranged from 0 to 68.2% and 0 to 25.5%, respectively, over a period of 3 yr. Within farm B, continuous fecal shedding of Salmonella spp. was observed with a prevalence ranging from 8 to 88%; Salmonella Cerro was the predominant serotype. Farm C appeared less contaminated with Salmonella and Listeria, although in the summer of 2005, 50 and 19.2% of fecal samples were positive for Listeria and L. monocytogenes, respectively. The high prevalence of E. coli (89 to 100%), Enterococcus (75 to 100%), and Campylobacter (0 to 81%) in feces suggested they were ubiquitous throughout the farm environment. Fecal culture and ELISA results indicated a low prevalence of Mycobacterium avium ssp. paratuberculosis infection in these farms (0 to 13.6% and 0 to 4.9% for culture-positive and ELISA-positive, respectively), although the occasional presence of high shedders was observed. Results have major implications for food safety and epidemiology by providing a better understanding of infectious disease dynamics on dairy farms. Comprehensive understanding of these infections may lead to better farm management practices and pathogen reduction programs to control and reduce the on-farm contamination of these pathogens and to prevent their further entry into the food-chain.

Healthcare burden of probable and proven invasive mucormycosis: a multi-centre cost-of-illness analysis of patients treated in tertiary care hospitals between 2003 and 2016.

BACKGROUND: Invasive mucormycosis (IM) is a rare invasive fungal infection with a high mortality rate. However, data concerning the clinical and economic burden of IM are scarce. AIM: To evaluate the direct treatment costs and additional expenditures of patients with IM. METHODS: A retrospective cost-of-illness analysis of cases with IM extracted from FungiScope - Global Registry for Emerging Fungal Infections, accessible through the epidemiological research platform www.ClinicalSurveys.net, was undertaken. Results of patients with IM were compared with those of matched patients with similar underlying conditions based on the German Diagnosis Related Group (G-DRG) coding. FINDINGS: Out of 46 patients with probable/proven IM, 31 (67%) patients were male and the median age was 53 years (range 11-88 years). Forty-two patients (92%) had haematological diseases as the most common risk factor. Analysis of cost factors identified antifungal treatment due to IM as the primary cost driver [€22,816, 95% confidence interval (CI) €15,036-32,346], with mean overall direct treatment costs of €53,261 (95% CI €39,660-68,825). Compared with matched patients, patients with IM were treated in hospital for 26.5 additional days (standard deviation 31.8 days; P < 0.001), resulting in mean additional costs of €32,991 (95% CI €21,558-46,613; P < 0.001). Probable IM, as well as absence of chemotherapy, surgical measures due to IM, and antifungal prophylaxis were associated with lower overall costs. Nineteen patients (41.3%) died during hospitalization. CONCLUSION: This study demonstrates the considerable healthcare burden of IM. The choice of antifungal agent for treatment of IM had no impact on overall cost.

The importance of culling in Johne's disease control.

Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection and results in economic losses in the dairy industry. To control MAP transmission in herds, test-based culling has been recommended and immediate culling of high shedding animals is typically implemented. In this study, we quantified the effects of MAP control in US dairy herds, using the basic reproduction ratio R(0). The effectiveness of culling strategies was evaluated for good and poor herd management (low- and high-transmission rates, respectively) by a phase diagram approach. To establish a quantitative relationship between culling rates and test properties, we defined the average detection times for low and high shedding animals. The effects of various culling strategies and test characteristics, such as test sensitivity, test turnaround time, and testing interval, were analyzed. To understand the overall effect of model parameters on R(0), we performed global uncertainty and sensitivity analyses. We also evaluated the effectiveness of culling only high shedding animals by comparing three test methods (fecal culture, fecal polymerase chain reaction, PCR, and enzyme-linked immunosorbent assay, ELISA). Our study shows that, in the case of good herd management, culling of only high shedding animals may be effective in controlling MAP transmission. However, in the case of poor management, in addition to immediate culling of high shedding animals, culling of low shedding animals (based on the fecal culture test) will be necessary. Culling of low shedding animals may be delayed 6-12 months, however, if a shorter testing interval is applied. This study suggests that if farmers prefer culling only high shedding animals, faster MAP detection tests (such as the fecal PCR and ELISA) of higher sensitivity should be applied with high testing frequency, particularly on farms with poor management. Culling of infectious animals with a longer testing interval is generally not effective to control MAP.

Environmental sampling to predict fecal prevalence of Salmonella in an intensively monitored dairy herd.

Although dairy cattle are known reservoirs for salmonellae, cattle that are shedding this organism are often asymptomatic and difficult to identify. A dairy herd that was experiencing a sustained, subclinical outbreak of Salmonella enterica subsp. enterica Cerro was monitored for 2 years. Fecal samples from the lactating cows were collected every 6 to 8 weeks and tested for the presence of Salmonella. Fecal prevalence of Salmonella fluctuated throughout the observation period and ranged from 8 to 88%. Manure composites and water trough samples were collected along with the fecal samples, and bulk milk and milk filters were cultured for the presence of Salmonella on a weekly basis. Over 90% of the manure composites--representing high-animal-traffic areas-were positive at each sampling. Salmonella was detected in 11% of milk samples and in 66% of the milk filters. Results of weekly bulk milk quality testing (i.e., bulk tank somatic cell score, standard plate count, preliminary incubation count) were typically well within acceptable ranges. Milk quality variables had low correlations with herd Salmonella fecal prevalence. When observed over time, sampling period average prevalence of Salmonella in milk filters closely paralleled fecal prevalence of Salmonella in the herd. Based on results of this study, milk filters appear to be an effective method for monitoring shedding prevalence at the herd level. In-line filter testing is also a more sensitive measure of Salmonella, and perhaps other pathogens, in raw milk than testing the milk alone.

Incidence of Escherichia coli O157:H7 and E. coli virulence factors in US bulk tank milk as determined by polymerase chain reaction.

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring Dairy 2002 survey, were analyzed for the presence of several genes encoding virulence factors associated with enterohemorrhagic forms of Escherichia coli (EHEC) using real-time and conventional PCR assays. Samples from 859 farms in 21 states were collected and enriched in EC medium at 42.5 degrees C to amplify any E. coli present, and DNA was isolated from the resulting biomass. The eaeA gene encoding intimin, a virulence factor associated with enteropathogenic forms of E. coli and EHEC, was detected in 199 (23%) of the samples. Thirty-six samples (4.2%) were positive for eaeA, the gamma allele of the translocated intimin receptor (gamma-tir), found in EHEC strains of O157:H7, and one or both shiga-like toxin genes (stx1 and stx2), a combination that may be indicative of the presence of O157:H7 EHEC. Testing these 36 samples with a commercially available real-time PCR kit for detection of O157:H7 indicated that 5 samples could be contaminated with O157:H7. A multiplex PCR to detect the presence of fliC, rfbE, and hlyA genes found in O157:H7 reduced to 2 (0.2% of all samples) the number of samples likely to be contaminated with this organism. A strain of O157:H7 (eaeA+, gamma-tir+, stx2+, rfbE+, fliC+, hlyA+) was subsequently isolated from one sample. Thirty-four eaeA-positive samples did not contain detectable gamma-tir but did contain one or both of the stx genes suggesting the presence of EHEC strains other than O157:H7. These results indicate a low incidence of O157:H7 in bulk tank milk but suggest that a risk from other enteropathogenic and EHEC forms of E. coli may exist and that PCR targeting virulence factors associated with highly pathogenic forms of E. coli may be an effective means of detecting potential dangers in raw milk.

Subtyping Listeria monocytogenes from bulk tank milk using automated repetitive element-based PCR.

Sixty-one Listeria monocytogenes strains from raw milk were analyzed with an automated repetitive element-based PCR (rep-PCR) system to examine the utility of this system for serotype grouping and to determine whether specific regional relationships could be identified. Results of the similarity analysis revealed two primary clusters of L. monocytogenes isolates. Cluster 2 exclusively contained serogroup 1/2a isolates; however, two 1/2a isolates were also found in cluster 1. Isolates of serogroups 1/2b, 4b, 3b, and 4c were also in cluster 1. Clusters 1 and 2 were separated at a relative similarity of 86%. Listeria species other than L. monocytogenes (L. ivanovii, L. seeligeri, L. welshimeri, L. grayi, and L. innocua) had similarity scores of less than 80% in pairwise comparisons with the L. monocytogenes isolates. Thus, this method may be useful for species identification once an isolate is characterized as Listeria. When rep-PCR fingerprints of the L. monocytogenes 1/2a isolates were compared, there was no apparent regional grouping. However, discrimination between isolates suggests that the rep-PCR assay might be useful for tracking L. monocytogenes 1/2a and for tracking isolates across regions or within smaller ecological niches. The automated rep-PCR method could not discriminate between serotypes 1/2b and 4b but may be useful for discriminating between 1/2a and other serotypes and for tracking isolates within serotype 1/2a.

Evaluation of quick tests for phosphorus determination in dairy manures.

Nutrients in animal manure are valuable inputs in agronomic crop production. Rapid and timely information about manure nutrient content are needed to minimize the risks of phosphorus (P) over-application and losses of dissolved P (DP) in runoff from fields treated with manure. We evaluated the suitability of a commercial hand-held reflectometer, a hydrometer, and an electrical conductivity (EC) meter for determining DP and total P (TP) in dairy manures. Bulk samples (n = 107) collected from farms across CT, MD, NY, PA, and VA were highly variable in total solids (TS) concentration, ranging from 11 to 213gL(-1), in suspensions' pH (6.3-9.2), and EC (6.2-53.3 dS m(-1)). Manure DP concentrations measured using the RQFlex reflectometer (RQFlex-DP(s)) were related to molybdate-reactive P (MRP(s)) concentrations as follows: RQFlex-DP(s) = 0.471 x MRP(s) + 1102 (r2 = 0.29). Inclusion of pH and squared-pH terms improved the prediction of manure DP from RQFlex results (r2 = 0.66). Excluding five outlier samples that had pH < or = 6.9 the coefficient of determination (r2) for the MRP(s) and RQFlex-DP(s) relationship was 0.83 for 95% of the samples. Manure TS were related to hydrometer specific gravity readings (r2 = 0.53) that were in turn related to TP (r2 = 0.34), but not to either RQFlex-DP or MRP. Relationships between suspensions' EC and DP or TP were non-significant. Therefore, the RQFlex method is the only viable option for on-site quick estimates of DP that can be made more robust when complemented with TS and pH measurements. The DP quick test can provide near real-time information on soluble manure nutrient content across a wide range of handling and storage conditions on dairy farms and quick estimates of potential soluble P losses in runoff following land applications of manure.

Prevalence of Salmonella enterica in bulk tank milk from US dairies as determined by polymerase chain reaction.

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring System Dairy 2002 survey, were analyzed for the presence of Salmonella enterica using a commercially available real-time polymerase chain reaction (PCR) kit. Samples from 854 farms in 21 states were collected and enriched in tetrathionate broth to amplify any salmonellae present, and DNA was isolated from the resulting biomass. One hundred one samples (11.8%) were shown to contain Salmonella enterica using the real-time PCR assay, whereas conventional culture techniques detected the pathogen in only 22 (2.6%) of the samples. A conventional PCR assay targeting a different gene from Salmonella enterica confirmed the presence of the organism in 94 of the real-time PCR-positive samples. Thus, assay of milk samples by real-time PCR indicates that the prevalence of Salmonella enterica in US bulk tank milk is substantially higher than previously reported.

Cerebrovascular, cardiovascular and renal hypertensive disease after hypertensive disorders of pregnancy.

BACKGROUND: Strong associations have been established in nationwide registry studies between hypertensive disorders of pregnancy (HDP) and later vascular morbidities and mortality. The aim of this case-control study is to examine the interdependent relationships of different predictive factors for vascular disease and HDP, because they are not clearly elucidated due to lack of detail in registries. METHODS AND RESULTS: We assembled three different case groups of women who had cerebrovascular, cardiovascular, or hypertensive kidney disease before the age of 55. The control group consisted of age-matched women who underwent hysterectomy for benign reasons. We assessed the occurrence of HDP in previous pregnancies. The strength of the association with vascular morbidities was tested with multivariable logistic regression in comparison with classic vascular risk factors. In all case groups, previous HDP occurred more frequent than in the control group. In logistic regression analysis, previous HDP were the strongest predictor in the cerebrovascular group (OR 4.2; 95% confidence interval [CI] 1.6-11.0). In the cardiovascular group and the kidney failure group a similar association was found, however, this was not statistically significant (OR 4.4 (95% CI 0.82-4) and 2.9 (95% CI 0.61-14), respectively). CONCLUSIONS: Previous hypertensive disorders of pregnancy are a strong predictor for later vascular morbidity. This is partially mediated through the presence of classic vascular risk factors, but our data suggest it is also an independent predictor.