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In a previous study, Multiplex-nanopore-sequencing based whole genome sequencing (WGS) allowed for accurate in silico serotype prediction of Salmonella within one day for five multiplexed isolates, using both SISTR and SeqSero2. Since only ten serotypes were tested in our previous study, the conclusions above were yet to be evaluated in a larger scale test. In the current study we evaluated this workflow with 69 Salmonella serotypes and also explored the feasibility of using multiplex-nanopore-sequencing based WGS for antimicrobial resistance gene (AMR) and virulence gene detection. We found that accurate in silico serotype prediction with nanopore-WGS data was achieved within about five hours of sequencing at a minimum of 30× Salmonella genome coverage, with SeqSero2 as the serotype prediction tool. For each tested isolate, small variations were observed between the AMR/virulence gene profiles from the Illumina and Nanopore sequencing platforms. Taking results generated using Illumina data as the benchmark, the average precision value per isolate was 0.99 for both AMR and virulence gene detection. We found that the resistance gene identifier – RGI identified AMR genes with nanopore data at a much lower accuracy compared to Abricate, possibly due to RGI’s less stringent minimum similarity and coverage by default for database matching. This study is an evaluation of multiplex-nanoporesequencing based WGS as a cost-efficient and rapid Salmonella classification method, and a starting point for future validation and verification of using it as a AMR/virulence gene profiling tool for the food industry. This study paves the way for the application of nanopore sequencing in surveillance, tracking, and risk assessment of Salmonella across the food supply chain. [ABSTRACT FROM AUTHOR]

The objective of this study was to validate two programs (SUPERSAT and EQUIL 2) for calculation of calcium oxalate (CaOx) and magnesium ammonium phosphate (struvite; MAP) relative supersaturation (RSS) in dog and cat urine. Healthy adult cats (n = 10) and dogs (n = 9) were fed standard diets for a 3-wk period. Urine was collected (24 h, dogs; 48 h, cats) and filtered, and the pH was measured. A 20-mL aliquot was titrated to pH 2 and frozen for analysis. Additional aliquots were incubated with 1 g seed crystals at 38[degree]C; CaOx for 24 h (cat) and 2, 6 and 9 d (dog); MAP for 48 h (dog) and 6 d (cat). Samples were analyzed for 10 substances. RSS was calculated using EQUIL 2 and SUPERSAT. CaOx RSS (SUPERSAT): dog urine was initially supersaturated, whereas cat urine was undersaturated with the diets used. Cat urine reached the solubility product (K[sub.sp]), (RSS = 1) after 24-h incubation, whereas dog urine was still approaching K[sub.sp] at 9 d. MAP RSS (SUPERSAT): urine from both species was undersaturated and increased toward K[sub.sp] during incubation. Final RSS values were compared for both programs. SUPERSAT resulted in values close to 1 for both CaOx and MAP; EQUIL 2 gave similar values for CaOx RSS, although MAP RSS values were considerably higher than 1. In conclusion, EQUIL 2 and SUPERSAT both calculated reasonably accurate RSS values for CaOx, whereas only SUPERSAT provided an accurate measure of MAP RSS. Key Words: * urine * cat * dog * supersaturation * computer program

Acidification of urine through dietary modification is considered to be the cornerstone for the management and prevention of struvite (magnesium ammonium phosphate) urolithiasis in cats (Taton et al. 1984). Urinary acidification, however, may be a risk factor for calcium oxalate urolithiaisis (Kirk et al. 1995, Osborne et al. 1995). The aim of this study was to compare the effect of commercial dry cat foods on urinary pH, and struvite and calcium oxalate relative supersaturation (RSS), the ultimate determinant of crystallization. Materials and methods. Nine different dry cat foods were fed to groups of six healthy, adult domestic short-haired cats 29 neutered females and 25 neutered males) for periods of between 14 and 21 d. Food allowances were calculated on the basis of an assumed maintenance requirement of 250 kJ/(kg body weight [multiplied by] d); the daily allowance was fed as two meals at 0800 and 1530 h. The cats were housed individually throughout each trial. All housing conditions and procedures fell within the UK Home Office regulations. During the last week of each trial, a 48-hour urine collection was made over dry ice to ensure rapid freezing of the samples. At the end of the collection period, the samples were thawed, and volume and pH were measured. The samples were acidified to pH 2 with hydrochloric acid and frozen for further analysis. Urine concentrations of sodium, potassium, magnesium, calcium, chloride, sulfate, phosphate, oxalate, citrate, pyrophosphate, ammonium and uric acid were analyzed by HPLC. A computer program, Equil 2 (Werness et al. 1985), was used to calculate urinary RSS for struvite and calcium oxalate from the concentrations of these analytes. This resulted in one struvite and one calcium oxalate RSS value for each cat during each trial. RSS is calculated from the activity product of the sample divided by the solubility product for the crystal in question; thus, values of KEY WORDS: cats, feline, urine pH, relative supersaturation, dry cat food

Urolithiasis, with or without urinary tract infection, is an important cause of lower urinary tract disease in dogs. Research has focused on gaining understanding of the factors that could either contribute toward urolith formation or be of value in the management of clinical cases associated with their presence. Work in this area has focused on the influences of urinary tract infection and the effects of dietary minerals, water turnover and urinary pH. A noninvasive system has been established at the Waltham Centre for Pet Nutrition to monitor various aspects of canine urinary tract health. A similar feline system has previously been described (Markwell and Smith 1993). Materials and methods. Dogs were individually housed in an environmentally controlled two-room pen consisting of an inner room (3.75 [m.sup.2]) entered from a central corridor and an outer room (2 [m.sup.2]). The floor covering in the inner room was heat-sealed sheet vinyl extending 40 cm up the walls; the outer room consisted of a fiberglass tray. A section of each inner pen had an underfloor warm bed area heated by electric cable. There was a separate air supply and extract for each pen with 12 air changes per hour. Warm air was supplied to the inner room and extracted from the outer conservatory. The temperature of the inner room was maintained at 22 [+ or -] 2 [degrees] C. In this study, a dry feline clinical diet (Waltham Veterinary Diet Feline Control pHormula, Effem Foods, Bolton, Cananda), designed for the treatment of struvite-associated urolithiasis, was fed to a panel of six dogs (one Labrador retriever, neutered female, age 5.8 y; two miniature schnauzers, one neutered male, one entire male, mean age 3.7 [+ or -] 0.1 y; three beagles, two neutered females, one neutered male, mean age 9.8 [+ or -] 2.5 y) for 42 d. The dogs were fed individually, to adult maintenance energy requirements (calculated as 460 body [weight.sup.0.75] kJ/d), three times daily at 0830, 1130 and 1530 h, and had free access to water. Blood samples were collected at the end of the study to assess the risk of metabolic acidosis. All housing conditions and procedures fell within the UK Home Office regulations. Urine samples were obtained by training dogs to urinate in the fiberglass tray. The urine rapidly drained into a glass U-tube that housed a combined pH and temperature electrode attached to a pH meter. The computer software (Signal Centre, Computer Park Software, Kettering, Northants, UK) recorded urine pH and temperature data from each pH meter every 30 s. Every 24 h, the data were analyzed by the software, which identifies urinations by an increase in the temperature of urine in the U-tube of at least 2 [degrees] C above ambient temperature. The software program was also designed to indicate freshly voided samples by auditory and visual means, allowing immediate sample collection when microscopic examination was required. The collection system can also be modified to freeze urine on voiding. Samples collected in this manner over a 48-h period are then defrosted, titrated to pH 2 with hydrochloric acid and analyzed for sodium, potassium, magnesium and calcium, chloride, sulfate, phosphate, oxalate, citrate, pyrophosphate, ammonia, uric acid and creatinine by HPLC. Urinary relative supersaturation (activity product/solubility product) for four urolith types are then calculated by computer program (Equil 89D, University of Florida, Gainesville, FL). A urinary relative supersaturation value of 1.0 indicates that the urine is oversaturated with respect to that urolith type. Results. Individual mean urine pH values in the dogs were all KEY WORDS: dogs, canine, urine pH, saturation, urinary tract health

Nutrient intake and urine composition were analyzed in calcium oxalate (CaOx)stone-forming and healthy control dogs to identify factors that contribute to CaOx urolithiasis. Stone-forming dogs had significantly lower intake of sodium, calcium, potassium, and phosphorus and significantly higher urinary calcium and oxalate concentrations, calcium excretion, and CaOx relative supersaturation (RSS). Feeding a diet used in the treatment of canine lower urinary tract disease for 1 month was associated with increased intake of moisture, sodium, and fat; reduced intake of potassium and calcium; and decreased urinary calcium and oxalate concentrations, calcium excretion, and CaOx RSS. No clinical signs of disease recurrence were observed in the stone-forming dogs when the diet was fed for an additional 11 months. The results suggest that hypercalciuria and hyperoxaluria contribute to the formation of CaOx uroliths in dogs and show that dietary modifications can alter this process.