Epidemiological Cutoff Values for Standard Broth Microdilution and Disk Diffusion Susceptibility Testing of Aeromonas hydrophila Isolated from Fish.

Aeromonas hydrophila and other closely related Aeromonas species cause motile aeromonad septicemia, a common fish disease. The disease affects many aquaculture sectors potentially requiring antimicrobial treatments. Therefore, researchers and laboratory diagnosticians need criteria called epidemiological cutoff values (ECVs) to determine whether a bacterial isolate has developed decreased susceptibility to an antimicrobial. To generate ECVs for this bacterium, we assembled a diverse collection of 245 isolates previously identified as A. hydrophila from fish. Using rpoD sequencing, we confirmed that 97 of the 245 isolates were A. hydrophila. We allocated the isolates among three laboratories and tested their susceptibility against eight antimicrobials using standard Clinical and Laboratory Standards Institute (CLSI) disk diffusion and broth microdilution methods. The resulting frequency distributions were statistically analyzed to determine wild-type cutoff estimates, which, along with scatterplots, were used to estimate potential ECVs. In collaboration with the CLSI, aquaculture working group, we proposed ECVs for six of the eight antimicrobials tested. Subsequently, the CLSI Subcommittee on Veterinary Antimicrobial Susceptibility Testing reviewed our data and approved the ECVs to be added to the 2020 edition of the VET04 performance standards for antimicrobial susceptibility testing of aquatic bacteria.

Pathology and Epidemiology of Oxalate Nephrosis in Cheetahs.

To investigate cases of acute oxalate nephrosis without evidence of ethylene glycol exposure, archived data and tissues from cheetahs ( Acinonyx jubatus) from North America ( n = 297), southern Africa ( n = 257), and France ( n = 40) were evaluated. Renal and gastrointestinal tract lesions were characterized in a subset of animals with ( n = 100) and without ( n = 165) oxalate crystals at death. Crystals were confirmed as calcium oxalate by Raman spectroscopy in 45 of 47 cheetahs tested. Crystals were present in cheetahs from 3.7 months to 15.9 years old. Cheetahs younger than 1.5 years were less likely to have oxalates than older cheetahs ( P = .034), but young cheetahs with oxalates had more oxalate crystals than older cheetahs ( P < .001). Cheetahs with oxalate crystals were more likely to have renal amyloidosis, interstitial nephritis, or colitis and less likely to have glomerular loop thickening or gastritis than those without oxalates. Crystal number was positively associated with renal tubular necrosis ( P ≤ .001), regeneration ( P = .015), and casts ( P ≤ .001) but inversely associated with glomerulosclerosis, renal amyloidosis, and interstitial nephritis. Crystal number was unrelated to the presence or absence of colitis and was lower in southern African than American and European animals ( P = .01). This study found no evidence that coexisting chronic renal disease (amyloidosis, interstitial nephritis, or glomerulosclerosis), veno-occlusive disease, gastritis, or enterocolitis contributed significantly to oxalate nephrosis. Oxalate-related renal disease should be considered as a potential cause of acute renal failure, especially in young captive cheetahs. The role of location, diet, stress, and genetic predisposition in the pathogenesis of oxalate nephrosis in cheetahs warrants further study.

Reproductive toxicity in rats with crystal nephropathy following high doses of oral melamine or cyanuric acid.

The industrial chemical melamine was used in 2007 and 2008 to raise the apparent protein content in pet feed and watered down milk, respectively. Because humans may be exposed to melamine via several different routes into the human diet as well as deliberate contamination, this study was designed to characterize the effect of high dose melamine or cyanuric acid oral exposure on the pregnant animal and developing fetus, including placental transfer. Clear rectangular crystals formed following a single triazine exposure which is a different morphology from the golden spherulites caused by combined exposure or the calculi formed when melamine combines with endogenous uric acid. Crystal nephropathy, regardless of cause, induces renal failure which in turn has reproductive sequelae. Specifically, melamine alone-treated dams had increased numbers of early and late fetal deaths compared to controls or cyanuric acid-treated dams. As melamine was found in the amniotic fluid, this study confirms transfer of melamine from mammalian mother to fetus and our study provides evidence that cyanuric acid also appears in the amniotic fluid if mothers are exposed to high doses.

Depletion of melamine and cyanuric acid in serum from catfish Ictalurus punctatus and rainbow trout Onchorhynchus mykiss.

Melamine and its triazine analogs, such as cyanuric acid, have been used to artificially inflate protein content both in animal feed ingredients, as well as in milk products produced for human consumption. We report here a LC-MS/MS method to quantify and confirm melamine and cyanuric acid in serum from channel catfish and rainbow trout with a limit of quantification of 0.8 µg/mL. The method was applied to serum samples from a residue depletion study in which fish were given a single oral dose of 20 mg/kg body weight melamine, cyanuric acid, or both compounds together. Samples were taken at 1, 3, 7, 14, and 28 days (an additional 42 day was added for trout). When given alone or in combination with cyanuric acid, melamine residues were highest on day 1 in both catfish and trout. Cyanuric acid was only quantifiable at day 1 in trout when given alone, and not at all in catfish. The serum half life of melamine in catfish was 1.50-1.62 days and 3.09-3.67 days in trout. This work highlights the differences of depletion kinetics in fish, which can be measured in days, as compared to the depletion in mammals, measured in hours.

A No Observable Adverse Effects Level (NOAEL) for pigs fed melamine and cyanuric acid.

Ingesting melamine adulterated milk products led to kidney stones in many infants in 2008. This differs from the renal failure caused by intratubular crystal formation after co-ingestion of melamine (MEL) and cyanuric acid (CYA) in adulterated pet foods in 2007. To better understand the potential risk of developing crystal nephropathy following co-ingestion of MEL and CYA, we fed 16 weanling pigs 0, 1, 3.3, 10, 33, or 100 mg/kg bw/day of each MEL and CYA, or 200 mg/kg bw/day of either compound individually for 7 days. Crystals were found in the renal medulla and cortex and urine sediments of all pigs fed both MEL and CYA each at 10 mg/kg bw/day (or greater). Crystals were also found in one of the two pigs fed 200 mg/kg bw/day MEL-only. In a 28 day study, 36 weanling pigs were fed 0, 1, or 3.3 mg/kg bw/day of MEL and CYA or 200 mg/kg bw/day MEL-only. Only one of the 3.3 mg/kg MEL and CYA pig kidneys contained crystals. The no-observed-adverse-effect level (NOAEL) for pigs fed MEL and CYA for 28 days was concluded to be 1.0 mg/kg bw/day corresponding to 25 mg/kg (ppm) MEL and 25 mg/kg (ppm) CYA in dry feed.