1. Crude salivary gland extract of the giant Amazon leech, Haementeria ghilianii, contains an inhibitor of plasma factor XIIIa. 2. The inhibitory agent was purified to homogeneity by anion-exchange, cation-exchange, gel-filtration and reverse-phase chromatography to yield a single band on SDS/PAGE with an apparent molecular mass of 7.3 kDa. It has been named tridegin. 3. Micro-sequencing of proteolytic fragments showed tridegin to be a peptide of 66 amino acids. The sequence is unique with little similarity to other leech-derived proteins. 4. Inhibition of plasma factor XIIIa activity was confirmed by four independent methods: tridegin increased the solubility of fibrin clots in urea, inhibited ammonia produced from the incorporation of ethylamine into casein, inhibited the incorporation of 5'-(biotinamido)pentylamine into casein and prevented gamma-dimer formation in clotting fibrinogen. 5. The IC50 of tridegin (approx. 9.2 nM) is very close to the concentration of factor XIIIa used in the assay and in fact depends on its concentration. This is the most potent inhibitor of factor XIIIa yet described. 6. Tridegin also inhibits platelet factor XIIIa (factor XIIIAa) with a similar potency to that of the plasma enzyme. 7. Tridegin also inhibits tissue transglutaminase but with lower potency and independently of the enzyme concentration. 8. Tridegin appears to be specific for transglutaminases, since it has no effect on the coagulation times of human plasma, on thrombin or factor Xa. Moreover it has no effect on other thiol-containing enzymes and has no ability to digest fibrinogen or cleave the isopeptide substrate, L-gamma-glutamyl-4-nitroanilide.

SummaryTridegin is a potent inhibitor of factor Xllla from the leech, Haementeria ghilianii, which inhibits protein cross-linking. It modifies plasmin-mediated fibrin degradation as shown by the absence of D-dimer and approximately halves the time for fibrinolysis. Plasma clots formed in the presence of Tridegin lyse more rapidly when either streptokinase, tissue plasminogen activator or hementin is added 2 h after clot formation. The effect of Tridegin is markedly increased if clots are formed from platelet-rich plasma. Platelet-rich plasma clots are lysed much more slowly by the fibrinolytic enzymes used and if Tridegin is present, the rate of lysis returns almost to that of platelet- free clots. These studies indicate the important role of platelets in conferring resistance to commonly used fibrinolytic enzymes and suggest that protein cross-linking is an important step in this effect. Moreover they indicate that Tridegin, a small polypeptide, may have potential as an adjunct to thrombolytic therapy.

Transmission of antimicrobial resistance (AMR) from animal production systems to humans through the food supply is a public health concern. Currently, little is known about the prevalence of AMR among veal calves in the United States. Therefore, the objective of this prospective cohort study was to estimate the prevalence of AMR and multidrug resistance (MDR) among Escherichia coli within a vertically integrated production system. In addition, this study aimed to identify genes associated with phenotypic resistance to third- and fourth-generation cephalosporins (3GC and 4GC). Calves from four veal cohorts were randomly sampled resulting in a total of 166 farm fecal samples, 159 harvest fecal swabs, 164 preevisceration swabs, and 122 final carcass swabs. The prevalence of MDR among random-pick E. coli isolates recovered from the respective samples was 97% (161/166), 35% (55/159), 61% (51/84), and 24% (5/21). A selective isolation protocol found cefotaxime (a 3GC)-resistant isolates in 91% (127/140) of farm fecal samples, 34% (55/164) of preevisceration swabs, and 19% (23/122) of final carcass swabs tested. Isolates resistant to cefepime, a 4GC, were found among 24% (33/140), 6.7% (11/164), and 0.8% (1/122) of the same, respective samples. Isolates resistant to ciprofloxacin, a fluoroquinolone, were recovered from 75% (73/98) of farm fecal samples, 23% (38/164) of preevisceration swabs, and 6.6% (8/122) of final carcass swabs. The bla and bla resistance genes were found in 89% (93/105) and 100% (42/42) of tested subsets of 3GC- and 4GC-resistant isolates, respectively. Pulsed-field gel electrophoresis (PFGE) analysis conducted on 3GC- and fluoroquinolone-resistant isolates showed three indistinguishable PFGE patterns from cefotaxime-resistant isolates recovered at farm and from two preevisceration carcass swabs. Although the prevalence of resistance declined between initial farm fecal samples and final carcass swabs, resistant bacteria recovered from carcasses illustrate the potential transmission of AMR to the human food supply.

The objective of this study was to determine the prevalence, serotypes, antimicrobial resistance phenotypes, and pulsed-field gel electrophoresis (PFGE) patterns of Salmonella recovered in feces and mesenteric and prefemoral lymph nodes (LNs) from cohorts of calves with and without a confirmed outbreak of salmonellosis. In a prospective cohort study, 160 calves from four farms without a reported outbreak (nonoutbreak farms) were sampled at farm and harvest. In addition, harvest samples from 80 calves of two farms with a confirmed outbreak (outbreak farms) were collected. A culture protocol for Salmonella isolation was applied for all samples and recovered isolates were further characterized by serotyping, antimicrobial susceptibility testing, and PFGE. Among nonoutbreak farms, Salmonella was recovered from 0% (0/160) farm fecal samples, 3.7% (6/160) harvest fecal swabs, 21.9% (35/160) mesenteric LNs, and 0.6% (1/160) prefemoral LNs. Serotypes identified in nonoutbreak herds included Salmonella Typhimurium, Cerro, Hartford, and Newport. Most isolates (64.3%, 27/42) exhibited a unique multidrug-resistant (MDR) phenotype, including resistance to extended-spectrum cephalosporins. Salmonella prevalence in harvest fecal samples and prefemoral LNs among calves from outbreak farms was numerically higher, but not significantly different than those without an outbreak. Serotypes recovered from outbreak farms included Salmonella Heidelberg and Typhimurium, and the monophasic Salmonella Typhimurium strains 4,5,12:i:- and 4,12:i:-, which have been also reported as highly pathogenic in humans. All isolates (33/33) exhibited an MDR phenotype. Salmonella strains recovered from ill calves in two outbreaks had indistinguishable PFGE patterns, suggesting between-farm transmission. In addition, the genotype of Salmonella Heidelberg causing an outbreak among calves was recovered from three prefemoral LNs of surviving members of the cohort at harvest. Implementation of preharvest biosecurity measures (limited personnel and visitor traffic, vehicle, footwear, and utensils disinfection) should be highly recommended to decrease the prevalence of Salmonella on farms and safeguard the food safety.

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