Surveillance of Salmonella prevalence in animal feeds and characterization of the Salmonella isolates by serotyping and antimicrobial susceptibility.

This article presents the surveillance data from the Feed Contaminants Program (2002-2009) and Salmonella Assignment (2007-2009) of the U.S. Food and Drug Administration (FDA), which monitor the trend of Salmonella contamination in animal feeds. A total of 2,058 samples were collected from complete animal feeds, feed ingredients, pet foods, pet treats, and supplements for pets in 2002-2009. These samples were tested for the presence of Salmonella. Those that were positive for Salmonella underwent serotyping and testing for antimicrobial susceptibility. Of the 2,058 samples, 257 were positive for Salmonella (12.5%). The results indicate a significant overall Salmonella reduction (p≤0.05) in animal feeds from 18.2% (187 samples tested) in 2002 to 8.0% (584 samples tested) in 2009. Among these samples, feed ingredients and pet foods/treats had the most significant reduction (p≤0.05). Of the 45 Salmonella serotypes identified, Salmonella Senftenberg and Salmonella Montevideo were the top two common serotypes (8.9%). Of the 257 Salmonella isolates obtained, 54 isolates (21%) were resistant to at least one antimicrobial. The findings provide the animal feed industries with Salmonella prevalence information that can be used to address Salmonella contamination problems. Our findings can also be used to educate pet owners when handling pet foods and treats at home to prevent salmonellosis.

Gonococcal pilus vaccine. Studies of antigenicity and inhibition of attachment.

A gonococcal pilus vaccine or placebo was injected subcutaneously or intramuscularly into 71 human volunteers. The vaccine was found to be safe. The principal adverse reaction was a complaint of a sore arm, which was caused, at least in part, to the volume of material injected. 6 of 64 (9%) volunteers receiving the larger doses also complained of malaise. The vaccine was found to be antigenic. All of the volunteers developed an immunoglobulin class-specific antibody response as measured by a solid phase radioimmunoassay. The antibody was capable of blocking the attachment of gonococci to epithelial cells. A slight antibody response was also demonstrated to gonococcal lipopolysaccharide but the antibody responsible for blocking attachment of gonococci was directed entirely at the pilus protein. The stimulated antibodies were shown to crossreact with isolated pili of heterologous gonococcal strains and to block the attachment of heterologous gonococci. Absorption of immune sera by a heterologous pilus reduced the inhibition of attachment antibodies to pre-immune level, suggesting that the immune response was directed at a common pilus determinant.

Effect of anticoagulants and heat on the detection of tumor necrosis factor in murine blood.

Assays for tumor necrosis factor (TNF) may be inhibited by nonspecific factors present in body fluids. We found that the ability to quantitate TNF is greatly affected by blood processing methods. Mice were anesthetized by inhalation of methoxyflurane before obtaining blood by cardiac puncture. The blood from a group of mice was allowed to clot before recovery of serum. Plasmas were obtained from three other groups of mice after collection of blood into tubes containing EDTA, sodium citrate, or preservative-free heparin. The pooled serum or plasmas were spiked with rhTNF to a final concentration of 1 microgram/ml and aliquoted for frozen storage. The serum and plasmas were divided into heated (56 degrees C for 30 min) and unheated portions prior to a standard L929 cytotoxicity assay. Comparison of absorbances at 595 nm after crystal violet staining of cells revealed differences in detection of TNF in plasmas compared to serum and in heated compared to nonheated samples. Citrated plasma clotted in the assay at dilutions at or below 1:25. EDTA plasma consistently produced unexplained lysis of L929 cells in both heated and unheated unspiked samples. We conclude that TNF levels should be determined only in heat treated serum samples, and that comparisons be made against both a TNF standard and a TNF standard prepared in normal homologous serum that can be heated and assayed in parallel with the test samples.

Trehalose dimycolate enhances survival of fission neutron-irradiated mice and Klebsiella pneumoniae-challenged irradiated mice.

The survival of B6D2F1 female mice exposed to lethal doses of fission neutron radiation is increased when trehalose dimycolate (TDM) preparations are given either 1 h after exposure or 1 day before exposure to radiation. TDM in an emulsion of squalene, Tween 80, and saline was the most effective formulation for increasing the 30-day survival of mice when given 1 day before (90%) or 1 h after (88%) exposure to radiation. An aqueous suspension of a synthetic analog of TDM was less effective at increasing 30-day survival (60%) when given 1 day prior to radiation exposure and not effective when given 1 h after radiation. Mice receiving a sublethal dose (3.5 Gy) of fission neutron radiation and either the TDM emulsion or synthetic TDM 1 h after irradiation were substantially more resistant to challenge with 10, 100, 1000, or 5000 times the LD50/30 dose of Klebsiella pneumoniae than untreated mice.

Brain CCK receptors: species differences in regional distribution and selectivity.

The binding of 125I-CCK-33 to its receptors prepared from cerebral cortex and cerebellum was studied in four species: mouse, rat, hamster, and guinea pig. Only the guinea pig showed significant binding to membranes from cerebellum and this binding was comparable to that observed for cerebral cortex. In all four species, the order of potency of unlabeled analogs to compete for the binding site was CCK-8 greater than CCK-33 greater than desulfated CCK-8 greater than CCK-4. While the affinity for CCK-8 and CCK-33 was similar in the various species, the relative affinity for desulfated CCK-8 and CCK-4 was less for hamster and guinea pig, indicating species differences in receptor specificity, as well as in regional localization.

Cholecystokinin induces the interaction of its receptor with a guanine nucleotide binding protein.

To evaluate the relation between the pancreatic cholecystokinin (CCK) receptor and guanine nucleotide-binding protein(s) we studied the effects of nucleotides on 125I-CCK binding to pancreatic acinar plasma membranes, 125I-CCK binding to solubilized 125I-CCK receptors, and the stability of the solubilized 125I-CCK-receptor complex. In plasma membranes, guanine nucleotides both inhibited CCK binding and increased the dissociation of CCK from its receptor. The potency of the nucleotides studied was GTP gamma S = GMP-PNP greater than GTP much greater than ATP. When membranes were solubilized with digitonin, subsequent binding of CCK was insensitive to guanine nucleotides including GTP, GMP-PNP and GTP gamma S. However, if CCK binding occurred before solubilization of the membranes, guanine nucleotides increased dissociation at concentrations and with a specificity similar to that observed for effects on intact pancreatic membranes. It is concluded that guanine nucleotides act via a protein which is separable from the receptor to induce dissociation of bound CCK. Moreover, CCK binding induces an association in the plasma membrane of the CCK receptor with this guanine nucleotide binding protein.

Synthetic trehalose dicorynomycolate (S-TDCM): behavioral effects and radioprotection.

This study evaluated synthetic trehalose dicorynomycolate (S-TDCM), an immunomodulator, for its survival enhancing capacity and behavioral toxicity in B6D2F1 female mice. In survival experiments, mice were administered S-TDCM (25-400 micrograms/mouse i.p.) 20-24 hr before 5.6 Gy mixed-field fission-neutron irradiation (n) and gamma-photon irradiation. The 30-day survival rates for mice treated with 100-400 micrograms/mouse S-TDCM were significantly enhanced compared to controls. Toxicity of S-TDCM was measured in nonirradiated mice by locomotor activity, food intake, water consumption, and alterations in body weight. A dose-dependent decrease was noted in all behavioral measures in mice treated with S-TDCM. Doses of 100 and 200 micrograms/mouse S-TDCM significantly reduced motor activity beginning 12 hr postinjection with recovery by 24 hr. A dose of 400 micrograms/mouse significantly decreased activity within the first 4 hr after administration and returned to control levels by 32 hr following injection. Food and water intake were significantly depressed at doses of 200 and 400 micrograms/mouse on the day following drug administration, and were recovered in 24 hr. Body weight was significantly decreased in the 200 micrograms/mouse group for 2 days and in the 400 micrograms/mouse group for 4 days following injection. A dose of 100 micrograms/mouse effectively enhanced survival after fission-neutron irradiation with no adverse effect on food consumption, water intake, or body weight and a minimal, short-term effect on locomotor activity.

Cancer incidence and mortality trends in Northeastern Ontario.

Over 629,000 people reside in the catchment area for the Northeastern Ontario Regional Cancer Centre. Historically, the area was renowned for employment in mining, forestry and lumbering, agriculture, the railway, and pulp and paper. At present, it is known for mining; community, business, and personal services; trade; manufacturing; and construction. Comparison of cancer incidence and mortality trends for two decades (1971-1980 and 1981-1990) with those of Ontario has revealed statistically significant excesses, at the 5% level or better, of trachea, bronchus, and lung cancer cases (SIR = 123 for 1971-1980 and 125 for 1981-1990) and deaths in men (SMR = 116 and 125, respectively); for women, excesses were observed for trachea, bronchus and lung cancer case (SIR = 114 and 118), and cervical cancer cases (SIR = 142 and 115) and deaths (SMR = 133 and 128). Enhanced recruitment strategies and early educational interventions are identified as priorities.

Naturally occurring toxins in feedstuffs: Center for Veterinary Medicine Perspective.

The objectives of this review are to provide 1) information on the FDA Feed Contaminants Program, 2) the legal history of aflatoxins and their current action levels, 3) a report on the levels of aflatoxins, fumonisins, vomitoxin, ochratoxin A, and zearalenone in domestic and import surveillance samples of feed during fiscal years 1989 through 1992, and 4) information on naturally occurring toxins encountered recently by the Center for Veterinary Medicine. Ten of 644 (1.6%) domestic corn samples and 7 of 106 (6.6%) domestic cottonseed samples contained aflatoxins at levels > 300 ppb. The mean fumonisin level in the 1990 survey of 85 corn screening samples was 12.1 ppm, and the values ranged from 2.6 to 32 ppm. The mean vomitoxin levels in the 1991 survey of 207 winter wheat samples and 206 spring wheat samples was 2.4 and .9 ppm, respectively. Ochratoxin A was not detected in 168 samples. Zearalenone was detected at levels > .15 ppm in only 1 of 161 samples. Cottonseed containing 13,000 ppm gossypol was recently implicated in the deaths of dairy cows. Crambe meal and canola meal are sanctioned for use in feed with certain restrictions, including the levels of glucosinolates. The FDA is continuing its surveillance and will strive to provide guidance on the increasing number of naturally occurring toxins.

Organohalogen and organophosphorus pesticides in mixed feed rations: findings from FDA's domestic surveillance during fiscal years 1989-1994.

During Fiscal Years 1989-1994, the U.S. Food and Drug Administration (FDA) collected and analyzed 545 domestic surveillance samples of mixed feed rations (172 for cattle, 125 for poultry, 83 for swine, 61 for pets, 56 for fish, and 48 miscellaneous). All samples were analyzed by gas-liquid chromatography for organohalogen and organophosphorus pesticides. Of the 545 samples, 88 (16.1%) did not contain detectable pesticide residues. In the 457 samples with detectable pesticide levels, 804 residues (654 quantitable and 150 trace) were found. None of these 804 residues exceeded regulatory guidance. Malathion, chlorpyrifos-methyl, diazinon, chlorpyrifos, and pirimiphos-methyl were the most commonly detected pesticides. These 5 organophosphorus pesticides accounted for 93.4% of all pesticide residues detected (malathion, 52.9%; chlorpyrifos-methyl, 25.2%; diazinon, 7.7%; chlorpyrifos, 4.9%; and pirimiphos-methyl, 2.7%). Their median values in samples containing quantitable levels ranged from 0.014 to 0.098 ppm. The most commonly detected organohalogen compounds were methoxychlor, DDE, PCB, dieldrin, pentachloronitrobenzene, and lindane. These 6 compounds combined accounted for only 4.1% of all residues detected. FDA is continuing its pesticide surveillance of feeds to help ensure animal safety and prevent violative residues in food derived from animals.

Sediment source tracing in a lowland agricultural catchment in southern England using a modified procedure combining statistical analysis and numerical modelling.

Catchment erosion, soil losses and resulting sediment pressures continue to represent cause for concern with respect to the ecological vitality and amenity value of riverine systems, including those in the agricultural catchments of southern England. Given that the sources of fine-grained sediment are typically diffuse in nature, it is essential to adopt a catchment-wide perspective to corresponding management strategies and sediment source tracing procedures have proved useful in assisting such planning. There remains, however, scope for further refining sediment sourcing procedures and on that basis, a recent study in the upper River Kennet (~214 km(2)) catchment in southern England, provided an opportunity for designing and testing a refined statistical procedure for sediment source discrimination with composite fingerprints using Genetic Algorithm (GA)-driven Discriminant Function Analysis, the Kruskal-Wallis H-test and Principal Components Analysis. The revised statistical verification of composite signatures was combined with numerical mass balance modelling using recent refinements including a range of tracer weightings and both local and GA optimisation. Comparison of the local and global optimisation increased confidence in the outputs of local optimisation and the goodness-of-fit for the predicted source contributions using the optimum composite signatures selected from the revised statistical testing ranged from 0.914 to 0.965. Overall relative frequency-weighted average median source type contributions were estimated to be 4% (agricultural topsoils; predicted deviate median inputs 1-19%), 55% (unmetalled farm track surfaces; predicted deviate median inputs 9-91%), 6% (damaged road verges; predicted deviate median inputs 4-42%), 31% (channel banks/subsurface sources; predicted deviate median inputs 5-41%) and 4% (urban street dust; predicted deviate median inputs 0-20%). The study provides further evidence of the importance of eroding farm tacks as a catchment scale sediment source and confirms the utility of tracing for assembling information on sediment inputs from both the agricultural and urban sectors.

Spectinomycin disk zone diameter as a predictor of outcome in clinical treatment of gonorrhea.

The MICs for 41 Neisseria gonorrhoeae strains from patients receiving spectinomycin treatment were determined by the agar dilution method and compared with the zones of inhibition produced by disks containing 100 micrograms of spectinomycin. Our data demonstrated a good correlation between the two methods. Moreover, a zone of inhibition of less than or equal to 15 mm was a good predictor of clinical treatment failures with spectinomycin.

Interaction of cholecystokinin and vasoactive intestinal polypeptide on function of mouse pancreatic acini in vitro.

In isolated mouse pancreatic acini, vasoactive intestinal polypeptide (VIP) and secretin potentiated amylase release stimulated by cholecystokinin (CCK). VIP (1-100 nM) or secretin (100-1000 nM) alone elicited a negligible secretory response, whereas in combination with CCK, these agents induced a significantly larger response. VIP increased maximal amylase release elicited by CCK without affecting the potency with which CCK stimulated secretion. The phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine (IBMX), from 0.03-1.0 mM had effects on secretion similar to those of VIP. VIP, IBMX and 8-Br-cyclic AMP, all of which act through or mimic the action of cyclic AMP, potentiated the secretory response to maximal concentrations of CCK, carbamylcholine and the ionophore A23187, all of which act via intracellular calcium. In contrast to amylase release, stimulation of acinar glucose transport by CCK or carbamylcholine was not augmented by VIP, secretin, IBMX or 8-Br-cyclic AMP. The results indicate that for amylase release from mouse pancreas, secretagogues acting via cyclic AMP potentiate those acting via calcium. However, potentiation does not apply to all biological responses of the pancreatic acinus and each response must be studied individually.

Expression of receptors for cholecystokinin and other Ca2+-mobilizing hormones in Xenopus oocytes.

The expression of receptors for cholecystokinin (CCK) and other similar acting Ca2+-mobilizing hormones was studied in Xenopus laevis oocytes. Poly(A)+ RNA was prepared from pancreatic AR42J cells, which normally express receptors for CCK and bombesin and the RNA injected into oocytes. The presence of these pancreatic receptors on the oocytes was then demonstrated by hormone-induced mobilization of 45Ca2+. CCK receptors were present 1 day (maximum, 2 days) after injection of RNA and were generally proportional to the amount of poly(A)+ RNA injected (1-50 ng). Oocyte CCK receptors retained selectivity for CCK analogs (CCK8 greater than unsulfated CCK8 greater than CCK4) and were blocked by the specific CCK receptor antagonist CR 1409. When poly(A)+ RNA was subjected to size fractionation on sucrose gradients, activity-inducing CCK receptors showed a single peak centered at 3 kilobases. The generality of this oocyte system for expressing Ca2+-mobilizing hormone receptors was further shown by expression of a response to bombesin after injection of AR42J cell RNA and a response to vasopressin and angiotensin II when poly(A)+ RNA from rat liver was injected. No response to CCK was demonstrable after injection of liver RNA, demonstrating the specificity of this assay.

The effect of cryosurgery and polymethylmethacrylate in dogs with experimental bone defects comparable to tumor defects.

The effects of liquid nitrogen (LN) and polymethylmethacrylate (PMMA) on normal bone, bone graft incorporation, and reossification were evaluated by simulating a tumor in dogs with experimental bone cavity. Ten skeletally mature mongrel dogs (20 femora) were divided into three groups: Group I, controls; Group II, LN (with and without bone graft); and Group III, PMMA (with and without LN). Roentgenograms, whole-mount histology, and tetracycline fluorescence studies were performed on the distal femur. Correlation of these studies showed that (1) marked trabecular and bone necrosis, extending 7-12 mm around the circumference of the cavity, developed by three and seven weeks after LN but no bony necrosis occurred after PMMA; (2) the pattern of reossification following cryosurgery was delayed and abnormal, demonstrating increased calcification and metaplastic bone formation; (3) cryosurgery decreased the rate of bone graft incorporation; (4) the cryonecrotic rim following cryosurgery correlated with an abortive attempt at peripheral reossification; and (5) cryosurgery had no effect on the articular cartilage. Cryosurgery is effective in causing bone necrosis, whereas PMMA is not, and the pattern of reossification is delayed and altered by freezing. This study suggests that microvascular thrombosis with subsequent ischemic infarction of bone is a major cause of bone necrosis following cryosurgery.

Quantitation of latent varicella-zoster virus and herpes simplex virus genomes in human trigeminal ganglia.

Using real-time fluorescence PCR, we quantitated the numbers of copies of latent varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) genomes in 15 human trigeminal ganglia. Eight (53%) and 1 (7%) of 15 ganglia were PCR positive for HSV-1 or -2 glycoprotein G genes, with means of 2,902 +/- 1,082 (standard error of the mean) or 109 genomes/10(5) cells, respectively. Eleven of 14 (79%) to 13 of 15 (87%) of the ganglia were PCR positive for VZV gene 29, 31, or 62. Pooling of the results for the three VZV genes yielded a mean of 258 +/- 38 genomes/10(5) ganglion cells. These levels of latent viral genome loads have implications for virus distribution in and reactivation from human sensory ganglia.

Genital antibody response to a parenteral gonococcal pilus vaccine.

A parenteral gonococcal pilus vaccine which has previously been shown to be safe and antigenic also results in the production of specific local genital antibody. All three major antibody classes were present in the local secretions, but immunoglobulin A predominated, a portion of which is dimeric 11S immunoglobulin A. This mucosal antibody is also capable of blocking the attachment of gonococci to epithelial cells. The antibody cross-reacted with five heterologous pili in a solid-phase radioimmunoassay. These results are encouraging and suggest that a gonococcal pilus vaccine may be efficacious in preventing gonorrhea.

Antigenic specificity of antibodies in vaginal secretions during infection with Neisseria gonorrhoeae.

Antibodies in genital secretions of patients with gonorrhea have been shown to inhibit the attachment of gonococci to epithelial cells. The gonococcal antigens for which these antibodies are specific were studied by adsorption of the genital secretions from a patient infected with gonorrhea with purified lipopolysaccharide, outer membrane complex, or purified pili of homologous Neisseria gonorrhoeae and measurement of the reduction of inhibition of attachment of the gonococci to epithelial cells. The removal of antibodies was documented with the use of a solid-phase radioimmunoassay in which the amount of antibody in the adsorbed secretions that bound to a specific gonococcal antigen was shown to be reduced as compared with the amount of antibody in unadsorbed secretions. The antibody in the secretions that inhibited attachment was removed primarily by adsorption with the homologous pili, not with homologous lipopolysaccharide. A preparation of the homologous outer membrane complex that contained pili, cell-wall proteins, and lipopolysaccharide also blocked the inhibitory antibody.

Efficacy trial of a parenteral gonococcal pilus vaccine in men.

A randomized, placebo-controlled, double-blind efficacy trial of a purified gonococcal pilus vaccine composed of a single pilus type was tested in 3123 men and 127 women volunteers. Either 100 micrograms of vaccine or a placebo was given intradermally on day 1 and day 14. Each group was evenly matched with respect to age, sex, prior history of a sexually transmitted disease, sexual exposure during the study and attrition from the study. None of the women volunteers acquired gonorrhoea during the trial. In the male volunteers, 108 vaccine and 102 placebo recipients acquired gonorrhoea 15 days or later after the initial immunization. Vaccines developed a sustained ELISA antibody response to homologous and heterologous pili, but the latter titres were approximately 40% as high as the homologous pilus antibody rises. There were, however, no increases in inhibition of attachment antibody (IEA) titres. Local antibodies (semen) against homologous and heterologous strains were also elicited (ELISA). The vaccine was safe and did not alter the clinical expression of disease. This gonococcal pilus vaccine composed of a single pilus type failed to protect men against gonococcal urethritis.