Diagnostic implications of antigen-induced gamma interferon, nitric oxide, and tumor necrosis factor alpha production by peripheral blood mononuclear cells from Mycobacterium bovis-infected cattle.

Bovine tuberculosis in the United States has proven costly to cattle producers as well as to government regulatory agencies. While in vivo responsiveness to mycobacterial antigens is the current standard for the diagnosis of tuberculosis, in vitro assays are gaining acceptance, especially as ancillary or complementary tests. To evaluate in vitro indices of cellular sensitization, antigen-induced gamma interferon (IFN-gamma), nitric oxide (NO), and tumor necrosis factor alpha (TNF-alpha) responses by blood mononuclear cells from Mycobacterium bovis-infected cattle were quantified and compared. Using an aerosol model of infection, two doses of each of two strains of M. bovis (95-1315 and HC-2045T) were used to induce a range of IFN-gamma, NO, and TNF-alpha responses. Infection-specific increases in NO, but not in IFN-gamma or TNF-alpha, were detected in nonstimulated cultures at 48 h, a finding that is indicative of nonspecific activation and spontaneous release of NO. The infective dose of M. bovis organisms also influenced responses. At 34 days postinfection, IFN-gamma, NO, and TNF-alpha responses in antigen-stimulated cells from cattle receiving 10(5) CFU of M. bovis organisms were greater than responses of cells from cattle infected with 10(3) CFU of M. bovis organisms. The NO response, but not the IFN-gamma and TNF-alpha responses, was influenced by infective strains of M. bovis. The TNF-alpha, NO, and IFN-gamma responses followed similar kinetics, with strong positive associations among the three readouts. Overall, these findings indicate that NO and TNF-alpha, like IFN-gamma, may prove useful as indices for the diagnosis of bovine tuberculosis.

Minimal prophylactic concentration of dietary zinc compounds in a mouse model of swine dysentery.

Dietary supplementation with 6000 mg of Zn2+/kg of feed has been shown to modify the clinicopathologic expression of Brachyspira hyodysenteriae infection in a laboratory mouse model of swine dysentery. However, this concentration impaired the body weight gain of the mice. The purpose of the present study was to determine a minimal prophylactic concentration of feed-grade zinc compounds that would not affect the growth of mice challenge-exposed with B. hyodysenteriae. A total of 440, 6- to 8-week-old, C3H/HeN mice were allocated randomly to groups and fed either a defined diet or a defined diet containing either 1000, 2000 or 4000 mg/kg ZnO, ZnSO4 or zinc-methionine for 7 days before intragastric inoculation with B. hyodysenteriae. From days 7 to 35 after inoculation, mice in each group were necropsied at weekly intervals for determination of body weight, presence of B. hyodysenteriae in the cecum, and histological assessment of cecal lesions. Only ZnO fed at 2000 mg/kg had a prophylactic effect against B. hyodysenteriae infection without affecting the body weight gain of the mice. The prophylactic effect of Zn2+ against infection with B. hyodysenteriae was also affected by the relative concentration of Fe2+ and Zn2+/Fe2+ ratio of the diet.

Effects of inorganic and organic copper supplemented to first-calf cows on cow reproduction and calf health and performance.

Two experiments were conducted to determine whether the supplementation of Cu in the organic or inorganic form to 2-yr-old cows, before and after calving, affects reproduction rate, calf health and performance, passive transfer of immunoglobulin, or liver and serum Cu concentrations compared with unsupplemented controls. Cows (n = 75 in 1997; n = 120 in 1998) were randomly assigned by estimated calving date and body condition score to one of three treatments: 1) Control, control; 2) Inorganic, inorganic Cu supplement (200 mg Cu from CuSO4); 3) Organic, organic Cu supplement (100 mg Cu from AvailaCu). In 1998, a fourth treatment was added; 4) CU-ZN, organic Cu and Zn (400 mg Zn from AvailaZn in the Organic diet). Cows were fed a hay-based diet and individually fed supplements for approximately 45 d before and 60 d after calving (approximately January 15 to May 15 each year). Liver biopsies were obtained from cows before supplementation began, and from cows and calves at 10 and 30 d after calving. Blood samples were obtained from both cows and calves at calving, and colostrum samples were collected for IgG and mineral content. Cow liver Cu concentrations before supplementation began were 58 mg/kg in 1997 and 40 mg/kg (DM basis) in 1998. By 10 d after calving, liver Cu concentrations of Control cows had decreased (P < 0.05) to 24 mg/kg (Cu deficient) in both years, whereas liver Cu concentrations of Cu-supplemented cows increased (P < 0.05) in both years. Calf liver Cu concentrations at 10 d of age were similar (P > 0.10) for all treatment groups. No differences (P > 0.10) were found in colostrum Cu concentrations, or in calf health among treatments. No differences (P > 0.10) were found in cow BW change, calf serum Cu concentrations, calf weaning weights, or in cow 60-d pregnancy rates among treatments in either year. In 1998, cows in the Organic group had higher (P < 0.05) 30-d pregnancy rate than Control cows. Neither serum samples nor placental tissue were reliable indicators of Cu status in cows. Feeding supplemental Cu (either inorganic, organic, or organic with extra Zn) to cows with liver Cu concentrations of approximately 50 mg/kg before calving did not improve cow 60-d pregnancy rates or the health and performance of their calves when compared with unsupplemented cows.

Gas chromatography/mass spectrometry identification and quantification of isazophos in a famphur pour-on and in bovine tissues after a toxic exposure.

A sample identified as "Warbex pour-on," expected to contain 13.2% famphur, and bovine tissue samples from 2 heifers that died after exhibiting signs of organophosphate intoxication were analyzed by gas chromatography/mass spectrometry (GC/MS). A product formulation problem was suspected because brain cholinesterase activities were depressed in both animals. Electron impact (EI) GC/MS of the pour-on revealed 9.7% famphur and an unidentified peak with approximately 76% of the peak area of the famphur. The unidentified peak showed a molecular ion at m/z 313, with a single Cl isotope cluster. Methane chemical ionization (MeCI) MS confirmed the molecular weight at 313 (1 Cl). A search on the molecular formula C9H17N3O3PSCl yielded a single match, isazophos. EI and MeCI GC/MS of reference isazophos confirmed the identity of the suspect peak. The concentration of isazophos in the pour-on was determined to be 6.0%. Famphur and isazophos were identified by their EI spectra and GC retention times in extracts of liver and brain from the 2 deceased animals. A GC/MS procedure utilizing selected ion monitoring (SIM) was developed for quantification of isazophos in liver, kidney, muscle, and fat of additional affected animals sacrificed at various times after exposure. Isazophos remained in animal tissues for as long as 94 days after topical exposure. Isazophos was present in fetal liver 70 days after exposure of the dam. High levels (6-3,500 ppm) of isazophos and famphur remained on the skin at 39 days postexposure.

Effects of supplementation of organic and inorganic combinations of copper, cobalt, manganese, and zinc above nutrient requirement levels on postpartum two-year-old cows.

The objective of this study was to determine whether a combination of Cu, Co, Mn, and Zn in an organic or inorganic form fed at higher than nutrient recommendations for 2-yr-old cows from calving to breeding would affect pregnancy rate, calving date, calf performance, and cow liver and serum mineral concentrations. Crossbred 2-yr-old cows were used after calving in 1994 (n = 127) and 1995 (n = 109). Cows were blocked by calving date to one of three treatments: 1) no supplemental minerals (CTL), 2) organic minerals (ORG), or 3) inorganic minerals (ING). Minerals were fed for the same daily intake for both organic and inorganic treatments: Cu (125 mg), Co (25 mg), Mn (200 mg), and Zn (360 mg). Cows were individually fed a mineral-protein supplement with grass hay from calving (February-March) to before breeding (May 15). Hay intakes were calculated using chromium oxide boluses to determine fecal output. Fecal excretion of minerals was calculated following trace element analysis of feces. Liver biopsies were obtained before calving, after calving (start of supplementation), at the end of supplementation, and in midsummer. Over 2 yr, more cows did not become pregnant (P < .01) in ORG (11/78) and ING (11/78) treatments than in CTL (0/80) treatments. A treatment x year interaction was found for day of conception. Cows in the ORG group conceived later (P < .01) than cows in the ING or CTL groups in 1994. In 1995, there was no difference (P > .10) in day of conception among groups. Liver Zn and Mn concentrations were not different (P > .10) and Cu concentrations increased (P < .01) for the ORG and ING groups. Cows in the ORG and ING groups had higher (P < .01) concentrations of Cu, Mn, and Zn in the feces than the CTL cows. Trace elements in the feces did not differ for ORG and ING groups. Results indicate that combinations of Cu, Co, Mn, and Zn fed at higher levels than are required reduced reproductive performance.

Screening nitrate in forages with a test strip: collaborative study.

A collaborative study was conducted for screening nitrate in forages with a commercially available test strip. The method involves extracting a finely ground sample with deionized water. The test strip is dipped in the sample extract. The color of the reaction zone on the test strip changes from white to pink or purple depending on the nitrate concentration in sample extract. The nitrate present in the extract is determined by comparing the color of the test strip to the color scale on the test strip container. Six blind quintuplicates of forage samples were analyzed by 20 collaborators. Nitrate concentrations in forage samples tested ranged from < 1000 ppm nitrate to > 10,000 ppm nitrate on dry matter basis. Each collaborator was asked to assign each sample to one of the 4 following nitrate concentration ranges: (1) < 1000 ppm, (2) 1000 to 5000 ppm, (3) > 5000 ppm to 10,000 ppm, and (4) > 10,000 ppm. Nineteen of 20 collaborators reported results. Results from 2 laboratories were rejected as outliers by inspection and chi 2 test. Sensitivity rates (p+) ranged from 0.965 to 0.998, with standard errors of 0.006 to 0.16. Specificity rates (p-) ranged from 0.991 to 0.997 for the 4 ranges, with standard errors of 0.003 to 0.006. False-positive rates (pf+) ranged from 0.006 to 0.046, with standard errors of 0.006 to 0.025. False-negative rates (pf-) ranged from 0.003 to 0.007, with standard errors of 0.003 to 0.006. Screening nitrate in forages with a test strip has been adopted first action by AOAC INTERNATIONAL.

Prophylactic effect of dietary zinc in a laboratory mouse model of swine dysentery.

Reduced prevalence of diarrhea and mortality has been reported after dietary supplementation with zinc compounds in swine with naturally acquired colibacillosis and those challenge-exposed with Serpulina hyodysenteriae; however, the usefulness of this approach for control of enteric diseases of swine remains to be determined. To examine the effect of dietary zinc-containing compounds on the colonization and development of cecal lesions associated with S hyodysenteriae infection, a defined diet alone or with added ZnO, ZnSO4, or Zn-methionine complex to a final concentration of approximately 6,000 mg of Zn2+/kg of complete feed was fed ad libitum to 156 female mice (strain C3H/HeN) for 10 days prior to oral inoculation either with S hyodysenteriae or sterile trypticase soy broth. Rations were continued for 42 days, while at weekly intervals, 3 mice/group were necropsied for determination of body weight, cecal weight, liver zinc concentration, presence of S hyodysenteriae in the cecum, and gross and histologic assessments of cecal lesions. From postinoculation day 0 to 42, the liver zinc concentration of mice fed the zinc-supplemented diets was approximately twice that of mice fed the basal diet, irrespective of the source of zinc. From postinoculation day 7 through 42, the overall recovery rate of S hyodysenteriae in infected mice fed the basal diet was 77.8%. In contrast, recovery rates of S hyodysenteriae from S hyodysenteriae-inoculated mice fed the zinc-supplemented diets were 0% for Zn-methionine and ZnO and 16.7% for ZnSO4. Mice fed the basal diet had significantly (P < 0.05) higher weight gain than mice fed the zinc-supplemented diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of divalent cations on hemolysin synthesis by Serpulina (Treponema) hyodysenteriae: inhibition induced by zinc and copper.

Serpulina hyodysenteriae produces an oxygen-stable heat-labile hemolysin that may be an important virulence factor in the pathogenesis of swine dysentery. We examined the effect of Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, and Zn2+ on the hemolytic activity of cell-free supernatant (CFS) from S. hyodysenteriae, isolate B204. Cells harvested from late logarithmic phase cultures were incubated in phosphate-buffered saline containing glucose and RNA-core (PBS-GR) with or without cations and the hemolytic activity of CFS obtained after successive 30 min incubation and washing cycles was determined. The addition of either ZnSO4 or CuSO4 to the PBS-GR caused complete inhibition of hemolytic activity after 3 cycles; other cations gave results similar to control extracts. Reduction in the concentration of Zn2+ in CFS by 60 to 80% after each incubation cycle and binding of Zn2+ by EDTA indicated that Zn2+ was associated with the cell fraction, and inhibition of hemolysin activity was specifically mediated by Zn2+. When the spirochetes were washed after incubation in the presence of ZnSO4 for 2 cycles and incubated in fresh PBS-GR without Zn2+, inhibition of hemolysin activity remained unchanged, indicating that the inhibitory effect of ZnSO4 was due to a direct action of ZnSO4 on the spirochetes. Since neither the viability of the spirochetes nor the activity of pre-formed hemolysin were affected by the presence of ZnSO4, the inhibitory effect of Zn2+ cations was attributed to reduced biosynthesis by viable S. hyodysenteriae cells rather than interference of Zn2+ cations with lysis of erythrocytes by the hemolysin. Transmission electron microscopic examination of spirochetes after incubation in PBS-GR containing ZnSO4 revealed clumping of ribosomes and clearing of cell cytoplasm.

Efficacy of an Actinobacillus pleuropneumoniae bacterin against serotypes 1, 3, 5 and 9.

A trial was performed in a swine research facility to ascertain the protection provided by a polyvalent Actinobacillus pleuropneumoniae (APP) bacterin containing serotypes 1, 3, 5 and 9. The test animals consisted of 60, eight-week-old, piglets, which were randomly divided into four main groups. The four main groups were further divided into three sub-groups (I, II, III) of five pigs each. Subgroup I was vaccinated intramuscularly, sub-group II was vaccinated subcutaneously, and sub-group III served as the unvaccinated control group. Each main group was challenged with a single APP serotype (1, 3, 5 or 9). Criteria for evaluation of the bacterin efficacy were mortality, lung lesions, pleural adhesions, and isolation of APP from tonsil or lung. Significant effects of vaccination over nonvaccination were reduced mortality, lung lesions, pleural adhesions, and isolations of APP from tonsil and lung. There were no significant differences between the intramuscular and subcutaneous routes of vaccination. It was concluded that the four-way APP bacterin used in this study provided satisfactory protection against homologous challenge. Evidence of protection was lower mortality and lung lesions and increased daily weight gains in vaccinates as compared with controls.

Trace element concentrations in perinatal beef calves from west central Nebraska.

Trace element analyses were performed on 144 livers from perinatal calves submitted to the Veterinary Science Laboratory, North Platte, NE. All calves originated from herds in west-central Nebraska and ranged in age from 8 months of gestation up to 6-week-old neonates. Calves were divided into 5 categories based on objectives gross examination and ocular fluid evaluation: abomasal ulcers, abomasal tympany, diarrhea, stillborn and excessive nitrate exposure. Excessive zinc concentrations were found in 4 of the 5 categories. Only in the abomasal ulcer category could a correlation be found; this category routinely was found to have deficient to low normal liver copper concentrations and highest liver molybdenum concentrations. Both excesses and deficiencies of selenium and iron occurred in all categories.

Apparent toxicosis associated with lily-of-the-valley (Convallaria majalis) ingestion in a dog.

Lily-of-the-valley (Convallaria majalis), an ornamental plant found in many urban settings, was believed responsible for the sudden collapse and death of a one-year-old mixed-breed dog. Postmortem examination revealed C majalis leaf fragments in the small intestine. The dog had gross and histologic lesions consistent with cardiac shock. Because C majalis contains cardiac glycosides that have digitalis-like activity, apparent C majalis intoxication was suspected in this dog on the basis of history, exposure to the plant, clinical signs of disease, postmortem lesions, and evidence of plant parts in the intestinal tract. A toxicologic assessment of excessive exposure to C majalis was based on the aforementioned criteria.

Hematology, blood chemistry and selenium values of captive pronghorn antelope, white-tailed deer and American bison.

Pronghorn were observed to have a significantly higher whole blood selenium concentration than either the white-tailed deer or bison. Pronghorn colloid values were significantly less than those of the bison, and approached statistical significance for the white-tailed deer. Differential white blood cell counts for the white-tailed deer were markedly different from those of the pronghorn and bison. The American bison had significantly higher cortisol values and lower T3 values than either the white-tailed deer or pronghorn.

Determination of nitrate in forages by using selective ion electrode: collaborative study.

Each of 10 collaborating laboratories analyzed 4 blind duplicate pairs of forage samples for nitrate, by using a potentiometric method. Two forage controls and a 100 000 mg KNO3/L standard were also provided. Nitrate was extracted into an aqueous Al2(SO4)3 solution containing 70 mg KNO3/L and quantitated with a nitrate-selective electrode. Standards were prepared using extracting solution as diluent. Nitrate concentrations in forage samples ranged from less than 0.50 to 4.35% KNO3. Repeatability coefficients of variation (CVo) ranged from 1.74 to 3.61%, and reproducibility coefficients of variation (CVx) ranged from 6.92 to 7.66%. Mean recovery of a 0.55% KNO3 spike was 94.5%. The method has been adopted official first action.