Short communication: Cold atmospheric plasma inactivation of Prototheca zopfii isolated from bovine milk.

Mastitis is a serious bovine diseases that can be caused by Prototheca zopfii, yeast-like algae belonging to the family Chlorellaceae. The substantial economic losses and health damage associated with bovine mastitis emphasize the need to develop effective strategies aimed at control of the infection. Unfortunately, P. zopfii is highly resistant to most common antibacterial and antifungal agents, as well as to heat treatment. We report here the first attempt to use cold atmospheric plasma to inactivate this pathogen. We studied 20 strains of P. zopfii isolated from milk samples taken from cows with clinical or subclinical mastitis. The studies confirmed the high level of resistance of P. zopfii to typical antifungal agents, such as voriconazole, fluconazole, amphotericin B, caspofungin, anidulafungin, and micafungin. In contrast, each of the strains revealed high susceptibility to cold atmospheric plasma, >2-fold higher compared with a reference strain of Candida albicans. The obtained results are promising and open up a new approach in the fight against P. zopfii.

Analysis of the relationship between fluconazole consumption and non-C. albicans Candida infections.

The effect of fluconazole consumption on the incidence of nosocomial non-C. albicans Candida infections remains unclear. In this study we investigated such a relationship in an intensive care unit (Poland) over an 11-year period (2002-2012). Statistics relating to the number of candidiasis cases and the number of defined daily doses of fluconazole showed that only a very weak and not statistically significant linear correlation existed between these two variables (r(2) = 0.36, P = 0.052). However, the assumption of a 1-year delay in the infection response to changes in fluconazole concentrations resulted in a strong and statistically significant linear correlation (r(2) = 0.64, P = 0.0053). To more accurately determine the nature of this relationship, a simple epidemiological model was proposed that provided a better than linear correlation (r(2) = 0.78, P = 0.00077). We successfully used this approach to analyze results from the literature that were interpreted as evidence that fluconazole use is not a risk factor for development of non-C. albicans Candida infections. If a time delay in the infection response was assumed, a strong and statistically significant correlation was obtained. These findings suggest the need for a closer look at fluconazole therapy as a possible risk factor for development of non-C. albicans Candida infections.

Growth promotion in chickens by interleukin-2.

Chicken interleukin-2 (cIL-2), which was prepared by sensitizing chicken lymphocytes with concanavalin A, was administered to fertile broiler eggs on Day 18 of embryonation (0.1 mg in 200 mL distilled water). Controls (CON) received distilled water. Hatched chicks were reared to 6 wk. Body weight (BW), as well as abdominal fat pad, liver, bursa of Fabricius, a thymic lobe, spleen, and gonads were excised and expressed relative to BW at 2, 4, and 6 wk of age. Additionally, hematocrit (HCT), hemoglobin (HGB), and plasma protein (PP) levels were determined at the three time intervals. Finally, chicks were sensitized against human gamma globulin (HGG) and challenged at 6 wk by intradermal injections into the wattles. Delayed type hypersensitivity (DTH) to HGG was used as a direct measure of cell-mediated immunity. In ovo cIL-2 increased BW consistently, relative fat pad weights at 2 wk, relative bursa and liver weights at 2 and 6 wk, HBG and relative thymic weight at 2 and 4 wk, and PP at 2 wk. Delayed type hypersensitivity to HGG was not affected by cIL-2. Potential metabolic and immunologic mechanisms to explain in ovo cIL-2 effects are discussed.

Applications in in ovo technology.

By mid-August 1995, 55% of broiler embryos in North America were vaccinated for Marek's disease using the INOVOJECT system, with 201 INOVOJECT machines placed with 16 of the top 25 poultry producers, providing the industry with the capacity to inject in excess of 400 million eggs per month or about 5 billion eggs per annum. In ovo administration of a bursal disease antibody-infectious bursal disease virus (BDA-IBDV) complexed vaccine to specific-pathogen-free (SPF) embryos was safer and more potent than conventional IBDV vaccine alone because it delayed the appearance of bursal lesions, produced no early mortality, produced higher geometric mean antibody titers against IBDV, and generated protective immunity against challenge. In ovo administration of a BDA-IBDV complexed vaccine to broiler embryos generated antibody titers against IBDV sooner than conventional virus vaccinates, and generated protective immunity against challenge Direct DNA injection of plasmid DNA encoding beta-galactosidase into breast muscle in ovo and posthatch was an effective means to achieve both gene transfer and expression, with potential for the development of gene vaccines using plasmids encoding protective antigens from poultry pathogens. In ovo administration of 800 U chicken myelomonocytic growth factor (cMGF), a chicken hematopoietic cytokine for cells of the monocytic-granulocytic lineages, significantly reduced mortality associated with Escherichia coli exposure within the hatcher when compared to PBS controls (6.1 vs 12.4, P < or = 0.05), but not when compared to a yeast expression control. A procedure was developed enabling injection prior to the onset of incubation without compromising embryo viability. This in ovo injection process has opened up the window of embryo development during incubation for intervention, as illustrated by the 100% male phenotype produced in chicks hatching from eggs injected with aromatase inhibitor prior to incubation. These data illustrate some of the in ovo applications presently in use by the poultry industry, and under development or in research at EMBREX.

Measurement of malabsorption of carotenoids in chickens with pale-bird syndrome.

Because pale-bird syndrome (PBS), defined as the failure of birds to realize the color potential of their diet, has been demonstrated to be caused by malabsorption or by hyperexcretion of carotenoids, a method for measuring malabsorption of carotenoids would be useful. The absorption of dietary canthaxanthin, a red diketocarotenoid, into serum during aflatoxicosis was measured in an experiment with a 2 x 9 factorial arrangement of treatments (0 and 5 micrograms of aflatoxin/g of diet; serum collected at 0, 2, 4, 6, 8, 10, 12, 14, and 24 h after a standard meal fed to four groups of 10 3-wk-old birds). Serum canthaxanthin levels determined by HPLC attained plateau values between 8 and 14 h after the meal. The absorption of canthaxanthin was depressed significantly (P less than .05) in birds with aflatoxicosis from 4 to 24 h after feeding the standard meal. Four field flocks diagnosed as having PBS were tested for malabsorption by intubating 10 birds with a standard amount of canthaxanthin and measuring serum canthaxanthin 12 h later. One flock had about 85% normally pigmented birds and 15% extremely pale birds, the second flock had a coccidiosis history, the third had a Newcastle disease history, and the fourth had a history of both coccidiosis and Newcastle disease. The flocks were 5- to 6-wk-old, received feed of the same manufacture, and their disease outbreaks had occurred 2 wk earlier.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatographic method for the determination of spectinomycin in turkey plasma.

A selective high-performance liquid chromatographic (HPLC) method with ultraviolet-visible (UV-VIS) detection was developed to measure therapeutic concentrations of spectinomycin in turkey plasma. Treatment of plasma samples with 3% trifluoroacetic acid in acetonitrile facilitated spectinomycin extraction and protein precipitation. After centrifugation, the stable derivatization reagent, 2,4-dinitrophenyl-hydrazine, was added to an aliquot of the supernatant, and the mixture was incubated for 30 min at 70 degrees C. Excess reagent was quenched with acetone and additional heating. The resulting derivative, a proposed spectinomycin-hydrazone, was separated from other compounds by reversed-phase HPLC during a short gradient run. The absorbance of the effluent was monitored spectrophotometrically with the UV-VIS detector set at 205 nm. The detector response was linear through the range of interest, 2-100 micrograms/ml.

Altered metabolism of carotenoids during pale-bird syndrome in chickens infected with Eimeria acervulina.

The progression of changes in carotenoid metabolism during pale-bird syndrome caused by a coccidial infection was investigated. Male broiler chickens 15 days of age on a yellow corn and soybean meal-based diet were infected with Eimeria acervulina oocysts and their serum, liver, and toe webs were sampled at 0, 4, 6, and 10 days postinfection for HPLC analysis of carotenoids. At 4 days postinfection a drastic reduction (71%) in serum lutein, the main body carotenoid, and smaller reductions in liver (58%) and toe webs (38%) occurred. Derivative forms of lutein, mainly esters, continued to be lost from tissues for 10 days postinfection. These carotenoids were apparently lost via the intestinal tract because birds placed on a white corn and soybean meal-based diet at time of infection had lutein in their jejunal contents even at 7 days postinfection. The loss of carotenoids from the body was accompanied by a decreasing ability to absorb canthaxanthin, a red carotenoid, from the intestinal contents. The absorption of canthaxanthin measured at 0, 3, 4, 5, 6, and 7 days reached its low point of 1% of preinfection ability on Day 5 before a slow recovery commenced. Thus, the pale-bird syndrome caused by E. acervulina appeared to be the result of a loss of previously absorbed carotenoids coupled with drastic malabsorption of dietary carotenoids.

Depletion of oxycarotenoid pigments in chickens and the failure of aflatoxin to alter it.

Aflatoxin, a demonstrated cause of pale bird syndrome in chickens, was investigated for its effects on the depigmentation of chickens placed on a diet low in carotenoids. Chickens were pigmented by feeding for 3 wk a white corn-soy diet supplemented with 50 micrograms free lutein and 0 or 4 micrograms aflatoxin/g diet. Then birds were switched to the same diets unsupplemented with lutein. At 0, 1, 2, 3, 6, and 9 days after switching, jejunal contents and mucosa, serum, liver, and toe web of 4 groups of 10 birds were removed for analysis of their carotenoids by high performance liquid chromatography. In control birds the order of decrease in total lutein was jejunal contents greater than jejunal mucosa greater than serum greater than liver greater than toe web. Aflatoxin did not alter the depletion process, except for minor retardation of lutein depletion in the mucosa and liver. Pharmacokinetic analysis of the data indicated that lutein depletion in the integument was accomplished through three sequential reactions (lutein diester----lutein monoester----lutein----serum lutein) and that aflatoxin had no effect on the reactions. These results imply that aflatoxin induces pale bird syndrome by interfering with the accumulation of pigment by chickens rather than by enhancing the depletion of pigment.

Metabolism of canthaxanthin, a red diketocarotenoid, by chickens.

Canthaxanthin, (4,4'-diketo-beta,beta-carotene), a red carotenoid used to extend the dominant wavelength of the yellow pigments in the skin and egg yolk of chickens, was fed (70 micrograms/g diet) to chicks depleted of normal tissue oxycarotenoids. High performance liquid chromatography analysis of tissues from such chicks revealed that a portion of canthaxanthin was reduced to 4-hydroxyechinenone (4-hydroxy-4'-keto-beta,beta-carotene) that in turn was reduced in part to isozeaxanthin (4,4'-dihydroxy-beta,beta-carotene). The alcohols thus formed were acylated in part to 4-hydroxyechinenone monoester and isozeaxanthin monoester and diester. Individual metabolites were identified by retention times, ratios A470/A430, and stopped-flow spectral analyses, which were identical in each case to authentic standards. Ratios of canthaxanthin to metabolites varied with the tissue, but in general metabolites were concentrated in the integument.

Carotenoid composition of serum and egg yolks of hens fed diets varying in carotenoid composition.

High performance liquid chromatography of yolks of hens fed a diet based on yellow corn, alfalfa, and soybeans revealed over 20 cartenoids. Lutein, lutein monester, lutein diester, 3'-oxolutein, cryptoxanthin, zeaxanthin, beta-carotene, and zeacarotene were identified by their retention times, visible absorption spectra, behavior on saponification, and their presence or absence when lutein was the primary carotenoid fed. Three weeks after placing the hens on a white corn-soy-based diet supplemented with lutein (20 micrograms/g diet), cryptoxanthin, zeaxanthin, and zeacarotene were undetectable in the yolk and lutein, lutein monoester, lutein diester, and 3'-oxolutein assumed new equilibrium concentrations. The data imply an esterification pathway and an oxidative pathway in laying hens for the metabolism of hydroxycarotenoids. Consideration of the concentrations and ratios of lutein and its metabolites in serum and yolk suggest a nonovarian site for the metabolism of lutein in laying hens.

Aflatoxin-impaired ability to accumulate oxycarotenoid pigments during restoration in young chickens.

The mechanism by which aflatoxin causes paling in chickens was investigated by measuring its effect on the restoration of pigments in 3-wk-old birds made pale by feeding a white corn-soy diet. Pigment restoration was accomplished by feeding the same diet supplemented with lutein (70 micrograms/g of diet), which is the major oxycarotenoid pigment in chicken diets and tissues. The oxycarotenoids (free, monoester, and diester forms of lutein) in the toe web, liver, serum, and jejunal mucosa of control and aflatoxin-fed (2 micrograms/g of diet) birds were measured by HPLC at 0, 1, 2, 3, 6, and 9 days of repletion. Aflatoxin caused a significant (P less than .05) depression of all forms of lutein in the toe web. In the liver, aflatoxin decreased lutein significantly (P less than .05) but increased lutein monoester and lutein diester. Lutein accumulation in serum and mucosa were inhibited significantly (P less than .05) starting on Days 2 and 3, respectively. These data imply that the normal accumulation of lutein from the diet proceeded into and through the mucosa to the serum to depot sites in the liver and integument, where lutein was acylated to its monoester, which was acylated to its diester. Further, aflatoxin inhibited, apparently independently, the accumulation of lutein by the mucosa, serum, liver, and integument. Pharmacokinetic analysis of the data indicated that both acylation steps in the integument were sensitive to aflatoxin, but the passage of lutein from serum into the integument was not affected.

Metabolism of lutein diester during aflatoxicosis in young chickens.

Young chickens were fed from hatching until 3 wk of age with a white corn-soy diet amended with lutein diester to supply 25 micrograms lutein/g diet and with varying amounts of aflatoxin (0, 1, 2, 4, and 8 micrograms/g diet). The lutein diester was added as a stabilized, microencapsulated extract of marigold (Tagetes erecta) petals. Aflatoxin had no significant effect on the partial conversion of lutein diester to lutein monoester and lutein that occurs in the jejunum of normal chickens. The concentration of lutein in the jejunal mucosa was increased slightly by intermediate levels of aflatoxin and depressed by higher levels. Aflatoxin depressed lutein (the dominant form) in serum by up to 40%. Aflatoxin enhanced the lutein accumulation in the liver as the monoester (four-fold) and diester (12-fold). Only minor effects of aflatoxin on the carotenoid content of the toe webs were noted in birds fed lutein diester. These results imply that the main effects of aflatoxin on the utilization of lutein diester are to impair the absorption of lutein, which is an intestinal product of dietary lutein diester, and to sequester lutein as lutein monoester and lutein diester in the liver.

Altered metabolism of carotenoids during aflatoxicosis in young chickens.

Young chickens were fed from hatching until 3 weeks of age with a white corn-soy diet (containing 1.36 micrograms total carotenoids per gram of diet) amended with a commercial preparation of lutein, a dihydroxycarotenoid, to supply 25 micrograms free lutein per gram diet. The diet which also contained 0, 1, 2, 4, or 8 micrograms aflatoxin per gram of diet was fed to four groups of ten chickens per aflatoxin treatment until they were 3 weeks old. Aflatoxin had no effect on the partial acylation of free lutein to lutein monoester that occurs in the jejunal contents of normal birds but it decreased significantly (P less than .05) the conversion of free lutein to lutein diester. Aflatoxin reduced up to 35% the lutein (94% free alcohol) content of the jejunal mucosa and the serum lutein (99% free alcohol) was reduced by up to 70%. Aflatoxin caused a slight (25%) decrease in the free lutein content of liver while increasing the monoester content 3.5-fold and the diester content 10-fold. This sequestering of lutein in the liver in esterified forms poorly transported to the integument presumably contributes to the poor pigmentation during aflatoxicosis. The forms of lutein in the toe web were diester (66%0, free alcohol (26%), and monoester (8%) and their sensitivity to aflatoxin followed the same order. These data offer clear, unequivocal proof that aflatoxin can cause poor pigmentation in birds, presumably by interfering with the absorption, transport, and deposition of carotenoids.

Alterations in carotenoid metabolism during ochratoxicosis in young broiler chickens.

The mechanism by which ochratoxin impairs the ability of chickens to utilize dietary carotenoids for carcass pigmentation was investigated. Graded doses of pure ochratoxin A (0, .5, 1.0, 2.0, and 4.0 micrograms of toxin/g of feed) were incorporated into a white corn-soy diet supplemented with an efficiently used oxycarotenoid (110 micrograms free lutein/g) and fed to broiler chicks from day of hatch to 3 weeks of age. Concentrations of free lutein and its metabolites, lutein diester, lutein monoester, and oxolutein, in the jejunal contents, jejunal mucosa, serum, liver, and toe web from these birds were measured by high performance liquid chromatography. Based on the threshold level of ochratoxin required for an effect on the concentrations of carotenoids and on the severity of the effect, five separate loci for the action of ochratoxin on carotenoid metabolism were detected: dilution of carotenoids in intestinal contents, depressed uptake by intestinal mucosa, depressed transport in serum, altered accumulation in liver, and altered acylation steps in the integument.

3'-Oxolutein, a metabolite of lutein in chickens.

3'-Oxolutein (3-hydroxy-3'-oxo-beta,epsilon-carotene) was isolated using high performance liquid chromatography (HPLC) from the tissues and egg yolks of chicken fed a diet high in lutein and free of detectable 3'-oxolutein. It was identified by HPLC retention time, absorption spectrum identical to lutein and its esters, disappearance under alkaline conditions without giving rise to lutein, formation of an alkali labile palmitate, formation of a 2,4-dinitrophenylhydrazone, and reduction to lutein from which the dipalmitate was prepared. In each instance the isolated compound behaved identically with authentic 3'-oxolutein prepared by nickel peroxide oxidation of lutein. The order of ratios of lutein to 3'-oxolutein in the various tissues from laboratory and field birds was, in general, intestinal contents greater than intestinal mucosa greater than serum greater than liver approximately toe web approximately egg yolk greater than bile. This order was consistent with the hypothesis that lutein was oxidized to 3'-oxolutein in the liver, which, at least in part, was excreted via the bile into the intestinal lumen where it was diluted with dietary lutein. The remainder of the 3'-oxolutein formed in the liver presumably went into the serum where it was transported to depot sites. The ratio of lutein to 3'-oxolutein in the toe webs of broilers varied with their diet and pigmentation status. Amounts of 3'-oxolutein were found in quail and turkey egg yolks. This previously unreported lutein oxidative capability of liver extends the previously known metabolic acylation and deacylation reactions in poultry.

Absorption, transport, and deposition in chickens of lutein diester, a carotenoid extracted from marigold (Tagetes erecta) petals.

Young broiler chickens were fed from hatching until 3 weeks of age with a white corn-soy diet amended with varying amounts of lutein diester to supply 0, 5, 10, 20, 40, and 80 micrograms free lutein/g diet. The lutein diester was added as a stabilized, microencapsulated extract of marigold (Tagetes erecta) petals. The concentrations of lutein diester, lutein monoester, and lutein in the contents of the jejunum and large intestine and in serum, liver, and toe web from these birds were measured by high pressure liquid chromatography. The contents of the jejunum and large intestine contained a mixture of lutein diester, lutein monoester, and lutein. The serum contained lutein (approximately 90%), lutein monoester (approximately 10%), and traces of lutein diester. The liver contained the three carotenoid classes in ratios reflecting the serum ratios. The ratios in toe web, an integumentary depot site, were reversed with lutein diester much greater than lutein monoester greater than lutein. The concentrations of each class in each tissue bore a linear relationship to the concentration of lutein diester in the diet. A simple explanation for these data is that the dietary lutein diester was hydrolyzed mainly to lutein, which was absorbed through the intestinal wall into the blood stream where it was transported to the liver, a storage site, and to the integumentary sites where it is esterified to lutein diester which is the main depot form.

Canthaxanthin as a model for the study of utilization of oxycarotenoids by chickens.

A white corn-soy diet amended with varying levels (0, 5, 10, 20, 40, and 80 ppm) of canthaxanthin, a red diketocarotenoid available by chemical synthesis, and fed to young broiler chickens for 3 weeks has the attributes of a useful experimental model for the study of absorption, transport, and deposition of oxycarotenoids. On high pressure liquid chromatography of extracts of the diet and tissues of the birds, canthaxanthin predominated over the background level of nonspecific carotenoids. The concentrations of canthaxanthin found in the contents of the jejunum and large intestine and in the serum, liver, and toe web were directly proportional to the dietary concentration. Departure from such linear relationships would signal loci of action for factors affecting pigmentation. Analysis of toe webs, an integumentary depot site for carotenoids, revealed that the concentrations of canthaxanthin and two other compounds, one more polar and the other less polar than canthaxanthin, were proportional to the dietary concentration of canthaxanthin. Saponification converted the less polar compound to the more polar compound. The behavior of these compounds in response to dietary canthaxanthin, to chromatography, and to saponification can be explained by assuming that canthaxanthin, a diketocarotenoid, was partly reduced to a ketohydroxy carotenoid (hydroxyechinenone) whose hydroxyl group is acylated to form an ester. These products represent new metabolic reactions in poultry.

Evidence for differential absorption of zeacarotene, cryptoxanthin, and lutein in young broiler chickens.

The intestinal tracts of young chicks, fed a yellow corn-soy-based diet from hatching until 3-weeks-old, were excised and divided into five segments equal in length. The contents of the segments were removed and analyzed for content of zeacarotene, cryptoxanthin (monohydroxycarotenoid), and lutein (dihydroxycarotenoid) by high pressure liquid chromatography. Cryptoxanthin, which is poorly absorbed by young chickens, served as a nonabsorbed reference material. The ratios of zeacarotene to cryptoxanthin were not reduced significantly (P less than .05) from that in the ingested feed until the ileal region was reached. The ratios of lutein to cryptoxanthin were reduced significantly (P less than .05) in the duodenal and upper jejunal region. The finding of different sites of absorption for zeacarotene and lutein, combined with the poor absorption of cryptoxanthin, suggests the existence in chickens of regulatory mechanisms for carotenoids.

Lutein as a model dihydroxycarotenoid for the study of pigmentation in chickens.

A model for the study of pigmentation in young chickens is described in which a white corn-soy based diet supplemented with varying amounts of free lutein (0, 5, 10, 20, 40, and 80 micrograms/g diet) was fed from hatching until 3 weeks of age. The carotenoid content of tissues dissected from chicks of the various groups was measured by high pressure liquid chromatography. In intestinal contents, three forms of lutein were found, with lutein monoester greater than free lutein greater than lutein diester. In the serum, free lutein (96%) and lutein monoester (4%) were found. In the liver, free lutein (80%), monoester (20%), and traces of diester were found. In the integument (toe web), diester greater than monoester approximately equal to free alcohol were found. In each tissue, the concentrations were directly proportional to the dietary concentration of free lutein. The simplest explanation of the data appears to be that part of the free lutein in the diet is esterified during its passage down the intestinal tract and, regardless of its status when absorbed, it is transported in the body as the free alcohol. When it enters depot sites such as the integument, lutein is deposited mainly as esters, presumably as the result of local enzymatic activity.