Host cytokine production, lymphoproliferation, and antibody responses during the course of Ancylostoma ceylanicum infection in the Golden Syrian hamster

The Syrian Golden hamster (Mesocricetus auratus) has been used to model infections with the hookworm Ancylostoma ceylanicum. New molecular immunological reagents to measure cellular immune responses in hamsters were developed and used to determine the impact of A. ceylanicum hookworm infection on host cytokine responses and lymphoproliferation. Initial larval infection with 100 third-stage A. ceylanicum larvae resulted in predominant Th1 responses (upregulation of proinflammatory cytokines) that lasted for the duration of larval migration and continued up to 14 days postinfection (prepatency). Subsequently, development of larvae into egg-laying adult hookworms (patency) coincided with a switch to Th2 predominant responses (interleukin-4 [IL-4]) as well as a marked increase in IL-10 production. This switch also concurred with reduced host lymphoproliferative responses to hookworm antigens. The findings demonstrate a similarity in immune responses between hamsters and humans infected with hookworms, suggesting that hamsters will be a useful animal model species for examining host immunity to human hookworm infections.     

Iron metabolism in Trypanosoma lewisi infection: Serum iron and serum iron-binding capacity

Summary Progressive changes in iron levels, total iron binding capacity and hematocrit values in sera of rats infected with Trypanosoma lewisi are described. The host dietary group were: (1) complete or full complement; (2) iron-deficient, and (3) pair-fed or calorically restricted.The hematocrit values of T. lewisi-infected rats given the various diets were not significantly different from those of the controls.The decrease in total iron binding capacity (TIBC) of rats inoculated with T. lewisi and fed complete and pair-fed diets ranged up to 15% over uninfected controls. TIBC levels in rats fed an iron-deficient diet and inoculated with T. lewisi ranged up to 32% over uninfected controls. TIBC levels of deficient infected rats were significantly different from the controls from day 90 of infection to the end of the observation period.Serum iron (SI) values of non-infected rats regardless of dietary regimen showed significantly higher values than T. lewisi-infected animals between days 95 and 120. The average SI value, for this period, in adequately fed control rats was 204±7 g/100 ml as compared to 172±5 g/100 for trypanosome-infected rats. SI levels of rats on a pair-fed diet and infected with T. lewisi decreased to 17% over uninfected controls. SI levels of animals on an iron-deficient diet and infected with T. lewisi decreased up to 76% over uninfected controls.     

Oral transfer of adult Ancylostoma ceylanicum hookworms into permissive and nonpermissive host species

Syrian hamsters become anemic and exhibit delayed growth following oral infection with third-stage Ancylostoma ceylanicum hookworm larvae. Here we describe experiments designed to determine the feasibility of adult worm transfer (AWT) between hosts, a technique that would facilitate the specific study of bloodfeeding hookworms in vivo without prior exposure of the host to larva-specific antigens, permit the ex vivo manipulation of adult parasites prior to reimplantation, and also allow for cross-species transfer of worms. Weanling hamsters given an oral AWT of 40 or 60 mixed-sex A. ceylanicum worms rapidly developed anemia; in the higher-dose group, hemoglobin levels declined from prechallenge levels by 44% within 4 days following AWT. Long-term survival of transferred worms was demonstrated by recovery of parasites from the intestines 42 days after AWT. AWT hamsters acquired humoral immune responses against soluble adult hookworm extracts and excretory-secretory products that were comparable in magnitude to those of animals given a typical infection with larvae. In AWT experiments employing the nonpermissive murine model, C57BL/6 mice given adult worms rapidly became anemic and lost weight in a manner similar to AWT hamsters. Infection of additional mouse strains demonstrated that while C57BL/10 and CD-1 mice also developed anemia following AWT, BALB/c mice were resistant. The technique of AWT to mice may further our understanding of hookworm pathogenesis by allowing the study of adult hookworm infections in a species with well-characterized genetics and an abundance of available reagents.     

Preventive effects of 5-fluorouridine and uridine on d-galactosamine-induced liver injury

Duplicate groups of rainbow trout fingerlings were fed diets containing a muxture of pyrrolizidine (Senecio) alkaloids (PAs), at levels of 20 and 100 ppm, for 12 months. The 100-ppm diet resulted in severe growth depression and mortalities and one group receiving that diet was terminated at 6 months. The other 100-ppm group was placed on the control diet after 2 months and held until termination at 12 months. Both levels of PAs resulted in severe hepatic lesions including megalocytosis, necrosis, and fibrosis. In addition venoocclusive disease was present in centrolobular and hepatic veins. The high dose of PAs also caused significant thickening of glomerular basement membranes. Removal of the fish from the 100-ppm diet at 2 months and feeding the control diet for 10 months resulted in only slight improvement in growth, and hepatic, vascular, and renal lesions persisted through 12 months. No evidence of neoplastic change was observed, although some nodular hyperplasia was seen in the fish fed 100 ppm PAs for 2 months and control diet for 10 months. In vitro studies with trout liver microsomes showed that control trout liver was capable of producing the toxic pyrrole metabolites at a rate comparable to uninduced rat liver microsomes.     

Energy and protein relations in the broiler chicken. 2. Effect of varied protein and constant carbohydrate levels on body composition and lipid metabolism

Male broiler chicks were fed diets containing 18, 23 or 30% protein on an ad libitum basis for 3 weeks. Chicks were also fed these diets on a restriction regime consisting of seven 3-day cycles (2 days with and 1 day without feed). Another group of chicks were fed diets containing 12, 23 or 30% protein on a 3-day rotation consisting of a daily change in dietary protein. Ad libitum. Feed intakes of chicks were inversely (P less than .05) related to the level of the dietary protein. Body composition data seemed to support a positive (P less than .05) relationship between dietary protein and total carcass protein and an inverse (P less than .05) relationship between dietary protein and carcass fat. In vitro lipogenesis was greater (P less than .05) in chicks fed an 18% protein diet than in chicks fed either 23 or 30% protein diets. Restricted. Protein levels had no effect on lipogenesis in chicks fed on a restriction regime but regulated lipogenesis in chicks fed on the rotational regime. Fatty acid synthetase, malic enzyme and isocitrate dehydrogenase activities, when measured during the rotational regime, were greatest following the 12% diet and least following the 30% diet. Enzyme activities, determined during the restriction regime, were greatest (P less than .05) after 1 day of refeeding and least (P less than .05) after 1 day of feed withdrawal. Measurement of net glucose production in vitro indicated that high-protein diets and feed withdrawal stimulated production of glucose.     

Protein selection, food intake, and body composition in response to the amount of dietary protein

Though not universally observed, moderately low-protein diets have been found to increase caloric intake and body fat. It appears that animals overeat in calories in order to obtain more dietary protein. For animals to control protein intake, they must be able to distinguish between two isocaloric diets containing different percentages of protein and make the appropriate dietary selection on the basis of their previous history of protein intake. Experiment 1 examined the 24-h diet selection (5 vs. 35% casein) of Sprague-Dawley rats that had been previously fed diets containing various percentages of dietary protein (5, 10, 20, 35, or 60% casein). Animals fed 5, 10, or 20% dietary protein showed a preference for the higher protein selection diet. In contrast, no significant diet preference was found in animals pre-fed the two higher levels of dietary protein (35 or 60% casein). In this study, daily food intake and body fat of rats fed the low-protein diets (5 and 10% casein) were similar to rats fed the 20% casein diet. Experiment 2 examined the effects of the level of methionine supplementation on rats fed 10% casein. In this study, food intake and body fat were increased by approximately 20% in rats fed 10% casein diets, regardless of the level of methionine supplementation (0.3 vs. 0.15%). Together, the results suggest that the presence of low-protein-induced hyperphagia helps maintain body protein levels in the face of moderately low dietary protein and promotes an increase in the amount of body fat and energy.     

Synergistic effects of T-2 toxin and a low protein diet on erythropoiesis in mice

Young male Swiss mice fed on a semipurified diet containing 8% protein and 10 ppm of T-2 toxin developed erythroid hypoplasia within 2 weeks. Red blood cell counts declined to 36% of control values by 6 weeks but had risen to 45% of control values by 8 weeks. Between 4 and 8 weeks, erythropoietic tissues regenerated and reticulocyte counts became greatly elevated. The toxin-free semipurified diet was adequate for normal growth and did not cause anemia in control mice fed either ad lib, or at a restricted rate. Anemia did not occur in mice fed the 10-ppm level to T-2 toxin in either a semipurified diet containing 16% protein, or in a balanced natural-ingredient mouse diet. These observations demonstrated that the inhibitory effect of T-2 toxin in erythropoiesis in mice was transient, and depended on the nutritional composition of the diet.     

The effect of consumption of the pyrrolizidine alkaloid-containing plant Senecio jacobaea on iron and copper metabolism in the rat

The effect of dietary tansy ragwort (Senecio jacobaea), a pyrrolizidine alkaloid (PA)-containing plant, on mineral metabolism in rats was studied. In experiment 1, rats were fed a dietary level of 5% tansy ragwort. At intervals of 1, 2, 4, 6 and 8 weeks animals were killed and tissue mineral levels determined. As compared to comparable controls, rats fed tansy ragwort showed by 6 weeks elevated liver and spleen copper and iron levels. Experiment 2 was a 3 X 4 factorial experiment with added dietary copper levels of 0, 50 and 250 ppm, and tansy ragwort levels of 0, 1, 2.5 and 5%. Liver copper levels increased markedly with increasing levels of tansy ragwort; at 0, 50 and 250 ppm added copper, liver copper levels were 4, 18 and 21 times greater in rats fed 5% tansy ragwort as compared to those with no tansy ragwort. Increases in liver iron and spleen copper were noted with consumption of tansy ragwort. Higher liver copper levels were observed when a casein-based diet rather than a soybean meal diet was used, suggesting an effect of phytate in soybean meal in reducing copper absorption. In the last experiment, 59Fe was administered to rats fed diets with or without tansy ragwort. After 5 weeks on tansy ragwort, rats showed very low levels of 59Fe in erythrocytes, tibia and liver, and elevated levels in spleen and kidney, suggesting either an impairment of hematopoesis or accelerated erythrocyte destruction as a result of PA consumption. These results indicate that PA's cause increased liver copper content, and disturbances in iron metabolism.     

Respiratory infection with attenuated Mycobacterium tuberculosis H37Ra in malnourished guinea pigs.

Specific pathogen-free guinea pigs were infected via the respiratory route with viable, attenuated Mycobacterium tuberculosis H37Ra and maintained on purified isocaloric diets. The control diet contained 30% protein (ovalbumin) and 50 ppm of added zinc (50 micrograms/g), the low protein diet contained 10% protein and 50 ppm of added zinc, and the low zinc diet contained 30% protein and no added zinc. Guinea pigs from each diet treatment were skin tested with purified protein derivative 48 h before sacrifice at 3, 4, and 5 weeks postinfection. Protein-deficient animals exhibited significantly reduced body weight, spleen weight, serum total proteins, and serum albumin. Zinc deficiency was characterized by loss of weight and progressive reductions in plasma zinc concentrations. The number of viable M. tuberculosis H37Ra cells was significantly higher in the lungs of both malnourished groups at 3 weeks, but fell below control viable counts by 5 weeks postinfection. A similar pattern was seen in the spleens and bronchotracheal lymph nodes. Both the proportion and intensity of delayed hypersensitivity reactions increased steadily between 3 and 5 weeks in control animals, whereas the two malnourished groups were essentially anergic at all intervals, despite systemic infection. These results demonstrate that both protein and zinc deficiencies exert a significant influence on the development of pulmonary tuberculosis but that the nature of the influence depends upon the interval studied. In both malnourished groups, the pulmonary infection tended to peak early and decline, whereas the disease developed more slowly in control animals. Apparent control of mycobacterial populations in the tissues was accomplished by malnourished animals in the absence of demonstrable delayed hypersensitivity.     

Ancylostoma ceylanicum anticoagulant peptide-1: role of the predicted reactive site amino acid in mediating inhibition of coagulation factors Xa and VIIa

Hookworm infection is a leading cause of gastrointestinal blood loss and iron deficiency anemia in developing countries. Ancylostoma hookworms secrete potent anticoagulants, which have been shown to target coagulation factors Xa and the factor VIIa/Tissue Factor complex, respectively. The goal of these experiments was to determine the mechanism of action of three recombinant hookworm anticoagulants using in vitro assays. Three hookworm coagulation inhibitors were expressed and purified, along with site directed mutants targeting each of the predicted P1 inhibitory reactive site amino acid residues. Using chromogenic assays, it has been confirmed that Ancylostoma caninum Anticoagulant Peptide 5 (AcAP5) inhibits coagulation factor Xa (fXa) by a canonical, substrate-like mechanism. In contrast, Ancylostoma ceylanicum Anticoagulant Peptide-1 (AceAP1) binds to and inhibits fXa by both active site and non-active site mediated interactions. Data from in vitro studies also demonstrates that AceAP1 inhibits the factor VIIa/Tissue complex (fVIIa/TF) and displays a distinct pattern of fXa binding. Together, these data suggest that the human hookworm A. ceylanicum has evolved a single anticoagulant that targets multiple components of the mammalian coagulation response, effectively mimicking the concerted action of the two related inhibitors from A. caninum. Despite the amino acid sequence similarity, AceAP1 appears to interact with coagulation proteases fXa and fVIIa by a novel mechanism, perhaps explaining its spectrum of inhibitory activity.     

The pathogenicity of an enteric Citrobacter rodentium Infection is enhanced by deficiencies in the antioxidants selenium and vitamin E

The pathogenesis of a Citrobacter rodentium infection was evaluated in mice fed diets with a single deficiency in either selenium or vitamin E or with a double deficiency in both selenium and vitamin E compared to mice on nutritionally adequate diets. Mice fed the selenium- and vitamin E-deficient diet for 6 weeks had increased loads of C. rodentium in the colon and spleen, which were not observed in mice fed either of the singly deficient diets or the adequate diet. Infected mice fed the doubly deficient diet had increased colon crypt hyperplasia and an influx of infiltrating cells along with gross changes to crypt architecture, including ulceration and denuding of the epithelial layer. Cytokine and chemokine mRNA levels in the colon were measured by real-time PCR. Expression of proinflammatory cytokines and chemokines was upregulated on day 12 after infection with C. rodentium in mice fed the doubly deficient diet compared to mice fed the control diet. Heme oxygenase 1, an enzyme upregulated by oxidative stress, also was more highly induced in infected mice fed the doubly deficient diet. Production of C. rodentium antigen-specific IgM and IgG antibodies was not affected by feeding the doubly deficient diet. The results indicated that selenium and vitamin E play an important role in host resistance and in the pathology induced by C. rodentium, an infection that mimics disease caused by common food-borne bacterial pathogens in humans.     

Maternal hemoglobin depletion in a settled Northern Kenyan pastoral population

Objectives: This study examines maternal hemoglobin depletion in a cross‐sectional sample of Ariaal women living in northern Kenya. Maternal hemoglobin depletion occurs when women do not have enough dietary iron to replace the high levels of iron allocated to the fetus during pregnancy. Methods: To study this phenomenon, reproductive histories, socioeconomic status, anthropometry, and hemoglobin levels were collected from a cross‐section of 200 lactating Ariaal women in northern Kenya. Results: Ariaal women show increasing levels of hemoglobin with increasing time since birth and lower hemoglobin levels with increasing parity, indicating an incomplete repletion of dietary iron over women's reproductive lifetime. Women who lived in a more livestock‐dependent village had higher hemoglobin levels and lower prevalence of clinical anemia than women who lived in villages more dependent on agriculture, indicating that differences in diet may alleviate the effects of iron depletion. Conclusions: These data demonstrate that Ariaal women are iron depleted due to pregnancy, incompletely replete hemoglobin during the course of lactation, and show depletion of hemoglobin with increasing parity. Women in this community may be able to improve their iron status through a greater reliance on food sources rich in dietary iron. Am. J. Hum. Biol., 2010. © 2010 Wiley‐Liss, Inc.     

Effects of infection with transmissible gastroenteritis virus on concomitant immune responses to dietary and injected antigens

Normal piglets weaned onto soy- or egg-based diets generated antibody responses to fed protein. Concurrent infection with transmissible gastroenteritis virus (TGEV) did not affect the responses to dietary antigens at weaning, nor did it affect the subsequent development of tolerance. However, TGEV infection did enhance the primary immunoglobulin M (IgM) and IgG1, but not IgG2, antibody responses to injected soy in comparison to those of uninfected animals. Paradoxically, TGEV-infected animals showed an enhanced primary IgG1 antibody response to injected soy at 4 weeks of age, but they subsequently showed a reduced secondary response after an intraperitoneal challenge at 9 weeks of age in comparison to uninfected animals. The results suggest that an enteric virus, either used as a vaccine vector or present as a subclinical infection, may not have significant effects on the development of dietary allergies but may have effects both on the primary response and on the subsequent recall response to systemic antigens to which the animal is exposed concurrently with virus antigens.     

Effects of Infection with Transmissible Gastroenteritis Virus on Concomitant Immune Responses to Dietary and Injected Antigens

Normal piglets weaned onto soy- or egg-based diets generated antibody responses to fed protein. Concurrent infection with transmissible gastroenteritis virus (TGEV) did not affect the responses to dietary antigens at weaning, nor did it affect the subsequent development of tolerance. However, TGEV infection did enhance the primary immunoglobulin M (IgM) and IgG1, but not IgG2, antibody responses to injected soy in comparison to those of uninfected animals. Paradoxically, TGEV-infected animals showed an enhanced primary IgG1 antibody response to injected soy at 4 weeks of age, but they subsequently showed a reduced secondary response after an intraperitoneal challenge at 9 weeks of age in comparison to uninfected animals. The results suggest that an enteric virus, either used as a vaccine vector or present as a subclinical infection, may not have significant effects on the development of dietary allergies but may have effects both on the primary response and on the subsequent recall response to systemic antigens to which the animal is exposed concurrently with virus antigens.     

Trypanosoma evansi infection on levels of copper, iron, and zinc in the plasma of rats

This study aimed to assess plasma concentrations of copper, iron, and zinc during the course of acute Trypanosoma evansi experimental infection in rats. Fifty male Wistar rats were randomly distributed into seven groups: three trypanosome-infected groups (T2, T4, and T6; n?=?10 animals per group) and four uninfected controls (C0, C2, C4, and C6; n?=?5 animals per group). Animals from trypanosome-infected groups were intraperitoneally injected with 10⁶ trypanosomes/animal. Blood samples were collected by cardiac puncture before infection (day 0; group C0) or on the second (C2 and T2), fourth (C4 and T4), and sixth (C6 and T6) day postinfection. Parasitemia and hematological evaluation were performed to assess the progression of the disease in animals. The difference between groups (control and infected) was evaluated on the same day postinfection. Plasma copper concentration increased in T4 and T6 groups (P?<?0.001) compared with the control group. Plasma iron concentration decreased only in group T2 (P?<?0.001). There was no significant difference in the plasma zinc concentration between groups. This study therefore demonstrates that high plasma copper concentration and depression in iron concentration is part of the acute phase response in rats infected with T. evansi.     

Influence of graded magnesium deficiencies on white blood cell counts and lymphocyte subpopulations in rats.

Increased white blood cell counts or leukocytosis have been observed primarily in young rats fed diets extremely low in Mg (< 60 ppm) and high in phosphorus (0.5 % P). We investigated the influence that acute and moderate Mg deficiencies have on blood leukocytes at high and low dietary phosphorus levels. For four weeks, male Sprague-Dawley rats (initially 7 weeks old) were fed diets containing 30, 60, 120, 208, or 850 ppm Mg and either 0.3 % or 0.5 % dietary phosphorus. Total leukocytes were increased in rats fed 30 ppm Mg (p < 0.0001), and the leukocyte subpopulation counts of lymphocytes, neutrophils, monocytes and eosinophils increased significantly only in the rats fed 30 ppm Mg (p < 0.0001). B-cells decreased significantly as a percentage of lymphocytes (p < 0.0093) as dietary Mg decreased. As total counts in blood, B-cells, CD4 and CD8 cells were significantly increased in the rats consuming the 30 ppm Mg diet. Dietary phosphorus only had an effect in combination with the lowest dietary Mg. These results demonstrate a threshold effect for increased leukocytes during a Mg deficiency of four weeks. A Mg deficiency of a longer duration may show different results.     

The effect of vitamin A deficiency and fluoride on glycosaminoglycan metabolism in bone

The effects of fluoride intake and vitamin A deficiency on glycosaminoglycan metabolism in vivo were investigated. Weanling female rats were fed either a vitamin A deficient diet ad libitum, a vitamin A supplemented diet pair-fed to the deficient animals, or the vitamin A supplemented diet ad libitum. Additionally, each vitamin A dietary group was divided into three subgroups with the animals receiving water containing 0, 10 or 50 ppm fluoride. The results showed that the groups receiving 10 and 50 ppm fluoride at all dietary levels of vitamin A had significantly higher in vivo 35SO4 incorporation in both the epiphyseal and diaphyseal regions of the bone than the animals receiving 0 ppm fluoride. The vitamin A deficient animals incorporated significantly less 35SO4 into glycosaminoglycans in the epiphyseal and diaphyseal regions of the bone compared to the pair-fed, vitamin A sufficient animals for all three fluoride receiving groups. There was no interaction between fluoride intake and dietary vitamin A levels on 35SO4 incorporation into glycosaminoglycans. Fluoride either increased sulfation or turnover of glycosaminoglycans.     

Subchronic Toxicity of 2,4,4'-Trichlorobiphenyl in the Rat Liver: An Ultrastructural and Biochemical Study

2,4,4'-Trichlorobiphenyl or PCB congener 28 was given to Sprague-Dawley weanling rats and the experimental diets were prepared by mixing the congener in 4% corn oil. The congener was administered to animals placed in four groups, each comprising 10 males or females. The diets contained 0.05, 0.5, 5, or 50 ppm congener. The fifth or control group comprised animals that received diets mixed with corn oil. Thirteen weeks after commencement of dosing, animals were euthanized and liver specimens were harvested from the animals and prepared for electron microscopy and biochemical analyses. The hepatocyte architectural modifications included an augmentation of SER profiles and an elevation of peroxisome numbers in animals regardless of gender, and mitochondrial abnormalities in the females only. Mitochondrial aberrations consisted of abnormal shapes and cristae in atypical orientation. The alterations were revealed in animals of the 5-and 50-ppm groups and were more extensive in the females. Ethoxyresorufin-O-de-ethylase activity was significantly high in the animals of the 50-ppm group. The results suggest that the female rats were more sensitive than the males to congener 28, and the no observable adverse effect level (NOAEL) was believed to be 0.5 ppm for the congener.     

Low dietary iron intake restrains the intestinal inflammatory response and pathology of enteric infection by food‐borne bacterial pathogens

Orally administrated iron is suspected to increase susceptibility to enteric infections among children in infection endemic regions. Here we investigated the effect of dietary iron on the pathology and local immune responses in intestinal infection models. Mice were held on iron‐deficient, normal iron, or high iron diets and after 2 weeks they were orally challenged with the pathogen Citrobacter rodentium. Microbiome analysis by pyrosequencing revealed profound iron‐ and infection‐induced shifts in microbiota composition. Fecal levels of the innate defensive molecules and markers of inflammation lipocalin‐2 and calprotectin were not influenced by dietary iron intervention alone, but were markedly lower in mice on the iron‐deficient diet after infection. Next, mice on the iron‐deficient diet tended to gain more weight and to have a lower grade of colon pathology. Furthermore, survival of the nematode Caenorhabditis elegans infected with Salmonella enterica serovar Typhimurium was prolonged after iron deprivation. Together, these data show that iron limitation restricts disease pathology upon bacterial infection. However, our data also showed decreased intestinal inflammatory responses of mice fed on high iron diets. Thus additionally, our study indicates that the effects of iron on processes at the intestinal host–pathogen interface may highly depend on host iron status, immune status, and gut microbiota composition.