Parasitol Res (2014) 113:267–274 DOI 10.1007/s00436-013-3653-3
ORIGINAL PAPER
Dietary selenium affects intestinal development of Eimeria papillata in mice Mohamed A. Dkhil & Abdel Azeem S. Abdel-Baki & Frank Wunderlich & Helmut Sies & Saleh Al-Quraishy
Received: 15 September 2013 / Accepted: 9 October 2013 / Published online: 13 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Here, we investigated the effect of the trace element selenium (Se) on course and outcome of Eimeria-paplllatainduced coccidiosis in mice. Male mice were fed on Seadequate (0.15 ppm), Se-deficient, and Se-high diets (1.0 ppm) for 6 weeks. Mice were orally infected with 1,000 oocysts. The prepatent period lasts for 3 days, but the course of infections varied. At Se-adequate diet, the maximum fecal output of oocysts amounted to 68,300 ooccysts/g feces on day 5 p.i.. However, fecal shedding of oocysts was accelerated in mice on Se-deficient diet and occurred already on day 4 p.i.. By contrast, maximal shedding is impaired in mice on high-Se diet, which takes place on day 5 p.i., but with a decreased output of only 7,300 oocysts/g feces. Light microscopy reveals that all developmental stages are affected: meronts, micro- and macrogamonts, and developing oocysts are increased in comparison with mice fed on seleniumadequate diet. At high Se, the number of parasitic stages in the jejunum is substantially higher than at Se-deficient diet. Se M. A. Dkhil : A. A. S. Abdel-Baki : F. Wunderlich : H. Sies : S. Al-Quraishy (*) Department of Zoology, College of Science, King Saud University, P.O. Box: 2455, Riyadh 11451, Saudi Arabia e-mail: [email protected] M. A. Dkhil Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo, Egypt A. A. S. Abdel-Baki Department of Zoology, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt F. Wunderlich Department of Biology, Heinrich Heine University, Duesseldorf, Germany H. Sies Department of Biochemistry and Molecular Biology I, Heinrich Heine University, Duesseldorf, Germany
does not affect the number of jejunal Alcian blue-stained goblet cells. Se deficiency increased the number of apoptotic cells in the jejunum. Substantially increased histological injury scores reveal more injuries in jejunum tissue infected by E. papillata. Our data indicate that high dietary Se exerts potential anticoccidial activity. This may be taken advantage of in control measures towards Eimeriosis as a feed additive, potentially alleviating the need for concomitantly utilized anticoccidial drugs in the feed.
Introduction Coccidiosis is an enteric disease caused by protozoan parasites of the genus Eimeria . There are known more than 800 different species of Eimeria (Mehlhorn 2008) that affect many vertebrates worldwide. In particular, different Eimerian species are threatening industrial farming of animals such as cattle, rabbits, and poultry (Naidoo et al. 2008; Veronesi et al. 2013). The typical pathological signs of Eimerian coccidiosis are hemorrhagic diarrhea and dehydration resulting in rapid weight loss, that is—if not medicated—followed by secondary septicemia and death, with a mortality rate of up to 80 % (Fossum et al. 2009). Since the disease rapidly spreads from one animal to the next by contact with infected feces, the economic losses in animal farms are enormous. The losses and the worldwide costs for control measures such as medication and prophylactic vaccination can be only estimated to be in the range of about 3 billion dollars per annum (Dalloul and Lillehoj 2006), The Eimeria parasites undergo a complex life cycle (López-Bernad et al. 1998; Mehlhorn 2008). Infection starts with the oral uptake of sporulated oocysts which release the sporozoites in the duodenal intestine. Sporozoites, in turn, invade enterocytes in which they develop schizogonally to meronts, releasing merozoites. These penetrate again host
268
cells and can develop to male microgamonts and female macrogamonts. Microgamonts produce microgametes which fertilize macrogamonts, and the resulting zygotes form unsporulated oocysts. After rupturing of the host cells, these are then expulsed from the intestine with the feces of the host animals. Final sporulation to infective oocysts largely occurs outside the host animals (Mehlhorn 2008). The development and multiplication of Eimeria parasites in intestinal cells cause enormous tissue damage not only by rupturing host cells but also by oxidative stress. Selenium is fundamental for an organism’s trace element/antioxidant state. It is incorporated as selenocysteine into Se-dependent enzymes such as glutathione peroxidases (GPx) and is involved in protection against oxidative stress (see Steinbrenner and Sies 2009). This prompted us to investigate the effect of dietary selenium on intestinal infections of mice with Eimeria papillata.
Materials and methods Animals Five-week-old male Swiss albino mice were obtained from the animal facilities of King Saud University (Riyadh) and fed the experimental diets and water ad libitum for 6 weeks. The experiments were performed and approved by State authorities and followed Saudi Arabian rules for animal protection. Diets The selenium-deficient diet C1045 was obtained from Altromin (Lage, Germany). It is based on the principle of a selenium-deficient diet first described by Burk (1987). It consists of 30 % Torula yeast, 57 % sucrose, 6.7 % corn oil, a vitamin mix, and a selenium-free mineral mix. The selenium-adequate and the selenium-high diets were produced by mixing sodium selenite into the selenium-deficient diet to yield a final selenium content of 0.15 and 1.0 ppm, respectively. Diets were fed as pellets for a period of 6 weeks. Thus, at the end of the experiment, animals were 11 weeks old. Infection of mice A self-healing strain of E. papillata was kindly provided by Prof. H. Mehlhorn (Heinrich Heine University, Duesseldorf, Germany). We maintained E. papillata in naturally infected mice, collected oocysts from feces, surface-sterilized the oocysts with sodium hypochlorite and washed at least four times with sterile saline before oral inoculation as described previously (Schito et al. 1996; Dkhil et al. 2011). Each mouse
Parasitol Res (2014) 113:267–274
was orally inoculated with 103 sporulated oocysts of E. papillata suspended in 100 μl sterile saline. Subsequently, fresh fecal pellets were collected every 24 h. The collected pellets from each mouse were weighed and the bedding was changed to eliminate reinfection. Oocyst output was measured as previously described (Schito et al. 1996; Dkhil et al. 2011). Fecal pellets were suspended in 2.5 % (w /v ) potassium dichromate and diluted in saturated sodium chloride for oocyst flotation. Oocysts were counted in a McMaster chamber and expressed as number of oocysts per gram of wet feces. Experimental design Animals were allocated to eight groups of eight male mice each. The first, second, and third groups are the non-infected group receiving Se-adequate (0.15 ppm), Se-deficient, and Sehigh (1.0 ppm) diet, respectively. The fourth, fifth, and sixth groups are the corresponding E.-papillata- infected groups that were fed on adequate, deficient, and high-selenium diet, respectively. Two further groups of mice were fed on standard laboratory chow and were used for infection with E. papillata and as non-infected controls. Histological analysis Pieces of jejunum were freshly prepared, fixed in 10 % neutral buffered formalin, and then embedded in paraffin. Sections were cut and stained with hematoxylin and eosin. According to Dommels et al. (2007), tissue sections were scored for inflammatory lesions, as indicated by infiltrations of mononuclear cells, neutrophils, eosinophils, and plasma cells, for tissue destruction and tissue repair. A rating score between 0 (no change from normal tissue) and 3 (lesions involved most areas and all the layers) was given for each aspect of inflammatory lesion, tissue destruction, and tissue repair. The sum of inflammatory lesions (doubled to give more weight to this parameter), tissue destruction, and tissue repair scores was used to represent the total histological injury score. Jejunal parasite number and goblet cells The number of parasites in villi was counted in sections stained with hematoxylin and eosin. Also, sections were stained with Alcian Blue for identification of goblet cells. For each animal, the number of goblet cells in the jejunum was counted on well-orientated villous-crypt units (VCU). Results were expressed as the mean number of goblet cells per 10 VCU (Allen et al. 1986). TUNEL assay for apoptosis Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling assay (TUNEL) staining was performed using
Parasitol Res (2014) 113:267–274
269
Results Intestinal infections with E. papillata normally take a selfhealing course and outcome. Thus, when male outbred Swiss albino mice fed standard laboratory diet are orally infected with 103 oocysts, the prepatent period lasts approximately 3 days before oocysts are beginning to be expulsed with feces on day 4 p.i. (Fig. 1, top panel). Maximum shedding of approximately 45×103 oocysts per gram feces occurs on day 5 p.i. before declining towards close to zero on day 7 p.i. Thereafter, the feces are free from oocysts on day 8 p.i. (Fig. 1). To detect any effects of nutrient selenium on the course and outcome of E. papillata infections, three different diets were prepared: a Se-deficient diet, an adequate diet (0.15 ppm), and a high-Se diet (1.0 ppm). These diets were given to three different groups of mice, each group consisting of eight mice, in the form of pellets for 6 weeks, before the mice were orally infected with 103 oocysts. The outcome of infections in all three groups is self-healing, but the time-course of infections is significantly different between the groups. In the Se-
Fig. 1 Time course of output of oocysts from mice infected with 103 sporulated E. papillata oocysts. Data are from animals fed for 6 weeks on standard laboratory diet (upper panel) or on the selenium-deficient diet C1045, C1045 with 0.15 ppm sodium selenite (Se-adequate) or C1045 with 1.0 ppm sodium selenite (Se high; lower panel). Values are means ± SD (n =8)
a TUNEL Apoptosis Detection Kit (GenScript, Piscataway, NJ, USA) according to the manufacturer’s protocol. Briefly, sections of paraffin-embedded jejunum were deparaffinised, rehydrated in graded ethanol solutions before digestion with proteinase K. Slides were mounted with 4′,6-diamidino-2phenylindole. Nuclei of apoptotic cells are stained dark brown. Sections were counterstained with hematoxylin. For each animal, the number of apoptotic cells in the jejunum was counted on well-orientated VCUs. Results were expressed as the mean number of apoptotic cells per 10 VCUs.
Statistical analysis One-way ANOVA was carried out, and the statistical comparisons among the groups were performed with Duncan’s test using a statistical package program (SPSS version 17.0). All p values are two-tailed and p
ORIGINAL PAPER
Dietary selenium affects intestinal development of Eimeria papillata in mice Mohamed A. Dkhil & Abdel Azeem S. Abdel-Baki & Frank Wunderlich & Helmut Sies & Saleh Al-Quraishy
Received: 15 September 2013 / Accepted: 9 October 2013 / Published online: 13 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Here, we investigated the effect of the trace element selenium (Se) on course and outcome of Eimeria-paplllatainduced coccidiosis in mice. Male mice were fed on Seadequate (0.15 ppm), Se-deficient, and Se-high diets (1.0 ppm) for 6 weeks. Mice were orally infected with 1,000 oocysts. The prepatent period lasts for 3 days, but the course of infections varied. At Se-adequate diet, the maximum fecal output of oocysts amounted to 68,300 ooccysts/g feces on day 5 p.i.. However, fecal shedding of oocysts was accelerated in mice on Se-deficient diet and occurred already on day 4 p.i.. By contrast, maximal shedding is impaired in mice on high-Se diet, which takes place on day 5 p.i., but with a decreased output of only 7,300 oocysts/g feces. Light microscopy reveals that all developmental stages are affected: meronts, micro- and macrogamonts, and developing oocysts are increased in comparison with mice fed on seleniumadequate diet. At high Se, the number of parasitic stages in the jejunum is substantially higher than at Se-deficient diet. Se M. A. Dkhil : A. A. S. Abdel-Baki : F. Wunderlich : H. Sies : S. Al-Quraishy (*) Department of Zoology, College of Science, King Saud University, P.O. Box: 2455, Riyadh 11451, Saudi Arabia e-mail: [email protected] M. A. Dkhil Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo, Egypt A. A. S. Abdel-Baki Department of Zoology, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt F. Wunderlich Department of Biology, Heinrich Heine University, Duesseldorf, Germany H. Sies Department of Biochemistry and Molecular Biology I, Heinrich Heine University, Duesseldorf, Germany
does not affect the number of jejunal Alcian blue-stained goblet cells. Se deficiency increased the number of apoptotic cells in the jejunum. Substantially increased histological injury scores reveal more injuries in jejunum tissue infected by E. papillata. Our data indicate that high dietary Se exerts potential anticoccidial activity. This may be taken advantage of in control measures towards Eimeriosis as a feed additive, potentially alleviating the need for concomitantly utilized anticoccidial drugs in the feed.
Introduction Coccidiosis is an enteric disease caused by protozoan parasites of the genus Eimeria . There are known more than 800 different species of Eimeria (Mehlhorn 2008) that affect many vertebrates worldwide. In particular, different Eimerian species are threatening industrial farming of animals such as cattle, rabbits, and poultry (Naidoo et al. 2008; Veronesi et al. 2013). The typical pathological signs of Eimerian coccidiosis are hemorrhagic diarrhea and dehydration resulting in rapid weight loss, that is—if not medicated—followed by secondary septicemia and death, with a mortality rate of up to 80 % (Fossum et al. 2009). Since the disease rapidly spreads from one animal to the next by contact with infected feces, the economic losses in animal farms are enormous. The losses and the worldwide costs for control measures such as medication and prophylactic vaccination can be only estimated to be in the range of about 3 billion dollars per annum (Dalloul and Lillehoj 2006), The Eimeria parasites undergo a complex life cycle (López-Bernad et al. 1998; Mehlhorn 2008). Infection starts with the oral uptake of sporulated oocysts which release the sporozoites in the duodenal intestine. Sporozoites, in turn, invade enterocytes in which they develop schizogonally to meronts, releasing merozoites. These penetrate again host
268
cells and can develop to male microgamonts and female macrogamonts. Microgamonts produce microgametes which fertilize macrogamonts, and the resulting zygotes form unsporulated oocysts. After rupturing of the host cells, these are then expulsed from the intestine with the feces of the host animals. Final sporulation to infective oocysts largely occurs outside the host animals (Mehlhorn 2008). The development and multiplication of Eimeria parasites in intestinal cells cause enormous tissue damage not only by rupturing host cells but also by oxidative stress. Selenium is fundamental for an organism’s trace element/antioxidant state. It is incorporated as selenocysteine into Se-dependent enzymes such as glutathione peroxidases (GPx) and is involved in protection against oxidative stress (see Steinbrenner and Sies 2009). This prompted us to investigate the effect of dietary selenium on intestinal infections of mice with Eimeria papillata.
Materials and methods Animals Five-week-old male Swiss albino mice were obtained from the animal facilities of King Saud University (Riyadh) and fed the experimental diets and water ad libitum for 6 weeks. The experiments were performed and approved by State authorities and followed Saudi Arabian rules for animal protection. Diets The selenium-deficient diet C1045 was obtained from Altromin (Lage, Germany). It is based on the principle of a selenium-deficient diet first described by Burk (1987). It consists of 30 % Torula yeast, 57 % sucrose, 6.7 % corn oil, a vitamin mix, and a selenium-free mineral mix. The selenium-adequate and the selenium-high diets were produced by mixing sodium selenite into the selenium-deficient diet to yield a final selenium content of 0.15 and 1.0 ppm, respectively. Diets were fed as pellets for a period of 6 weeks. Thus, at the end of the experiment, animals were 11 weeks old. Infection of mice A self-healing strain of E. papillata was kindly provided by Prof. H. Mehlhorn (Heinrich Heine University, Duesseldorf, Germany). We maintained E. papillata in naturally infected mice, collected oocysts from feces, surface-sterilized the oocysts with sodium hypochlorite and washed at least four times with sterile saline before oral inoculation as described previously (Schito et al. 1996; Dkhil et al. 2011). Each mouse
Parasitol Res (2014) 113:267–274
was orally inoculated with 103 sporulated oocysts of E. papillata suspended in 100 μl sterile saline. Subsequently, fresh fecal pellets were collected every 24 h. The collected pellets from each mouse were weighed and the bedding was changed to eliminate reinfection. Oocyst output was measured as previously described (Schito et al. 1996; Dkhil et al. 2011). Fecal pellets were suspended in 2.5 % (w /v ) potassium dichromate and diluted in saturated sodium chloride for oocyst flotation. Oocysts were counted in a McMaster chamber and expressed as number of oocysts per gram of wet feces. Experimental design Animals were allocated to eight groups of eight male mice each. The first, second, and third groups are the non-infected group receiving Se-adequate (0.15 ppm), Se-deficient, and Sehigh (1.0 ppm) diet, respectively. The fourth, fifth, and sixth groups are the corresponding E.-papillata- infected groups that were fed on adequate, deficient, and high-selenium diet, respectively. Two further groups of mice were fed on standard laboratory chow and were used for infection with E. papillata and as non-infected controls. Histological analysis Pieces of jejunum were freshly prepared, fixed in 10 % neutral buffered formalin, and then embedded in paraffin. Sections were cut and stained with hematoxylin and eosin. According to Dommels et al. (2007), tissue sections were scored for inflammatory lesions, as indicated by infiltrations of mononuclear cells, neutrophils, eosinophils, and plasma cells, for tissue destruction and tissue repair. A rating score between 0 (no change from normal tissue) and 3 (lesions involved most areas and all the layers) was given for each aspect of inflammatory lesion, tissue destruction, and tissue repair. The sum of inflammatory lesions (doubled to give more weight to this parameter), tissue destruction, and tissue repair scores was used to represent the total histological injury score. Jejunal parasite number and goblet cells The number of parasites in villi was counted in sections stained with hematoxylin and eosin. Also, sections were stained with Alcian Blue for identification of goblet cells. For each animal, the number of goblet cells in the jejunum was counted on well-orientated villous-crypt units (VCU). Results were expressed as the mean number of goblet cells per 10 VCU (Allen et al. 1986). TUNEL assay for apoptosis Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling assay (TUNEL) staining was performed using
Parasitol Res (2014) 113:267–274
269
Results Intestinal infections with E. papillata normally take a selfhealing course and outcome. Thus, when male outbred Swiss albino mice fed standard laboratory diet are orally infected with 103 oocysts, the prepatent period lasts approximately 3 days before oocysts are beginning to be expulsed with feces on day 4 p.i. (Fig. 1, top panel). Maximum shedding of approximately 45×103 oocysts per gram feces occurs on day 5 p.i. before declining towards close to zero on day 7 p.i. Thereafter, the feces are free from oocysts on day 8 p.i. (Fig. 1). To detect any effects of nutrient selenium on the course and outcome of E. papillata infections, three different diets were prepared: a Se-deficient diet, an adequate diet (0.15 ppm), and a high-Se diet (1.0 ppm). These diets were given to three different groups of mice, each group consisting of eight mice, in the form of pellets for 6 weeks, before the mice were orally infected with 103 oocysts. The outcome of infections in all three groups is self-healing, but the time-course of infections is significantly different between the groups. In the Se-
Fig. 1 Time course of output of oocysts from mice infected with 103 sporulated E. papillata oocysts. Data are from animals fed for 6 weeks on standard laboratory diet (upper panel) or on the selenium-deficient diet C1045, C1045 with 0.15 ppm sodium selenite (Se-adequate) or C1045 with 1.0 ppm sodium selenite (Se high; lower panel). Values are means ± SD (n =8)
a TUNEL Apoptosis Detection Kit (GenScript, Piscataway, NJ, USA) according to the manufacturer’s protocol. Briefly, sections of paraffin-embedded jejunum were deparaffinised, rehydrated in graded ethanol solutions before digestion with proteinase K. Slides were mounted with 4′,6-diamidino-2phenylindole. Nuclei of apoptotic cells are stained dark brown. Sections were counterstained with hematoxylin. For each animal, the number of apoptotic cells in the jejunum was counted on well-orientated VCUs. Results were expressed as the mean number of apoptotic cells per 10 VCUs.
Statistical analysis One-way ANOVA was carried out, and the statistical comparisons among the groups were performed with Duncan’s test using a statistical package program (SPSS version 17.0). All p values are two-tailed and p
