Density of selected enzymes in the goblet cells of the small intestine in piglets experimentally infected with the coccidium Isospora suis

The density of selected enzymes in the goblet cells of the mucous membrane of the small intestine was studied in a group of 12 gnotobiotic piglets experimentally infected with the coccidium Isospora suis one day after parturition (DPP), using the Vickers M-786 scanning and integrating microdensity meter. At an infecting dose of 100,000 oocysts of I. suis, the histochemistry of the goblet cells of the mucous membrane of the piglets changed significantly in the period of 4 to 10 days after infection (DPI). Increases occur in the density of non-specific esterase (EC. 3.1.1.1.) and acid phosphatase (EC. 3.13.2.). The density of acid and neutral muco-substances declines and the densities of alkaline phosphatase (EC. 3.1.3.1.) and aminopeptidase M (EC. 3.4.11.2) are significantly high. The goblet cells of the mid and posterior parts of jejunum are very similar in their histochemistry in the experimentally infected gnotobiotic piglets. In the duodenum and ileum the histochemical picture of the goblet cells shows no substantial difference from the data recorded in the goblet cells of the mucous membrane of the small intestine of the four control piglets at an age of two to seven days.     

Morphometric analysis of the activity of alkaline phosphatase in the small intestine of piglets infected with the coccidium Isospora suis

Gnotobiotical one-day-old piglets were infected with 100,000 Isospora suis coccidia oocysts, and were immediately killed. In piglets killed on the 3rd to 11th day after infection (DAI), the morphometric analysis of alkaline phosphatase activity was performed in the area of the microvillus zone of small intestine. In the control 2-7 days old animals, the small intestine was not equally supplied with alkaline phosphatase. In duodenum the activity reached 88.37 per cent of the active length of absorbent surface (% LAac), in the middle jejunum 95.98 per cent LAac, in the dorsal jejunum 78.63% LAac and the ileum 90.55 % LAac. The width of the active area was more balanced and ranged from 5.003 microM in the ileum to 6.129 microM in the dorsal jejunum. In infected gnotobiotical piglets the lowest activity was found out on the 3rd to 4th DAI, with a greater decline on the 9th day after infection. The range from 25.99 to 40.50 per cent LAac with minimum in the duodenum and maximum in the ileum was observed on the 3rd DAI. In the middle and dorsal ileum the activity was nearly equal (28.34 and 27.69 per cent LAac). nI the dorsal jejunum a moderate increasing was up to 47.13% LAac on the 4th DAI, with the exception of the ileum, where the activity of alkaline phosphatase decreased to 24.96% LAac. On the 9th DAI the activity of alkaline phosphatase was nearly equal in the whole small intestine (from 55.70 to 60.01% LAac) with the maximum in the middle jejunum. In the width of the reaction product a direct dependence on the total activity of alkaline phosphatase was evident only in the segment of the middle and dorsal jejunum and ileum, but merely on the period of the 3rd to 4th DAI. The lowest values were measured in the middle jejunum (0.982 micron on the 3rd DAI and 0.709 micron on the 4th DAI). No dependence was observed between the total activity and the reaction product in the middle jejunum (0.982 micron on the 3rd DAI and 0.709 micron on the 4th DAI), there was no general stabilisation of the activity of alkaline phosphatase.     

Lysosomal activity in enterocytes in the small intestine in piglets experimentally infected with Isospora suis

The lysosomal activity of enterocytes of the small intestine mucosa was investigated in gnotobiotic and conventional piglets experimentally infected on the first day after birth (DAB) by the oocysts of the coccidia Isospora suis. A method of the proof of beta-D-glucuronidase (EC.3.2.1.31.) activity was used to demonstrate lysosomes. The piglets were infected by different infection doses of oocysts (100,000 oocysts in gnotobiotic piglets and 200,000 oocysts in conventional piglets). In the gnotobiotic infected piglets the activity of beta-D-glucuronidase in enterocyte lysosomes was investigated in the period from day 3 to day 11 after infection (DAI) and in the infected conventional piglets in the period from day 2 to day 10 after infection. Comparing the control piglets, the group of gnotobiotic piglets at the age of 2-5 days and the group of conventional piglets at the age of 4-7 days, the higher activity of beta-D-glucuronidase was demonstrated in the lysosomes of intestinal mucosa enterocytes in the gnotobiotic control piglets (+5.30 of the average density value, Dx). In the infected gnotobiotic and conventional piglets the pattern of beta-D-glucuronidase activity was found to have three stages in the course of this infection. Two stages can be characterized by a great increase in the enzyme activity (DAI 3-9 in gnotobiotic piglets, DAI 2-3 and 7-9 in conventional piglets. The third stage, which is manifest mainly in the conventional infected piglets, is characterized by a marked decrease in the activity of beta-D-glucuronidase, reaching the level of control findings (DAI 10 and mainly 11 in gnotobiotic piglets. DAI 4-6 and 10 in conventional piglets). A topographical picture shows that the two stages of increase and the stage of beta-D-glucuronidase activity decrease occur in the whole small intestine without any predisposition defect of the enzyme in the different sections of the small intestine.     

Mucus synthesis in the goblet cells of the small intestine in experimental infection with the coccidium Isospora suis in piglets

The state of mucus synthesis in the goblet cells of the small intestine was studied in conventional piglets infected with a dose of 200,000 oocytes of the coccidium Isospora suis the first and fifth day after parturition. The synthesis of mucus and its chemical characteristics undergo significant changes during the third and fourth day after infection. The activity of acid and neutral mucous substances declines; their level and the physiological synthetic function of goblet cells begin to return to the normal during the period starting on the eight to tenth day after infection. However, there were no fully functioning goblet cells in the broken numerical ratio even at the end of the period of investigation, i.e. the 13th day after infection. The thin surface layer of mucus remained almost unchanged within the whole extent of the small intestine parts studied.     

Densitometric analysis of aminopeptidase M activity in small intestine mucosa in piglets experimentally infected with Isospora suis

In gnotobiotical and conventional piglets infected a day post partum (DPP) with oocysts of the coccidium Isospora suis, densitometrical analysis of the activity of aminopeptidase M (EC.3.4.11.2; APM) was performed in the area of microvillous zone of the small intestine. Piglets were infected with different infection doses of oocysts (100,000 oocysts) in gnotobiotical piglets and 200,000 oocysts in conventional piglets). In infected gnotobiotical piglets, the APM activity was studied in the period from the 3rd to 11th day after infection (DAI) and in infected conventional piglets in the period of to the 2nd to 10th day after infection (DAI). Control piglets, in the group of the gnotobiotical animals at the age of 2 to 5 days in the group of the conventional animals at the age of 4 to 7 days, had different APM activity in the microvillous zone of the intestinal mucosa. It was stated that the microvillous zone of the intestinal mucosa gained higher values in control conventional piglets (+7.01 mean values of density). In infected gnotobiotical piglets the density fall of the reaction product of APM was demonstrated already on the third day with further marked reduction of APM density on the 4th day after infection in the whole small intestine with predominance of the persisting APM activity in ileum. Even despite the slight increase in the density of the reaction product of APM in the period from the 5th to 7th DAI (the highest increase in APM density on the 6th DAI), a further decrease of the activity was recorded again on the 8th and namely the 9th DAI in the whole small intestine (the lowest value of density was found in the rear jejunum), the ileum mucosa being affected, too. A slightly higher density of the reaction product of APM was found in the duodenum. On the 10th DAI the APM density started to change and on the 11th DAI in the duodenum and in the middle jejunum it even reached higher values in comparison with the control data. Some differences were proved in the infected conventional piglets in comparison with the development of the APM activity in the small intestine mucosa in the infected gnotobiotical piglets. On the 3rd and 4th DAI APM defect occurred in the whole small intestine, with APM density prevailing in the ileum mucosa (like in the group of infected gnotobiotical piglets). The second period of decrease in APM activity lasted for almost four days (6th to 9th DAI).(ABSTRACT TRUNCATED AT 400 WORDS)     

Activity of mucosubstances and the goblet cell count in the large intestine of piglets infected with the coccidium, Isospora suis

In a group of conventional and gnotobiotic piglets experimentally infected with the Isospora suis coccidia the quantitative presence of acid and neutral mucous substances in the large intestine and the counts of goblet cells in the surface mucosa and in Lieberkühnis crypts (in the following text called just the crypts) were investigated. In conventional piglets infected with the dose of 200,000 oocysts of I. suis coccidia the lowest content of acid mucous substances was recorded from the eighth to tenth day after infection (DAI). A decrease in the activity of neutral mucous substances was somewhat slower. The lowest count of goblet cells was found on DAI 9, especially on the surface mucosa (4.89 to 4.91 goblet cells per 10 enterocytes). There was observed no difference in the piglets infected the first and fifth day after parturition (DAP). Gnotobiotic piglets infected with the dose of 100,000 oocysts of I. suis coccidia on DAP 1 showed the lowest content of mucous substances in the large intestine from the ninth to tenth day after infection. Unlike the conventional piglets, in gnotobiotic piglets there was recorded a decrease in the content of acid and neutral mucous substances. The gnotobiotic piglets had the lowest counts of goblet cells in the surface mucosa (10:4.57) and in the crypts (10:7.71) on DAI 9. As to the quantitative proportions, in the conventional and gnotobiotic piglets neutral mucous substances prevailed on the other days (DAI 3-7 and DAI 11), similarly like on DAI 8. The results of this investigation revealed a functional disease of the large intestine in conventional and gnotobiotic piglets infected experimentally with the Isospora suis coccidia.     

Histochemistry of selected peptidases in small intestine mucosa in piglets experimentally infected with Isospora suis

In the small intestine mucosa of 24 gnotobiotical piglets experimentally infected the first day post partum with oocysts of the coccidium Isospora suis, the activities of dipeptidylpeptidase IV (EC.3.4.14.5.; DAP IV) and gamma-glutamyl transferase (EC.2.3.2.2.; GGT) in the microvillous zone of enterocytes were evaluated by scanning densitometry. The tissue of the small intestine in piglets infected with a dose of 100,000 oocysts of the coccidia of I. suis was examined in the period from the first till the eleventh day post infection (DPI). In the control piglets at the age of 2-5 days it was found that most of the DAP IV activity was located in the microvillous zone of the enterocytes of the middle jejunum, rear jejunum and ileum. The DAP IV activity of duodenum mucosa was lower; as compared with the activity in the mucosa of the jejunum and ileum it reached 53-57%. In the case of GGT activity, the highest density values of the reaction product were recorded in the microvillous zone of enterocytes of the duodenum and the whole jejunum, the lowest in the ileum mucosa (86-89%) of the activity found in the duodenum and jejunum). During the experimental infection the infected piglets had a significant deficit of both peptidases, especially DAP IV (the whole studied period). The development of GGT activity was slightly different with the onset of the marked decline of the enzyme activity only on the fifth DPI. The lower GGT activity persisted till the eighth DPI. The density of the GGT reaction product began to return to the normal on the ninth to eleventh DPI. No predisposition in the location of the deficit was observed in the peptidases studied during the infection. The decline of the activity of both enzymes influenced also the mucosa of all studied parts of the small intenstine. The difference lay in the relevance of lowering of the density of reaction product of DAP IV and GGT on other DPI and in the different intensities of the return of the activity to the physiological normal.     

The ratio of enterocytes and goblet cells in the mucosa of the small intestine in experimental infection of piglets with the coccidium Isospora suis

The proportions of cup-shaped cells and enterocytes were studied in piglets infected the first and fifth day after birth with 200,000 oocysts of Isospora suis. The greatest imbalance was found in the ratio of the cells of the intestinal mucous membrane in the region of middle and lower jejunum (ratio of 10:1.75)--this was recorded the third to fourth day after infection when the ratio made 10:0.62, whereas in the control animals it was 10:5.7. On the eighth to ninth day after infection the number of cup-shaped cells began to increase, the ratio reaching the values of 10:2.62. Even the 13th day after infection the ratio remained far from normal (10:2.50).     

Activity of selected dehydrogenases and monoamine oxidases in the small intestine of gnotobiotic piglets infected with the coccidium Isospora suis

In the small intestine of 16 gnotobiotic piglets infected a day post partum (DPP) by Isospora suis coccidia the activities were studied of selected dehydrogenases and monoaminoxidase (O2 oxidoreductase, MAOx, EC 1.4.3.4.). The following dehydrogenases were investigated: succinate dehydrogenase (SDH, EC 1.3.99.1.), glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:menadion oxidoreductase, GPOX, EC 1.1.99.5.) and tetrazolium oxidoreductase (NADH, ES 1.6.99.3.). The activities of NADH and GPOX were found to decrease, a decrease being somewhat milder in MAOx, at a high infection dose of I. suis oocysts (750,000 oocysts), in comparison with the control, already on the first day after infection (DAI). The SDH levels did not change. In piglets infected by a low infection dose of I. suis oocysts a double marked decrease (negative to slightly positive finding) was recorded in the period from the third to the eighth day after infection (DAI). A similar pattern with a longer time interval between the decreases was observed in GPOX (4th to 11th day after infection). The findings of SDH and MAOx activities were different. The SDH activity is maintained at the same level (++) for the whole period of investigation and there occurs a decrease (+) only on the 9th day after infection, persisting until 11 DAI. The MAOx activity and its change correspond to the SDH activity; the difference being that in the second group the starting level is high ( ) and on the eleventh day after infection it is low or medium (0-++), in comparison with the standard. This variability is discernible from 8th DAI.     

Alkaline phosphatase activity levels during infection with the coccidia Isospora suis in piglets

The activity of alkaline phosphatase (EC 3.1.3.1.) was studied in the small intestines of piglets infected with the coccidia Isospora suis on the first and fifth day of age. Alkaline phosphatase activity is reduced significantly the third to fourth day after infection (DAI) throughout the small intestine in piglets infected on the fifth day of age. Similar results were obtained in the piglets killed the eighth to tenth DAI which had also been infected on the fifth day of their age. Piglets infected the first day of age and killed on the 8th to 13th DAI exhibited no larger differences from the above-mentioned groups, but alkaline phosphatase activity was distributed more regularly all over the surface of the villi. The normal values, comparable with the controls, were not obtained within 13 days after infection (piglet age 14 days).     

Lectin histochemistry for glycoconjugates in the small intestines of piglets naturally infected with Isospora suis

Composition of glycoconjugates was examined in small intestines naturally infected with Isospora suis in preweaned pigs by use of 21 biotinylated-labeled lectins with avidin-biotin-peroxidase complex method. As compared with control pig, staining of 18 lectins altered in jejunal villus brush border and goblet cells of pigs naturally infected with I. suis. These results indicate that I. suis infection alters carbohydrate residues on the jejunal intestines.     

Pathogenicity of Isospora suis in gnotobiotic and conventionalised piglets

Isospora suis is unequivocally a primary pathogen of swine. Inoculation of I suis in conventionalised and germ-free piglets caused a biphasic disease course with marked diarrhoea, villous atrophy and necrosis of the intestinal epithelium at four to six and eight to 10 days after inoculation. The presence of a normal bacterial flora markedly (P less than 0.05) influenced the survival rate of piglets but did not appear to affect the histopathological changes observed. Mild limited focal necrosis and bile stasis were present in the liver at eight to 10 days after inoculation. In this period there was also ectasia of lymph vessels in the intestinal lymph nodes.     

Ultrastructure of developing Isospora suis in cultured cells

The ultrastructure of Isospora suis sporozoites, type-1 meronts, and type-1 merozoites was examined, using transmission electron microscopy of infected cultured cells. The ultrastructure of sporozoites and type-1 merozoites was similar. Each possessed trimembranous pellicles, subpellicular microtubules, a conoid, anterior and posterior polar rings, rhoptries, micronemes, a single vesicular nucleus, tubular mitochondria, Golgi complexes, ribosomes endoplasmic reticula, inactive micropores, amylopectin bodies, lipid bodies, dense bodies, and crystalloid bodies. Merozoites were produced by endodyogeny. Ultrastructural events associated with merozoite production by type-1 meronts are described.     

Pathogenesis of experimental combined infections with Isospora suis and rotavirus in conventional and gnotobiotic piglets

51 gnotobiotic and 63 conventional, one-, or two-days-old piglets were divided into five groups and infected orally either with Isospora suis or rotavirus alone, or with both agents simultaneously or successively with alternative sequences and various intervals. 15 gnotobiotic and 10 conventional piglets served as controls. The development of small intestinal lesions after infection with I. suis was biphasic. The dominant alteration resulting from rotavirus infection was villus atrophy, considerably more pronounced and extensive in gnotobiotic than in conventional piglets. Synergistic action of I. suis and rotavirus was manifested both clinically, and morphologically. This action culminated at the time of the actual, or presumed development of merogony of I. suis, i.e. on DPI 3 to 5. The action develops only if the intestinal epithelium is damaged functionally and morphologically by a preceding rotavirus infection. It is concluded that the synergistic action is based on a competition of rotavirus and I. suis for mature, enzymatically active absorptive cells.     

猪附红细胞体人工感染小鼠的病理学试验

附红细胞体是寄生于人、猪以及其他动物的红细胞表面、血浆以及骨髓内的一群多形态微生物.猪附红细胞体病缺乏特征性临床症状和病理变化,使发病率和死亡率大大升高,给养殖户造成很大经济损失.本试验旨在研究猪附红细胞体病对小鼠血液生理指标的影响,及对小鼠各脏器的致病程度,为附红细胞体病的诊断及深入研究提供基础试验依据.     

Detection of rotavirus in experimentally infected piglets.

Scouring and vomiting was induced in piglets by experimental infection with a field strain of rotavirus. Virus or viral antigen was detected in the small intestine by the fluorescent antibody technique and virus could also be demonstrated in infected tissue culture cells by immuno-fluorescence. Progeny particles in the epithelial layer of the small intestine were identified by electron microscopy. Three morphological types could be distinguished, often associated with electron-dense inclusion, and infected cells, though small in number, were present throughout the length of the small intestine.     

Comparison of different methods for examining the feces of suckling piglets for Isospora suis

A highly sensitive diagnostic method is of great importance for detection of I. suis (Isospora suis). In the present study KSFV (combined sedimentation-flotation method) using different flotation solutions (NaCl, ZnSO4, NaCl-sucrose) was investigated regarding sensitivity, costs and expenditure of human labour in comparison to FV (flotation method) using NaCl-sucrose solution and to AM (autofluorescence microscopy). Ninety faecal samples were examined. Using AM, oocysts were detected in 83.3% of the samples. In cases of KSFV and FV, respectively, 41.1% to 53.3% of samples were tested positive. The quantity of detected oocysts was markedly higher in AM than in KSFV or FV. Therefore AM is the most sensitive of the comprised methods. The expenditure of labour is lowest in AM, time exposure is lower in FV than in KSFV. For performance of AM a fluorescence microscope is required contrary to KSFV and FV.     

Histochemistry of acid phosphatase in small intestine mucosa in experimental coccidiosis in suckling piglets]

The activity of acid phosphatase (phosphohydrolase of orthophosphate monoesters; EC. 3.1.3.2) was evaluated densitometrically in the mucosa of duodenum, jejunum and ileum of 22 conventional piglets which were experimentally infected by oocysts of the coccidiae Isospora suis (infection dose of 200,000 oocysts) on day one after parturition (DAP). The activity of the studied hydrolase was investigated in the infected piglets during days two to ten after infection (DAI) in the intestinal mucosa (enterocytes) and in goblet cells. The density of the reaction product of acid phosphatase was simultaneously determined in the same mucosal cells of different sections of the small intestine in five control conventional piglets at the age of 2-14 days. In the small intestine mucosa of control piglets the activity of acid phosphatase was demonstrated to be located especially in the supranuclear zone of enterocytes. As for goblet cells, the reaction product of acid phosphatase is distributed in all zones (supra-, para-, infranuclear zones); the lowest density of this enzyme was found in the infranuclear zone. The activity of acid phosphatase is also localized in intestinal crypts: in their cells the enzyme concentration is decreasing from duodenum to caudal sections. Important changes were revealed, in comparison with the control data, in the development of the activity of acid phosphatase in the intestinal mucosa cells in the experimentally infected piglets. In the period of investigation (DAI 2-10) there were two stages of the development of the density of the enzyme reaction product. The first stage can be characterized by an increase, the other by a decrease in the level of acid phosphatase activity. Enterocytes are influenced in both stages, but the decrease in the density of the reaction product of acid phosphatase was observed only in absorption cells, and not in goblet cells. The increase in the activity of acid phosphatase occurs in the periods of DAI 4 and 9-10. Enzymatic deviations occur mainly in the absorption cells of the mucosa of duodenum and middle jejunum; in the cells of posterior jejunum and ileum an increase in the density of the reaction product of acid phosphatase was also demonstrated, but at the lower quantitative level (especially on DAI 4). The decrease in the activity of acid phosphatase has a protracted development and it takes place on DAI 5 to 8.(ABSTRACT TRUNCATED AT 400 WORDS)     

仔猪实验性感染新疆猪瘟病毒野毒株的病理学观察

猪瘟 ( Hog cholera,HC)是由猪瘟病毒 ( HCV)引起猪的一种急性、热性、败血性、高度接触性传染病。猪是惟一在自然条件下对 HCV易感的动物 ,该病流行广泛、发病率和病死率高、危害极大 ,因而在国际兽疫局制定的《国际动物卫生法典》中 ,将它列为 A类 1 6种法定传染病之一 ,我国     

Prevalence of Isospora suis and Eimeria spp. in suckling piglets and sows in Poland

The aim of this investigation was to determine the prevalence of coccidian infections in suckling piglets and sows in Poland. The research was carried out in 14 out of 16 Polish provinces in the years 2003-2005. The investigation was conducted on three types of farms: large farms (>100 sows), medium farms (25-100 sows) and small farms (<25 sows). Diarrhoea of unweaned piglets was observed on all the examined farms. Overall, 780 litters of suckling piglets from 104 farms and 267 mother sows were examined. The faeces were analyzed with the modified McMaster method. Isopsora suis was found in 217 (27.8%) litters from 70 (66.7%) farms. Eimeria spp. was detected only in 20 (2.6%) litters from 12 (11.5%) farms. On the large farms I. suis infection was detected in 31.7% of litters whereas Eimeria spp. in 1.4% of them. On the medium sized farms I. suis was found in 18.1% of litters and Eimeria spp. in 0.6%. On the small farms I. suis was detected in only 13.2% of litters, whereas Eimeria spp. in as many as 28.9%. I. suis and Eimeria spp. oocysts were found in 18 (6.7%) and 16 (6%) sows respectively. From 72 sows producing I. suis infected piglets only 12 (16.7%) shed I. suis oocysts and as little as 4 (5.6%) shed Eimeria oocysts. In the remaining 56 sows (77.8%) no cases of coccidian infections were detected. The results of this investigation demonstrate the high prevalence of I. suis in suckling piglets on the large swine farms in Poland.