Veterinary Parasitology, 35 (1990) 71-77 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
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Concurrent Infection of E n t e r o c y t e s with E i m e r i a s c a b r a and Other E n t e r o p a t h o g e n s in S w i n e B. KOUDELA 1, J. V[TOVEC 1 and J. STI~RBA2
1Institute of Parasitology, Czechoslovak Academy of Sciences, Branigovskd 31, 370 05 C'eskOBudOjovice (Czechoslovakia) 2Laboratory of Electron Microscopy, Branigovskd 31, South Bohemian Biological Center, Czechoslovak Academy of Sciences, CeskO BudOjovice (Czechoslovakia) (Received for publication 29 June 1989 )
ABSTRACT Koudela, B., Vitovec, J. and St~rba, J., 1990. Concurrent infection of enterocytes with Eimeria scabra and other enteropathogens in swine. Vet. Parasitol., 35: 71-77. Bacteria were detected in the enterocytes of the distal jejunum in weaned pigs on Days 7 and 9 post-infection (DPI) with Eimeria scabra in addition to the developmental stages of the coccidia. Short rod-shaped bacteria were identified in ~ 60% of the enterocytes that contained developmental stages of E. scabra. No such bacteria were observed in cells where coccidia were absent. Gamonts of cryptosporidia were also observed within the microvillous zone of the enterocytes in the distal jejunum of weaned pigs on DPI 9 with E. scabra. Cryptosporidia were present only in enterocytes harbouring stages of E. scabra. Chlamydial particles were also found in the cytoplasm of enterocytes 7 DPI with E. scabra. The presence of other enteropathogens exclusively in the enterocytes containing developmental stages of coccidia suggests that the coccidium E. scabra facilitates the invasion and development of bacteria, cryptosporidia and chlamydia in the enterocytes.
INTRODUCTION
The life cycle ofEimeria scabra has been described by Rommel and Ipczynski (1967). Pathogenicity and pathological alterations, consequent to the experimental infection of weaned pigs with E. scabra were studied by V/tovec et al. (1987). The present report describes concurrent infections of enterocytes in the distal jejunum of weaned pigs with E. scabra and other infectious agents. MATERIALS AND METHODS
The method and course of experimental infection with E. scabra was performed as described by V/tovec et al. (1987). Briefly, 22 coccidia-free weaned 0304-4017/90/$03.50
© 1990 Elsevier Science Publishers B.V.
72
B. KOUDELAETAL.
pigs aged 32-70 days were inoculated orally with two different doses of E. scabra (200 000 and 4 X 10Goocysts) using a stomach tube. Pigs were killed 3-18 days post-infection (DPI) and necropsies were performed. Tissue specimens from their intestines for histological investigations were fixed in formol saline and processed routinely. Sections were cut and stained with haematoxylin and eosin, and with azur-eosin to identify coccidia in the sections. Similar intestinal tissue samples for transmission electron microscopy (TEM) were fixed in formaldehyde-glutaraldehydeas described by Karnovsky (1965) and specimens for scanning electron microscopy (SEM) were fixed in 4% buffered paraformaldehyde. Material for TEM was post-fixed in osmium tetroxide, dehydrated and embedded in resin. Ultrathin sections were cut and stained with uranyl acetate and lead citrate, and examined with a Philips EM 420 electron microscope. For SEM, the tissue specimens were dehydrated and critical point dried under CO2 before being mounted and sputter coated with gold, and viewed in the Tesla BS 300. RESULTS AND DISCUSSION
Upon studying the ultrastructure of the distal jejunum in weaned pigs 7 and 9 DPI, it was observed that the cytoplasm of enterocytes contained bacteria in addition to developmental stages of the coccidia (Fig. 1A-C). Short rod-shaped bacteria were found in ~ 60% of enterocytes which contained developmental stages ofE. scabra. No such bacteria were observed in cells where coccidia were absent. Within enterocytes infected with E. scabra and bacteria, ultrastructural changes were more severe than in cells infected with coccidia alone. In enterocytes infected with both agents, the cytoplasm contained reduced amounts of the intracellular organelles; mitochondria were scarce and dilated, the rough and smooth endoplasmic reticulum was dilated, and there was an increased number of residual bodies (Fig. 1B ). The most severe changes in the infected enterocytes were observed in the apical portion. The microvilli were bulbous and irregular, and the plasma membrane had ruptured (Figs. 1A and C, and 2C). Intercellular junctions between infected and neighbouring enterocytes were often disconnected (Fig. 1B). Coccidial stages within such cells displayed unequal maturity. By 7 DPI, enterocytes infected with both bacteria and E. scabra contained mostly mature meronts, and by 9 DPI stages of gametogony were observed (Fig. 1A). In the enterocytes infected solely with E. scabra, the cytoplasm also contained dilated endoplasmic reticulum of both round and swollen mitochondria. The microvilli were shorter and more robust than those of the uninfected enterocytes. Sheppard (1974) reported similar ultrastructural changes in rats experimentally infected with Eimeria nieschulzi. He found these changes in entero-
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Fig. 1. (A) Enterocyte of the distal jejunum concurrently infected with bacteria and Eimeria scabra. MG = macrogamont. TEM; scale bar= 1 ttm. (B) Enterocyte with a macrogamont of E. scabra in the cytoplasm together with bacteria. MG ---macrogamont of E. scabra; B = bacteria; arrows-- loosening of the intercellular junction. TEM; scale bar = 0.5/zm. (C) Inner surface of the distal jejunum of weaned pigs; enterocytes concurrently infected with bacteria and E. scabra. MR=merozoites of E. scabra; MG=macrogamont of E. scabra; arrow = bacteria. SEM; scale bar= 3/lm. (D) Enterocytes of distal jejunum concurrently infected with cryptosporidia and E. scabra. MG ---macrogamont of E. scabra; Cr = cryptosporidia. TEM; scale bar = 2 ttm.
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Fig. 2. (A) Enterocytes of distal jejunum infected concurrently with chlamydia and E. scabra. CH = chlamydia; M R = merozoites of E. scabra; N = nucleus of the host cell. TEM; scale bar = 1 /lm. (B) Chlamydial particles at different phases of development. R B = r e t i c u l a t e bodies: IB = intermediate chlamydial forms; EB = elementary bodies of chlamydia. TEM; scale bar = 0.3 ,am. ( C ) Morphology of the microvilli of an enterocyte infected with E. scabra compared to the micro villi of an uninfected enterocyte. MG = macrogamont of E. scabra. TEM; scale bar = 1 !nn.
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cytes invaded by the coccidia as well as in uninvaded enterocytes. In the present investigations, only enterocytes infected by E. scabra were altered. The postmortem examination of the pigs used in the present investigation included routine bacteriological examination of the intestine and some parenchymal organs (Vftovec et al., 1987). In animals whose enterocytes contained bacteria, no specific infectious pathogenic organisms were isolated. A strain of Escherichia coli from the intestine was found not serotyped. Among members of the family Enterobacteriaceae, species of the genera Salmonella, Yersinia and Shigella, and enteroinvasive strains of E. coli are capable of entering the cytoplasm of enterocytes (Finlay and Falkow, 1988). The bacteria detected in the enterocytes in conjunction with E. scabra were probably an enteroinvasive strain of E. coli (EIEC). Gamonts of cryptosporidia (Fig. 1D ) were observed within the microvillous zone of enterocytes in the distal part of the jejunum of the pigs 9 DPI with E. scabra (Fig. 1D ). The type and degree of alterations in these cells were similar in enterocytes infected with the two coccidial species and in those invaded with E. scabra alone. The present experiments provided evidence that pigs were also susceptible to infections with cryptosporidia, as has been proven in humans and calves (Moon and Bemrick, 1981; Tzipori et al., 1982). Recently, Tacal et al. (1987) demonstrated by faecal examination that 5% of the pigs were naturally infected with cryptosporidia and Sanford (1987) identified cryptosporidia in histological sections of the intestine in 184 of 3491 piglets examined (5.3 % ). The infections were most frequent in piglets aged 6-12 weeks. In the present observations, cryptosporidial infections were mild and none were found on faecal examination. Light microscopy detected no individual cryptosporidia, which could have been due to the dominance of pathological alterations caused by E. scabra observed 9 DPI. Chlamydia were detected in the cytoplasm of enterocytes 7 DPI with E. scabra (Fig. 2A ). Chlamydial particles were seen as reticular bodies ( 500-700 nm ) in the cytoplasmic vacuole. They were small electron-dense elementary bodies (250-400 n m ) and transient bodies with an electron-dense centre (Fig. 2B). No chlamydia were observed in enterocytes which contained no stages of E. scabra. Ultrastructural changes in enterocytes infected with chlamydia and E. scabra were of the same nature and intensity as those in enterocytes infected with E. scabra alone. The isolation of chlamydia from the intestines of healthy pigs (KSlbl, 1969) and the presence of chlamydial antibodies in most pigs that did not have signs of disease (Wilson and Plummer, 1966) suggest that these organisms have low pathogenicity in pigs when present alone. Pospischil and Wood (1987) recently identified chlamydia in the intestines of pigs which had lesions caused by Salmonella typhimurium. The presence of chlamydia exclusively in enter-
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ocytes containing mature meronts of E. scabra suggests that the coccidium E. scabra enables chalmydia to invade and develop in enterocytes. In enterocytes infected concurrently by E. scabra and other enteropathogens, alterations of the cytoplasmic membrane were apparently induced by the coccidia; the penetration of other infectious agents was probably a secondary phenomenon. Morphologically, the changes were in the plasma membranes and specifically in the microvillous zone. They consisted of reduced numbers of microvilli, their shortening and thickening, and altered terminal web of the microfilaments (Fig. 2C). According to the working hypothesis of Pasternak and Fernando (1984), coccidial infection induces accumulation of the gel-phase lipid of the host cell membrane. Exogenous trypsin could then enter the infected cell and trigger the release of degradative enzymes that eventually lyse the infected cell from within. This sequence of events would ensure that coccidial stages are not released prematurely and that the host cell membrane becomes susceptible to lysis without the direct intervention of an elaborate mechanism that is governed by the parasite. In the present study, degradation of the host cell could have been by another infectious agent. More than one organism within one enterocyte would result in a more rapid degradation of the cell than an exclusively coccidial infection. This hypothesis is supported by the fact that concurrent infection of the enterocytes with E. scabra and bacteria resulted in more severe pathological alterations in the ultrastructure than with E. scabra alone. ACKNOWLEDGEMENTS
We thank Mrs. Martina Doudov~ and Mr. P~emysl Mil~Sek for excellent technical assistance. We owe special thanks to Miss Jana Kopetov~ for her assistance in the preparation of this manuscript.
REFERENCES Finlay, B.B. and Falkow, S., 1988. A comparison of microbial strategies of Salmonella, Shigella and Yersinia species. In: Bacteria Host Cell Interaction, Allan R. Liss Inc., New York, pp. 227243. Karnovsky, M.J., 1965. A fbrmaldehyde-paraformaldehyde fixative of high osmolarity for use in electron microscopy. J. Cell. Biol., 27: 137a-138a. KSlbl, V., 1969. Untersuchungen tiber des Vorkommen von Miyagawanellen beim Schwein. Wien. Tieraerztl. Monatsschr., 56: 332-335. Moon, H.W. and Bemrick, W.J., 1981. Fecal transmission of calf cryptosporidia between calves and pigs. Vet. Pathol., 18: 248-255. Pasternak, J. and Fernando, M.A., 1984. Host cell response to coccidian infection: an introspective survey. Parasitology, 88: 555-563. Pospischil, A. and Wood, R.L., 1987. Intestinal chlamydia in pigs. Vet. Pathol., 25: 568-570.
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Rommel, M. and Ipczynski, V., 1967. Der Lebenszyklus des Schweinekokzids Eimeria scabra (Henry 1931 ). Berl. Muench. Tieraerztl. Wochenschr., 80: 65-70. Sanford, S.E., 1987. Enteric cryptosporidial infection in pigs: 184 cases (1981-1985). J. Am. Vet. Med. Assoc., 190: 695-698. Sheppard, A.M., 1974. Ultrastructuralpathology ofcoccidian infection. J. Parasitol., 60:369 371. Tacal, J.V., Jr., Sobieh, M. and El-Ahraf, A,, 1987. Cryptosporidium in marked pigs in southern California, USA. Vet. Rec., 120:615 616. Tzipori, S., Smith, M., Makin, T. and Halpin, C., 1982. Enterocolitis in piglets caused by Cryp tosporidium sp. purified from calf faeces. Vet. Parasitol., 11: 121-126. Vitovec, J., Koudela, B. and St~rba, J., 1987. Pathology and pathogenicity of Eimeria scabra (Henry 1931 ) in experimentally infected pigs. Folia Parasitol., 34: 299-304. Wilson, M.K. and Plummer, P., 1966, A survey of pig sera of the presence of antibodies to the psittacosis lymphogranuloma venereum group of organisms. J. Comp. Pathol., 76: 427-435.
71
Concurrent Infection of E n t e r o c y t e s with E i m e r i a s c a b r a and Other E n t e r o p a t h o g e n s in S w i n e B. KOUDELA 1, J. V[TOVEC 1 and J. STI~RBA2
1Institute of Parasitology, Czechoslovak Academy of Sciences, Branigovskd 31, 370 05 C'eskOBudOjovice (Czechoslovakia) 2Laboratory of Electron Microscopy, Branigovskd 31, South Bohemian Biological Center, Czechoslovak Academy of Sciences, CeskO BudOjovice (Czechoslovakia) (Received for publication 29 June 1989 )
ABSTRACT Koudela, B., Vitovec, J. and St~rba, J., 1990. Concurrent infection of enterocytes with Eimeria scabra and other enteropathogens in swine. Vet. Parasitol., 35: 71-77. Bacteria were detected in the enterocytes of the distal jejunum in weaned pigs on Days 7 and 9 post-infection (DPI) with Eimeria scabra in addition to the developmental stages of the coccidia. Short rod-shaped bacteria were identified in ~ 60% of the enterocytes that contained developmental stages of E. scabra. No such bacteria were observed in cells where coccidia were absent. Gamonts of cryptosporidia were also observed within the microvillous zone of the enterocytes in the distal jejunum of weaned pigs on DPI 9 with E. scabra. Cryptosporidia were present only in enterocytes harbouring stages of E. scabra. Chlamydial particles were also found in the cytoplasm of enterocytes 7 DPI with E. scabra. The presence of other enteropathogens exclusively in the enterocytes containing developmental stages of coccidia suggests that the coccidium E. scabra facilitates the invasion and development of bacteria, cryptosporidia and chlamydia in the enterocytes.
INTRODUCTION
The life cycle ofEimeria scabra has been described by Rommel and Ipczynski (1967). Pathogenicity and pathological alterations, consequent to the experimental infection of weaned pigs with E. scabra were studied by V/tovec et al. (1987). The present report describes concurrent infections of enterocytes in the distal jejunum of weaned pigs with E. scabra and other infectious agents. MATERIALS AND METHODS
The method and course of experimental infection with E. scabra was performed as described by V/tovec et al. (1987). Briefly, 22 coccidia-free weaned 0304-4017/90/$03.50
© 1990 Elsevier Science Publishers B.V.
72
B. KOUDELAETAL.
pigs aged 32-70 days were inoculated orally with two different doses of E. scabra (200 000 and 4 X 10Goocysts) using a stomach tube. Pigs were killed 3-18 days post-infection (DPI) and necropsies were performed. Tissue specimens from their intestines for histological investigations were fixed in formol saline and processed routinely. Sections were cut and stained with haematoxylin and eosin, and with azur-eosin to identify coccidia in the sections. Similar intestinal tissue samples for transmission electron microscopy (TEM) were fixed in formaldehyde-glutaraldehydeas described by Karnovsky (1965) and specimens for scanning electron microscopy (SEM) were fixed in 4% buffered paraformaldehyde. Material for TEM was post-fixed in osmium tetroxide, dehydrated and embedded in resin. Ultrathin sections were cut and stained with uranyl acetate and lead citrate, and examined with a Philips EM 420 electron microscope. For SEM, the tissue specimens were dehydrated and critical point dried under CO2 before being mounted and sputter coated with gold, and viewed in the Tesla BS 300. RESULTS AND DISCUSSION
Upon studying the ultrastructure of the distal jejunum in weaned pigs 7 and 9 DPI, it was observed that the cytoplasm of enterocytes contained bacteria in addition to developmental stages of the coccidia (Fig. 1A-C). Short rod-shaped bacteria were found in ~ 60% of enterocytes which contained developmental stages ofE. scabra. No such bacteria were observed in cells where coccidia were absent. Within enterocytes infected with E. scabra and bacteria, ultrastructural changes were more severe than in cells infected with coccidia alone. In enterocytes infected with both agents, the cytoplasm contained reduced amounts of the intracellular organelles; mitochondria were scarce and dilated, the rough and smooth endoplasmic reticulum was dilated, and there was an increased number of residual bodies (Fig. 1B ). The most severe changes in the infected enterocytes were observed in the apical portion. The microvilli were bulbous and irregular, and the plasma membrane had ruptured (Figs. 1A and C, and 2C). Intercellular junctions between infected and neighbouring enterocytes were often disconnected (Fig. 1B). Coccidial stages within such cells displayed unequal maturity. By 7 DPI, enterocytes infected with both bacteria and E. scabra contained mostly mature meronts, and by 9 DPI stages of gametogony were observed (Fig. 1A). In the enterocytes infected solely with E. scabra, the cytoplasm also contained dilated endoplasmic reticulum of both round and swollen mitochondria. The microvilli were shorter and more robust than those of the uninfected enterocytes. Sheppard (1974) reported similar ultrastructural changes in rats experimentally infected with Eimeria nieschulzi. He found these changes in entero-
INFECTIONOF ENTEROCYTESWITHEIMER1A SCABRA
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Fig. 1. (A) Enterocyte of the distal jejunum concurrently infected with bacteria and Eimeria scabra. MG = macrogamont. TEM; scale bar= 1 ttm. (B) Enterocyte with a macrogamont of E. scabra in the cytoplasm together with bacteria. MG ---macrogamont of E. scabra; B = bacteria; arrows-- loosening of the intercellular junction. TEM; scale bar = 0.5/zm. (C) Inner surface of the distal jejunum of weaned pigs; enterocytes concurrently infected with bacteria and E. scabra. MR=merozoites of E. scabra; MG=macrogamont of E. scabra; arrow = bacteria. SEM; scale bar= 3/lm. (D) Enterocytes of distal jejunum concurrently infected with cryptosporidia and E. scabra. MG ---macrogamont of E. scabra; Cr = cryptosporidia. TEM; scale bar = 2 ttm.
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Fig. 2. (A) Enterocytes of distal jejunum infected concurrently with chlamydia and E. scabra. CH = chlamydia; M R = merozoites of E. scabra; N = nucleus of the host cell. TEM; scale bar = 1 /lm. (B) Chlamydial particles at different phases of development. R B = r e t i c u l a t e bodies: IB = intermediate chlamydial forms; EB = elementary bodies of chlamydia. TEM; scale bar = 0.3 ,am. ( C ) Morphology of the microvilli of an enterocyte infected with E. scabra compared to the micro villi of an uninfected enterocyte. MG = macrogamont of E. scabra. TEM; scale bar = 1 !nn.
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cytes invaded by the coccidia as well as in uninvaded enterocytes. In the present investigations, only enterocytes infected by E. scabra were altered. The postmortem examination of the pigs used in the present investigation included routine bacteriological examination of the intestine and some parenchymal organs (Vftovec et al., 1987). In animals whose enterocytes contained bacteria, no specific infectious pathogenic organisms were isolated. A strain of Escherichia coli from the intestine was found not serotyped. Among members of the family Enterobacteriaceae, species of the genera Salmonella, Yersinia and Shigella, and enteroinvasive strains of E. coli are capable of entering the cytoplasm of enterocytes (Finlay and Falkow, 1988). The bacteria detected in the enterocytes in conjunction with E. scabra were probably an enteroinvasive strain of E. coli (EIEC). Gamonts of cryptosporidia (Fig. 1D ) were observed within the microvillous zone of enterocytes in the distal part of the jejunum of the pigs 9 DPI with E. scabra (Fig. 1D ). The type and degree of alterations in these cells were similar in enterocytes infected with the two coccidial species and in those invaded with E. scabra alone. The present experiments provided evidence that pigs were also susceptible to infections with cryptosporidia, as has been proven in humans and calves (Moon and Bemrick, 1981; Tzipori et al., 1982). Recently, Tacal et al. (1987) demonstrated by faecal examination that 5% of the pigs were naturally infected with cryptosporidia and Sanford (1987) identified cryptosporidia in histological sections of the intestine in 184 of 3491 piglets examined (5.3 % ). The infections were most frequent in piglets aged 6-12 weeks. In the present observations, cryptosporidial infections were mild and none were found on faecal examination. Light microscopy detected no individual cryptosporidia, which could have been due to the dominance of pathological alterations caused by E. scabra observed 9 DPI. Chlamydia were detected in the cytoplasm of enterocytes 7 DPI with E. scabra (Fig. 2A ). Chlamydial particles were seen as reticular bodies ( 500-700 nm ) in the cytoplasmic vacuole. They were small electron-dense elementary bodies (250-400 n m ) and transient bodies with an electron-dense centre (Fig. 2B). No chlamydia were observed in enterocytes which contained no stages of E. scabra. Ultrastructural changes in enterocytes infected with chlamydia and E. scabra were of the same nature and intensity as those in enterocytes infected with E. scabra alone. The isolation of chlamydia from the intestines of healthy pigs (KSlbl, 1969) and the presence of chlamydial antibodies in most pigs that did not have signs of disease (Wilson and Plummer, 1966) suggest that these organisms have low pathogenicity in pigs when present alone. Pospischil and Wood (1987) recently identified chlamydia in the intestines of pigs which had lesions caused by Salmonella typhimurium. The presence of chlamydia exclusively in enter-
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ocytes containing mature meronts of E. scabra suggests that the coccidium E. scabra enables chalmydia to invade and develop in enterocytes. In enterocytes infected concurrently by E. scabra and other enteropathogens, alterations of the cytoplasmic membrane were apparently induced by the coccidia; the penetration of other infectious agents was probably a secondary phenomenon. Morphologically, the changes were in the plasma membranes and specifically in the microvillous zone. They consisted of reduced numbers of microvilli, their shortening and thickening, and altered terminal web of the microfilaments (Fig. 2C). According to the working hypothesis of Pasternak and Fernando (1984), coccidial infection induces accumulation of the gel-phase lipid of the host cell membrane. Exogenous trypsin could then enter the infected cell and trigger the release of degradative enzymes that eventually lyse the infected cell from within. This sequence of events would ensure that coccidial stages are not released prematurely and that the host cell membrane becomes susceptible to lysis without the direct intervention of an elaborate mechanism that is governed by the parasite. In the present study, degradation of the host cell could have been by another infectious agent. More than one organism within one enterocyte would result in a more rapid degradation of the cell than an exclusively coccidial infection. This hypothesis is supported by the fact that concurrent infection of the enterocytes with E. scabra and bacteria resulted in more severe pathological alterations in the ultrastructure than with E. scabra alone. ACKNOWLEDGEMENTS
We thank Mrs. Martina Doudov~ and Mr. P~emysl Mil~Sek for excellent technical assistance. We owe special thanks to Miss Jana Kopetov~ for her assistance in the preparation of this manuscript.
REFERENCES Finlay, B.B. and Falkow, S., 1988. A comparison of microbial strategies of Salmonella, Shigella and Yersinia species. In: Bacteria Host Cell Interaction, Allan R. Liss Inc., New York, pp. 227243. Karnovsky, M.J., 1965. A fbrmaldehyde-paraformaldehyde fixative of high osmolarity for use in electron microscopy. J. Cell. Biol., 27: 137a-138a. KSlbl, V., 1969. Untersuchungen tiber des Vorkommen von Miyagawanellen beim Schwein. Wien. Tieraerztl. Monatsschr., 56: 332-335. Moon, H.W. and Bemrick, W.J., 1981. Fecal transmission of calf cryptosporidia between calves and pigs. Vet. Pathol., 18: 248-255. Pasternak, J. and Fernando, M.A., 1984. Host cell response to coccidian infection: an introspective survey. Parasitology, 88: 555-563. Pospischil, A. and Wood, R.L., 1987. Intestinal chlamydia in pigs. Vet. Pathol., 25: 568-570.
INFECTIONOFENTEROCYTESWITHEIMERIA SCABRA
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Rommel, M. and Ipczynski, V., 1967. Der Lebenszyklus des Schweinekokzids Eimeria scabra (Henry 1931 ). Berl. Muench. Tieraerztl. Wochenschr., 80: 65-70. Sanford, S.E., 1987. Enteric cryptosporidial infection in pigs: 184 cases (1981-1985). J. Am. Vet. Med. Assoc., 190: 695-698. Sheppard, A.M., 1974. Ultrastructuralpathology ofcoccidian infection. J. Parasitol., 60:369 371. Tacal, J.V., Jr., Sobieh, M. and El-Ahraf, A,, 1987. Cryptosporidium in marked pigs in southern California, USA. Vet. Rec., 120:615 616. Tzipori, S., Smith, M., Makin, T. and Halpin, C., 1982. Enterocolitis in piglets caused by Cryp tosporidium sp. purified from calf faeces. Vet. Parasitol., 11: 121-126. Vitovec, J., Koudela, B. and St~rba, J., 1987. Pathology and pathogenicity of Eimeria scabra (Henry 1931 ) in experimentally infected pigs. Folia Parasitol., 34: 299-304. Wilson, M.K. and Plummer, P., 1966, A survey of pig sera of the presence of antibodies to the psittacosis lymphogranuloma venereum group of organisms. J. Comp. Pathol., 76: 427-435.
