Br. J. exp. Path. (1975) 56, 373
PATHOLOGICAL CHANGES IN PREGNANT MICE INFECTED WITH COXSACKIEVIRUS B3 AND GIVEN DIETARY CASEIN HYDROLYSATE SUPPLEMENT A. B. G. LANSDOWN From the Clinical Re8earch Centre, Watford Road, Harrow, Middle8ex, HAI 3UJ Received for publication April 7, 1975
Summary.-Coxsackievirus B3 infection in pregnant mice leads to a severe pancreatic exocrine insufficiency in the mothers and retarded foetal growth. As a consequence of the pancreatic damage, the animals are rendered incapable of digesting sufficient amounts of dietary proteins to allow maternal liver development to proceed as normal for the pregnant rodent. Faecal nitrogen was increased and the maternal livers were small for the weights of the animals and exhibited a lower than normal nitrogen content. Feeding of additional amino acids and simple peptides in the diet in the form of casein hydrolysate either from before or after virus injection appeared to compensate for the inability of these animals to digest dietary protein nitrogen and allowed maternal liver development and foetal growth to proceed at a rate not significantly different from normal. Although these results apply to infections with Coxsackievirus B3 in pregnancy, they may be relevant to other infections which adversely affect foetal growth by their pathological effects on maternal organs necessary for maintaining optimal foetal growth.
COXSACKIEVIRUS B infections inhuman pregnancy commonly result in mild or subclinical illness in the mother and may be responsible for congenital heart disease, spontaneous abortions, stillbirth and intrauterine growth retardation (Kilbrick and Benirischke, 1958; Czeizel, 1967; Brown and Karunas, 1972; Fruhling et al., 1962). Experimental studies in mice using Coxsackievirus B3 have shown that foetal growth retardation occurs and that the maturation of foetal plasma proteins is delayed (Coid and Ramsden, 1973; Coid, Ramsden and Healy, 1974; Lansdown, 1975). In a study of the pathology of Coxsackievirus B3 infections in pregnancy, in an experiment to elucidate the mechanism of foetal growth impairment, Lansdown and Coid (1974) suggested that whereas the possibility of the virus exerting a direct effect on the foetus cannot be discounted (Soike, 1967; Droughet and Levantis, 1968), foetal growth retardation
is largely due to a maternal protein deficiency resulting from virus induced pancreatic exocrine insufficiency. Subsequent studies have shown that if the diets of Coxsackievirus B3 infected pregnant mice are supplemented with casein hydrolysate, foetal growth and plasma protein development are not significantly impaired (Lansdown, Coid and Ramsden, 1975). In that work, attention was focused principally on the influence of the added casein hydrolysate on foetal growth. In the present communication, the influence of this dietary supplement on the pathological changes elicited in the mothers by the Coxsackievirus is described and consideration given to the possible mechanism by which the administration of casein hydrolysate in the diet mitigates foetal growth impairment. MATERIALS AND METHODS Viru.S stock.-The Coxsackievirus B3 used in these studies was derived from the original
A. B. G. LANSDOWN
374
Nancy strain. It had been passaged through suckling mice, isolated in primary rhesus monkey kidney cells and then through vervet monkey kidney cells (VERO). The virus suspension was diluted 1 in 10 with Hanks' buffered salt solution to give a tissue culture infective dose (TCID50) of 105 85/ml. Animal8.-Mice of the " TO " outbred strain from the original Swiss stock (Theiler) were used throughout this work. Virgin females (20-25 g) were placed overnight with proven males and the morning when vaginal plugs were detected was designated Day 1 of pregnancy. Inoculation of virus.-Pregnant mice were inoculated intramuscularly with 0-15 ml of virus suspension into each hind leg on the 8th day of gestation. Control mice were inoculated with an equivalent dose of virus suspension that had been inactivated by heating to 560 for 30 min. Experimenntal.-In an initial series of experiments, mice were given a standard rodent breeder diet (Spiller's Laboratory 1 diet) in powdered forn and containing 0 or 10% enzymic casein hydrolysate (Sigma Chemical Co., St Louis, U.S.A.) from Day 1 of pregnancy through until Day 18, when all animals were killed by cervical dislocation. In the second series the standard powdered diet was fed from Days 1 to 10 of gestation and then a diet supplemented with casein hydrolysate was given until Day 18. At autopsy, the animals were weighed with foetuses in utero. Maternal livers were also weighed and representative samples of this tissue (approximately 0 5 g) were stored at - 700 for total nitrogen analysis by the standard microKjeldahl technique. The remaining tissue was routinely fixed in phosphate buffered formalin for histological examination. Histological sections were stained by haematoxylin and eosin (H. and E.), PAS for glycogen, and by Oil red-O for neutral fat. Sections of maternal pancreas
were stained by H. and E. and by Gomori's
aldehyde fuchsin method for beta cell granules. Maternal faecal samples were collected during the 3 periods-0-8 days (pre-infection), 9-14 days (infective phase) and 15-18 (post-infective stage) and analysed for nitrogen content using the Kjeldahl estimation. RESULTS
On the 18th day of gestation mice in all groups, including those given virus and casein hydrolysate, appeared to be in a good state of health although some animals injected with live virus and not receiving the casein hydrolysate weighed less than those of other groups and on occasions had slightly ruffled coats. Animals ate and drank well at all stages of the experiments and the diet supplemented with casein hydrolysate did not appear to be unpalatable. At autopsy, all mice injected with live virus, irrespective of the constitution of their diets, exhibited a marked reduction in the size of the pancreas, visceral adhesions and evidence of fatty tissue degeneration in the visceral fat pads. The liver was small in live virus treated mice fed no casein hydrolysate and the ratio of liver weight to the total body weight of the animal was lower than in other groups (see Table). When the liver samples were analysed for their total nitrogen content, the nitrogen level in the livers of infected mice fed no supplement
TABLE.-Maternal Liver Weights Relative to Body Weights in Mice Infected with Cox8ackievirus B3 and Given Dietary Supplements of Casein Hydroly8ate Days of pregnancy when casein hydrolysate given 1-18 1-18
1-18
10-18 10-18 10-18
Average body weight
Nitrogen Liver weight Liver weight X 100 content (g) (g) Body weight (mg/g) 45B926± 1-823 2-19±0 082 4 791±0v139 29 860±0i916 31-987±1-057 1220±0 044 3-852+0.132 25-971±0 606
Treatment Heat killed virus Live virus + 0% casein hy. Live virus + 10% 40 casein hy.
200±1*520
1 906±0 097
Heatkilledvirus 47-990+1-600 2 357±0 065 Live virus + 0% 36-742±1700 1 580±0 123 casein hy. Live virus + 10% 38-251±1448 2 003+0070 casein hy.
4-742±0-225 29 750±0 857 4-986±0-118 31-290±0-989 4
255+0
191
27-120±0-626
5-234-4-0 158 30 040±0-648
DIET IN COXSACKIEVIRUS B3 INFECTIONS IN PREGNANCY
was significantly less than in either the heat-killed virus treated controls or in the live virus casein hydrolysate treated animals. In these latter 2 groups the total liver nitrogen levels did not differ significantly. Histologically, the livers from infected mice fed an unsupplemented diet exhibited a consistent pattern of pathological changes, including a dilatation and vacuolation of periportal cells, nuclear pyknosis and neutral fat deposition as described previously (Lansdown and Coid, 1974; Lansdown and Ellaby, 1974). Liver glycogen was minimal and did not differ according to the treatment given. In infected animals given casein hydrolysate, changes of this type were rarely seen. Histological changes in the pancreas in all mice injected with live Coxsackievirus B3 consisted of widespread acinar degeneration and necrosis, loss of zymogen granules and an infiltration of mononuclear and plasma cells. Cells of the Islets of Langerhans appeared to be morphologically normal and no changes in the distribution patterns of beta cell granules were evident. Addition of casein hydrolysate to the diet did not influence the severity of the pancreatic changes brought about by the action of the virus. Analysis of faecal samples taken from 60
ch5 dE 50 E
c a
40' 3
Days
0 8
9:14
15 18
I nac. Cox. B3 + 0%Casein hydrolysate in diet Live Cox. B3 + 0% Casein hydrolysate in diet Uve Cox. B3 + 10% Casein hydrolysate in diet FIG.-Fecal nitrogen excretion in mice infected with Coxsackievirus B3 and given casein hydrolysate in diet.
375
mice before injection of virus showed that the total nitrogen content was approximately 32 mg/g solid weight (Figure). In the infective and post-infective stages in mice injected with live Coxsackievirus the total nitrogen level was significantly increased such that in the 15-18 day period of gestation the total nitrogen content was more than double that seen in the control animals. Addition of casein hydrolysate to the diet did not significantly alter the amount of nitrogenous material excreted. This observation suggests that most of this supplementary nitrogen is absorbed intestinally. DISCUSSION
Coxsackievirus B3 infection in pregnant mice causes severe pancreatic exocrine insufficiency and, as a consequence, important dietary constituents, notably proteins, are not digested and metabolized as normal (Lansdown and Coid, 1974). The pathological signs of protein deficiency in these pregnant animals included a suppression of hepatic protein synthesis and a mobilization of the reserves of labile protein in this organ. In the present work, this loss of labile protein from the liver was identified by a reduced level of total nitrogen. Also, as a result of the decreased digestion and metabolism of nitrogen containing substances in the intestine, the amount of nitrogenous substances excreted in the faeces was significantly increased. Administration of casein hydrolysate in the diet of Coxsackievirus B3 infected mice appeared to mitigate the effects of the virus induced pancreatic insufficiency in the mothers. Despite the infection, the livers of these animals appeared to develop normally for the period of gestation, showing a normal histological pattern and nitrogen content. This observation suggests that the supplementary nitrogen administered in the form of casein hydrolysate compensates for the inability of these animals to utilize the nitrogenous compounds present in the standard diet.
376
A. B. G. LANSDOWN
Thus, the liver is able to maintain a normal level of protein synthesis and storage as required for normal foetal development as shown in our previous communication (Lansdown et al., 1975). Although these observations support our original view (Lansdown and Coid, 1974) that in Coxsackievirus B3 infected mice, foetal growth retardation is due largely to a maternal protein deficiency resulting from the damage caused by the virus in the pancreas rather than to any direct effects that the virus may have in the foetus, further studies are in progress to examine in detail whether Coxsackievirus B infections do have a direct effect on foetal tissues, resulting in subtle changes leading to alterations in the foetal plasma protein profiles as reported previously by Coid and Ramsden (1973). I wish to thank Miss Susan Ellaby for her skilled technical assistance and Dr C. R. Coid for his interest and constructive advice throughout. REFERENCES BROWN, G. C. & KARuINAS, R. S. (1972) Relationship
of Congenital Anomalies and Maternal Infection with Selected Enteroviruses. Am. J. Epidemiol., 95, 207.
COID, C. R. & RAMSDEN, D. B. (1973) Retardation of Foetal Growth and Plasma Protein Development in Foetuses from Mice Injected with Coxsackie B3 Virus. Nature, Lond., 241, 460. COID, C. R., RAMSDEN, D. B. & HEALY, M. J. R. (1974) Foetal Mouse Weights and Albumin/a,fetoprotein Ratios after Maternal Infection with Coxsackievirus B3. Med. microbiol. Immunol., 159, 285. CZEIZEL, A. (1967) Coxsackievirus and Congenital Malformation. J. Am. med. A88., 201, 156. DROUGHET, V. & LEvANTIs, F. (1968) Le passage transplacentaire du virus Coxsackie B3 chez la souris gestante et la contamination foetale in utero. Ann. In8t. Pa8teur, 114, 249. FRUHLING, L., KORN, R., LAVILLAUREIX, J., SURJus, A. & FOUSSEREAU, S. (1962) La myoendocardite chronique fibro-elastique du nouveaune et du nourrisson. Ann. anat. Path., 7, 227. KILBRICK, S. & BENIRSCHKE, K. (1958) Severe Generalized Disease (Encephalohepatomyocarditis) occurring in the Newborn Period and due to Infection with Coxsackievirus, Group B. Pediatric8, 22, 857. LANSDOWN, A. B. G. (1975) Influence of Time of Maternal Infection with Coxsackievirus B3 on Foetal Growth in Mice. Br. J. exp. Path. 56, 119. LANSDOwN, A. B. G. & COID, C. R. (1974) Pathological Changes in Pregnant Mice Infected with Coxsackievirus B3 as a Possible Cause of Retarded Foetal Development. Br. J. exp. Path., 55, 101. LANSDowN, A. B. G., COID, C. R. & RAMSDEN, D. B. (1975) Mitigation of Coxsackievirus B3-induced Fetal Growth Retardation in Mice by the Administration of Dietary Casein Hydrolysate. Nature, Lond., 254, 599. LANSDOwN, A. B. G. & ELLA Y, S. J. (1974) Histochemical Demonstration of Changes in Liver Cell Enzymes in Pregnant Mice Infected with Coxsackie B3 Virus. Hi8tochemi8try, 40, 175. SoixE1, K. (1967) Coxsackie B3 Virus Infection in the Pregnant Mouse. J. infect. Di8., 117, 203.
PATHOLOGICAL CHANGES IN PREGNANT MICE INFECTED WITH COXSACKIEVIRUS B3 AND GIVEN DIETARY CASEIN HYDROLYSATE SUPPLEMENT A. B. G. LANSDOWN From the Clinical Re8earch Centre, Watford Road, Harrow, Middle8ex, HAI 3UJ Received for publication April 7, 1975
Summary.-Coxsackievirus B3 infection in pregnant mice leads to a severe pancreatic exocrine insufficiency in the mothers and retarded foetal growth. As a consequence of the pancreatic damage, the animals are rendered incapable of digesting sufficient amounts of dietary proteins to allow maternal liver development to proceed as normal for the pregnant rodent. Faecal nitrogen was increased and the maternal livers were small for the weights of the animals and exhibited a lower than normal nitrogen content. Feeding of additional amino acids and simple peptides in the diet in the form of casein hydrolysate either from before or after virus injection appeared to compensate for the inability of these animals to digest dietary protein nitrogen and allowed maternal liver development and foetal growth to proceed at a rate not significantly different from normal. Although these results apply to infections with Coxsackievirus B3 in pregnancy, they may be relevant to other infections which adversely affect foetal growth by their pathological effects on maternal organs necessary for maintaining optimal foetal growth.
COXSACKIEVIRUS B infections inhuman pregnancy commonly result in mild or subclinical illness in the mother and may be responsible for congenital heart disease, spontaneous abortions, stillbirth and intrauterine growth retardation (Kilbrick and Benirischke, 1958; Czeizel, 1967; Brown and Karunas, 1972; Fruhling et al., 1962). Experimental studies in mice using Coxsackievirus B3 have shown that foetal growth retardation occurs and that the maturation of foetal plasma proteins is delayed (Coid and Ramsden, 1973; Coid, Ramsden and Healy, 1974; Lansdown, 1975). In a study of the pathology of Coxsackievirus B3 infections in pregnancy, in an experiment to elucidate the mechanism of foetal growth impairment, Lansdown and Coid (1974) suggested that whereas the possibility of the virus exerting a direct effect on the foetus cannot be discounted (Soike, 1967; Droughet and Levantis, 1968), foetal growth retardation
is largely due to a maternal protein deficiency resulting from virus induced pancreatic exocrine insufficiency. Subsequent studies have shown that if the diets of Coxsackievirus B3 infected pregnant mice are supplemented with casein hydrolysate, foetal growth and plasma protein development are not significantly impaired (Lansdown, Coid and Ramsden, 1975). In that work, attention was focused principally on the influence of the added casein hydrolysate on foetal growth. In the present communication, the influence of this dietary supplement on the pathological changes elicited in the mothers by the Coxsackievirus is described and consideration given to the possible mechanism by which the administration of casein hydrolysate in the diet mitigates foetal growth impairment. MATERIALS AND METHODS Viru.S stock.-The Coxsackievirus B3 used in these studies was derived from the original
A. B. G. LANSDOWN
374
Nancy strain. It had been passaged through suckling mice, isolated in primary rhesus monkey kidney cells and then through vervet monkey kidney cells (VERO). The virus suspension was diluted 1 in 10 with Hanks' buffered salt solution to give a tissue culture infective dose (TCID50) of 105 85/ml. Animal8.-Mice of the " TO " outbred strain from the original Swiss stock (Theiler) were used throughout this work. Virgin females (20-25 g) were placed overnight with proven males and the morning when vaginal plugs were detected was designated Day 1 of pregnancy. Inoculation of virus.-Pregnant mice were inoculated intramuscularly with 0-15 ml of virus suspension into each hind leg on the 8th day of gestation. Control mice were inoculated with an equivalent dose of virus suspension that had been inactivated by heating to 560 for 30 min. Experimenntal.-In an initial series of experiments, mice were given a standard rodent breeder diet (Spiller's Laboratory 1 diet) in powdered forn and containing 0 or 10% enzymic casein hydrolysate (Sigma Chemical Co., St Louis, U.S.A.) from Day 1 of pregnancy through until Day 18, when all animals were killed by cervical dislocation. In the second series the standard powdered diet was fed from Days 1 to 10 of gestation and then a diet supplemented with casein hydrolysate was given until Day 18. At autopsy, the animals were weighed with foetuses in utero. Maternal livers were also weighed and representative samples of this tissue (approximately 0 5 g) were stored at - 700 for total nitrogen analysis by the standard microKjeldahl technique. The remaining tissue was routinely fixed in phosphate buffered formalin for histological examination. Histological sections were stained by haematoxylin and eosin (H. and E.), PAS for glycogen, and by Oil red-O for neutral fat. Sections of maternal pancreas
were stained by H. and E. and by Gomori's
aldehyde fuchsin method for beta cell granules. Maternal faecal samples were collected during the 3 periods-0-8 days (pre-infection), 9-14 days (infective phase) and 15-18 (post-infective stage) and analysed for nitrogen content using the Kjeldahl estimation. RESULTS
On the 18th day of gestation mice in all groups, including those given virus and casein hydrolysate, appeared to be in a good state of health although some animals injected with live virus and not receiving the casein hydrolysate weighed less than those of other groups and on occasions had slightly ruffled coats. Animals ate and drank well at all stages of the experiments and the diet supplemented with casein hydrolysate did not appear to be unpalatable. At autopsy, all mice injected with live virus, irrespective of the constitution of their diets, exhibited a marked reduction in the size of the pancreas, visceral adhesions and evidence of fatty tissue degeneration in the visceral fat pads. The liver was small in live virus treated mice fed no casein hydrolysate and the ratio of liver weight to the total body weight of the animal was lower than in other groups (see Table). When the liver samples were analysed for their total nitrogen content, the nitrogen level in the livers of infected mice fed no supplement
TABLE.-Maternal Liver Weights Relative to Body Weights in Mice Infected with Cox8ackievirus B3 and Given Dietary Supplements of Casein Hydroly8ate Days of pregnancy when casein hydrolysate given 1-18 1-18
1-18
10-18 10-18 10-18
Average body weight
Nitrogen Liver weight Liver weight X 100 content (g) (g) Body weight (mg/g) 45B926± 1-823 2-19±0 082 4 791±0v139 29 860±0i916 31-987±1-057 1220±0 044 3-852+0.132 25-971±0 606
Treatment Heat killed virus Live virus + 0% casein hy. Live virus + 10% 40 casein hy.
200±1*520
1 906±0 097
Heatkilledvirus 47-990+1-600 2 357±0 065 Live virus + 0% 36-742±1700 1 580±0 123 casein hy. Live virus + 10% 38-251±1448 2 003+0070 casein hy.
4-742±0-225 29 750±0 857 4-986±0-118 31-290±0-989 4
255+0
191
27-120±0-626
5-234-4-0 158 30 040±0-648
DIET IN COXSACKIEVIRUS B3 INFECTIONS IN PREGNANCY
was significantly less than in either the heat-killed virus treated controls or in the live virus casein hydrolysate treated animals. In these latter 2 groups the total liver nitrogen levels did not differ significantly. Histologically, the livers from infected mice fed an unsupplemented diet exhibited a consistent pattern of pathological changes, including a dilatation and vacuolation of periportal cells, nuclear pyknosis and neutral fat deposition as described previously (Lansdown and Coid, 1974; Lansdown and Ellaby, 1974). Liver glycogen was minimal and did not differ according to the treatment given. In infected animals given casein hydrolysate, changes of this type were rarely seen. Histological changes in the pancreas in all mice injected with live Coxsackievirus B3 consisted of widespread acinar degeneration and necrosis, loss of zymogen granules and an infiltration of mononuclear and plasma cells. Cells of the Islets of Langerhans appeared to be morphologically normal and no changes in the distribution patterns of beta cell granules were evident. Addition of casein hydrolysate to the diet did not influence the severity of the pancreatic changes brought about by the action of the virus. Analysis of faecal samples taken from 60
ch5 dE 50 E
c a
40' 3
Days
0 8
9:14
15 18
I nac. Cox. B3 + 0%Casein hydrolysate in diet Live Cox. B3 + 0% Casein hydrolysate in diet Uve Cox. B3 + 10% Casein hydrolysate in diet FIG.-Fecal nitrogen excretion in mice infected with Coxsackievirus B3 and given casein hydrolysate in diet.
375
mice before injection of virus showed that the total nitrogen content was approximately 32 mg/g solid weight (Figure). In the infective and post-infective stages in mice injected with live Coxsackievirus the total nitrogen level was significantly increased such that in the 15-18 day period of gestation the total nitrogen content was more than double that seen in the control animals. Addition of casein hydrolysate to the diet did not significantly alter the amount of nitrogenous material excreted. This observation suggests that most of this supplementary nitrogen is absorbed intestinally. DISCUSSION
Coxsackievirus B3 infection in pregnant mice causes severe pancreatic exocrine insufficiency and, as a consequence, important dietary constituents, notably proteins, are not digested and metabolized as normal (Lansdown and Coid, 1974). The pathological signs of protein deficiency in these pregnant animals included a suppression of hepatic protein synthesis and a mobilization of the reserves of labile protein in this organ. In the present work, this loss of labile protein from the liver was identified by a reduced level of total nitrogen. Also, as a result of the decreased digestion and metabolism of nitrogen containing substances in the intestine, the amount of nitrogenous substances excreted in the faeces was significantly increased. Administration of casein hydrolysate in the diet of Coxsackievirus B3 infected mice appeared to mitigate the effects of the virus induced pancreatic insufficiency in the mothers. Despite the infection, the livers of these animals appeared to develop normally for the period of gestation, showing a normal histological pattern and nitrogen content. This observation suggests that the supplementary nitrogen administered in the form of casein hydrolysate compensates for the inability of these animals to utilize the nitrogenous compounds present in the standard diet.
376
A. B. G. LANSDOWN
Thus, the liver is able to maintain a normal level of protein synthesis and storage as required for normal foetal development as shown in our previous communication (Lansdown et al., 1975). Although these observations support our original view (Lansdown and Coid, 1974) that in Coxsackievirus B3 infected mice, foetal growth retardation is due largely to a maternal protein deficiency resulting from the damage caused by the virus in the pancreas rather than to any direct effects that the virus may have in the foetus, further studies are in progress to examine in detail whether Coxsackievirus B infections do have a direct effect on foetal tissues, resulting in subtle changes leading to alterations in the foetal plasma protein profiles as reported previously by Coid and Ramsden (1973). I wish to thank Miss Susan Ellaby for her skilled technical assistance and Dr C. R. Coid for his interest and constructive advice throughout. REFERENCES BROWN, G. C. & KARuINAS, R. S. (1972) Relationship
of Congenital Anomalies and Maternal Infection with Selected Enteroviruses. Am. J. Epidemiol., 95, 207.
COID, C. R. & RAMSDEN, D. B. (1973) Retardation of Foetal Growth and Plasma Protein Development in Foetuses from Mice Injected with Coxsackie B3 Virus. Nature, Lond., 241, 460. COID, C. R., RAMSDEN, D. B. & HEALY, M. J. R. (1974) Foetal Mouse Weights and Albumin/a,fetoprotein Ratios after Maternal Infection with Coxsackievirus B3. Med. microbiol. Immunol., 159, 285. CZEIZEL, A. (1967) Coxsackievirus and Congenital Malformation. J. Am. med. A88., 201, 156. DROUGHET, V. & LEvANTIs, F. (1968) Le passage transplacentaire du virus Coxsackie B3 chez la souris gestante et la contamination foetale in utero. Ann. In8t. Pa8teur, 114, 249. FRUHLING, L., KORN, R., LAVILLAUREIX, J., SURJus, A. & FOUSSEREAU, S. (1962) La myoendocardite chronique fibro-elastique du nouveaune et du nourrisson. Ann. anat. Path., 7, 227. KILBRICK, S. & BENIRSCHKE, K. (1958) Severe Generalized Disease (Encephalohepatomyocarditis) occurring in the Newborn Period and due to Infection with Coxsackievirus, Group B. Pediatric8, 22, 857. LANSDOWN, A. B. G. (1975) Influence of Time of Maternal Infection with Coxsackievirus B3 on Foetal Growth in Mice. Br. J. exp. Path. 56, 119. LANSDOwN, A. B. G. & COID, C. R. (1974) Pathological Changes in Pregnant Mice Infected with Coxsackievirus B3 as a Possible Cause of Retarded Foetal Development. Br. J. exp. Path., 55, 101. LANSDowN, A. B. G., COID, C. R. & RAMSDEN, D. B. (1975) Mitigation of Coxsackievirus B3-induced Fetal Growth Retardation in Mice by the Administration of Dietary Casein Hydrolysate. Nature, Lond., 254, 599. LANSDOwN, A. B. G. & ELLA Y, S. J. (1974) Histochemical Demonstration of Changes in Liver Cell Enzymes in Pregnant Mice Infected with Coxsackie B3 Virus. Hi8tochemi8try, 40, 175. SoixE1, K. (1967) Coxsackie B3 Virus Infection in the Pregnant Mouse. J. infect. Di8., 117, 203.
