Br. J. exp. Path. (1976) 57, 645
EXPERIMENTAL CANINE DISTEMPER INFECTION AS A MEANS OF DEMONSTRATING LATENT EFFECTS OF SUBACUTE LEAD INTOXICATION D. J. WHITE* AND S. McLEOD Fromb the WVellcome Research Laboratories, Langley Court, Beckenhanl, Kent Received for publication June 16, 1976
Summary.-Observations on the response of the body to experimental infection with distemper virus in dogs previously dosed subacutely with lead have demonstrated a latent effect of lead on several body systems. Effects which indicated a relationship to earlier treatment with lead included evidence for stimulation of haemoglobin synthesis, changes to red blood cells resulting in increased destruction, increased vulnerability of the parenchymatous cells of the liver to damage, reduction in the weight of the skeleton and thyroid, an increase in weight of the thymus and brain and histopathological changes in the thymus.
THE TOXIC EFFECTS of exposure to large amounts of lead on the blood system, kidney and brain are well documented (Goyer and Rhyne, 1973). There is considerable concern, however, as to the possible effects of mild or continuous exposure to increased amounts of lead on biological systems. In this direction particular attention has been paid to the investigation of very early effects of lead on haem synthesis and the measurement of the enzyme d-amino-laevulinic acid dehydrogenase has been found to be a particularly sensitive indicator of early lead effect (Hernberg and Nikkanen, 1970). The long-term significance of the intake of subclinical levels of lead on bodily functions has proved difficult to assess and the present report describes an experiment designed to investigate the pathological effects of subclinical lead intoxication in the dog. This has been done by assessing response of the body to infection with canine distemper virus in dogs previously given lead compared with *
dogs similarly infected with distemper but not given lead. MATERIALS AND METHODS Animals.-16 pure-bred Beagle dogs of less than 6 months of age were divided into 4 groups of 2 ,3 and 2 Y and kennelled individually under strict quarantine conditions. The diet was proprietary tinned dog food and biscuit meal; tap water was available ad lib. Canine distemper virus.-The Snyder Hill strain of distemper virus was obtained as a 20% lyophilized suspension of dog brain and further passaged in a susceptible dog by intracerebral inoculation. The dog was killed after 8 days and a 10% (w/v) suspension of the brain prepared in phosphate-buffered saline and used immediately at a dose rate of 0-2 ml injected i.v. Serum neutralizing antibody levels for infected dogs at death were determined by the method of Prydie (1968) and expressed as the logl0 reciprocal of serum dilution giving 5000 neutralization. Treatment.-Lead as lead carbonate was given orally to 2 groups of dogs (Groups 2 and 4) at a dose rate of 50 mg/kg body wt. daily for 3 weeks. Six weeks after the end of the dosing period, dogs in one group given lead (Group 4) and one undosed group (Group 3) were injected i.v. with distemper virus. Dogs in Group 1 remained undosed to act as controls.
Present address: Beecham Pharmaceuticals (Research
Division), Harlow, Essex.
646
D. J. WHITE AND S. McLEOD
Clinical examinations.-Alterations in food and water consumption and in clinical appearance were recorded daily. Body weight was measured once a week and rectal temperature daily. Haematology.-Blood samples were taken from the jugular vein, using EDTA as anticoagulant, before dosing commenced and then once weekly. Packed cell volume (PCV, ml%) was calculated using a Haematocrit Computer; red cell count (RBC, 106/mm3) using a Coulter Counter Model 5; white cell count (WBC, 103/mm3) using a Coulter Counter Model A; mean cell volume (MCV, fl) using a Mean Cell Computer and haemoglobin (Hb, g/100 ml) using an E.E.L. Haemoglobinometer. Mean corpuscular haemoglobin (MCH, pg) and mean corpuscular haemoglobin concentation (MCHC, g%) were calculated from the above values. Differential white cell count, reticulocyte count and incidence of basophilic stippling of red cells were done using suitably stained smears. Biochemistry.-Serum samples were obtained on 4 occasions for measurement of the following parameters using a Technicon Auto-Analyser I: glutamic pyruvic transaminase (SGPT), Technicon method N-54 (Henry et al., 1960); glutamic oxaloacetic transaminase (SGOT), method N-25b (Morgenstern et al., 1966); alkaline phosphatase (SAP), method N-6b (Bessey, Lowry and Brock, 1946), urea nitrogen (BUN), method N-ic (Marsh, Fingerhut and Miller, 1965) and cholesterol by the method of Levine and Zak (1964). Terminal procedures.-Dogs were deeply anaesthetized by i.v. injection of pentobarbitone and the brain perfused with 1% acetic acid in 10% formalin. All organs were examined macroscopically and weighed; the skin, muscle and skeleton were also weighed. Samples of the heart, lungs, liver, kidneys, spleen, pancreas, thyroid, thymus, adrenals, stomach, duodenum, ileum, colon, testes or ovaries, eyes, brain, pituitary and sciatic nerve were placed in 10% phosphate-buffered formalin at pH 7; samples of pancreas were also fixed in Bouin's fluid. Tissues were routinely processed, embedded in paraffin wax and sections cut at 5 ,um and stained with haematoxylin and eosin. In addition, frozen sections of liver, kidney and adrenal were stained with Oil-red-O for fat, the liver was also stained with PAS for glycogen; paraffin sections of the spleen and thymus were stained with Perls' method for ferric iron. Sections of brain were stained with Luxol fast blue for myelin and with Glees-Marsland's silver stain for neurons and nerve fibres. Statistical analyses.-Analysis of variance was carried out on log-transformed data for the haematological values measured on Day 70. This enabled a comparison of the results to be made between the controls and dogs given lead
only and distemper only and those given both lead and distemper. RESULTS
During the 3-week period of lead dosing no significant effects on food and water consumption or on body weight gain were observed in the lead dosed dogs of Groups 2 and 4. Their clinical condition did however alter slightly with the appearance of some harshness of the coat and a tendency towards the passage of either loose or hard faeces. The appearance of the coat and of the faeces returned to normal following cessation of dosing except for one dog of Group 2 in which clinical condition never fully returned to normal up to the time of death. No significant differences from control values were seen in any group in the period of dosing and during the withdrawal period in haematological or biochemical indices, except for evidence for the appearance in the peripheral blood during the period of lead dosing of small numbers of immature red cells in Groups 2 and 4, and for slight increases in cholesterol in these groups. The incidence of basophilic stippling of red cells in these groups reached a peak at the end of the dosing period and then gradually returned to normal during the following 6 weeks. Infection of Groups 3 and 4 with canine distemper virus was carried out on Day 63. The incubation period was short, 36-48 h, before the onset of typical clinical signs of the disease accompanied by pyrexia; there was no difference between the groups in the severity of the infection but 2 dogs in Group 3 died before the end of the experiment: 1 dog was killed on the 7th day post-infection due to rapidly worsening condition and the second dog was found dead on the 9th day post-infection. All the remaining dogs including those of Groups 1 and 2 were killed on Day 73 due to the deteriorating clinical condition of the distemper-infected dogs. The mean serum neutralizing antibody titre at death for Group 3 was 3-55 and for Group 4 was 3-3.
647
EXPERIMENTAL CANINE DISTEMPER INFECTION 0 75
PCV
1 70
1-65-
log 1-60
L+D-
RBC
D- o
L+D-
0 70log 0-65
L-D-0. LL0
9*.
L
D
LL- D+
1-55-
L-D--0*
L+
L-;--
L+D+
0o60
MCH
Hb
L-D+ 0 D+
MCH~~
1-50log
L-D- E.
15 1 45-
DL+D-
L-
L+D+
.D+ *-.
1 40-* L-D +
1 32:0 log
1 955
L+D+
MCV
,
L-
L-D-
D-
0----
:
L-D+
L: .
.
L+D-
1901
I L-D+
1 21 1-
log 1 0-
D+ L+D+ L+
090 8-
FiG. 1.-Results of haemabological examination on Day 70 plotted from analysis of variance of logtransformed data for treated and untreated dogs, and combinations of treatments. L - D = Group 1 untreated; L - = Groups 1 and 3 not given lead; L + = Groups 2 and 4-given lead; D -= Groups 1 and 2 not given dist emper;D += Groups 3 and 4-given distemper; L -D + = Group 3- given distemper but not lead; L + D + = Group 4-given distemper and lead.
Blood samples were obtained for haematology on the 7th day after infection from all dogs and the results are summarized in Fig. 1. It can be seen that distemper infection resulted in marked effects with significant differences between the 2 infected groups. These effects in both groups consisted of a reduction in PCV, RBC and Hb and an increase in MCV and WBC. In the case of PCV and Hb the decrease was much less marked in the group previously dosed with lead; however, the latter group showed a more marked decrease in RBCs. The change seen in MCH was directly opposite in the 2 groups, the lead-dosed infected dogs showing a substantial increase and the
group given distemper only showing a marked reduction. MCV in Group 3 increased only slightly but in Group 4 a marked increase was seen; there was, however, no evidence for this increase in peripheral blood smears where the numbers of immature cells showed no change. The results of biochemical analyses performed during the experiment are shown in Fig. 2. No significant alterations were seen during the lead-dosing period except for marginal increases in SAP and cholesterol in Group 2. Distemper infection resulted in a moderate rise in BUN and cholesterol of similar proportions in both infected groups, no doubt reflecting the response of the organism to a severe
648
D. J. WHITE AND S. McLEOD SGOT
SGPT
50-
40-
I.u.3S 30-
10 -12
140-
C
28 63 Days
70
-12
63
28
Days
BUN
SAP
120-
I.U
1008063 28 Days
63
28
70
70
Days
CHOLESTEROL
200175-
mg% 150125
-12
63
28
70
D ay s
FIG. 2.-Graphs of the mean values for SGPT, SGOT and SAP (all in international units) and cholesterol and BUN (both mg/100 ml) for Group 1 (undosed controls) O--- -O; Group 2 (lead only) 0; Group 3 (infected with distemper on Day 63) O---- [1; Group 4 (lead-dosed and infected * with distemper on Day 63) * *.
pyrexic disease. A slight increase in SGOT was also seen in both groups, being more marked in Group 4; the latter group also showed increases in SGPT and SAP, values for Group 3 remaining similar to those of controls. None of the biochemical changes recorded on Day 70 was statistically significant. Physical examination post mortem showed 1 dog of Group 2 to have a loose, harsh coat; on macroscopic examination of the organs of this dog the spleen was found to be slightly enlarged and small areas of consolidation were present in the lungs. No abnormalities were seen in the
organs of the other animals in Group 2 or in the controls. In Groups 3 and 4 typical clinical signs of canine distemper were present and macroscopic examination of the carcases showed similar abnormalities to be present in all the dogs; these included a harsh, dry coat and loose skin, reduction in body fat, congestion of the spleen, distension of the gall bladder with bile and lack of food material in the alimentary tract. The group mean organ and tissue weights, both absolute and related to body weight, together with the body weight just prior to lead dosing and again
649
EXPERIMENTAL CANINE DISTEMPER INFECTION
TABLE.-Mean Organ and Tissue Weights for Groups 1-4 Expressed as Absolute Values (g) and as Percentage of Body Weight and also Body Weight (kg) Before Treatment and at Death Group 4
3 _~
Tissue Liver
Kidney
Spleen Heart Pancreas
Absolute (g) Body wt. 282- 8 3-60 ±8-7 ±0 83 44- 13 0-56 ±2-08 ±0-23 29- 75 0-38 ±6-92 ±0- 88 73- 75 0 94 ±4-26 ±0 04 25-5 0-32
±2-33 Adrenals
1- 18
±0-15 Thyroids
1- 76
±0 39 Thymus
Brain
11 2 ±1 89 71-12
±1-19 Skin
1472
±82 Muscle
3338
Skeleton
1282
±40
±43
.~~~~~~~~~~~~~- .~~~~~~~~~~-
Absolute (g) 299 8
% Body wt.
±6-5
±0-22
44-13 ±0 94 33- 88 ±7-48 61 -0 ±5 05 *17-75 ±1 65 1-16
±0-02 0-015 ±0-001 0-022 ±0005 0-14
3*92 0 58
±004 0-46 ±0 13 *0 79 ±0 03
*0.23
±0O01
0.015
±0-14
±0O001
1*49 ±0-23 13 87
±0-002
±0-023
±2-29
±0-26
0-91
*78- 37 ±2-39 1481
1-03 ±0 076 19- 04 ±1-17
±0O019 18-8 ±1-16 42-6 ±0 7 16-35 ±0 43
±176 3223 ±289 1171 ± 74 5-35
5 75 Body weight before treatment (kg) 7 84 Body weight at at death (kg) * P = < 0 05 using Student's t test. tP = < 0X01.
7-74
at death are shown in the Table. Distemper infection caused a number of alterations in both infected groups but with some intergroup differences. A comparison of the results for Group 2 with the controls of Group 1 shows that the body weight at death was similar but that the absolute weight of the skeleton was reduced in the lead-dosed group; a similar reduction in skeletal weight was also seen in Group 4. Other changes in Group 2 included a reduction in heart weight, the result for the relative weight being significant; the weight of the pancreas was also significantly lower and
0-019 0-18
41- 5 ±0- 97 15-18
±0-38
Absolute (g) 291 3 ±18-9 *53.33 ±2 99 19 25 ±4-84 61-13 ±7-29 19-25 ±3-66 1 40 ±0 16 1-64 ±0-75 5 94 ±1-18 71-12 ±2-27 *999 ±104 *2210 ±278 1226 ±43 5-45 6 15
Body wt. t4.75 ±0 14
*0.88 ±0- 84 0 3 ±0 57 0.99 ±0-08 0-32 ±0-06 0 023 ±0 003 0-025
Absolute (g) 260*8 ±19-6 46- 13 ±1b82 18-5 ±3-13 56 63 ±2-85 20 0 ±2 58 1-42
±0O11 1-08
±0O009
±0O9
0 096 ±0-019 *1-17
±2 98
±0 065 16-26
8*82 74- 25 ±5-36 1191
±1 36
+69
*35-5 ±1-7
*2194 ±233 1155
*20.2
±1-16
±44
% Body wt. t4-33 ±0-06 *0.77
±0O59 0 3 ±3-13 0.95 O0- 04 0*32 ±2 58 *0-024 ±0-002 0-018 ±0-002 0*14 ±0 039
*1.24 ±0095 19* 84 ±0 33 *36-1 ±1b 33 *19.3 ±0 71
5.5
6-05
a slight reduction in thyroid weight was recorded; the thymus and the brain both showed a slight increase in weight compared with those of controls and higher values for these organs were also observed in Group 4 when compared with Group 3. From the results in Groups 2 and 4 the alterations which were suggestive of an effect of lead were the reduction in weight of the skeleton, heart and thyroid and the increase in weight ofthe thymus and brain. No significant differences were seen between any of the groups on microscopic examination of sections of the heart, pancreas, thyroid, adrenals, testes, ovaries,
650
D. J. WHITE AND S. McLEOD
eyes, brain and pituitary gland. In the stomach of all the dogs infected with distemper, eosinophilic, intracytoplasmic inclusion bodies typical of distemper were seen in the mucosal cells; similar inclusion bodies were also seen in the bronchiolar epithelium of the lungs. Single dogs from Groups 3 and 4 showed degenerative changes in the intestinal wall. The kidneys of the infected dogs were congested and in 2 dogs of Group 4 localized lesions of interstitial nephritis were seen; no evidence of either intranuclear or intracytoplasmic inclusion bodies was seen in the epithelium of the tubules. The main changes in all treated groups related to the amount of ferric iron present in the liver and spleen and to the appearance of the thymus and spleen in the distemper-infected dogs. In the liver sections of 2 dogs of Group 2 very occasional Kupffer cells contained ferric iron and in all dogs of this group a slight increase in iron deposition compared with the controls was seen in the red pulp of the spleen; in 1 dog of Group 4 the splenic white pulp was markedly reduced and several germinal centres had a vacuolated appearance. In the liver and spleen of almost all the distemper-infected dogs a significant increase in ferric iron of similar degree was seen; in 2 dogs from each infected group germinal centres of the splenic white pulp contained small lakes of brick-red material subsequently identified as haemoglobin by staining by Okajima's method. In the thymus of 3 dogs of Group 3 and all the dogs of Group 4 there was marked atrophy with a reduction in mature lymphocytes and a lack of Hassall's corpuscles; in addition, in 3 of the dogs of Group 4, groups of large vacuolated cells resembling macrophages and containing fat were seen throughout the thymic tissue.
would give rise to only mild clinical signs of toxic effect. This was largely achieved in that the only clinical evidence of lead poisoning was a slight fall in bodily condition. This was accompanied by a mild effect on red blood cell production; no tendency towards anaemia was seen and no biochemical evidence of liver or kidney damage was observed. During the 6-week period which was allowed to elapse following cessation of dosing and prior to infection with distemper, the clinical condition and effect on the red cells returned to normal with the exception of one dog which never fully recovered its bodily condition. It has however been shown, by employing the experimental model of infection with distemper virus, that an underlying effect of the earlier intake of lead was present in these dogs. This effect was demonstrated by alterations in the response of the erythropoietic system, evidence of damage to the liver, alterations in the weight of the skeleton, thyroid, brain and thymus and histological evidence of an altered response of the thymus to distemper infection. The latent effect of lead on the red cells was of 2 types. Firstly, lead appeared to have had a protective or even stimulatory effect on haemoglobin production, this was accompanied by an increase in the index of red cell size but no evidence for an increase in the release of immature red cells was seen in the peripheral blood. Secondly, although distemper infection gave rise to lysis of red cells as judged by increased iron in the liver and spleen and free haemoglobin in the splenic white pulp, the effect was more marked in dogs previously given lead. Lead is known to have a deleterious effect on red cell survival (Waldron, 1966) and the findings here suggest the presence of a continuing effect of lead during the formation of red cells in the bone marrow. The best-documented pathological DISCUSSION effect of lead on the organs occurs in the In the first part of this experiment an kidneys (Goyer and Rhyne, 1973) and it attempt was made to give large amounts might have been expected that some of lead to dogs over a short period which evidence of abnormal kidney function,
EXPERIMENTAL CANINE DISTEMPER INFECTION
either biochemical or histological, would have been seen. However, no effect on the kidney was observed during the period of lead dosing nor was a prior effect of lead demonstrated by subsequent distemper infection. A latent effect of lead on the liver was however suggested on biochemical evidence by the modest increase in SGPT, SGOT and SAP in the lead-dosed dogs infected with distemper. The liver is not generally recognized as a prime site for the toxic effects of lead and the majority of reports are concerned with changes in ultrastructure (Fiaccavento, Arrigoni and Chiappino, 1968; Hoffman et al., 1974). However, Marsden and Wilson (1955) described moderate liver damage in 2 cases of childhood poisoning and there are a number of reports of biochemical evidence of liver damage in experimental animals (Secchi, Alessio and Spreafico, 1971; Trejo et at., 1972). In the present experiment a skeletal origin for SAP cannot be excluded since distemper produced marked alterations in the mineral status of bone as reported elsewhere (White, Marshall and McLeod, 1975); similarly the raised SGOT levels may have resulted from the increased destruction of red cells. However, in the dog, increased levels of SGPT have been reported to be highly specific for liver damage (Zinkl et al., 1971) and on balance it is likely that the observed increases in enzymes seen here indicate a mild toxic effect on the liver parenchyma, the response of which has been affected by earlier contact with lead. Alterations in the weight of body tissues indicative of an effect of lead included the slightly reduced weight of the skeleton, the reduction in thyroid weight and the increase in weight of the thymus and particularly of the brain. The increase in brain weight, still present almost 8 weeks after cessation of lead dosing, is of particular interest since the most distressing symrptoms of lead poisoning in both man and animals involve the nervous system. The neurological symptoms of lead poisoning in children 43
651
and dogs are throught to be mainly due to an increase in intracrannial pressure as a result of oedema of the brain (Clasen et al., 1974; Stowe et at., 1973).- In the present experiment no histological evidence of oedema was observed but the possibility remains that the increase in brain weight was due to the presence of an increased amount of brain water. The increased weight of the thymus in both lead-dosed groups when compared with their respective control group indicates a stimulatory effect of lead. This finding, together with the histological observation of vacuolation in lead-dosed, infected dogs suggests higher reactivity of the thymus. Vacuolation is thought to represent sites of death of rapidly dividing lymphocytes and the appearance observed here is very similar to that seen in the involutionary changes in the thymus described by Henry (1967). At first sight these changes are incompatible with the immunosuppressive effects of lead which have been reported by Hemphill-, Kaeberle and Buck (1973) and by Koller (1973); the latter author inoculated rabbits with pseudorabies virus and found significantly lower serum neutralizing antibody titres in lead-dosed animals. The neutralizing antibody titres to distemper virus reported in the present paper were very similar in both groups of infected dogs, although marginally lower in those previously given lead. An explanation for the difference in results may lie in the length of time elapsing since the end of lead dosing and challenge with distemper virus. The observations made in this experiment provide evidence for the existence of a latent effect of lead on a number of body systems. The practical importance of these findings may lie in the possibility of altered responses of the body to other disease processes as a result of the residual effects of lead.
We acknowledge with thanks the help of Mr R. B. Clampitt with the biochemical analyses and Mr P. A. Young with the statistics.
652
D. J. WHITE AND S. McLEOD
REFERENCES BESSEY, 0. A., LowRy, 0. H. & BROCK, M. J. (1946) Method for the Rapid Determination of Alkaline Phosphatase with 5 cubic millimetres of Serum. J. Biol. Chem., 164, 321. CLASEN, R. A., HARTMANN, J. F., STARR, A. J., COOGAN, P. S., PANDOLFI, S., LAING, I., BECKER, R. & HASS, G. M. (1974) Electron Microscopic and Chemical Studies of the Vascular Changes and Edema of Lead Encephalopathy. Am. J. Path., 74, 215. FIACCAVENTO, S., ARRIGONI, G. & CHIAPPINO, G. (1968) Aspetti Morfologi Utrastructurali della Cellula Epatica Nella Intossicazione Saturnina. Lav. Med., 22, 219. GOYER, R. A. & RHYNE, B. C. (1973) Pathological Effects of Lead. In International Review of Experimental Pathology, Vol. 12. Ed. G. W. Richter and M. A. Epstein. New York: Academic Press. HEMPHILL, F. E., KAEBERLE, M. L. & BUCK, W. B. (1973) Lead-Induced Alterations of Immunologic Reactivity in Mice. J. Am. Vet. Med. A88., 163, 1191. HENRY, L. (1967) Involution of the Human Thymus. J. Path. Bact., 93, 661. HENRY, R. J., CHIAMORI, N., GOLUB, 0. J. & BERKMAN, S. (1960) Revised Spectrophotometric Methods for the Determination of GlutamicOxaloacetic Transaminase, Glutamic-Pyruvic Transaminase and Lactic Acid Dehydrogenase. Tech. Bull. Reg. Med. Tech., 30, 149. HERNBERG, S. & NIKKANEN, J. (1970) Enzyme Inhibition by Lead Under Normal Urban Conditions. Lancet, i, 63. HOFFMAN, E. O., DILtTzIo, N. R., HOLPER, K., BRETTSCHNEIDER, L. & COOVER, J. (1974) Ultrastructural Changes in the Liver of Baboons Following Lead and Endotoxin Administration. Lab. Inve8t., 30, 311. KOLLER, L. D. (1973) Immunosuppression Produced by Lead, Cadmium and Mercury. Am. J. vet. Re8., 34, 14,57.
LEVINE, J. B. & ZAK, B. (1964) Automated Determination of Serum Total Cholesterol. Clin. chim. Acta, 10, 381. MARSDEN, H. B. & WILSON, V. K. (1955) LeadPoisoning in Children: Correlation of Clinical and Pathological Findings. Br. med. J., i, 324. MARSH, W. H., FINGERHUT, B. & MILLER, H. (1965) Automated and Manual Direct Methods for the Determination of Blood Urea. Clin. Chem., 11, 624. MORGENSTERN, S., OKLANDER, M., AUERBACH, J., KAUFMAN, J. & KLEIN, B. (1966) Automated Determination of Serum Glutamic Oxaloacetic Transaminase. Clin. Chem., 12, 95. PRYDIE, J. (1968) Studies with an Attenuated Canine Distemper Vaccine Derived from Chick Embryo Cultures. Res. vet. Sci., 9, 443. SECCHI, G. C., ALESSIO, L. & SPREAFICO, F. (1971) Serum Enzymatic Activities in Experimental Lead Poisoning. Enzyme, 12, 63. STOWE, H. D., GOYER, R. A., KRIGMAN, M. M., WILSON, M. & CATES, M. (1973) Experimental Oral Lead Toxicity in Young Dogs. Archs Path., 95, 106. TREJO, R. A., DILuzIo, N. R., LoOSE, L. D. & HOFFMAN, E. (1972) Reticuloendothelial and Hepatic Functional Alterations Following Lead Acetate Administration. Exp. mol. Path., 17, 145. WALDRON, H. A. (1966) The Anaemia of Lead Poisoning: a Review. Br. J. indu8st. Med., 23, 83. WHITE, D. J., MARSHALL, A. J. & McLEOD, S. (1975) The Influence of Experimental Distemper Infection on the Distribution of Lead in Dogs Previously Subacutely Intoxicated with Lead Carbonate. Br. J. exp. Path., 56, 544. ZINKL, J. G., BUSH, R. M., CORNELIUS, C. E. & FREEDLAND, R. A. (1971) Comparative Studies on Plasma and Tissue Sorbitol, Glutamic, Lactic and Hydroxybutyric Dehydrogenase and Transaminase Activities in the Dog. Re8. vet. Sci., 12, 211.
EXPERIMENTAL CANINE DISTEMPER INFECTION AS A MEANS OF DEMONSTRATING LATENT EFFECTS OF SUBACUTE LEAD INTOXICATION D. J. WHITE* AND S. McLEOD Fromb the WVellcome Research Laboratories, Langley Court, Beckenhanl, Kent Received for publication June 16, 1976
Summary.-Observations on the response of the body to experimental infection with distemper virus in dogs previously dosed subacutely with lead have demonstrated a latent effect of lead on several body systems. Effects which indicated a relationship to earlier treatment with lead included evidence for stimulation of haemoglobin synthesis, changes to red blood cells resulting in increased destruction, increased vulnerability of the parenchymatous cells of the liver to damage, reduction in the weight of the skeleton and thyroid, an increase in weight of the thymus and brain and histopathological changes in the thymus.
THE TOXIC EFFECTS of exposure to large amounts of lead on the blood system, kidney and brain are well documented (Goyer and Rhyne, 1973). There is considerable concern, however, as to the possible effects of mild or continuous exposure to increased amounts of lead on biological systems. In this direction particular attention has been paid to the investigation of very early effects of lead on haem synthesis and the measurement of the enzyme d-amino-laevulinic acid dehydrogenase has been found to be a particularly sensitive indicator of early lead effect (Hernberg and Nikkanen, 1970). The long-term significance of the intake of subclinical levels of lead on bodily functions has proved difficult to assess and the present report describes an experiment designed to investigate the pathological effects of subclinical lead intoxication in the dog. This has been done by assessing response of the body to infection with canine distemper virus in dogs previously given lead compared with *
dogs similarly infected with distemper but not given lead. MATERIALS AND METHODS Animals.-16 pure-bred Beagle dogs of less than 6 months of age were divided into 4 groups of 2 ,3 and 2 Y and kennelled individually under strict quarantine conditions. The diet was proprietary tinned dog food and biscuit meal; tap water was available ad lib. Canine distemper virus.-The Snyder Hill strain of distemper virus was obtained as a 20% lyophilized suspension of dog brain and further passaged in a susceptible dog by intracerebral inoculation. The dog was killed after 8 days and a 10% (w/v) suspension of the brain prepared in phosphate-buffered saline and used immediately at a dose rate of 0-2 ml injected i.v. Serum neutralizing antibody levels for infected dogs at death were determined by the method of Prydie (1968) and expressed as the logl0 reciprocal of serum dilution giving 5000 neutralization. Treatment.-Lead as lead carbonate was given orally to 2 groups of dogs (Groups 2 and 4) at a dose rate of 50 mg/kg body wt. daily for 3 weeks. Six weeks after the end of the dosing period, dogs in one group given lead (Group 4) and one undosed group (Group 3) were injected i.v. with distemper virus. Dogs in Group 1 remained undosed to act as controls.
Present address: Beecham Pharmaceuticals (Research
Division), Harlow, Essex.
646
D. J. WHITE AND S. McLEOD
Clinical examinations.-Alterations in food and water consumption and in clinical appearance were recorded daily. Body weight was measured once a week and rectal temperature daily. Haematology.-Blood samples were taken from the jugular vein, using EDTA as anticoagulant, before dosing commenced and then once weekly. Packed cell volume (PCV, ml%) was calculated using a Haematocrit Computer; red cell count (RBC, 106/mm3) using a Coulter Counter Model 5; white cell count (WBC, 103/mm3) using a Coulter Counter Model A; mean cell volume (MCV, fl) using a Mean Cell Computer and haemoglobin (Hb, g/100 ml) using an E.E.L. Haemoglobinometer. Mean corpuscular haemoglobin (MCH, pg) and mean corpuscular haemoglobin concentation (MCHC, g%) were calculated from the above values. Differential white cell count, reticulocyte count and incidence of basophilic stippling of red cells were done using suitably stained smears. Biochemistry.-Serum samples were obtained on 4 occasions for measurement of the following parameters using a Technicon Auto-Analyser I: glutamic pyruvic transaminase (SGPT), Technicon method N-54 (Henry et al., 1960); glutamic oxaloacetic transaminase (SGOT), method N-25b (Morgenstern et al., 1966); alkaline phosphatase (SAP), method N-6b (Bessey, Lowry and Brock, 1946), urea nitrogen (BUN), method N-ic (Marsh, Fingerhut and Miller, 1965) and cholesterol by the method of Levine and Zak (1964). Terminal procedures.-Dogs were deeply anaesthetized by i.v. injection of pentobarbitone and the brain perfused with 1% acetic acid in 10% formalin. All organs were examined macroscopically and weighed; the skin, muscle and skeleton were also weighed. Samples of the heart, lungs, liver, kidneys, spleen, pancreas, thyroid, thymus, adrenals, stomach, duodenum, ileum, colon, testes or ovaries, eyes, brain, pituitary and sciatic nerve were placed in 10% phosphate-buffered formalin at pH 7; samples of pancreas were also fixed in Bouin's fluid. Tissues were routinely processed, embedded in paraffin wax and sections cut at 5 ,um and stained with haematoxylin and eosin. In addition, frozen sections of liver, kidney and adrenal were stained with Oil-red-O for fat, the liver was also stained with PAS for glycogen; paraffin sections of the spleen and thymus were stained with Perls' method for ferric iron. Sections of brain were stained with Luxol fast blue for myelin and with Glees-Marsland's silver stain for neurons and nerve fibres. Statistical analyses.-Analysis of variance was carried out on log-transformed data for the haematological values measured on Day 70. This enabled a comparison of the results to be made between the controls and dogs given lead
only and distemper only and those given both lead and distemper. RESULTS
During the 3-week period of lead dosing no significant effects on food and water consumption or on body weight gain were observed in the lead dosed dogs of Groups 2 and 4. Their clinical condition did however alter slightly with the appearance of some harshness of the coat and a tendency towards the passage of either loose or hard faeces. The appearance of the coat and of the faeces returned to normal following cessation of dosing except for one dog of Group 2 in which clinical condition never fully returned to normal up to the time of death. No significant differences from control values were seen in any group in the period of dosing and during the withdrawal period in haematological or biochemical indices, except for evidence for the appearance in the peripheral blood during the period of lead dosing of small numbers of immature red cells in Groups 2 and 4, and for slight increases in cholesterol in these groups. The incidence of basophilic stippling of red cells in these groups reached a peak at the end of the dosing period and then gradually returned to normal during the following 6 weeks. Infection of Groups 3 and 4 with canine distemper virus was carried out on Day 63. The incubation period was short, 36-48 h, before the onset of typical clinical signs of the disease accompanied by pyrexia; there was no difference between the groups in the severity of the infection but 2 dogs in Group 3 died before the end of the experiment: 1 dog was killed on the 7th day post-infection due to rapidly worsening condition and the second dog was found dead on the 9th day post-infection. All the remaining dogs including those of Groups 1 and 2 were killed on Day 73 due to the deteriorating clinical condition of the distemper-infected dogs. The mean serum neutralizing antibody titre at death for Group 3 was 3-55 and for Group 4 was 3-3.
647
EXPERIMENTAL CANINE DISTEMPER INFECTION 0 75
PCV
1 70
1-65-
log 1-60
L+D-
RBC
D- o
L+D-
0 70log 0-65
L-D-0. LL0
9*.
L
D
LL- D+
1-55-
L-D--0*
L+
L-;--
L+D+
0o60
MCH
Hb
L-D+ 0 D+
MCH~~
1-50log
L-D- E.
15 1 45-
DL+D-
L-
L+D+
.D+ *-.
1 40-* L-D +
1 32:0 log
1 955
L+D+
MCV
,
L-
L-D-
D-
0----
:
L-D+
L: .
.
L+D-
1901
I L-D+
1 21 1-
log 1 0-
D+ L+D+ L+
090 8-
FiG. 1.-Results of haemabological examination on Day 70 plotted from analysis of variance of logtransformed data for treated and untreated dogs, and combinations of treatments. L - D = Group 1 untreated; L - = Groups 1 and 3 not given lead; L + = Groups 2 and 4-given lead; D -= Groups 1 and 2 not given dist emper;D += Groups 3 and 4-given distemper; L -D + = Group 3- given distemper but not lead; L + D + = Group 4-given distemper and lead.
Blood samples were obtained for haematology on the 7th day after infection from all dogs and the results are summarized in Fig. 1. It can be seen that distemper infection resulted in marked effects with significant differences between the 2 infected groups. These effects in both groups consisted of a reduction in PCV, RBC and Hb and an increase in MCV and WBC. In the case of PCV and Hb the decrease was much less marked in the group previously dosed with lead; however, the latter group showed a more marked decrease in RBCs. The change seen in MCH was directly opposite in the 2 groups, the lead-dosed infected dogs showing a substantial increase and the
group given distemper only showing a marked reduction. MCV in Group 3 increased only slightly but in Group 4 a marked increase was seen; there was, however, no evidence for this increase in peripheral blood smears where the numbers of immature cells showed no change. The results of biochemical analyses performed during the experiment are shown in Fig. 2. No significant alterations were seen during the lead-dosing period except for marginal increases in SAP and cholesterol in Group 2. Distemper infection resulted in a moderate rise in BUN and cholesterol of similar proportions in both infected groups, no doubt reflecting the response of the organism to a severe
648
D. J. WHITE AND S. McLEOD SGOT
SGPT
50-
40-
I.u.3S 30-
10 -12
140-
C
28 63 Days
70
-12
63
28
Days
BUN
SAP
120-
I.U
1008063 28 Days
63
28
70
70
Days
CHOLESTEROL
200175-
mg% 150125
-12
63
28
70
D ay s
FIG. 2.-Graphs of the mean values for SGPT, SGOT and SAP (all in international units) and cholesterol and BUN (both mg/100 ml) for Group 1 (undosed controls) O--- -O; Group 2 (lead only) 0; Group 3 (infected with distemper on Day 63) O---- [1; Group 4 (lead-dosed and infected * with distemper on Day 63) * *.
pyrexic disease. A slight increase in SGOT was also seen in both groups, being more marked in Group 4; the latter group also showed increases in SGPT and SAP, values for Group 3 remaining similar to those of controls. None of the biochemical changes recorded on Day 70 was statistically significant. Physical examination post mortem showed 1 dog of Group 2 to have a loose, harsh coat; on macroscopic examination of the organs of this dog the spleen was found to be slightly enlarged and small areas of consolidation were present in the lungs. No abnormalities were seen in the
organs of the other animals in Group 2 or in the controls. In Groups 3 and 4 typical clinical signs of canine distemper were present and macroscopic examination of the carcases showed similar abnormalities to be present in all the dogs; these included a harsh, dry coat and loose skin, reduction in body fat, congestion of the spleen, distension of the gall bladder with bile and lack of food material in the alimentary tract. The group mean organ and tissue weights, both absolute and related to body weight, together with the body weight just prior to lead dosing and again
649
EXPERIMENTAL CANINE DISTEMPER INFECTION
TABLE.-Mean Organ and Tissue Weights for Groups 1-4 Expressed as Absolute Values (g) and as Percentage of Body Weight and also Body Weight (kg) Before Treatment and at Death Group 4
3 _~
Tissue Liver
Kidney
Spleen Heart Pancreas
Absolute (g) Body wt. 282- 8 3-60 ±8-7 ±0 83 44- 13 0-56 ±2-08 ±0-23 29- 75 0-38 ±6-92 ±0- 88 73- 75 0 94 ±4-26 ±0 04 25-5 0-32
±2-33 Adrenals
1- 18
±0-15 Thyroids
1- 76
±0 39 Thymus
Brain
11 2 ±1 89 71-12
±1-19 Skin
1472
±82 Muscle
3338
Skeleton
1282
±40
±43
.~~~~~~~~~~~~~- .~~~~~~~~~~-
Absolute (g) 299 8
% Body wt.
±6-5
±0-22
44-13 ±0 94 33- 88 ±7-48 61 -0 ±5 05 *17-75 ±1 65 1-16
±0-02 0-015 ±0-001 0-022 ±0005 0-14
3*92 0 58
±004 0-46 ±0 13 *0 79 ±0 03
*0.23
±0O01
0.015
±0-14
±0O001
1*49 ±0-23 13 87
±0-002
±0-023
±2-29
±0-26
0-91
*78- 37 ±2-39 1481
1-03 ±0 076 19- 04 ±1-17
±0O019 18-8 ±1-16 42-6 ±0 7 16-35 ±0 43
±176 3223 ±289 1171 ± 74 5-35
5 75 Body weight before treatment (kg) 7 84 Body weight at at death (kg) * P = < 0 05 using Student's t test. tP = < 0X01.
7-74
at death are shown in the Table. Distemper infection caused a number of alterations in both infected groups but with some intergroup differences. A comparison of the results for Group 2 with the controls of Group 1 shows that the body weight at death was similar but that the absolute weight of the skeleton was reduced in the lead-dosed group; a similar reduction in skeletal weight was also seen in Group 4. Other changes in Group 2 included a reduction in heart weight, the result for the relative weight being significant; the weight of the pancreas was also significantly lower and
0-019 0-18
41- 5 ±0- 97 15-18
±0-38
Absolute (g) 291 3 ±18-9 *53.33 ±2 99 19 25 ±4-84 61-13 ±7-29 19-25 ±3-66 1 40 ±0 16 1-64 ±0-75 5 94 ±1-18 71-12 ±2-27 *999 ±104 *2210 ±278 1226 ±43 5-45 6 15
Body wt. t4.75 ±0 14
*0.88 ±0- 84 0 3 ±0 57 0.99 ±0-08 0-32 ±0-06 0 023 ±0 003 0-025
Absolute (g) 260*8 ±19-6 46- 13 ±1b82 18-5 ±3-13 56 63 ±2-85 20 0 ±2 58 1-42
±0O11 1-08
±0O009
±0O9
0 096 ±0-019 *1-17
±2 98
±0 065 16-26
8*82 74- 25 ±5-36 1191
±1 36
+69
*35-5 ±1-7
*2194 ±233 1155
*20.2
±1-16
±44
% Body wt. t4-33 ±0-06 *0.77
±0O59 0 3 ±3-13 0.95 O0- 04 0*32 ±2 58 *0-024 ±0-002 0-018 ±0-002 0*14 ±0 039
*1.24 ±0095 19* 84 ±0 33 *36-1 ±1b 33 *19.3 ±0 71
5.5
6-05
a slight reduction in thyroid weight was recorded; the thymus and the brain both showed a slight increase in weight compared with those of controls and higher values for these organs were also observed in Group 4 when compared with Group 3. From the results in Groups 2 and 4 the alterations which were suggestive of an effect of lead were the reduction in weight of the skeleton, heart and thyroid and the increase in weight ofthe thymus and brain. No significant differences were seen between any of the groups on microscopic examination of sections of the heart, pancreas, thyroid, adrenals, testes, ovaries,
650
D. J. WHITE AND S. McLEOD
eyes, brain and pituitary gland. In the stomach of all the dogs infected with distemper, eosinophilic, intracytoplasmic inclusion bodies typical of distemper were seen in the mucosal cells; similar inclusion bodies were also seen in the bronchiolar epithelium of the lungs. Single dogs from Groups 3 and 4 showed degenerative changes in the intestinal wall. The kidneys of the infected dogs were congested and in 2 dogs of Group 4 localized lesions of interstitial nephritis were seen; no evidence of either intranuclear or intracytoplasmic inclusion bodies was seen in the epithelium of the tubules. The main changes in all treated groups related to the amount of ferric iron present in the liver and spleen and to the appearance of the thymus and spleen in the distemper-infected dogs. In the liver sections of 2 dogs of Group 2 very occasional Kupffer cells contained ferric iron and in all dogs of this group a slight increase in iron deposition compared with the controls was seen in the red pulp of the spleen; in 1 dog of Group 4 the splenic white pulp was markedly reduced and several germinal centres had a vacuolated appearance. In the liver and spleen of almost all the distemper-infected dogs a significant increase in ferric iron of similar degree was seen; in 2 dogs from each infected group germinal centres of the splenic white pulp contained small lakes of brick-red material subsequently identified as haemoglobin by staining by Okajima's method. In the thymus of 3 dogs of Group 3 and all the dogs of Group 4 there was marked atrophy with a reduction in mature lymphocytes and a lack of Hassall's corpuscles; in addition, in 3 of the dogs of Group 4, groups of large vacuolated cells resembling macrophages and containing fat were seen throughout the thymic tissue.
would give rise to only mild clinical signs of toxic effect. This was largely achieved in that the only clinical evidence of lead poisoning was a slight fall in bodily condition. This was accompanied by a mild effect on red blood cell production; no tendency towards anaemia was seen and no biochemical evidence of liver or kidney damage was observed. During the 6-week period which was allowed to elapse following cessation of dosing and prior to infection with distemper, the clinical condition and effect on the red cells returned to normal with the exception of one dog which never fully recovered its bodily condition. It has however been shown, by employing the experimental model of infection with distemper virus, that an underlying effect of the earlier intake of lead was present in these dogs. This effect was demonstrated by alterations in the response of the erythropoietic system, evidence of damage to the liver, alterations in the weight of the skeleton, thyroid, brain and thymus and histological evidence of an altered response of the thymus to distemper infection. The latent effect of lead on the red cells was of 2 types. Firstly, lead appeared to have had a protective or even stimulatory effect on haemoglobin production, this was accompanied by an increase in the index of red cell size but no evidence for an increase in the release of immature red cells was seen in the peripheral blood. Secondly, although distemper infection gave rise to lysis of red cells as judged by increased iron in the liver and spleen and free haemoglobin in the splenic white pulp, the effect was more marked in dogs previously given lead. Lead is known to have a deleterious effect on red cell survival (Waldron, 1966) and the findings here suggest the presence of a continuing effect of lead during the formation of red cells in the bone marrow. The best-documented pathological DISCUSSION effect of lead on the organs occurs in the In the first part of this experiment an kidneys (Goyer and Rhyne, 1973) and it attempt was made to give large amounts might have been expected that some of lead to dogs over a short period which evidence of abnormal kidney function,
EXPERIMENTAL CANINE DISTEMPER INFECTION
either biochemical or histological, would have been seen. However, no effect on the kidney was observed during the period of lead dosing nor was a prior effect of lead demonstrated by subsequent distemper infection. A latent effect of lead on the liver was however suggested on biochemical evidence by the modest increase in SGPT, SGOT and SAP in the lead-dosed dogs infected with distemper. The liver is not generally recognized as a prime site for the toxic effects of lead and the majority of reports are concerned with changes in ultrastructure (Fiaccavento, Arrigoni and Chiappino, 1968; Hoffman et al., 1974). However, Marsden and Wilson (1955) described moderate liver damage in 2 cases of childhood poisoning and there are a number of reports of biochemical evidence of liver damage in experimental animals (Secchi, Alessio and Spreafico, 1971; Trejo et at., 1972). In the present experiment a skeletal origin for SAP cannot be excluded since distemper produced marked alterations in the mineral status of bone as reported elsewhere (White, Marshall and McLeod, 1975); similarly the raised SGOT levels may have resulted from the increased destruction of red cells. However, in the dog, increased levels of SGPT have been reported to be highly specific for liver damage (Zinkl et al., 1971) and on balance it is likely that the observed increases in enzymes seen here indicate a mild toxic effect on the liver parenchyma, the response of which has been affected by earlier contact with lead. Alterations in the weight of body tissues indicative of an effect of lead included the slightly reduced weight of the skeleton, the reduction in thyroid weight and the increase in weight of the thymus and particularly of the brain. The increase in brain weight, still present almost 8 weeks after cessation of lead dosing, is of particular interest since the most distressing symrptoms of lead poisoning in both man and animals involve the nervous system. The neurological symptoms of lead poisoning in children 43
651
and dogs are throught to be mainly due to an increase in intracrannial pressure as a result of oedema of the brain (Clasen et al., 1974; Stowe et at., 1973).- In the present experiment no histological evidence of oedema was observed but the possibility remains that the increase in brain weight was due to the presence of an increased amount of brain water. The increased weight of the thymus in both lead-dosed groups when compared with their respective control group indicates a stimulatory effect of lead. This finding, together with the histological observation of vacuolation in lead-dosed, infected dogs suggests higher reactivity of the thymus. Vacuolation is thought to represent sites of death of rapidly dividing lymphocytes and the appearance observed here is very similar to that seen in the involutionary changes in the thymus described by Henry (1967). At first sight these changes are incompatible with the immunosuppressive effects of lead which have been reported by Hemphill-, Kaeberle and Buck (1973) and by Koller (1973); the latter author inoculated rabbits with pseudorabies virus and found significantly lower serum neutralizing antibody titres in lead-dosed animals. The neutralizing antibody titres to distemper virus reported in the present paper were very similar in both groups of infected dogs, although marginally lower in those previously given lead. An explanation for the difference in results may lie in the length of time elapsing since the end of lead dosing and challenge with distemper virus. The observations made in this experiment provide evidence for the existence of a latent effect of lead on a number of body systems. The practical importance of these findings may lie in the possibility of altered responses of the body to other disease processes as a result of the residual effects of lead.
We acknowledge with thanks the help of Mr R. B. Clampitt with the biochemical analyses and Mr P. A. Young with the statistics.
652
D. J. WHITE AND S. McLEOD
REFERENCES BESSEY, 0. A., LowRy, 0. H. & BROCK, M. J. (1946) Method for the Rapid Determination of Alkaline Phosphatase with 5 cubic millimetres of Serum. J. Biol. Chem., 164, 321. CLASEN, R. A., HARTMANN, J. F., STARR, A. J., COOGAN, P. S., PANDOLFI, S., LAING, I., BECKER, R. & HASS, G. M. (1974) Electron Microscopic and Chemical Studies of the Vascular Changes and Edema of Lead Encephalopathy. Am. J. Path., 74, 215. FIACCAVENTO, S., ARRIGONI, G. & CHIAPPINO, G. (1968) Aspetti Morfologi Utrastructurali della Cellula Epatica Nella Intossicazione Saturnina. Lav. Med., 22, 219. GOYER, R. A. & RHYNE, B. C. (1973) Pathological Effects of Lead. In International Review of Experimental Pathology, Vol. 12. Ed. G. W. Richter and M. A. Epstein. New York: Academic Press. HEMPHILL, F. E., KAEBERLE, M. L. & BUCK, W. B. (1973) Lead-Induced Alterations of Immunologic Reactivity in Mice. J. Am. Vet. Med. A88., 163, 1191. HENRY, L. (1967) Involution of the Human Thymus. J. Path. Bact., 93, 661. HENRY, R. J., CHIAMORI, N., GOLUB, 0. J. & BERKMAN, S. (1960) Revised Spectrophotometric Methods for the Determination of GlutamicOxaloacetic Transaminase, Glutamic-Pyruvic Transaminase and Lactic Acid Dehydrogenase. Tech. Bull. Reg. Med. Tech., 30, 149. HERNBERG, S. & NIKKANEN, J. (1970) Enzyme Inhibition by Lead Under Normal Urban Conditions. Lancet, i, 63. HOFFMAN, E. O., DILtTzIo, N. R., HOLPER, K., BRETTSCHNEIDER, L. & COOVER, J. (1974) Ultrastructural Changes in the Liver of Baboons Following Lead and Endotoxin Administration. Lab. Inve8t., 30, 311. KOLLER, L. D. (1973) Immunosuppression Produced by Lead, Cadmium and Mercury. Am. J. vet. Re8., 34, 14,57.
LEVINE, J. B. & ZAK, B. (1964) Automated Determination of Serum Total Cholesterol. Clin. chim. Acta, 10, 381. MARSDEN, H. B. & WILSON, V. K. (1955) LeadPoisoning in Children: Correlation of Clinical and Pathological Findings. Br. med. J., i, 324. MARSH, W. H., FINGERHUT, B. & MILLER, H. (1965) Automated and Manual Direct Methods for the Determination of Blood Urea. Clin. Chem., 11, 624. MORGENSTERN, S., OKLANDER, M., AUERBACH, J., KAUFMAN, J. & KLEIN, B. (1966) Automated Determination of Serum Glutamic Oxaloacetic Transaminase. Clin. Chem., 12, 95. PRYDIE, J. (1968) Studies with an Attenuated Canine Distemper Vaccine Derived from Chick Embryo Cultures. Res. vet. Sci., 9, 443. SECCHI, G. C., ALESSIO, L. & SPREAFICO, F. (1971) Serum Enzymatic Activities in Experimental Lead Poisoning. Enzyme, 12, 63. STOWE, H. D., GOYER, R. A., KRIGMAN, M. M., WILSON, M. & CATES, M. (1973) Experimental Oral Lead Toxicity in Young Dogs. Archs Path., 95, 106. TREJO, R. A., DILuzIo, N. R., LoOSE, L. D. & HOFFMAN, E. (1972) Reticuloendothelial and Hepatic Functional Alterations Following Lead Acetate Administration. Exp. mol. Path., 17, 145. WALDRON, H. A. (1966) The Anaemia of Lead Poisoning: a Review. Br. J. indu8st. Med., 23, 83. WHITE, D. J., MARSHALL, A. J. & McLEOD, S. (1975) The Influence of Experimental Distemper Infection on the Distribution of Lead in Dogs Previously Subacutely Intoxicated with Lead Carbonate. Br. J. exp. Path., 56, 544. ZINKL, J. G., BUSH, R. M., CORNELIUS, C. E. & FREEDLAND, R. A. (1971) Comparative Studies on Plasma and Tissue Sorbitol, Glutamic, Lactic and Hydroxybutyric Dehydrogenase and Transaminase Activities in the Dog. Re8. vet. Sci., 12, 211.
