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This is to certify that the
thesis entitled

Lead Shot Poisoning in Red-Tailed Hawks

presented by

Susan Stein

has been accepted towards fulfillment
of the requirements for

M. S . degree in EathnlagL

 

J.D. Krehbiel, D.V.M., Ph.D.

 

Major professor

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LEAD SHOT POISONING IN RED-TAILED HAWKS

BY

Susan Stein

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathology

1979

ABSTRACT

LEAD SHOT POISONING IN RED-TAILED HAWKS

BY

Susan Stein

Research was conducted to duplicate lead poisoning as seen in
living birds of prey. Nine birds were divided into 3 groups.

Group 1 consisted of control birds which received no lead. Birds
in the second group received 4 sequential doses of 10 number 5 lead
shot. Individuals in the third group were dosed 4 times with 30
number 5 shot.

Birds were evaluated for clinical evidence of lead poisoning,
blood and tissue lead levels, total leukocyte numbers, hemoglobin,
hematocrit, osmotic erythrocyte fragility, and gross and microscopic
lesions.

Apparent clinical signs were not observed in any bird on experi—
ment. Analyses of repeated blood samples from all dosed birds were
indicative of acute exposure to lead. Tissue lead levels did not
indicate toxicosis. Blood parameters monitored in this study were
found not to be predictable for lead intoxication. Spontaneous
erythrocyte fluorescence occurred on a non-uniform basis in birds
from groups 2 and 3. Gross observation and microscopic examination
of selected tissues revealed no pathological changes associated with

plumbism.

Susan Stein
Birds of prey which were able to egest lead shot showed neither
gross nor microscopic evidence of lead poisoning but, in fact, did

absorb lead during instances of acute exposure.

".....The greatest importance of lead poisoning under natural
conditions may not be direct mortality, but subclinical disease and its
subsequent effect on susceptibility to disease, predators, and natural

elements as well as its effect on reproduction."

- R. S. Cook and D. 0. Trainer,

Journal of Wildlife Management,

Volume 30, No. 1, January 1966, p.8.

ii

ACKNOWLEDGMENTS

The author wishes to express her appreciation to the following:

To Dr. J. Krehbiel, my major professor, for guidance and direc-
tion during the course of my degree program.

To Dr. J. Stuht for counseling and assistance in conducting this
research.

To Dr. A. Trapp for advice and information regarding avian
pathology.

To the Michigan Department of Natural Resources at Rose Lake for
providing experimental animals, facilities, and financial support to
pursue graduate research.

To Shirley Howard, B.S., M.S., for superior technical assistance
and moral support.

To my dear husband, Ron, for sympathy and empathy during my

graduate studies.

iii

TABLE OF CONTENTS

INTRODUCTION. . . . . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . . . . .

Clinical Signs and Gross Lesions. . .
Microscopic Lesions . . . . . . . . .
Diagnostic Technique. . . . . .

Other Blood Values. . . . . . . . .
Erythrocyte Fragility . . . . . . . .
Blood Values. . . . . . . . . . . . .
Tissue Lead Levels. . . . . . . . . .
Spontaneous Erythrocyte Fluorescence.
Summary of Literature Review. . . . .

OBJECTIVES. . . . . . . . . . . . . . . .

MATERIALS AND METHODS . . . . . . . . . .

Sources and Care of Experimental Animals

Experimental Method . . . . . . . . .
Procedures. . . . . . . . . . . . . .
Hematologic Procedures. . . . . . . .
Spontaneous Erythrocyte Fluorescence.
Erythrocyte Fragility . . . . . . . .
Blood Lead Determinations . . . . . .
Tissue Handling . . . . . . . . . . .
Tissue Lead Determinations. . . . . .

RESULTS . . . . . . . . . . . . . . . . .

weight 0 0 O O O O O O 0 O O O O O O 0
Blood Lead Levels . . . . . . . . . .

Hemoglobin, Hematocrit, and Mean Corpuscular

Hemoglobin Concentration . . . .
Leukocytes. . . . . . . . . . . . . .
Tissue Lead Levels. . . . . . . . . .
Erythrocyte Fragility . . . . . . . .
Spontaneous Erythrocyte Fluorescence.

Radiographic Findings and Shot Retention Times.
Pathologic Changes - Gross and Microscopic Lesions.

DISCUSSION. . . . . . . . . . . . . . .

iv

Page

24

24
24
25
26
26
27
28
28
29

31

31
33

34
37
39
42
42
48
S4

58

SUMMARY . . . . . .

LIST OF REFERENCES.

61

62

LIST OF TABLES

Table Page
1 Average Blood Lead Levels (ppm) . . . . . . . . . . . . . 33
2 Changes in Blood Lead Levels (%). . . . . . . . . . . . . 34

3 Hemoglobin (Hb), Hematocrit (HCT), and Mean
Corpuscular Hemoglobin Concentrations (MCHC). . . . . . . 35

4 Total Leukocyte Counts (mms). . . . . . . . . . . . . . . 37
5 Average Tissue Lead Levels (ppm). . . . . . . . . . . . . 39

6 Changes in Tissue Lead Levels (%) . . . . . . . . . . . . 40

vi

LIST OF FIGURES

Figure Page
1 Body Weight. . . . . . . . . . . . . . . . . . . . . . 32
2 Tissue Lead Concentrations (ppm) . . . . . . . . . . . 41
3 Osmotic Erythrocyte Fragility in Birds 4, S, 10

(CODtrOlS) 0 o o o e o e o o o o o o o o o o o o o o o 43
4 Osmotic Erythrocyte Fragility in Birds 2, 6, 8

(10 Shot) 0 I O O O O O O O O O O O O O O O O O O O O O 44
S Osmotic Erythrocyte Fragility in Birds 3, 7, 9

(30 shot). . . . . . . . . . . . . . . . . . . . . . . 4S
6 Spontaneous Erythrocyte Fluorescence . . . . . . . . .46-47
7 Spontaneous Erythrocyte Fluorescence in Bird 2 . . . . 49
8 Spontaneous Erythrocyte Fluorescence in Bird 3 . . . . 49

vii

INTRODUCTION

Lead is a recognized constituent of the food chain of all birds.
Sublethal exposure by ingestion of plants and other living tissues
produces natural background lead levels in raptors and non-raptors
alike (Bagley et a1., 1967).

Excessive ingestion of lead, especially in the form of spent shot,
has been a major factor in North American waterfowl poisonings since
the late 1800's (Cook et a1., 1966). These migratory individuals with
ingested lead shot and high tissue lead levels are often food sources
for predatory birds.

Non-digested material forms a pellet in the muscular stomach of the
raptor. This material is periodically egested in the normal individual
(Duke, 1978). Digestible muscle and viscera from crippled ducks and
small mammals with high tissue lead levels would not be egested and may
provide a means of secondary toxicosis.

The quantity of lead ingested, the duration, and the frequency of
exposure to lead required to produce toxicosis are unknown in the
raptor, although well documented in waterfowl (Coburn et a1., 1951).

Plumbism in raptors has been diagnosed at necr0psy since severely
debilitated birds die shortly after presentation.

The objectives of this research were to attempt to duplicate the
natural occurrence of lead poisoning in raptors using red-tailed hawks,
and to evaluate the clinical and subclinical changes in the experi-

mental birds.

LITERATURE REVIEW

Lead poisoning has been noted as a factor in waterfowl mortality
since 1874 (Phillips and Lincoln, 1930). Grinnell (1901) observed
physical signs of plumbism in ducks, geese, and swans naturally poi-
soned on the North Carolina coast during the winter of 1893-4. Bowles
(1908) reported a condition in mallards found in marshy areas on the
Puget Sound. The birds had no wounds or visible trauma, but contained
quantities of lead shot in the stomach. The causes of death were not
positively determined as many of these animals had recently been eaten
by hawks or owls.

The possibility of death due to ingestion of spent shot has been
recorded in scaled and bobwhite quail (Campbell, 1950; Westemeier,
1965) and mourning doves (Locke et al., 1967).

Birds affected by lead show pronounced weight loss, difficulty in
flying and walking, and severe muscle degeneration (Linduska, United
States Department of the Interior, 1964). The inability for flight
escape enables these birds to become sitting ducks for hunting raptors.

The dietary preferences of raptors vary. Red-tailed hawks, for
example, survive on rodents, rabbits, insects, and snakes, as well as
medium or large birds caught on the ground (Brown et a1., 1968).
Research on captive birds of prey indicates superior performance and
maintenance when a diet of larger mammals is supplemented with smaller
mammals and birds (Mendelssohn et a1., 1970). Red-tails in harsh

winter climates expand their diets to include poultry, game birds and

song birds (Hausman, 1948). The golden eagle, another raptor, feeds on
ducks, geese and other waterfowl, grouse, wild turkeys, herons, other
birds, mammals and carrion. The sea eagle has a predilection for a
seafowl diet in addition to mammals. Other raptors fond of waterfowl
are the black and white gyrfalcon, peregrine falcon, and the large
white snowy owl (Hausman, 1948). Studies by Craighead and Craighead in
1942 and 1948 of raptor winter diets in southern Michigan showed that
the red-tailed hawk was more flexible in its tastes than some of the
other buteos. A red-tailed hawk's summer diet in Superior Township of
Michigan in 1942 and 1948 included adult ring-necked pheasants, as did
that of Cooper's hawks and horned owls (Craighead and Craighead,

1956).

Benson et a1. (1974) reported on the death of a prairie falcon fed
ground duck heads which had detectable lead levels. The author felt
these levels were high enough to cause plumbism and subsequent death.
This method of acquired lead toxicosis falls into a classification of
secondary poisoning (e.g., lead poisoning due to the ingestion of
tissues from an animal sick or dead from lead poisoning). Jacobson et
a1. (1977) described a case of lead poisoning in an immature bald
eagle which on postmortem had a total of 75 lead shot in the gastro-
intestinal tract. Redig (1979) diagnosed clinical lead poisoning in
2 prairie falcons and 1 goshawk. He also suspected lead as being the
primary cause of death in l prairie falcon, l bald eagle, and 2 red-
tailed hawks.

Unpublished information from the logs of the Michigan State Univer-

sity Animal Health Diagnostic Laboratory lists 32 avians as having died

since 1968 either from lead poisoning alone or in conjunction with
other disease processes. Fourteen of these 32 birds (ranging from

ducks to bustards) still retained eroded lead shot in the gizzard.

Clinical Signs and Gross Lesions

 

Grinnell (1901) in his observations of waterfowl in Texas and North
Carolina made note of the clinical signs of lead poisoning. He found
birds free from external lesions, but unable to fly. His initial de-
scriptions were of a "croupy" (his terminology) condition with the bird
having labored, rattling respirations and a yellow discharge from the
bill. His dissection of an affected goose revealed particles of pitted
lead shot in the gizzard. The gizzard had a decayed, greenish inner
membrane not seen in a healthy individual. There was marked inflamma-
tion of the intestine and the rectum. One liver lobe was discolored.
This animal had a healthy external appearance. Similar lesions
appeared in a swan necropsied by Grinnell a few days later.

Additional information in 1950 (Jordon et a1.) included severe
weight loss as a common finding in lead poisoned ducks.

Robert Stroud, writing from Alcatraz in 1939, observed awkward
movements, blindness and digestive disorders in some of his domestic
fowl which he later concluded had lead toxicosis. His microscopic
evaluations of livers and kidneys from poisoned juvenile birds revealed
"lipid degeneration" (his terminology). There were no macroscopic
lesions in these tissues. Another bird had jerky movements, vocal-
ized, and eventually convulsed and died. The kidneys again appeared
grossly normal and healthy, although the liver was discolored and

yellow and the spleen shrunken. Beer and Stanley (1964) necr0psied 74

birds (swans, ducks, geese) suspected of having lead poisoning. Two-
thirds of these birds were emaciated, having little or no subcutaneous
or visceral fat. Other findings noted were gizzard impaction, anemia,
enlarged gallbladders, hepatic atrophy and deterioration. At least

63 percent of these wildfowl had recognizable lead in the gizzard.
Bellrose, in a 1964 United States Department of the Interior publi-
cation, described lead poisoning symptoms. He believed the initial
sign of toxicosis was inappetcnce due to partial paralysis of the
gizzard musculature. His view was that birds with prolonged cases of 2
or 3 weeks' duration either recovered or died after impaction of the
stomach. The critical time period for retaining or voiding this shot
was 20 days (Bellrose, 1964). Birds did not linger in acute toxicity
cases, but succumbed in l to 2 weeks depending upon the quantity of
lead ingested (Bellrose, 1964). Chronic victims had atrophied flight
muscles and green-stained vents. They were less likely to escape
hunters and predators.

The average period of shot retention in mallards was 18 days
(Bellrose, 1964). Waterfowl eliminated the shot by defecation.
Raptors had a much slower course of disease (Redig, 1979). The
clinical signs were not pronounced and sometimes were nothing more
than depression or weakness. The mutes often became runny as the
disease progressed (Redig, 1979).

Raptors had an additional mechanism for eliminating non-digestible
material. Raptors egested this material in the form of a pellet.

This regurgitation was considered a physiologic requirement for birds
of prey (Mendelssohn, et a1., 1970). Predicting the regularity of

this egestion was rather difficult. The time period from eating to

egestion for owls, for example, was about 12 hours. Hawks had an
interval of 19.5 hours to 23.5 hours, supposedly triggered by light
(Duke et a1., 1976). The quantity of food consumed influenced the
rate of egestion in owls. Meal size apparently did not have the same
effect in hawks. Duke calculated that hawks cast pellets from approx-
imately 80% of their meals. The specific diet, as well as the health
of the raptor, apparently influenced the egestion rate (Duke et a1.,
1976). Consumption of lead shot, or of prey with high tissue lead
levels, did not result in the same rate of casting for owls as for
hawks. Hawks continued to eat with retained stomach pellets, while
owls did not (Duke et a1., 1976).

The number of retained lead shot fatal to birds varied among
individuals (Bellrose, 1964). The variability in the number of shot
required for poisoning and the course of disease were influenced by
the type and quantity of food consumed. Birds with ingested shot,
continuing to eat as they normally would, had excellent chances for
survival (Bellrose, 1964). The gizzard in these birds also continued
to function as did the egestion, or casting, mechanism.

Coburn et a1. (1951) dosed wild caught mallards with lead to
simulate field poisonings and to clinically observe the birds. These
birds did not demonstrate the classic mammalian signs seen with lead
poisoning. They initially displayed lethargy and weakness. Progress-
ive paralysis of leg and wing musculature was observed in most birds.
Green bile staining the vent area was a common finding. The gross
lesions included emaciation, liver atrophy, gizzard atrophy, and
flabby heart muscles. Roscoe et a1. (1977) found ruffled feathers,

head pressing, and diarrhea in addition to previously observed clinical

signs. His gross lesions duplicated those previously mentioned.
Published descriptions of the clinical signs of lead poisoning

in specific birds of prey mentioned incoordination in flight, ataxia,

apparent problems in visual perception (Benson et a1., 1974), head

tilts, and yellowish material lining the oral cavity (Jacobson et a1.,

1977).

Microscopic Lesions

 

Buck et a1. (1973) indicated that certain changes were consistent
with exposure to excessive lead. Acute exposures resulted in mild
gastritis, pale livers with centrilobular degeneration, occasional
acid-fast intranuclear inclusions in the renal tubular epithelium,
renal hemorrhage, and degeneration or necrosis of the renal tubular
epithelium. A thickened glomerular capsule was often present.

Stroud (1939) investigated microscopic sections of liver and
kidney which grossly appeared unaffected, but had marked "lipoid
degeneration" (his terminology).

Canada geese fatally dosed with number 4 lead pellets had such
lesions as hepatic congestion, brownish pigment in the hepatic and
Kupffer cells, necrosis of the renal tubular epithelial cells, myo-
cardial necrosis, hyperemia and hemorrhage in the lungs, muscle ne-
crosis in the gizzard, and irregular necrotic areas in the pancreas
(Cook and Trainer, 1965). Locke and Bagley (1967) likewise found
focal hepatic necrosis and pigment deposition in lead poisoned mourn-
ing doves. They saw acid-fast intranuclear inclusion bodies in the
epithelium of the proximal convoluted tubules of the kidney as did

Locke et al. (1966) and Grandy et a1. (1968). Canada geese dying from

lead intoxication did not consistently have acid-fast intranuclear
inclusion bodies in the renal tubular epithelium (Bagley and Locke,
1967).

Roscoe et al. (1977) examined tissues from mallards dosed with
predetermined numbers of number 4 lead shot. There were acid-fast
intranuclear inclusions in the proximal convoluted tubular epithelium
of the kidney in 70% of the birds that died during the study. Other
lesions included bile pigment accumulation in the hepatocytes, degen-
eration of the proventricular plica, edema of the gizzard mucosa, and
lymphocytic and heterophilic infiltration into the gizzard mucosa.

The histopathologic changes seen in the tissues of lead poisoned
birds necropsied since 1968 at the Michigan State University Animal
Health Diagnostic Laboratory ranged from hepatic hydropic degeneration
and fatty metamorphosis (cases 103557-560, 119047, 111423) to mild
toxic tubular nephrosis and renal tubular epithelial degeneration
(cases 103557-60, 111243-4, 111423, 119047, 135898).

Histopathologic changes in various raptors with lead poisoning
included degenerative changes in the renal tubular epithelial cells,
acid-fast intranuclear inclusion bodies in those same cells, and
cloudy swelling of hepatocytes (Locke et a1., 1969). Examination of
tissues from an affected bald eagle revealed focal hepatic necrosis,
pulmonary congestion, mild catarrhal enteritis, and mild interstitial
nephritis. The bald eagle had no lead inclusions in the renal tubular

epithelium (Jacobson et a1., 1977).

Diagnostic Techniques

 

A blood lead determination has been the best antemortem diagnostic
technique for evaluating acute lead poisoning in domestic mammals
(Hernberg et a1., 1970; Buck et a1., 1973). Values for blood lead
levels in the chicken ranged from 8 parts per million (ppm) without
clinical manifestations of poisoning to 13 ppm just before death
(personal communication of Buck et al. to V. E. Vengris, College of
Veterinary Medicine, Iowa State University, 1972). Cattle, horses,
waterfowl, and dogs apparently are more frequently affected by lead
poisoning, while goats, chickens, and pigs are less susceptible (Buck
et a1., 1973).

Cook and Trainer (1966) dosed Canada geese with 2, 5, 10, 25, 50
and 100 number 4 lead pellets. All geese had rapid rises in blood
lead levels within 3 days which reflected a direct relationship
between the quantity of lead ingested and the blood lead levels.
Predosing blood lead levels ranged from .018 milligrams per 100 grams
(mg/100 gm) to .037 mg/lOO gm. These were normal values for geese.
Postdosing levels ranged from .081 mg/lOO gm to 1.020 mg/lOO gm.

Barrett and Karstad (1971) monitored 6 mallard ducks receiving
8 number 6 lead shot. The ducks had pretrial blood lead levels ranging
from .6 ppm to 1.8 ppm. The lead levels ranged from 7 ppm to 19.1 ppm
by the third day after dosing. Another group of mallards receiving 6,
12, and 18 pellets had markedly increased blood lead levels by 10
hours after dosing. The data generated by Barrett and Karstad did not
reflect a direct relationship between the quantity of lead shot ingested
and the resulting blood lead levels. Additional birds receiving 15 or

25 pellets had elevated blood lead levels by the first day after

10

exposure.

Longcore et al. (1974) dosed each of 100 four-month-old drake
mallards with a single number 4 lead shot. Blood lead levels ranged
from 2.7 ppm to 20 ppm in the birds that died. Birds euthanized
during the study had blood lead levels from 3.7 ppm to 22.9 ppm. A
blood lead level of .02 ppm was considered a normal background level
for wild mallards (Finley and Locke, 1976).

A study by Finley et al. (1976) in which six-month-old mallards
were dosed with a single number 4 lead shot had the following results.
The first day after dosing the mean blood lead level reached 1.66 ppm.
The blood lead level peaked at 1 week postdosing at 2.36 ppm and
remained elevated at .64 ppm through the fourth week. Additional
birds treated in a similar fashion averaged .64 ppm of blood lead over
a 4 week period. Control birds had blood lead levels of .05 ppm
during the same period. Ten ppm has been considered indicative of an
acute exposure to lead despite the wide range of blood lead levels
obtained by various authors (Longcore et al., 1974).

Clinical cases of apparent lead poisoning in 2 Florida cranes and
2 greater sandhill cranes were reported in 1977 by Kennedy et al. The
blood lead levels in these cranes ranged from 1.46 ppm to 3.78 ppm

before treatment was initiated.

Other Blood Values

 

Kennedy (1977) determined no clear relationship between blood lead
levels and leukocyte numbers in lead poisoned cranes. The hematocrits
of these cranes did not reflect depressed red cell counts although 3

of the 4 cranes appeared clinically anemic.

11

Redig (1979) stated that a lead poisoned raptor usually will have
a pronounced leukopenia, but insignificant changes in total protein
and hematocrit.

A group of mallards given 8 number 4 lead pellets by Roscoe et al.
(1976) exhibited an average mean corpuscular hemoglobin concentration
of 26 grams percent (g%) as compared to 3lg% seen in control birds
8 days after poisoning. Coburn et al. (1951) dosed 12 mallards with 8
and 12 milligrams of lead per kilogram of body weight over periods
ranging from 19 days to 41 days. They found consistent decreases in
hemoglobin values (e.g., average initial hemoglobin value for all
birds was 107.9% and final average hemoglobin value for all birds was
47.6%). The red cell counts dropped in most of Coburn's experimental
individuals during the first 10 to 14 days of lead administration.

The total leukocyte counts varied from predosage counts of 19,000 to
90,000.

Changes in the hemoglobin levels and erythrocyte numbers were
further substantiated by de Bruin (1971). He rationalized that lead
was partial to and interacted with the erythrocyte membrane. The
erythrocyte then lost potassium and water and had an altered phosphor—
ous uptake. These changes accounted for the shortened lifespan of
circulating red blood cells. At the same time hemoglobin synthesis
was occurring primarily in the erythroblasts of the bone marrow.
Several of the enzymes in the hemoglobin synthesis pathway were in-
hibited due to lead's affinity for sulfhydryl groups. These negative
influences on circulating erythrocytes and erythroid elements in the
bone marrow perhaps accounted for the shortened lifespan of erythro-

cytes in lead poisoned individuals (de Bruin, 1971).

12

Erythrocyte Fragility

 

Griggs (1964) found the red cells from lead poisoned human subjects
increasingly resistant to hypotonic sodium chloride solutions. Incuba-
tion of the collected samples further increased the osmotic erythrocyte
resistance. Griggs concluded that as a result of lead poisoning the
red blood cell shrank, or lost corpuscular volume, and consequently
swelled to a greater critical size in hypotonic solution before lysis
occurred. Wintrobe et al. (1974),in discussing the anemia of lead
poisoning, also made mention of the decreased osmotic fragility and the
increased mechanical fragility of the erythrocytes in affected humans.

Data summarized and presented by Aub et al. (1925) indicated a
species variation in erythrocyte osmotic fragility after exposure to
lead. Man, rabbits, guinea pigs and rats apparently had character-
istic changes in red cell resistance while horses, chickens, dogs and
catsclhlnot display this phenomenon. Aub et al. (1925) specifically
demonstrated that erythrocytes from lead poisoned rabbits had increased
resistance to lysis in hypotonic saline solution. Schalm et al. (1975)
concluded that the pronounced blood loss anemia in a lead poisoned
heifer resulted from the increased erythrocyte destruction directly
due to decreased osmotic resistance - in opposition to Aub's rabbit
research. Frankel et al. (1970) stated that conditions causing
spherocytosis of the erythrocytes (e.g., most hemolytic anemias)
resulted in increased osmotic fragility while thin, flat target cells

were less susceptible to hypotonic saline solutions.

13

Blood Values1

 

Lucas and Jamroz (1961) commented on the intrinsic value of mon-
itoring hematologic parameters in birds. They concluded that total
white cell counts and differential counts on a single bird were of
limited value. Hemoglobin and hematocrit values, on the other hand,
had a very limited range of variability and would be indicative of
problems in an individual bird. They also recommended the minimum

numbers of birds required for studying each blood parameter.

Tissue Lead Levels

 

Adler (1947) analyzed the tissue lead concentrations of 4 lead
poisoned Canada geese. He concluded from his data that the liver was
the organ of choice for chemical analysis to diagnose lead poisoning.
This hepatic lead level represented an ongoing toxicosis, while an
analysis of bone indicated chronic poisoning. His control birds had
liver lead levels of 0 ppm to 1 ppm. The concentrations of lead in
the livers of the 4 poisoned geese ranged from 9 ppm to 27 ppm.
Kidney lead levels for the controls were 0 ppm to 6 ppm. The lead
levels in kidney tissue from the poisoned birds ranged from 12 ppm to
57 ppm. Adler's 2 control birds had no measurable lead in the lung.
Lead levels of 2 ppm to 9 ppm were found in the lungs of the affected

geese. The heart muscles of the control birds had no detectable

1Alfred M. Lucas and Casimir Jamroz, Atlas of Avian Hematology (United
States Department of Agriculture, 1961), p. 217.

 

14

quantities of lead upon analysis. Corresponding data for the poisoned
geese was not available.

Buck et a1. (1973) established liver lead levels of 10 ppm and
kidney cortex levels of 15 ppm as consistent with lead poisoning.
Control liver lead levels of less than 3 ppm (often ranging from .3 to
1.5 ppm) and control kidney lead levels of 1 ppm to 3 ppm were also
established.

Coburn et al. (1951) determined that any one of liver, skeleton,
or soft tissues might be used as field samples for chemical analyses
to determine lead poisoning. He found average liver lead values 40
times greater in his poisoned mallards than in the corresponding
control birds. The poisoned birds' livers had 12 to 36 ppm of lead.
The rate of lead deposition in any tissue in this same experiment did
not directly reflect the dosage level of lead nor the length of the
test period.

A 1965 report by Hunter and Rosen on a lead poisoned wild pheasant
stated that 42 ppm of lead were found in breast tissue and 168 ppm in
liver tissue. Normal lead values were set at 1 ppm. Experimentally
poisoned pheasants had breast and liver lead levels of 50 ppm and
143 ppm respectively. Cook and Trainer (1966) found liver lead levels
ranging from 5 ppm to 32 ppm in experimentally poisoned Canada geese.
These levels were compatible with those derived by Adler (1947) and
Coburn (1951).

A mourning dove with clinical plumbism had lead concentrations of
72 ppm and 187 ppm in the liver and tibia respectively (Locke and
Bagely, 1967). Further field investigations at this same time gener-

ated data on 40 dove livers. The lead content of these samples ranged

15

from .4 ppm to 14 ppm. The dove having a liver lead concentration of
14 ppm had 2 lead shot in its gizzard. Bagley and Locke (1967) ana-
lyzed samples from 483 Canada geese collected during a massive die-off
on the Atlantic Coast which lasted from November, 1965, to April,
1966. Chemical analyses on the livers and tibias of 20 of these birds
yielded the following data: livers had lead concentrations from 6 ppm
to 53 ppm and tibias had lead levels from 12 ppm to 102 ppm. Kidneys
from 7 geese had lead levels from 8 ppm to 32 ppm. Eleven control
geese had liver lead levels of .4 ppm to .8 ppm. Bagley and Locke
(1967) subsequently undertook a study of liver lead levels in 28
species of wild birds and bone lead levels in 13 species. They con-
cluded that lead was a regular constitutent in the tissues of wild
birds. Periods of active exposure were reflected by elevated liver
lead concentrations, while bone lead levels were indicative of chronic
toxicity. The danger of lead mobilization from bone sites during
stress situations was also noted.

A clinically affected Andean condor was found to have 38 ppm of
lead in its liver (Locke et al., 1969). No controls were available to
evaluate the lead levels in livers of healthy condors, but this level
was high enough to indicate lead poisoning. The condor had been fed
hunter-killed carcasses and apparently had ingested some of the lead
shot in the carcasses.

Longcore et al. (1974) compared tissue lead levels in mallards
dosed with a single number 4 lead shot. He concluded that while a
specific lead level was not diagnostic for plumbism, lead levels in
the brain, liver, and kidneys were indicative of fatal lead poisoning.

The lead levels in the tibias of euthanized birds in this study ranged

16

from 57 to 212 ppm. Birds that died as a result of lead poisoning had
tibial lead levels of 87 to 209 ppm. Liver lead levels in euthanized
birds were 26 to 65 ppm. Fatally affected birds had liver lead levels
ranging from 32 to 83 ppm. The researchers concluded that lead levels
from 6 to 20 ppm in the liver indicated acute lead intoxication.
Kidney lead levels were measured at 53 to 408 ppm for mallards that
died due to lead poisoning. Euthanized birds had kidney lead levels
of 22 to 243 ppm. It was suggested that the kidney tissues might be
vital indicators of lead toxicity inasmuch as they function to excrete
body wastes and ingested poisons. Twenty ppm lead in the kidney
tissues was indicative of a serious lead toxicosis. Heart muscle from
euthanized mallards in this same study had .4 ppm to 46 ppm lead.
Dying birds had cardiac lead levels of 1.3 to 4.8 ppm. Lastly, lead
levels in the lungs of euthanized ducks ranged from 1.7 to 15 ppm.
Ducks that died on experiment had lung lead levels of 2.5 to 8.2 ppm.
The lung lead levels in euthanized birds were initially higher, but
birds euthanized at longer intervals from the time of dosing had
decreasing lung lead levels.

Longcore et a1. (1974) concluded that the most reliable test for
diagnosing lead poisoning was the analysis of soft tissues from
affected birds. They also concluded that continuous oral ingestion of
lead resulted in high residues in bones and lower residues in livers
and kidneys. The smallest amounts of lead were accumulated by the
heart, lung, muscle and brain. The authors speculated that tissues
involved with circulatory function (e.g., heart, lung, and blood)
might have lead levels which decreased with time since circulatory

lead levels decreased as lead was stored in such other tissue sites as

l7

bone.

A report of lead poisoning in a prairie falcon in 1974 (Benson et
a1.) cited the feeding of duck heads with high lead levels as the
source of the poisoning. Lead residues in the affected falcon were
3.9 ppm in the heart muscle, 6.0 ppm in the kidney, 12.9 ppm in the
brain tissue, 17.4 ppm in the liver, and 36.0 ppm in the humerus and
femur. It was felt that the liver lead level of 17.4 ppm was highly
significant and indicated fatal lead poisoning.

A bald eagle with numerous lead shot in the gizzard was submitted
to the University of Maryland for clinical examination (Jacobson et
al., 1977). The bird was presented alive and died shortly thereafter
in acute respiratory distress. Chemical analyses of the liver and
kidney yielded lead concentrations of 22.9 ppm and 11.3 ppm respec-
tively. These values were considered to be consistent with lead
toxicosis.

Analyses of the liver and kidney from a fatally affected greater
sandhill crane resulted in lead levels of 29 ppm and 18.6 ppm respect-
ively. It was noted that these tissue lead levels were significant for
lead poisoning (Kennedy et a1., 1977).

Birds presented to the Animal Health Diagnostic Laboratory at Mich-
igan State University since May, 1968, had the following tissue lead
levels:

1. Four pelicans had liver and kidney levels of 32 ppm.

(Case 103557-560).
2. One swan had liver and kidney levels of 33.3 ppm.

(Case 104655)

18

3. Two ducks had combined stomach contents, liver and kidney
levels of 31 ppm. (Case 105917-919)

4. A bustard had liver and kidney levels of 100 ppm.
(Case 107035)

5. Three pelicans had composite liver and kidney samples of
44 ppm. (Case 111243-4)

6. One duck had liver and kidney lead levels measured at
12.8 ppm. (Case 111423)

7. An Emperor penguin had liver levels of 85 ppm lead.
(Case 113188)

8. Two ducks had liver lead levels of 95 ppm and kidney lead
levels of 91 ppm. (Case 181748-51)

All of the above birds were reported as confirmed cases of lead poison-

ing either clinically affected or dead at presentation.

Spontaneous Erythrocyte Fluorescence

 

Low concentrations of lead inhibit several enzymes in heme syn-
thesis. Measurement of heme precursors in the blood indicates the
effects of lead absorption (Baloh, 1974). One step of heme synthesis
impaired by lead is the insertion of iron into the protoporphyrin
moiety (Sassa et al., 1972). The enzyme heme synthetase, which has a
thiol (-SH) group, is needed for this step in the pathway (Buck et al.,
1973). Lead apparently has an affinity for essential sulfhydryl or
thiol groups (de Bruin, 1971). There is a subsequent accumulation of
the hemoglobin precursors within the tissues and body fluids of indiv-
iduals with adequate lead absorption (de Bruin, 1971). One such

precursor, protoporphyrin, is formed in the mitochondria of the

19

immature erythrocyte during its stay in the bone marrow. Lead in-
hibits the use of this protoporphyrin, so as the maturing erythrocyte
loses its Initochondria and enters the circulation, it has the same
protoporphyrin content as it had in the bone marrow (Sassa et al.,
1973). This increased protoporphyrin, also known as free erythrocyte
protoporphyrin, enables erythrocytes to fluoresce red when excited by
blue light (Vannotti et al., 1949).

Whitaker and Vietti in 1959, screened blood smears from several
children with lead poisoning. They examined the slide preparations
under ultraviolet light and found red fluorescence in 75% to 100% of
the erythrocytes. They concluded that the fluorescence was apparently
related to the quantities of free protoporphyrin in the erythrocytes.
Nelson et al. (1968) examined blood smears from human patients with
lead poisoning and estimated the number of fluorescing erythrocytes in
25 to 50 oil immersion fields viewed under a Leitz fluorescent micro-
scope. The authors then quantitated erythrocyte free protoporphyrin
levels and measured urinary lead excretion in their patients. They
concluded that their "fluorescyte" preps had a high degree of correl-
ation with the quantitated erythrocyte protoporphyrin concentrations
and the urinary lead levels. Their findings confirmed the assumption
that peripheral erythrocytes fluoresce due to increased protoporphyrin
levels.

These increased levels of protoporphyrin, specifically proto-
porphyrin IX, were also noted in patients with iron deficiency anemia
(Lamola et a1., 1974). Lamola demonstrated that protoporphyrin in the
blood of humans with lead poisoning, or severe iron deficiency anemia,

was complexed with zinc and not truly free erythrocyte protoporphyrin.

20

Lamola et al. (1974) concluded that measuring whole blood for fluor-
escence at 594 nm was the simplest and most direct screening test for
lead poisoning.

Chisolm (1973) noted that the fluorescence seen with accumulations
of protoporphyrin during lead poisoning resulted from a delayed
reaction due to interference with erythropoiesis. The fluorescence
appeared at some later time after chronic lead absorption (Chisolm,
1973).

Barrett et a1. (1971) decided to employ spontaneous erythrocyte
fluorescence as a rapid test to diagnose lead poisoning and estimate
its incidence in waterfowl. Mallard ducks and Canada geese were
experimentally poisoned with various quantities of number 6 lead shot.
Blood from the birds was collected in heparin. Each sample was
screened for fluorescence within 48 hours of collection under 400x
magnification by dark field microscopy. The combined ultraviolet and
blue light illuminating the viewing field had a wavelength of 400 nm.
One hundred and forty-one of the 176 blood samples fluoresced. The
intensity of fluorescence varied from pale pink to bright red and
lasted for from 5 seconds to several minutes. The longest and bright-
est fluorescence was observed in erythrocytes collected 3 to 9 days
after the birds had been exposed to lead. Fluorescence diminished as
each mallard duck and goose returned to a state of health (Barret et
al., 1971).

It has been observed that other disease conditions, namely, severe
iron—deficiency anemia, hemolytic anemia and pernicious anemia, may
cause increases in protoporphyrin and perhaps spontaneous erythrocyte

fluorescence; however, these conditions have been unknown in wild

21

waterfowl (Barret et a1., 1971). The finding of fluorescent eryth-
rocytes in blood smears from lead poisoned birds may not indicate
fatal poisoning, but perhaps an increased vulnerability to predation
and hunting (Barrett et al., 1971). Fewer than 1% fluorescytes on
blood smears treated as previously described were assumed to be normal;
more than 10%umnxeabnormal (Nelson et al., 1968). Buck et al. (1973),
using the 1971 data obtained by Barrett and Karstad, stated: "In
waterfowl, a simple diagnostic test involving microscopic examination
of red blood cells for red fluorescence has been shown to be of
value."2

It was determined in 1976 that fluorescent erythrocytes from lead
poisoned waterfowl have emission and excitation maxima of 635 nm and
408 nm respectively (Roscoe et al., 1976). In a study by Roscoe et
a1. (1976) 40 mallards had the maximum accumulation of protoporphy-
rin IX on day 8 after treatment with lead. This protoporphyrin IX had
the same maxima as that previously determined for fluorescent erythro—
cytes (Lamola et al., 1975). Roscoe et a1. (1976) also concluded that
the protoporphyrin levels were related to the number of shot each bird
ingested, and these levels, measured in micrograms per deciliter
(ug/dl), were inversely proportional to the mean corpuscular hemo-

globin concentrations. This group of investigators analyzed the

2William B. Buck, Gary D. Osweiler, and Gary A. Van Gelder: Clinical
and Diagnostic Toxicology. Kendall Hunt Publishing Company, Dubuque,
Iowa, (1973), p. 196.

 

22

spectra of blood samples from their research birds and determined that
the fluorescing compound in lead poisoned birds was not complexed with
zinc as in man. Further studies by Roscoe et a1. (1977) in which
mallard ducks were divided into an acutely poisoned group and a
chronically poisoned group confirmed the previous findings. Roscoe et
al. (1979), using a modified hematofluorometer, were able to measure
protoporphyrin levels in lead poisoned mallards. The data collected
demonstrated a lag period of approximately 6 days between peak blood
lead concentrations and peak blood protoporphyrin IX concentrations.
They speculated that an anemia resulting from lead intoxication would
increase erythropoiesis and increase the synthesis of protoporphyrin IX

by immature erythrocytes.

Summary of Literature Review

 

The literature cited in this section indicates that lead poisoning
is a serious problem among wildfowl. There is no reason to believe
that the inclusion of lead in the food chain of raptors is not also
a very real threat. The clinical signs and gross and microscopic
lesions characteristic of lead toxicosis have been documented at
length. The analyses of selected tissues for lead concentrations
serve as confirmation of other diagnostic methods (e.g., spontaneous
erythrocyte fluorescence) which are gaining wider field acceptance.

It is apparent that further investigations are needed to define the
problem of lead toxicosis in those avian species feeding on poisoned

birds or mammals.

OBJECTIVES

The objectives of this research were:
1. To reproduce lead poisoning, as seen in the wild, in a

representative raptor - the red-tailed hawk (Buteo jamaicensis).

 

2. To define and enumerate clinical signs, if any, resulting
from lead administration to these birds.
3. To define any gross or microscopic changes, in selected

avian tissues, which were caused by the lead.

23

MATERIALS AND METHODS

This research was initiated in January 1978. The birds were
maintained at the Rose Lake Wildlife Station, Stoll Road, East
Lansing, Michigan. Analyses were conducted at the Department of

Pathology, Michigan State University, East Fee Hall.

Sources and Care of Experimental Animals

 

Red-tailed hawks which had sustained injuries, and were unsuitable
for release into the wild, were supplied by the Michigan Department
of Natural Resources. All birds were maintained indoors in suitable
caging and standardized on a diet of freshly killed laboratory mice
(outbred Swiss—Webster ICR mice supplied by Spartan Research Animals,
Haslett, MI). Birds were housed 1 to a cage and had no visual contact
with one another. The cages were monitored daily and cleaned as

needed. Fresh water in large crocks was available at all times.

Experimental Method

 

The birds were randomly divided into 3 representative groups
after a standard period of acclimation to food and housing. One
group of birds (birds 2, 6, and 8) were sequentially fed freshly
killed mice with 10 number 5 lead shot hidden in the brain tissue.
Another group of birds (birds 3, 7, and 9) received 30 number 5 lead
pellets at each dosing. Birds 4, 5, and 10 were controls and as

such received mice with no lead pellets.

24

25

Procedures

 

A feasibility study was conducted using a single red-tailed hawk.
The diet, dosing schedule, blood collection techniques and methodology
were standardized using this individual (bird 1).

On day 1 all birds were weighed and bled. Blood was obtained from
the alar vein by fresh venipuncture with a 25 gauge 5/8 inch needle.
Approximately 1.25 milliliters (ml) to 1.5 ml of blood were collected
from each bird in a heparinized plastic syringe (Crisler et al.,
1972). Three blood smears were immediately made from each sample and
allowed to air dry. The remaining blood samples were transferred to
sterile heparinized plastic tubes and refrigerated. On day 1 all
birds were radiographed (Department of Radiology, Veterinary Clinical
Center, Michigan State University) for the presence of any retained
shot acquired in the wild. Birds were then offered the appropriately
dosed freshly killed mice. On day 4 each bird was again weighed,
bled, radiographed, and redosed with lead-primed mice. On day 7 all
birds were again weighed, radiographed, and dosed. On day 10 birds
were weighed, bled, and dosed. Birds were weighed, bled, and radio-
graphed on day 17. On day 24 each individual was weighed, bled, and
euthanized.

Each bird in the 3 replicate trials was observed daily for the
following clinical signs:

1. lethargy

2. inappetence

3. green diarrhea
4. facial edema

5. wing droop

26

6. feather loss
7. death
A daily record was kept of the casts from each bird. The mutes
were examined daily for any lead shot lost through the digestive
system. All expelled casts were retrieved and examined. Lead pellets

adhering to or housed inside cast material were salvaged.

Hematologic Procedures

 

An aliquot of blood was taken for microhematocrit and hemoglobin
(AO hemoglobinometer) determination (Benjamin, 1961).

Blood smears were stained with Wright's stain while being pro—
cessed through an automatic stainer (Ames Hema-Tek SlidestainerR).

Microscopic examination of the prepared smears was performed to
evaluate relative changes in the numbers of monocytes, heterophils,
eosinophils, lymphocytes and basophils. Absolute leukocyte counts as
well, were performed on blood obtained from birds 5, 6, 7, 8, 9, 10
(hemocytometer). A dilution factor of 200 was used for all white cell

counts .

Spontaneous Erythrocyte Fluorescence

 

A drop of heparinized blood was placed on a microscope slide and
coverslipped. The wet mount was examined at 400K magnification through
a Zeiss binocular microscope equipped with a dark field condenser. A
fluoroisothiocyanate (FITC) filter was used as the primary (excitation)
filter. The wavelength of the excitation filter peaked at 495 nm. A
secondary (barrier) filter of 530 nm wavelength was also used. An

additonal filter, a BC 38 red-suppressor filter, excluded any red

27

light except that originating from the fluorescing erythrocytes.
Fifty oil-immersion fields were examined on each slide. The number of
fluorescing erythrocytes, the brilliance (pink to red), and the dura-

tion of fluorescence were noted for each slide.

Erythrocyte Fragility

 

The osmotic fragility of the erythrocytes was determined using the
Dacie method.3 A phosphate buffered stock solution osmotically equal
to 10% saline was prepared. The stock sample was diluted 1:10 with
normal saline to prepare a working solution. Dilutions ranging from
.1% to .85% were made by adding calculated quantities of distilled
water to the working solution. Units of .05 m1 of heparinized venous
blood were added to tubes containing 5 m1 of each dilution. The
solutions were mixed, incubated for one-half hour at room temperature,
remixed, and centrifuged at 2000 rpm for 5 minutes. The supernatant
from each solution was collected and inserted into a colorimeter
(Perkin Elmer spectrophotometer) set at 545 nm to determine optical
density. The .85% solution supernatant was used as a blank, and the
.l% saline solution supernatant was used as an indicator of 100% hemo-
lysis. The percent hemolysis was calculated from the highest theoret-

ical optical density reading (.1% saline).

3Gradwohl's Clinical Laboratory Methods and Diagnosis. (ed. by

S. Frankel, S. Reitman, and A. C. Sonnenwirth). C. V. Mosby Co.,
St. Louis, 1, (1970), p. 572-574.

 

28

Percent hemolysis in each tube plotted against the corresponding

saline concentrations graphically depicted osmotic fragility.

Blood Lead Determinations

Blood was kept frozen prior to preparation for lead level deter-
minations. All determinations were made on a Perkin Elmer 360 atomic
absorption spectrophotometer set at 283.3 nm. The Delves technique
was used to analyze the blood (Delves, 1970). Each sample of 10 micro--
liters (pl) of whole heparinized blood was transferred to a nickel-
alloy (Delves) cup for this technique. One set of standards con-
taining .4 ng/ml lead and .8 ug/ml lead was used. All unknowns were
analyzed in triplicate. The blood samples were dried at 140°C, and
20 ul of 30% hydrogen peroxide was added to each sample. The samples
were then oxidized at 140°C for approximately 2 minutes. Each cooled
sample was placed in the spectrophotometer, which had been refitted
with a 3 slot burner head, and atomized in an air-acetylene flame.
The air to acetylene ratio was 70 to 45. An attached strip chart
recorder recorded peak heights. The following formulae were used to

calculate blood lead concentrations:

pg Pb/ml of blood average sample peak height X concentration

average standard peak height of standard

pg Pb/dl of blood ug Pb/ml X 100

Tissue Handling

 

All birds were euthanized with carbon dioxide gas and necropsied
at the termination of each trial. Selected tissues (e.g., gizzard,

spleen, proventriculus, crop, kidney, lung, liver, bone, and heart)

29

were saved for chemical analysis and histopathologic preparation.
Tissues for chemical analysis were weighed and frozen until required.
Tissues for microscopic examination were fixed in 10% buffered forma-
lin, embedded in paraffin, sectioned, and stained with hematoxylin and

eosin or Ziehl-Neilsen acid fast stains.

Tissue Lead Determinations4

 

All tissues were ashed with magnesium acetate to facilitate
removal of organic material. A gram of each tissue, except bone, and
1 ml of magnesium acetate were dried in a 56°C oven overnight. The
samples were then ashed in a muffle furnace at 450°C to 500°C for 4 to
5 hours. The ashed samples were dissolved in hydrochloric acid solution
(2N HCl), complexed by the addition of ammonium pyrrolidine dithio-
carbamate (AFDC) and freed from any residual tissue matrices by par—
titioning into an organic solvent, methyl isobutyl ketone (MIBK). The
organic supernatant was aspirated after centrifugation and analyzed
against an MIBK blank.

The ashing process for bone material was done as follows. The
right femur from each experimental bird was rinsed with distilled
water and dried in a 100°C oven. Two milliliters of magnesium acetate

were added to a 1 gram sample of bone. This mixture was then dried in

4W. Hyde, J. Kiesey, P. F. Ross, and H. M. Stahr: Analytical

Toxigology Methods Manual. Iowa State University Press, Ames, Iowa,
(1977), p. 42-44.

 

 

30

a 100°C oven. All material was ashed for 16 hours in a 550°C muffle
furnace after drying. The residues were dissolved in 2N HCl and

transferred to labelled tubes. The original crucibles were washed

with 2 rinses of distilled water to insure the transfer of all ashed
materials. The extraction procedure at this point continued as

previously described.

RESULTS

Lead shot was successfully administered to the experimental
birds. Overt clinical signs of lead poisoning were never noted in
dosed individuals. The parameters measured were treated in two
different fashions. Individual data were presented where appropriate,
while other data were more readily understood if presented in a
composite form as controls, 10 pellet dosed, or 30 pellet dosed

birds.

Weight
All birds were weighed on days 1, 4, 7, 10, 17 and 24 of the

research protocol. The weight graph in Figure 1 shows all 3 groups
of birds having had a demonstrable weight gain after an initial
period of stabilization. This period of gain was followed by a
pronounced weight loss in all 3 groups. However, at the end of

the experimental period all birds had a net weight gain.

31

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33

Blood Lead Levels

 

The average blood lead levels for each group of birds are presented

 

 

in Table 1.
Table 1. Average Blood Lead Levels (ppm)
Birds Dosed Birds Dosed
Days on Experiment Control Birds With 10 Pellets With 30 Pellets
1 (Baseline Values) .163 ppm .820 ppm .375 ppm
4 .170 ppm 2.053 ppm 2.767 ppm
10 .203 ppm 3.693 ppm 9.693 ppm
17 .203 ppm 2.963 ppm 8.960 ppm
24 .167 ppm 1.67 ppm 4.340 ppm
Each group of birds had increased blood lead levels. Machine

variability or sensitivity may have accounted for changes in the

control group.

The standards used during blood analyses reflected

very slight changes in mechanical operation of the atomic absorption

spectrophotometer.

Table 2 shows the percentage increase from baseline, or original

blood lead levels, in each group over the duration of the experiments.

34

 

Table 2. Changes in Blood Lead Levels (%)
Birds Dosed Birds Dosed
Days on Experiment Control Birds With 10 Pellets With 30 Pellets
1 Baseline 0 Baseline 0 Baseline 0
4 4% 150% 638%
10 24% 350% 2485%
17 24% 261% 2289%
24 2% 104% 1057%

 

The group of birds receiving 30 number 5 lead shot at each dosing
demonstrated the most marked increases in blood lead levels. The
hawks that received less lead had measurable increases in blood lead

levels, but not as pronounced as the more heavily dosed individuals.

Hemoglobin, Hematocrit, and Mean Corpuscular Hemoglobin Concentration

 

The average composite values for hemoglobin (Hb), hematocrit (HCT)
and mean corpuscular hemoglobin concentration (MCHC) are presented in

Table 3.

35

 

 

 

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36

No consistent conclusions were drawn from this data due to fluc-
tuations in the values for Hb, HCT and thus the calculated MCHC.
Graphic depictions of this same data did not present any uniform
direction for each measurement and are therefore not included in the

results.

37

Leukocytes

 

The total leukocyte counts, or total white cell counts, for each
group of birds are presented in Table 4. The initial total white cell

count on day l was taken as a zero point.

Table 4. Total Leukocyte Counts (mms)

 

Birds Dosed With Birds Dosed With
Day Control Birds 10 Shot 30 Shot
1 17, 139 33, 778 24, 556
4 16, 750 26, 222 21, 917
10 21, 167 25, 445 21, 278
17 35, 334 27, 111 28, 000
24 24, 278 22, 889 21, 566

Each group of birds had a decrease in total leukocyte numbers
after the initial blood collection. The lead dosed groups had more
marked declines in leukocyte numbers after the initial dosing than did
the control group. The control group had a rise in total cell numbers
at the time of the third bleeding. Birds in the lead dosed groups
continued to experience declining cell numbers. Sample 4 on day 17
showed the control group responding with a dramatic increase in leukocyte
numbers. The birds in the group receiving 30 lead pellets at each
dosing had a marked rise in total leukocyte numbers, while the group
receiving 10 pellets at each dosing also had a perceptible increase in
cell numbers. This rebound in total leukocyte numbers occurred 1 week
after the last lead was fed. None of the birds retained shot at the

time of this blood collection. Blood lead levels had decreased by

38

day 17 as previously noted in Table 1. All 3 groups had decreased

total white cell counts at the time of the last blood sampling.

39

Tissue Lead Levels

 

The average tissue lead levels for each group of birds are pre-

sented in Table 5.

 

Table 5. Average Tissue Lead Levels (ppm)

Birds Dosed Birds Dosed
Tissue Control Birds With 10 Pellets With 30 Pellets
kidney .163 ppm .420 ppm .933 ppm
spleen .300 ppm .961 ppm 1.193 ppm
gizzard .291 ppm .665 ppm 1.332 ppm
proventriculus .258 ppm .521 ppm .490 ppm
crop .551 ppm .521 ppm .567 ppm
heart .414 ppm .644 ppm .757 ppm
lung .560 ppm .840 ppm 1.990 ppm
liver .350 ppm .433 ppm 1.100 ppm
bone .953 ppm 1.06 ppm 3.617 ppm

Table 6 presents the tissue lead values from the experimentally
dosed birds as percent change from the baseline values from the
control birds. These baseline values represent background lead levels

which an individual could accumulate in his or her natural environment.

40

 

Table 6. Changes in Tissue Lead Levels (%)

Tissue Birds Dosed With 10 Pellets Birds Dosed With 30 Pellets
kidney 157.20% 471.42%
spleen 120.44% 397.77%
gizzard 136.80% 374.58%
proventriculus 102.37% 90.22%
crop - 7.36% 2.91%
heart 55.61% 83.03%
lung 50.00% 255.36%
liver 23.8% 214.29%
bone 11.19% 279.38%

Figure 2 pictorially reflects these changes. The kidneys, giz-
zards and spleens from the dosed birds apparently concentrated the
highest levels of lead when compared to the same tissues in undosed
individuals. The gizzard, which concentrates the ingesta for cast
formation and mechanically abrades the material, had a level of lead
far above the control levels. The proventriculus and crop are essent-
ially rapid transit areas for food in raptors. The proventricular
lead levels were markedly higher than those of the control birds, but
crop lead levels for the dosed groups were unremarkable. The birds in
the group receiving 10 pellets at each dosing had a composite crop
lead level below the control level. The heart, constantly in contact
with the elevated blood lead, had measurable increases in ppm of lead
per gram of muscle mass in both dosed groups. Birds receiving higher

doses of lead had higher tissue lead levels than the group receiving

3.

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Tissue Lead Concentrations (ppm)

spleen proventriculus

42

lower doses in almost all cases. All dosed birds similarly had higher
tissue lead levels than undosed birds in every instance save one.
Tissue lead levels remained elevated despite a 2 week period from the

day of the last dosing to the day of euthanasia.

Erythrocyte Fragility

 

The 3 graphs in Figures 3, 4, and 5 represent the osmotic erythro-
cyte fragility curves for all 3 groups of birds. The 5 curves on each
graph depict areas of maximum hemolysis occurring at salt concentra-
tions between .15% and .25%. Slight rises in osmotic fragility were
seen at salt solutions of .4% to 5.5% for the first blood sample from
both control birds and heavily dosed birds. The control birds and the
high dose group of birds again had slight increases in osmotic fra-
gility between salt concentrations of .3% to .35% at the fourth

bleeding.

Spontaneous Erythrocyte Fluorescence

 

Spontaneous erythrocyte fluorescence was only observed in lead
dosed birds. The predose blood samples from individuals in each group
contained no fluorescent erythrocytes when viewed by fluorescent micro-
scopy. The quality and quantity of fluorescence were of such variable
natures that individuals from each group are represented as single

units in Figure 6.

43

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Bird 2 (10 pellet)
Days

Number of
Fluorescing Cells
per 50 High Power
Fields (hpf)

Color of Cells

Duration of
Fluorescence

Bird 6 (10 Pellet)
Days

Number of
Fluorescing Cells
per 50 hpf

Color of Cells
Duration of
Fluorescence

Bird 8 (10 Pellet)
Days

Number of
Fluorescing Cells
per 50 hpf

Color of Cells
Duration of

Fluorescence

Figure 6.

46

l 4

O 6

- faint
pink

- 5
seconds

1 4

0 0

1 4

0 0

10

245

red and
pink -
faint

15
seconds

10

pale
red

5
seconds

10

17

1718

red and
pink-mixed
brightness

15
seconds

17

bright
red

12
seconds

17

Spontaneous Erythrocyte Fluorescence

24

2871

faint

pink

5
seconds

24

24

Figure 6 (cont'd).
Bird 3 (30 Pellet)
Days

Number of
Fluorescing Cells

per 50 hpf

Color of Cells

Duration of
Fluorescence

Bird 7 (30 Pellet)
Days

Number of
Fluorescing Cells
per 50 hpf

Color of Cells
Duration of
Fluorescence

Bird 9 (30 Pellet)
Days

Number of Fluorescing
Cells per 50 hpf

Color of Cells

Duration of
Fluorescence

47

bright
red

5
seconds

10

184

faint pink
to bright
red

10
seconds

10

faint
red

5
seconds

10

17

17

faint
red

5
seconds

17

10

pink

2

seconds

24

357

faint pink
to bright
red

10
seconds

24

24

numerous

very faint faint

pink

too brief
to count

Not all dosed birds developed fluorescing erythrocytes as seen

from the above chart.

The quantity of lead the bird received appar-

ently did not correspond to the quantity or quality of erythrocyte

48

fluorescence. Bird 2, which received 10 lead pellets at each dosing,
had the most fluorescing erythrocytes with the longest duration of
fluorescence. Bird 8 never developed any fluorescing erythrocytes.
Birds 2, 3 and 9 retained varying qualities of fluorescence despite
being pellet-free since the 17th day on experiment. Birds 6 and 7
returned to a nonfluorescent state by the termination of the experi-

ment. Examples of this fluorescence are seen in Figures 7 and 8.

Radiographic Findings and Shot Retention Times

 

Birds 2, 3, and 4 comprised the first experimental group. A

preliminary radiograph was taken of each individual.

An intramedullary pin, which had not stabilized 2 fracture sites
of the right humerus, was seen on the preliminary radiograph. Bird 2
was dosed with 10 number 5 lead shot on day 1 of which 8 were recov-
ered from casts and mutes found on the cage floor on days 2 and 3.
Radiographs taken on day 4 revealed 2 pellets remaining in the gizzard.
The bird ate 10 more lead shot on day 4, for a total of 12 internally
located lead shot. A cast egested on day 6 contained 7 pellets.
Mutes and casts recovered on day 7 contained the 5 remaining shot and
radiographs taken that same day revealed no retained lead pellets.
The bird was again fed 10 shot on day 7, cast 7 of these shot by the
following day, and retained 3 other shot. The bird was dosed on day 10
with 10 more pellets. Bird 2 eliminated all remaining 13 lead

pellets by day 11. Bird 2 retained 2 lead shot for 7 days and 3 shot

49

 

Figure 7. Spontaneous erythrocyte fluorescence
in bird 2. Photographed 1 week after
last dosing.

 

Figure 8. Spontaneous erythrocyte fluorescence in
bird 3. Photographed 2 weeks after last
dosing.

50

for 4 days.

Bird 3

Bird 3 had cerclage wires visible at 2 sites of a nonunion frac-
ture of the left humerus on the preliminary radiograph. The bird was
fed 30 number 5 lead shot in a mouse meal on day 1. Several shot were
passed on day 2, and by day 3 all the pellets had been either egested
or defecated by the bird. Radiographs on day 4 showed no retained
lead shot, and the bird was redosed with an additional 30 pellets on
that same day. He egested a cast containing 28 pellets on day 5 and
passed an additional pellet on each of days 6 and 7. Radiographs on
day 7 showed no pellets in the gastrointestinal tract, and the bird
was redosed that same day. A cast containing 23 pellets was found on
the cage floor on day 8. The remaining 7 lead shot were recovered
from the cage floor on day 10, at which time the bird was radio-
graphically free from lead shot. Bird 3 retained 7 lead shot for 3

days and 1 pellet for 3 days.

Bird 4

Bird 4 was the control bird for this group and received a mouse
diet without lead shot. This bird was free from any previously
ingested pellets, but had radiographic evidence of metallic foreign
bodies in 5 locations. What appeared to be lead pellets were found in
the muscle masses adjacent to both left and right tibias. Another
pellet was superimposed on the pelvis. Two additional pellets were

located near the left radius. The left proximal ulna had been

51

fractured as an apparent consequence of being shot. These pellets

never changed position at any time in the sequence of radiographs.

The second group of experimental birds included birds 5, 6, and 7.
Bird 5 was the control individual in this group. Radiographs
taken on days 1 and 4 showed no evidence of either ingested lead shot

or pellets embedded in bone or soft tissue.

Bird 6

The initial radiograph of bird 6 showed no lead pellets in any
location. This bird received 10 number 5 lead shot in a mouse meal on
day 1. A single pellet was found in the cage on day 2, and 9 more
lead shot were recovered from a cast egested on day 3. The bird was
radiographically free from lead on day 4, and on that day was again
fed 10 lead shot. Single shot were passed by the bird on days 5 and
6 with the 8 remaining pellets found in mutes and casts littering the
cage floor on day 7. Radiographs on day 7 confirmed the abscence of
retained lead shot. A 10 pellet dosing was repeated at that time.
A cast egested on day 8 contained 3 of those pellets. One pellet was
found on the cage bottom on day 9, and the remaining pellets were
retrieved from a cast and mutes examined on day 10. A subsequent
radiograph demonstrated the absence of lead in this bird on the same
day just prior to redosing with 10 lead shot in a mouse meal. Three
shot were found in the bird's water dish, 3 in a cast, and the
remaining shot on the cage floor on day 11. The bird was radiograph-

ically free from lead pellets on that day and received no additional

52

shot. Bird 6 retained 9 lead shot, 8 shot, and 6 shot each for

periods as long as 3 days.

The preliminary radiographs taken of bird 7 showed no lead pellets
located either in the gastrointestinal tract or soft tissues. The
bird was dosed on day l with 30 number 5 lead shot and cast 28 of
these pellets on day 2. No pellets were egested on day 3. A single
pellet was found in the cage on day 4,2nuiradiographs taken on that
same day showed a single shot retained in the gizzard. The bird ate
another 30 pellets on day 4. Combined casts from days 6 and 7 con-
tained all 31 pellets. Radiographs on day 7 demonstrated no lead shot
in the bird. Thirty additional pellets were fed to the bird on day 7,
and he egested 28 of these pellets on day 8. Two pellets were found
on the cage floor on day 10,znulradiographs taken on that day revealed
no retained lead shot. The last dose of lead shot was administered at
this time. Bird 7 cast 21 of these pellets on day 11, egested 2 lead
shot on day 12, and egested the remaining 7 pellets on day 13. Bird 7
retained a single lead pellet for 6 days, 2 pellets for 3 days and

7 pellets for 3 days.

Birds 8, 9 and 10 completed the last replicate.

Bird 8 had no shot in any tissue prior to the start of this exper-
iment as determined by radiographic technique. This bird ate 10
number 5 lead shot offered in a mouse meal on day l. Undigested

material cast late on day 2 and early on day 3 contained all 10 shot.

53

The bird was fed another 10 lead pellets on day 4, and 3 of these
pellets were passed on day 5. A cast egested on day 6 contained 7
additional lead pellets. A radiograph taken on day 7 demonstrated no
remaining lead shot,znu1an additional 10 pellets were consumed by the
bird on that day. One lead shot was found in the mutes on day 8, and
9 more shot were cast by the bird on day 10. A radiograph taken on
day 10 demonstrated no lead shot. This bird was dosed for the last
time on day 10. Examination of the cage floor on day 11 yielded 9 of
these 10 pellets. The last lead pellet was found in a cast on day 13
at which time a final radiograph showed the bird to be free from all
fed lead shot. This bird retained 9 lead pellets for 3 days, and 1

lead pellet for 3 days.

An initial radiograph of bird 9 showed a metallic foreign object
in the soft tissue immediately ventral to the left femur. The object
appeared to be a lead pellet. This bird was fed 30 number 5 lead shot
in a mouse meal on day l. Twenty-six shot were recovered from mater—
ial on the cage floor on day 2, 1 shot on day 3, and 3 shot on day 4.
A radiograph taken on that day showed only the lead shot in the left
thigh area. An additional 30 pellets were fed on day 4. One lead
shot was recovered on day 5. A cast containing 18 pellets was re—
trieved from the cage on day 6 along with an additional 7 pellets re-
covered from the cage floor. A radiograph taken on day 7 showed 4
lead shot remaining in the gizzard. Thirty more pellets were consumed
by bird 9 on day 7. Nine shot were removed from the floor of the cage

on the following day,£fi5well as a cast containing 22 more shot. A

54

single lead pellet was found on the cage floor on day 9. Radiographs
on day 10 showed 2 lead shot remaining in the gizzard. The bird was
dosed with lead shot for the last time on day 10. There were 4 pellets
in the mutes and 28 in a cast from the bird on day 12. Radiographic
examination of the bird at this time demonstrated only the initial

lead pellet seen in the femoral area. This bird retained 3 lead shot
in the gizzard for 4 days, 4 shot for 3 days, and 2 shot for 8 days.

No other bird in the study retained lead shot for as long, however, as
individual lead shot were not identified it was impossible to deter-

mine if the same shot were retained throughout the entire period.
Bird 10
Bird 10 was an undosed control bird. Radiographic examination

showed no evidence of lead pellets in any tissues or organs.

Pathologic Changes - Gross and Microscopic Lesions

 

Bird 2

It was observed on gross examination that an intramedullary pin
joined the fractured halves of the right ulna, but not the radius.
Fetid green caseated material surrounded the fracture site. No
additional gross lesions were observed in any other tissues. In-
flammatory cells were seen on microscopic examination to have infil-
trated the submucosal and subepithelial layers of the organs of the
upper digestive tract (esophagus, crop, proventriculus). The esophagus
appeared to be most markedly infiltrated by these cells which were

primarily eosinophils and heterophils.

55

Bird 3

Gross examination of the left radius and ulna revealed midshaft
fractures with pieces of cerclage wire attached to the distal portions
of both bones. A hard yellowish-green mass surround the proximal ends
of the fracture sites. No other gross or microscopic lesions were

seen.

Bird 4

What resembled 2 number 5 lead shot were found on gross inspection.
One pellet was harbored in an area of yellow, flaky fat in the omentum
and the second was lodged in the right tibio-tarsal joint. No gross
tissue proliferation had occurred around the shot in the joint.
Microscopic examination of the tissues revealed no microscopic

abnormality.

Bird 5

The only visible change at necropsy was what appeared to be a
slight thickening of the proventricular mucosa. Microscopic exam-
ination of this area, as well as the other tissues collected, showed
no significant pathologic changes. There was congestion and hemor-
rhageixlthe peribronchiolar areas of the lung which most likely

resulted from death due to asphyxiation.

Bird 6
No significant gross lesions were found in this bird. Microscopic
areas of peribronchiolar hemorrhage, most likely due to death by

asphyxiation, were seen in the lungs.

56

Bird 7

This was a one-legged bird. Gross inspection of the internal
structures showed a whitish area 3 millimeters wide on the ventral
pole of the dorsal lobe of the right kidney. This proved to be an
area of degeneration on histopathologic examination. The submucosa of
the gizzard and especially the lamina propria of the crop had areas
of heterophilic and eosinophilic infiltration when viewed microscop-
ically. Bird 7 also had areas of peribronchiolar hemorrhage in the

lung.

Bird 8
Small red foci less than 1 millimeter in diameter were grossly

visible scattered throughout the pancreas. These spots appeared to be
areas of congestion when viewed microscopically. The crop had marked
eosinophilic infiltration into the lamina propria, noticeable vascular
congestion, and mononuclear infiltration into the muscularis on micro-
scopic examination. Several bi-operculate parasite ova had penetrated
the epithelial layer of the crop. These ova could best be identified

as Capillaria spp. A similar, but milder, cellular response was

 

seen in the submucosa of the gizzard.

Bird 9

A single shot was lodged in the muscle mass of the left thigh
immediately ventral to the femur. No gross tissue response to this
foreign body was observed. Microscopic findings were as follows.
The lungs had areas of vascular congestion and peribronchiolar hem-

orrhage. Sections of esophagus and crop had marked eosinophilic

57

infiltration into the lamina propria with parasitic ova similar to
those seen in bird 8 present at the epithelial surface of the esoph-
agus. Small collections of eosinophils and heterophils were seen in

the lamina propria of the gizzard.

Bird 10

No gross lesions were observed in this bird. The lung was the
only tissue with pronounced histopathologic changes, which included
marked areas of peribronchiolar hemorrhage, vascular congestion, and
edema. A granuloma with some accompanying heterophils was observed
in this tissue. Acid-fast stains revealed no causative agent in or

near the granuloma.

DISCUSSION

Ingested lead shot has been a mortality factor in upland game
birds (Campbell, 1950; Westemeier, 1965), waterfowl (Phillips et al.,
1930; Grinnell, 1901), and raptors (Locke et a1., 1969; Jacobson et
al., 1977; Redig, 1979).

Clinical signs and gross and microscopic pathology have been
described by numerous investigators (Cook et al., 1966; Coburn et al.,
1951; Bagley et al., 1967; Bellrose, 1964; Grinnell, 1901).

Acid—fast inclusion bodies in the epithelial cells of the proximal
convoluted tubules have been reported in kidney sections from a vari-
ety of birds exposed to lead, including mourning doves (Locke et al.,
1967), Canada geese (Bagley et al., 1967), ducks (Locke et al., 1966),
and raptors (Locke et a1., 1969). These inclusions have not been
found consistently in every bird having lead poisoning.

The literature describes most birds dying from lead poisoning as
having characteristic clinical signs and histopathologic lesions.

None of the birds used in this study displayed either the gross signs
or microscopic lesions associated with plumbism or died as a result
of lead poisoning. Length of exposure to lead and opportunity for
its prolonged absorption from the gastrointestinal tract apparently
are important factors in fatal poisoning.

Tissue lead levels indicative of toxicosis have been established
by previous investigators (Adler, 1974; Buck et al., 1973; Coburn et

al., 1951; Longcore et al., 1974) and were markedly higher than those

58

50

for 4 days.

Bird 3

Bird 3 had cerclage wires visible at 2 sites of a nonunion frac-
ture of the left humerus on the preliminary radiograph. The bird was
fed 30 number 5 lead shot in a mouse meal on day 1. Several shot were
passed on day 2, and by day 3 all the pellets had been either egested
or defecated by the bird. Radiographs on day 4 showed no retained
lead shot, and the bird was redosed with an additional 30 pellets on
that same day. He egested a cast containing 28 pellets on day 5 and
passed an additional pellet on each of days 6 and 7. Radiographs on
day 7 showed no pellets in the gastrointestinal tract, and the bird
was redosed that same day. A cast containing 23 pellets was found on
the cage floor on day 8. The remaining 7 lead shot were recovered
from the cage floor on day 10, at which time the bird was radio-
graphically free from lead shot. Bird 3 retained 7 lead shot for 3

days and l pellet for 3 days.

Bird 4

Bird 4 was the control bird for this group and received a mouse
diet without lead shot. This bird was free from any previously
ingested pellets, but had radiographic evidence of metallic foreign
bodies in 5 locations. What appeared to be lead pellets were found in
the muscle masses adjacent to both left and right tibias. Another
pellet was superimposed on the pelvis. Two additional pellets were

located near the left radius. The left proximal ulna had been

51

fractured as an apparent consequence of being shot. These pellets

never changed position at any time in the sequence of radiographs.

The second group of experimental birds included birds 5, 6, and 7.
Bird 5

Bird 5 was the control individual in this group. Radiographs
taken on days 1 and 4 showed no evidence of either ingested lead shot

or pellets embedded in bone or soft tissue.

Bird 6

The initial radiograph of bird 6 showed no lead pellets in any
location. This bird received 10 number 5 lead shot in a mouse meal on
day l. A single pellet was found in the cage on day 2, and 9 more
lead shot were recovered from a cast egested on day 3. The bird was
radiographically free from lead on day 4, and on that day was again
fed 10 lead shot. Single shot were passed by the bird on days 5 and
6 with the 8 remaining pellets found in mutes and casts littering the
cage floor on day 7. Radiographs on day 7 confirmed the abscence of
retained lead shot. A 10 pellet dosing was repeated at that time.
A cast egested on day 8 contained 3 of those pellets. One pellet was
found on the cage bottom on day 9, and the remaining pellets were
retrieved from a cast and mutes examined on day 10. A subsequent
radiograph demonstrated the absence of lead in this bird on the same
day just prior to redosing with 10 lead shot in a mouse meal. Three
shot were found in the bird's water dish, 3 in a cast, and the
remaining shot on the cage floor on day 11. The bird was radiograph-

ically free from lead pellets_on that day and received no additional

52

shot. Bird 6 retained 9 lead shot, 8 shot, and 6 shot each for

periods as long as 3 days.

The preliminary radiographs taken of bird 7 showed no lead pellets
located either in the gastrointestinal tract or soft tissues. The
bird was dosed on day 1 with 30 number 5 lead shot and cast 28 of
these pellets on day 2. No pellets were egested on day 3. A single
pellet was found in the cage on day 4,2nu1radiographs taken on that
same day showed a single shot retained in the gizzard. The bird ate
another 30 pellets on day 4. Combined casts from days 6 and 7 con-
tained all 31 pellets. Radiographs on day 7 demonstrated no lead shot
in the bird. Thirty additional pellets were fed to the bird on day 7,
and he egested 28 of these pellets on day 8. Two pellets were found
on the cage floor on day 10,2nuiradiographs taken on that day revealed
no retained lead shot. The last dose of lead shot was administered at
this time. Bird 7 cast 21 of these pellets on day 11, egested 2 lead
shot on day 12, and egested the remaining 7 pellets on day 13. Bird 7
retained a single lead pellet for 6 days, 2 pellets for 3 days and

7 pellets for 3 days.

Birds 8, 9 and 10 completed the last replicate.

Bird 8 had no shot in any tissue prior to the start of this exper-
iment as determined by radiographic technique. This bird ate 10
number 5 lead shot offered in a mouse meal on day l. Undigested

material cast late on day 2 and early on day 3 contained all 10 shot.

53

The bird was fed another 10 lead pellets on day 4, and 3 of these
pellets were passed on day 5. A cast egested on day 6 contained 7
additional lead pellets. A radiograph taken on day 7 demonstrated no
remaining lead shot,znulan additional 10 pellets were consumed by the
bird on that day. One lead shot was found in the mutes on day 8, and
9 more shot were cast by the bird on day 10. A radiograph taken on
day 10 demonstrated no lead shot. This bird was dosed for the last
time on day 10. Examination of the cage floor on day 11 yielded 9 of
these 10 pellets. The last lead pellet was found in a cast on day 13
at which time a final radiograph showed the bird to be free from all
fed lead shot. This bird retained 9 lead pellets for 3 days, and 1

lead pellet for 3 days.

Bird 9

An initial radiograph of bird 9 showed a metallic foreign object
in the soft tissue immediately ventral to the left femur. The object
appeared to be a lead pellet. This bird was fed 30 number 5 lead shot
in a mouse meal on day l. Twenty-six shot were recovered from mater-
ial on the cage floor on day 2, 1 shot on day 3, and 3 shot on day 4.
A radiograph taken on that day showed only the lead shot in the left
thigh area. An additional 30 pellets were fed on day 4. One lead
shot was recovered on day 5. A cast containing 18 pellets was re-
trieved from the cage on day 6 along with an additional 7 pellets re-
covered from the cage floor. A radiograph taken on day 7 showed 4
lead shot remaining in the gizzard. Thirty more pellets were consumed
by bird 9 on day 7. Nine shot were removed from the floor of the cage

on the following day,zu5we11 as a cast containing 22 more shot. A

54

single lead pellet was found on the cage floor on day 9. Radiographs
on day 10 showed 2 lead shot remaining in the gizzard. The bird was
dosed with lead shot for the last time on day 10. There were 4 pellets
in the mutes and 28 in a cast from the bird on day 12. Radiographic
examination of the bird at this time demonstrated only the initial

lead pellet seen in the femoral area. This bird retained 3 lead shot
in the gizzard for 4 days, 4 shot for 3 days, and 2 shot for 8 days.

No other bird in the study retained lead shot for as long, however, as
individual lead shot were not identified it was impossible to deter—

mine if the same shot were retained throughout the entire period.
Bird 10
Bird 10 was an undosed control bird. Radiographic examination

showed no evidence of lead pellets in any tissues or organs.

Pathologic Changes - Gross and Microscopic Lesions

 

Bird 2

It was observed on gross examination that an intramedullary pin
joined the fractured halves of the right ulna, but not the radius.
Fetid green caseated material surrounded the fracture site. No
additional gross lesions were observed in any other tissues. In-
flammatory cells were seen on microscopic examination to have infil-
trated the submucosal and subepithelial layers of the organs of the
upper digestive tract (esophagus, crop, proventriculus). The esophagus
appeared to be most markedly infiltrated by these cells which were

primarily eosinophils and heterophils.

55

Bird 3

Gross examination of the left radius and ulna revealed midshaft
fractures with pieces of cerclage wire attached to the distal portions
of both bones. A hard yellowish-green mass surround the proximal ends
of the fracture sites. No other gross or microscopic lesions were

seen.

Bird 4

What resembled 2 number 5 lead shot were found on gross inspection.
One pellet was harbored in an area of yellow, flaky fat in the omentum
and the second was lodged in the right tibio-tarsal joint. No gross
tissue proliferation had occurred around the shot in the joint.
Microscopic examination of the tissues revealed no microscopic

abnormality.

Bird 5

The only visible change at necropsy was what appeared to be a
slight thickening of the proventricular mucosa. Microscopic exam-
ination of this area, as well as the other tissues collected, showed
no significant pathologic changes. There was congestion and hemor-
rhage:hithe peribronchiolar areas of the lung which most likely

resulted from death due to asphyxiation.

Bird 6
No significant gross lesions were found in this bird. Microscopic
areas of peribronchiolar hemorrhage, most likely due to death by

asphyxiation, were seen in the lungs.

56

Bird 7

This was a one-legged bird. Gross inspection of the internal
structures showed a whitish area 3 millimeters wide on the ventral
pole of the dorsal lobe of the right kidney. This proved to be an
area of degeneration on histopathologic examination. The submucosa of
the gizzard and especially the lamina propria of the crop had areas
of heterophilic and eosinophilic infiltration when viewed microscop-
ically. Bird 7 also had areas of peribronchiolar hemorrhage in the

lung.

Bird 8
Small red foci less than 1 millimeter in diameter were grossly

visible scattered throughout the pancreas. These spots appeared to be
areas of congestion when viewed microscopically. The crop had marked
eosinophilic infiltration into the lamina propria, noticeable vascular
congestion, and mononuclear infiltration into the muscularis on micro-
scopic examination. Several bi-operculate parasite ova had penetrated
the epithelial layer of the crop. These ova could best be identified

as Capillaria spp. A similar, but milder, cellular response was

 

seen in the submucosa of the gizzard.

Bird 9

A single shot was lodged in the muscle mass of the left thigh
immediately ventral to the femur. No gross tissue response to this
foreign body was observed. Microscopic findings were as follows.
The lungs had areas of vascular congestion and peribronchiolar hem—

orrhage. Sections of esophagus and crop had marked eosinophilic

57

infiltration into the lamina propria with parasitic ova similar to
those seen in bird 8 present at the epithelial surface of the esoph-
agus. Small collections of eosinophils and heterophils were seen in

the lamina propria of the gizzard.

Bird 10

No gross lesions were observed in this bird. The lung was the
only tissue with pronounced histopathologic changes, which included
marked areas of peribronchiolar hemorrhage, vascular congestion, and
edema. A granuloma with some accompanying heterophils was observed
in this tissue. Acid-fast stains revealed no causative agent in or

near the granuloma.

DISCUSSION

Ingested lead shot has been a mortality factor in upland game
birds (Campbell, 1950; Westemeier, 1965), waterfowl (Phillips et al.,
1930; Grinnell, 1901), and raptors (Locke et al., 1969; Jacobson et
al., 1977; Redig, 1979).

Clinical signs and gross and microscopic pathology have been
described by numerous investigators (Cook et al., 1966; Coburn et al.,
1951; Bagley et al., 1967; Bellrose, 1964; Grinnell, 1901).

Acid-fast inclusion bodies in the epithelial cells of the proximal
convoluted tubules have been reported in kidney sections from a vari-
ety of birds exposed to lead, including mourning doves (Locke et al.,
1967), Canada geese (Bagley et al., 1967), ducks (Locke et al., 1966),
and raptors (Locke et al., 1969). These inclusions have not been
found consistently in every bird having lead poisoning.

The literature describes most birds dying from lead poisoning as
having characteristic clinical signs and histopathologic lesions.

None of the birds used in this study displayed either the gross signs
or microscopic lesions associated with plumbism or died as a result
of lead poisoning. Length of exposure to lead and opportunity for
its prolonged absorption from the gastrointestinal tract apparently
are important factors in fatal poisoning.

Tissue lead levels indicative of toxicosis have been established
by previous investigators (Adler, 1974; Buck et a1., 1973; Coburn et

al., 1951; Longcore et al., 1974) and were markedly higher than those

58

59

obtained in this study. There were increases in tissue lead levels
over the control values obtained from the red—tailed hawks, but these
were not indicative of fatal toxicosis.

Published blood lead levels in birds with fatal lead poisoning
range from 2.7 to 20 ppm, with 10 ppm suggestive of acute exposure
(Longcore et al., 1971). This correlates with the blood lead values
of 1.67 to 9.69 ppm generated by this study.

There have been few published reports of hematologic observations
on lead poisoned birds. Inconsistencies in total leukocyte numbers seen
in lead poisoned birds, as previously reported, were also observed in
this study. The only published information on the hematocrit in lead
poisoned raptors described alterations in the hematocrit as being insig—
nificant (Redig, 1979). Changes in hematocrit, hemoglobin values,
and erythrocyte fragility were not useful in monitoring the progress
of lead absorption in the red-tailed hawks. The insignificant changs
in erythrocyte fragility were reflective of the material presented
by Aub et al. (1925) in which species variation existed with regard
to changes in red cell osmotic resistance. The chicken erythrocyte
did not display any characteristic changes in osmotic resistance in
Aub‘s work.

Spontaneous erythrocyte fluorescence was observed only in those
birds receiving lead shot; however, not all birds receiving lead shot
developed fluorescing erythrocytes. The number and intensity of the
fluorescing erythrocytes did not directly correlate with either the
quantity of lead ingested nor the retention time of the pellets in
the birds. This fluorescent phenomenon may assist in the recognition
of lead poisoned birds, although other diagnostic tests might provide

more information.

60

It may be concluded from this research that repeated ingestion
of lead shot by red-tailed hawks and other raptors may not produce
clinical signs or result in fatal lead poisoning. The raptor's
ability to egest lead pellets significantly decreases shot retention
time in the bird, and apparently minimizes the opportunity for
absorption of the toxic material from the gastrointestinal tract.
Interference with this casting mechanism may be of primary importance
in pellet retention, lead absorption, and fatal poisoning in birds

of prey.

SUMMARY

Ten red-tailed hawks were used in this investigation. Research
was conducted to duplicate lead poisoning as seen in free living birds
of prey. The first bird was used for a feasibility study. The re-
maining 9 birds were divided into the following groups of 3
birds each:

(1) control, un-dosed birds, (2) birds receiving 4 sequential

doses of 10 number 5 lead shot, and (3) birds receiving 4

sequential doses of 30 number 5 lead shot.

Birds were evaluated for clinical evidence of lead poisoning,
blood and tissue lead levels, changes in total leukocyte numbers,
hemoglobin, hematocrit, and osmotic erythrocyte fragility, and gross
and microscopic lesions.

Clinical signs were not observed in any of the birds during the
course of experimentation. Analyses of repeated blood samples from
all dosed birds indicated increases in blood lead levels consistent
with acute exposure to lead. Tissue lead levels, although elevated,
were not indicative of toxicosis. Blood values monitored in this
study were found not to be predictable for lead intoxication. Spon-
taneous erythrocyte fluorescence occurred on a nonuniform basis in
lead dosed birds from groups (2) and (3). Gross observation and
microscopic examination of selected tissues revealed no pathologic
changes associated with plumbism.

Birds of prey which were able to egest lead shot showed neither
gross nor microscopic evidence of lead poisoning, but did absorb lead
during instances of acute exposure based on the findings under this

protocol.

61

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