Quantitative determinations of normal horse, cat, and dog haptoglobins

QU~TITATI~ NORMAL HORSE,

~ETE~INATIONS OF CAT, AND DOG HAFTOGLOBINS

John W. Harvey College of Veterinary Medicine University of Florida Gainesville, Florida 32610

ABSTRACT A haptogZobinassay method, based on the principle that cyanmethemoglobin is protected from acid de~t~t~on when bowed to haptogtobin, was euakzted and found to be weZI suited for haptogkbin deterWide haptoglobin ranges tJere minations in horses, cats and okgs. differences were observed in each species. Inasmuch as no significant observed between species, a normal range of 20 to 190 mg cyanmethemoglobin bandit capacity/d2 serum or plasma is reco~s~ed for use in atI 3 species.

INTRODUCTION Haptoglobin is an "2 plasma glycoprotein that forms a stable complex with hemoglobin. The resultant complex is rapidly remaved from the circulation by the reticuloendothelial system (1,2). Consequently, low plasma or serum haptoglobin is usually indicative of recent intravascular hemolysis and haptoglobin determinations have provided useful information in the assessment of hemolytic diseases in man (2). The purpose of the present study was twofold. First, a new quantitative haptoglobin assay method (3), based on the principle that cyanmet; hemoglobin is protected from denaturation in acidic medium (pH 3.7) when complexed with haptoglobin, was evaluated for its applicability in animals. Second, haptoglobin was assayed from normal horses, cats and dogs to see if species differences in haptoglobin content occur and to report normal values for these species.

MATERIALS AND METHODS Blood samples were obtained from normal dogs and cats of mixed breed ing, from thoroughbred horses, quarter horses, and ponies, and from healthy laboratory personnel. Physical examinations and CBC's were used to gauge clinical normalcy.

The author thanks Sharon A. Zahner for technical assistance and Drs. Mark W. Coleman and Richard L. Asquith for normal blood samples. Florida Agricultural Experiment Station Journal Series No. 6121.

AUGUST-SEPTEMBER

1976

VOL. 6 NO. 2-3

133

THERIOGENOLOGY

Stock cyanmethemoglobin solutions were prepared from erythrocytes washed three times with isotonic saline solution. Packed erythrocytes were lysed by dilution (approximately 1 to 250) in Drabkin's solution (4), and stroma were removed by centrifugation at 25,000 x g for 15 minutes. Resulting cyanmethemoglobin solutions were diluted to 60 mg/dl with additional Drabkin's solution, using a 4 mM extinction coefficient (540 nm) for cyanmethemoglobin of 11.0 and a value of 64,458 for the molecular weight of the hemoglobin tetramer (5). Absorbance measurements and absorption spectral scans were performed using a double beam spectrophotometer (a) with l-cm light path and 0.2 nm resolution. Wavelength calibration was checked by using a holmium oxide filter as a wavelength standard. Test and blank incubations were conducted and haptoglobin values were calculated as described by Elson (3), except that an absorbance measurement was made at.405 nm rather than 407 nm. Cyanmethemoglobin solutions were stored at 5'C and discarded after two weeks. Aliquots of normal human serum (stored at -2O'C) were used as quality controls.

RESULTS The serum haptoglobin assay (3) was evaluated first using serum fron normal people. The Soret peak of the cyanmethemoglobin-haptoglobin complex (pH 3.7) in man (and subsequently in animals) ranged from 405 nm to 406 nm. In the majority of cases the peak was at 405 nm, compared to 407 nm reported previously (3). The differential extinction coefficient was determined by varying the concentration of cyanmethemoglobin in the presence of excess haptoglobin and found to be identical to that previously reported (3). Haptoglobin values determined using plasma (EDTA or heparin) were the same as those obtained with serum. Duplicate values rarely varied by more than 10%. Serum haptoglobin values in 11 normal people (Table I) ranged from 30 to 161 mg cyanmethemoglobin binding capacityldl. This compares favorably with a normal range of 23 to 141 mg/dl proposed by Elson (3). Cyanmethemoglobin solutions were also prepared from washed horse, cat, and dog erythrocytes as described in the methods section. Human serum haptoglobin was measured using cyanmethemoglobin solutions from each of the animals studied and values were in close agreement with those determined when human haptoglobin was assayed using human cyanmethemoglobin. In addition, no differences were observed when serum haptoglobins from each animal species were measured using human cyanmethemoglobin compared to those measured using cyanmethemoglobin prepared from each respective species. As a result of these findings, human cyanmethemoglobin solution was used to measure serum or plasma haptoglobin in all species. The haptoglobin values determined in normal horses, cats and dogs are given for comparison to man (Table I). There were no significant differences (P>0.05) between species or between thoroughbreds, quarter horses, and ponies. Of 83 individuals studied, only 3 (1 dog and 2 cats) had values above 190 mg/dl, and only two (1 dog and 1 horse) had values below 20 mg/dl.

al3eckmanModel 25, Beckman Instruments, Inc., Fullerton, CA.

134

AUGUST-SEPTEMBER

1976

VOL. 6 NO. 2.3

THERIOGENOLOGY

Serum or plasma samples obtained for haptoglobin assays may at times contain hemoglobin. Clinically, it is important to know whether its presence is an artifact (mechanical hemolysis of erythrocytes during sample collection and/or handling) or is indicative of intravascular hemolysis. The method presented herein measures only unbound (free) haptoglobin. If a reasonable amount of free haptoglobin is still present in a sample containing hemoglobin, intravascular hemolysis is unlikely, since the total haptoglobin binding capacity of plasma is small compared to the total amount of hemoglobin within circulating erythrocytes. Although test and blank absorption spectra are essentially superimposed when no free haptoglobin is present in plasma or serum samples, qualitative information, with regard to the presence or absence of bound haptoglobin, can still be obtained from the shape of the curves. When bound haptoglobin is present, distinct absorbance peaks are observed at 405 nm (Fig. 1A). In the absence of bound haptoglobin, the curves tend to be flat in the Soret band region (Fig. 1B). Wavelength scans from an assay of normal serum containing free haptoglobin is shown for comparison (Fig. 1C).

DISCUSSION Haptoglobin binds so tightly to hemoglobin that some have termed the reaction irreversible. The reaction of human haptoglobin with hemoglobin has a very broad species specificity. Horse, rabbit, bovine and chicken hemoglobins bind human haptoglobin with the same rate as does human hemoglobin (1). Sheep and goat hemoglobins react even faster than human hemoglobin and no dissociation of the hemoglobin-haptoglobin complexes has been detected using sensitive displacement assays (1). It is, therefore, not surprising that no differences were observed in the present study when haptoglobin from one species was assayed with cyanmethemoglobins prepared from other mammalian species. In plasma and serum samples that are visibly free of hemolysis, absorbance scanning is unnecessary. Absorbance measurements of test and blank solutions are made at only two wavelengths (405 nm and 380 nm). Two wavelength determinations are essential since Elson (3) observed that test and blank absorbance spectra, from samples containing no demonstrable haptoglobin, were at times identical in shape but displaced variably from one another, sometimes as much as 0.060 absorbance units. This appeared to be due to a difference in turbidity from test to blank and the AA at 380 nm adjustment was utilized to compensate for the nonspecific absorbance. This same phenomena of test curve versus blank curve displacement was also observed in the present study, in samples when all of the haptoglobin was bound by hemoglobin present in the sample. The AA at 380 nm correction may be either positive or negative. When hemoglobin is present in serum or plasma samples, test and blank solutions should be scanned (420 nm to 380 nm) against distilled water, so that absorption spectra of the test and blank can be visualized. A distinct absorbance peak at 405 nm in the blank, as well as test solution, is suggestive of the presence of bound haptoglobin. If no absorbance peak is present in the Soret band region of either test or blank scans, then all of the cyanmethemoglobin has been denatured and no haptoglobin (bound or unbound) is present.

AUGUST-SEPTEMBER

1976 VOL. 6 NO. 2-3

THERIOGENOLOGY

The quantitative haptoglobin method evaluated in the present study is simple, rapid (compared to other methods), reproducible, and well suited for use in animals. Reagents are inexpensive and easily obtained. Only one cyanmethemoglobin solution is needed to measure horse, cat, dog and human haptoglobins. A narrow bandwidth spectrophotometer is, however, required, and wavelength scanning capabilities are desirable. Wide ranges in haptoglobin concentrations were observed in all species. Inasmuch as no significant differences were observed between species, a normal range of 20 to 190 mg/dl is suggested for the three animal species reported herein. Although the measurement of serum or plasma haptoglobin has not been used as a clinical tool to any extent in veterinary medicine, this assay is potentially useful and should be considered when various infectious, autoimmune and toxic anemias, etc., are suspected (2,6,7). Zero or low haptoglobin content is usually indicative of recent intravascular hemolysis, although anhaptoglobinemia may be associated with severe liver injury (2). Haptoglobin and fibrinogen are synthesized in the liver (8). Like fibrinogen, haptoglobin synthesis and plasma concentration increases markedly in the presence of inflammation or tissue necrosis (2,8,9). Increases in plasma haptoglobin also occur occasionally in human cancer patients (2). Although these increases are nonspecific, they can provide information comparable to that obtained by measuring fibrinogen or the erythrocyte sedimentation rate. The erythrocyte sedimentation rate is clinically useful only in the dog (10). Haptoglobin measurement might be particularly useful in some species (e.g. cat), where the fibrinogen content has not always been a reliable indicator of inflammation (10). Ongoing therapeutic regimens (particularly with regard to steroid therapy) must be considered when evaluating haptoglobin in clinical cases, since corticosteroid and ACTH administration have resulted in increased haptoglobin values in man and animals (2,ll).

REFERENCES 1.

Cohen-Dix, P., Nobel, R.W., and Reichlin, M. Comparative Binding Studies of the Hemoglobin-Haptoglobin and the Hemoglobin-Anithemoglobin Reactions. Biochemistry -12:3744-3751 (1973).

2.

Laurell, C.-B., Griinvall,C. 135-173 (1962).

3.

Elson, E.C. Quantitative Determination of Serum Haptoglobin. A Simple and Rapid Method. Am. J. Clin. Path. -62:655-663 (1974). Drabkin, D.L., Austin, J.H. Spectrophotometric Studies. II. Prepa rations from Washed Blood Cells; Nitric Oxide and Sulfhemoglobin. J. Biol. Chem. -112:51-65 (1935).

4.

Haptoglobins.

Adv. Clin. Chem. -5:

5.

Van Assendelft, O.W. Spectrophotometry of Haemoglobin Derivatives. Charles C. Thomas, 1970 p 100.

6.

McGuire, T.C., Henson, J.B., and Quist, S.E. Viral-Induced Hemolysis in Equine Infectious Anemia. Am. J. Vet. Res. -30:2091-2097 (1969).

136

AIJGUST-SEPTEMBER

1976

VOL. 6 NO. 2-3

THERIOGENOLOGY

7.

Lumsden, J.H., Valli, V.E., McSherry, B.J., Robinson, G.A., and Claxton, M.J. The Kinetics of Hematopoesis in the Light Horse III. The Hematologic Response to Hemolytic Anemia. Can. J. Comp. Med. -39:332-339 (1975).

8.

Alper, C.A., Peters, J.H., Birtch, A.G., and Gardner, F.H. Haptoglobin Synthesis. I. -In Vitro Studies of the Production of Haptoglobin, Fibrinogen, and y-Globulin by the Canine Liver. 3. Clin. Invest. -44:574-581 (1965).

9.

Ganrot, K. Plasma Protein Response in Experimental Inflammation in the Dog. Res. Exp. Med. -161:251-261 (1973).

10. Schalm, O.W., Jain, N.C., and Carroll, E.J. Veterinary Hematology. Lea & Febiger, 3rd edition 1975 pp 40-41, 120. 11. Jarret, I.G. A Polymeric Form of Haemoglobin-Binding Protein in Sheep Following Metabolic and Hormonal Disturbance. Aust. J. Biol. Sci. -25:941-948 (1972).

Table 1.

Haptoglobin Determinations in Animals and Man*

Species

Mean

Horse (30)

Range

88

19 - 177

Cat (23)

111

31 - 216

Dog (19)

104

14 - 2521

Man (11)

89

*

30 - 161

mg cyanmethemoglobin binding capacity/d1 serum or plasma. Number of individuals given in parentheses.

AUGUST-SEPTEMBER

1976

VOL.

6 NO. 2-3

137

THERIOGENOLOGY

0.8

i

0.8

f m 0" t a

0.4

A

38,

400

C

e

420

38'

400

420

WAVELENGTH

380

400

420

Cnml

Fig. I.. Absorption spectra of haptoglobin test (solid lines) and blank (dotted lines) solutions. Test and blank absorption spectra from two cat serum samples containing hemoglobin and no unbound (free) haptoglobin (A and B) are shown in comparison to those from a normal cat serum (C). One serum sample (A) contained bound haptoglobin (hemoglobin-haptoglobin complex) and the other (8) contained neither bound nor free haptoglobin.

138

AUGUST-SEPTEMBER

1974

VOL. 6 NO. 2-3

THERIOGENOLOGY

HERPESVIRUS SAIMIRI LYMPHOMA IN OWL MONKEYS (AOTUS TRIVIRGATUS): SUSCEPTIBILITY, LATENT PERIOD, HEMATOLOGIC PICTURE AND COURSE R.D. Hunt, B.3. Blake, M.D. Daniel New England Regional Primate Research Center One Pine Hill Drive Southborough, Massachusetts 01772

A critical examination of 100 owl monkeys experimentally infected with Herpesvirus saimiri has demonstrated a 67% incidence of lymphoma based on histopathological examination. Leukemia developed in 36 animals with malignant lymphoma with total peripheral lymphocyte counts of 28,640 to 173,760/mm3. The first signs of leukemia were detected from 30 to 362 days post-inoculation with a mean of 141 days. The duration of leukemia before death ranged from 1 day to 231 days with a mean of 42 days. The interval from inoculation to death of the 67 animals with lymphoma ranged from 13 days to 539 days with a mean of 139 days. Evidence is presented which implicates variability of inocula and variability within the owl monkey to explain the diversity of responses. The susceptibility of owl monkeys to Herpesvirus saimiri induced malignant lymphoma is well established; but it is clear from several reports from this and other laboratories that their susceptibility to this oncogenic virus is variable and that the course of the disease following inoculation is inconstant (l-3). Most reports, however, concern observations on small numbers of animals. To more clearly define this irregularity, we have examined data collected from 100 owl monkeys inoculated with H. saimiri. Certain of these have been the subject of earlier reports but the clinicopathologic features of most have not been reported. Materials and Methods The 100 monkeys are from 15 separate studies in which various inocula were employed (Table I). In 8 studies the inoculum was obtained from owl monkey kidney cell passages of an original Herpesvirus saimiri isolate (Nos. 59, 96, 102, 103, 136, 150, 151, 248). In AE 125 a second inoculum was employed on day 180. In AE 157 the inoculum was whole blood from a leukemic monkey from AE 136 and in AB 199 the inoculum was whole blood from a leukemic monkey from AE 173.

This study was conducted in part under Contract No. 1 CP 33390 within the Virus Cancer Program of the National Cancer Institute, National Institutes of Health, Public Health Service.

AUGUST-SEPTEMBER

1976

VOL.

6 NO. 2-3

139

THERIOGENOLOGY

In AE 173 the monkeys were inoculated with H. saimiri which had been passed 5 times in Dog Fetal Lung (DFL) tissue culture and challenged on day 30 with a parent virus. In AE 254 a twenty-first DFL passed virus was inoculated on day 1 and 30 and the monkeys were challenged on day 45 with a parent virus. In AE 268 a twenty-second DFL passed virus was inoculated on day 1 and 30 and a parent virus on day 45 and day 374. In AE 269 ultraviolet inactivated virus was inoculated on day 1 and 21 and a parent virus challenge on day 45. Susceptibility and Survival Time Of the 100 monkeys, 67 (67%), 41 (71.0%) males and 26 (65%) females, developed malignant lymphoma (Table II). When examined by individual study, the incidence varied from 25% to lOO%, (33.3 t,o 100% males and 0 to 100% females). The sex of the animals appeared to have no significant influence on either the development or course of the disease. Fourteen animals died of other causes between 14 days and 585 days following inoculation. Eight of these died between 14 and 86 days which is well prior to the mean survival time of 139 days for animals with malignant lymphoma. If these animals were excluded, the incidence of lymphoma would be 75%. Eighteen animals were killed in the absence of disease at the termination of various studies between 100 and 720 days past inoculation. Only one of these was killed prior to the mean survival time for animals with lymphoma. The interval from inoculation to death of the 67 animals with lymphoma ranged from 13 days to 593 days (mean 139 days). The distribution of deaths in time, which is illustrated in Text-figure 1, appears to be random. When examined by individual study, the great variation in survival time, as well as susceptibility, did not appear until the fifth experiment (AE 125). In the first 4 studies, comprising 12 animals, the incidence was 100% and all animals died between 13 and 28 days. In all subsequent studies, except AE 248, there was a great range in survival time. In AE 248 the viral inoculum was an isolate obtained from a naturally occurring case of Herpesvirus saimiri lymphoma. This will be discussed further subsequently. It seems apparent, however, that in most studies the survival time is not determined by a property of the inoculum, as this variation is present within individual studies where each animal received the same inoculum. As indicated earlier, multiple injections of virus were employed in 5 studies. It is not possible to determine for most animals in these studies which inoculum induced the disease or whether, in some animals, the development of lymphoma required multiple hits of virus. In one study it was evident that the original inoculum induced lymphoma, as in AE 125, 4 animals died of lymphoma prior to the second injection. Obviously in the other 4 multiple injection studies the survival time would be influenced by which inoculum induced the disease. However, the survival time following the last injection before death in these studies was extremely variable ranging from 36 to 328 days. The studies with DFL passed virus (AE 173, 254, 268) and ultraviolet inactivated virus (AE 269) indicate that the injection(s) of altered virus did not afford protection.

141)

AUGUST-SEPTEMBER

1976

VOL. 6 NO. 2-3

The incidence and survival time in owl monkeys is in dramatic contrast to our experience with cotton-topped marmosets. Of 42 male and 43 female animals divided into 14 studies, 39 (93%) males and 41 (95%) females died of malignant lymphoma within 70 days. Of the 5 negative animals, 3 died of other causes at 10, 15 and 34 days and 2 were killed at 76 days. Occurrence of Leukemia Peripheral blood studies were conducted on 93 animals of which 61 developed malignant lymphoma. Leukemia was detected in 25 (64%) males and 11 (42%) females totalling 36 (or 59%) of the 61 animals. The first signs of leukemia were detected from 30 days to 345 days post injection (Text-figure 2). The duration of leukemia before death ranged from 1 day to 330 days with a mean of 42 days (Text-figure 3). Over 60% of leukemic animals died within 30 days of onset. Only 3 animals were leukemic for over 4 months. There was no correlation between time of onset of leukemia and its duration (Table III). Nor was there any correlation between time of onset or duration with the leukemic peak (Table III). Typical courses for leukemia are depicted in Text-figures 4, 5. The peak total peripheral lymphocyte counts ranged from 28,640 to 173,76O/mm3. Inoculation of Leukemic Blood vs Virus Two groups of animals were inoculated with whole blood obtained from monkeys in another study; AE 157 received 1 ml of blood intravenously from animal No. 398-69 of AE 136; and AS 199 received 2.5 ml of blood intravenously from animal No. 554-70 from AE 173. The total lymphocyte counts of the donor animals on the day of injection were: animal No. 398-69 - 173,76O/mm3 and animal No. 554-70 33,05o/mm3. These studies were conducted to determine if the inoculation of viral genome in peripheral lymphocytes would behave any differently from J& saimiri passed in owl monkey kidney cells with the hope of helping to understand the variability in response of owl monkeys. The incidence, occurrence of leukemia and duration had the same variability as seen with the injection of virions. Interestingly only one of 3 animals developed lymphoma in Study No. 157 which received about twice as many lymphocytes as in Study No. 199. In AE 157 two animals died without lymphoma at 31 and 62 days after injection due to other causes. One animal died of lymphoma at 225 days after a 191 day course of leukemia. In AE 199 both animals died of lymphoma; one at 154 days after a 28 day course of leukemia and one at 186 days after a 1 day course of leukemia.

AUGUST-SEPTEMBER

1976

VOL. 6 NO. 2-3

141

THERIOGENOLOGY

Spontaneous H. saimiri Lymphoma We have previously described 3 examples of spontaneous lymphoma in owl monkeys (4). $. saimiri was recovered by co-cultivation from 2 of these and was not attempted on the third. None of these animals are included in the 100 inoculated monkeys discussed throughout this report. Whole blood from one of these monkeys and virus recovered from this same animal were inoculated into additional owl monkeys (AE 248). The monkey which received whole blood (1.5 ml, intravenously; lymphocyte count of 50,190/mm3) was leukemic for 27 days and died of malignant lymphoma at 38 days. The 4 monkeys injected with virus all developed malignant lymphoma and died between 22 and 34 days post inoculation. Two were leukemic for 1 and 4 days. This is the first and only study since the original 4 described earlier, in which a variable incidence and course was not seen-. This suggests that this particular isolate may be "more oncogenic" than others. We are currently retrieving this viral isolate to examine this hypothesis further. Histopathology As previously described, H. saimiri malignant lymphoma is characterized by diffuse infiltration of most organs and tissues by lymphoblasts and less often lymphocytes or stem cells. The extent of the infiltration varies between animals and within a single animal from tissue to tissue. The severity of the disease could not be correlated with the survival time, however, in general the infiltrate was less extensive in animals who survived less than 40 days, although there were exceptions to this. There were both extensive and minimal lymphomatous infiltrates in animals surviving for a longer period. This suggests that the time between inoculation of virus and transformation of lymphocytes to malignant cells is variable. The only positive correlation was that the longer the duration of leukemia (when present) the more extensive the histopathologic lesions. This is not surprising as it indicates a longer course following transformation. Comment The data summarized in this report define the clinicopathologic response of owl monkeys following the injection of Herpesvirus saimiri. It is important to be cognizant of the variable response to properly interpret any study employing J!. saimiri in this species, especially in studies of prophylaxis and therapy. Of equal, or perhaps greater, importance is the understanding of the biological basis for the variability. This knowledge could have great impact in understanding the natural biology of cancer on many species including human beings. The answer, however, will probably not be easily obtained, no more so than understanding similar variabilities with other infectious diseases. The owl monkey does, however, provide a laboratory model to address this important question with respect to malignant lymphoma.

142

AUGUST-SEPTEMBER

1976

VOL. 6 NO. 2-3

THERIOGENOLOGY

The data suggest two sources of variability: (1) within the owl monkey and (2) properties of the virus. The presence of a variable response within a single study employing a single inoculum clearly indicates that at least, in part, the underlying basis for this variability lies within the owl monkey itself. Pearson et al. (5) reported that owl monkeys who do not develop malignant lymphoma do not develop an antibody response to H. saimiri early antigens, whereas those succumbing to infection do. -This observation may provide a clue for fruitful study. The control of the mechanism(s) leading to the outcome of the host-virus interaction would probably be genetic and the variable response may reflect the fact that there are 7 karyotypes of owl monkeys (6). These are reflected in slight phenotypic variations but to date have not been correlated with differences in their response to infectious agents. Unfortunately, most of the animals discussed in this report were studied prior to the demonstration of multiple genetic types. We are currently specifically addressing this question. In addition to searching for the control of variability in the monkey, the studies reported here also suggest that there may be variability in the oncogenicity of the viral inoculum. As indicated in results, the first 4 studies we conducted gave consistent results. In each of the remaining 11 studies, except one (AE 248), a variable response resulted. In the one study with a uniform response, the viral inoculum was prepared from a recently isolated H. saimiri from a spontaneous lymphoma in an owl monkey. We are currently conducting studies with this isolate to determine if the observation is reproducible.

AtJGUST-SEPTEMBER

1976

VOL. 6 NO. 2-3

143

THERIOGENOLOGY

References

1.

Ablashi, D.V., Loeb, C.R., Bennett, D.G., Lymphocytic Leukemia J. Nat. Cancer Inst.

2.

Cicmanec, J.L., Loeb, W.F., and Valerio, M.G. Lymphoma in Owl Monkeys (Atous trivirgatus) Inoculated with Herpesvirus saimiri: Clinical, Hematologic and Pathologic Findings. J. Med. Primatol. 3: 8-17 (1974).

3.

Melendez, L.V., Hunt, R.D., Daniel, M.D., Blake, B. Joan, and Garcia, F.G. Acute Lymphocytic Leukemia in Owl Monkeys Inoculated with Herpesvirus saimiri. Science -171: 1161-1163 (1971).

4.

Hunt, R.D., Barahona, H-H., King, N.W., Fraser, C.E.O., Garcia, F.G., and Melendez, L.V. Spontaneous Herpesvirus saimiri Lymphomas in Owl Monkeys in Leukemogenesis, Ed. Y. Ito and R.M. Dutcher, Univ. Tokyo Press TokyofKarger, Base1 1975 pp 351-355.

5.

Pearson, G.R., Orr, T., Rabin, H., Cicmanec, J., Ablashi, D., and Armstrong, G. Antibody Patterns to Herpesvirus saimiri-Induced Antigens in Owl Monkeys. J. Nat. Cancer Inst. -51: 1939-1944 (1973).

6.

Ma, N.S.F., Jones, T.C., Miller, A.C., Morgan, L.M., and Adams, E.A. Chromosome Polymorphism and Banding Patterns in Owl Monkeys (Aotus). J. Lab. Anim. Sci., accepted for publication.

144

W.F., Valerio, M.G., Adamson, R.H., Armstrong, and Heine, U. Malignant Lymphoma with Induced in Owl Monkeys by Herpesvirus saimiri. 47: 837-855 (1971). -

AUGUST-SEPTEMBER

1976

VOL. 6 NO. 2-3

b

N

?

of

3

d

5;

2

.

I!P:S 8%

Ill

Iii I+

!-‘*

!B

%

+!

t

8

.

.

R,

Ii

c/J

D -c

0

IO

341-350

241-250 251-260 26l- 270

240

I-

1

s

-

23l-

m

I91 161-190 171-160-200 201-210

-

21 I-220 221-230

ISI -160 161-170

I31 I21 I41 -130 -140 -150

IO1 -I Ill-120

El-90 9 I -100

61 51 --60TO7l60_

31-40 41 - 50

NO.

ANIMALS

(D P .

m

161-190

b

371-360

351-360 361-370

l6l-l70171-160-

Ill-120121-130 131-140 141-150 151-160

91-100 101-l IO r

71-00 El90

61 - 70

41-50 51 -60

21 -ii\ 31-40

0

w

2. NO.

ANIMALS

THERIOGENOLOGY

DAYS

Figure

3.

Days survival 36 ad

146

after detection

of leukemia

in

n-wnkeys.

AUGUST-SEPTEMBER

1976

VOL. 6 NO. 2-3

THERIOGENOLOGY

180160.

W B C

--AE266 -AE266 ----AIL254 ----AEl73

I

An.M.213-73 AnNoZCii-73 AnNo. 92-73 AnNa646-70

140I20-

103/mm3 , o.

24

72

120

168

216

264

312

360

406

456

504

552

600

OPI

____

-_I -.-

AE 125 An. No 639-69 AE 136 396-69 AE246 An.No 646-72 AE266 An.NePlZ-?3

An. No.

I

W B

c 103/lIlN13

! _,‘d

! I i

! !

i i i i

24

72

120

168

216

264

312

360

408

456

504

552

t

600

OPI

Figure4.

AUGUST-SEPTEMBER

CZcxxseofleukeniainbo select&cwlmmkeys illustratizig the diversityof mspnse.

1976

VOL. 6 NO. 2-3

147

THERIOGENOLOGY

TABLE I INOCULAEMPLOYED IN EACH STUDI Experiment

of Animals ---

NO.

Source of Virus

i'ropagated In

N*, of Passages

NO. nf Injections

59

SM

"Ki

96

SM

OXR

5

1

102

SM

OMX

5

1

OMK

3

OM with lymphoma

103

SM

125

2

OMX

2

OM with lymphoma

OMK

2

136

OM with lymphoma

OMX

2

:50

OM with lymphoma

OMK

3

SM

OMX

2

SM

OMK

3

SM

OMK

4

SM

OMK

3

SM

WI.

5

SM

om

5

6

151

Other

1

1

157 173

199

Leukemic OM blood

248

OMK

2

SM

DFL

21

SM

OMK

SPI

DFL

SM

om

5

2

SM

OMK-UV

5

2

OM with lymphoma

254

17

26R

12

269

9

5'

SM

SM =

OM OMX DFL w

148

= = = =

5 22

Squirrelmonkey Owl monkey Owl monkey kidney Dog fetal lung ultravioletinactivated

AUGUST-SEPTEMBER

1976

VOL. 6 NO. 2.3

Animals

91-285

6-330 32-55

96-263 102-132

2 2

2

2 4

2 4 7 2 1

9 6 6

3

7

2

4

8

6

3

157

173

199

248

254

268

269

58

39-373 102-593

7-38 52-345

4

5

7

7

1

2

151

40

1

1

4

10

150

41

26

1

25

2

2

5

i

6

7

1

1

I

11

1

30

126-185

40-263

34

221

87-187

54

l-4

l-28

6-93

191

69

7-28

72

8-109

2

3

1 69-189

2

1 4

2

136

2

3

31-34

154-186

49-356

225

290

24-221

126

43-251

17

lb-19

1

1

9

1

1

lb-28

13-18

125

0

Survival p.i. (Range)

103

0

Days

1

0

Duration of Leukemia (Days)

1

4

(Days)

of Leukemia

onset

102

2

of Animals With Leukemia Female Male No.

4

TOTAL

-.

Of

Malignant Lymphoma Male Female

NO. With

SAIMIRI

2

2

No. of Animals Mall? FelMle

"IT" HERPESVIRUS

96

59

Experiment No.

INOCULATED

II

BY STUDY OF OWL MONKEYS

TABLE

SUMHARY OF FINDINGS

103 263 257 250 40 82

537-70 548-70 554-70 556-70 626-70 707-70

173

M M M M F M

34

93-69

157 52 93 6 21 12 22

191

277 157 144 356 257 250 40 95

63.72 40.93 48.03 35.42 28.64 35.38 60.07

87 117 187 118

124

72 195 201 238

43.67

69

221

266-69 M

151

73.0 51.9 27.41 28.81

24 7 28 22

87 117 187 118

M M M M

78-69 17-70 20-70 21-70

150

87.54 80.65 35.02 153.50

Peak Lymphocyte Count 103/nml3 DPI

173.76

8 31 27 109

Duration Leukemia (Days)

72

54

398-69 M

69 167 189 139

136

M M F F

DPI Onset Leukemia

621-69 622-69 638-69 639-69

Animal No.

125

Study No.

DURATION OF LEUKEMIA AND LEUKEMIC PEAK

COMPARISON OF THE TIME OF ONSET OF LEUKEMIA,

TABLE III

578-72 M 582-72 M M F F F M M M

201-72 67-73 71-73 72-73 73-73 75-73 78-73

248

254

200-73 F 203-73 F

269

DPI = Days post inoculation

201-73 M 210-73 F 212-73 M 213-73 F

268

88-73 F 92-73 M

538-70 M 694-70 M

Animal No.

199

Study No.

Table III continued

132 102

96 110 263 152

52 147 92 78 175 53 53 345 206

30 30

126 185

DPI Onset Leukemia

147 116

131.60 164.67 54.80 33.73

55 32

96

79.35 97.92

152 570 361

52 147 120 78 191 53 78 345 232

30 30

140 185

25.19 26.28 28.78 59.18 28.81 69.43 66.13 34.40 154.84

87.85 37.95

57.85 38.92

Peak Lymphocyte count 103/mn3 DPI

6 49 330 209

25 38 31 14 28 7 28 28 27

4 1

28 1

Duration Leukemia (Days)