Attempts to transmit bovine anaplasmosis to small laboratory animals

EXPERIMENTAL

PARASITOLOGY 16, 57-63 (1965)

Attempts

William

to Transmit Bovine Anaplasmosis to Small Laboratory Animals A.

Summers

and

Lilliam

L. Gonzalez

Department of Microbiology, School of Medicine, Indiana University, Indianapolis, Indiana (Submitted

for

publication,

16 September

1963)

SUMMERS, W. A., AND GONZALEZ, L. L. 1965. Attempts to transmit bovine anaplasmosis to small laboratory animals. Experimental Parasitology 16, 57-63. The susceptibility to infection with Anaplasma marginale was studied in several species of normal and experimentally altered nonruminant mammals. The methods used to alter the susceptibility of experimental animals included: (1) splenectomy, (2) adrenalectomy, (3) X-irradiation, administration of (4) mucin, (5) zymosan, (6) cortisone, and (7) bovine serum, and the production of (8) low blood glucose and (9) riboflavin deficiency. No microscope evidence was obtained to indicate that this organism grew in such animals, but young mice and splenectomized guinea pigs died following challenge under conditions which suggested an infectious process. Further investigation is being made to trace the fate of A. marginale in the tissues of these species. The toxicity of heavily infected bovine anaplasma blood was noted in young rats and gerbils.

Among the many unsolved problems relating to the biology of Anaplasma marginale Theiler 1910 is that of its apparent inability to infect nonruminant mammals (Smith and Kilborne, 1893; Stiles, 1929; Dikmans, 1933a; Dykstra et al., 1938, 1948). Of equal interest and importance is the fact that all reported attempts to propagate A. marginale in artificial medium have proved unsuccessful in the final analysis (Veglia, 1915 ; de Faria, 1928; Dikmans, 1933b; Rossi and Triozon, 19.53; Dimopoullos, 1959 as reported by Ristic, 1960). Were either or both methods of propagation possible, investigators working in the field of bovine anaplasmosis would gain several important advantages: (a) lessened cost and maintenance of experimental animals inasmuch as cattle and deer are presently being widely used; (b) better opportunity for the study of life cycle, metabolism, morphology, and immunology; and (c) greater accuracy in the study of potential chemotherapeutic agents and methods of immunization.

Investigations into the propagation of A. in small animals and artificial culture were begun here in 1959. The present report deals with a portion of the results having to do (a) with susceptibility of normal animals and (b) the susceptibility of animals whose natural defense mechanisms we attempted to alter or partially eliminate. The empirical nature of these methods is recognized. marginale

MATERIALS

AND

METHODS

was used One strain of A. marginale throughout this investigation. Initially, large quantities of heavily infected bovine whole blood were sent to this laboratory by air.l Later the same strain of organism was passaged in calves and was maintained thereafter in our laboratory. 1 Supplied by Dr. T. 0. Roby, Beltsville Parasitological Laboratory, Animal Disease and Parasite Research Division, U. S. Department of Agriculture, Beltsville, Maryland. 57

58

SUMMERS

AND

When a supply of heavily infected bovine erythrocytes was required, the organism was transmitted to a splenectomized calf by the subcutaneous inoculation of 5.0 ml of carrier blood. When the parasitemia in the splenectomized calf reached 70-8Or/o, blood was drawn and prepared for use in the experiments. Portions of the blood were frozen at -70°C. Anaplasma marginale retains its infectivity for cattle for 56 days when stored at -66” (Bedell and Dimopoullos, 1962). Anaplasma organisms were prepared for inoculation into experimental animals by three

Results of Injection

rlnimal Mice Mice Mice Mice Mice Mice Mice

of Anaplasma

Age or weight Newborn 1Ogm 18-20 gm 18-20 gm 1X-20 gm 18-20 gm 18-20 gm

GONZALEZ

methods: (a) Freshly drawn infected blood was defibrinated by shaking with glass beads. Blood cells prepared in this manner were always used within 24 hours. (b) Infected erythrocyte stromata which had been obtained either by dehemoglobinization in distilled water or by freezing were suspended in phosphate buffered saline after removal of free hemoglobin by centrifugation (il. mayginale is retained within the stromata). (c) A mixture of free anaplasma organisms and disrupted stromata was obtained by sonication of infected, dehemoglobinized erythrocytes. Son-

TABLE I marginale into Normal Mice, Rats, Guinea Pigs, Cats, Rabbits, Gerbils. and Hamsters

NO.

utilized 18

52 100 50 50

50 25 12 12

Amount (ml), route of administration, and method of preparation of inoculum 0.05 i.p.a 0.05 i.p.

0.2 i.p. 0.1 icut 0.1 i.v. 0.03 i.cer 0.03 im. 1.0 i.p. 0.2 i.cer 0.05 i.cer 0.1 icer 0.5 i.p. 0.1 i.p. 0.5 i.p.

Rats Rats Rats Rats Rats Rats Rats

7 days 7 days 10 days 10 days 2 1 days 35 days 120 days

Cats Cats Cats

3 days 28 days 3-4 kg

12

0.5 i.p. 10.0 i.p. 5.0 i.p.

350 gm

20

0.2 i.p.

5

0.1 iv.

Guinea pigs Rabbits

3 kg

15 15

24 24 24 5

Gerbils Gerbils Gerbils

40-50 gm 40-50 gm 40-50 gm

12

4 4

1.0 i.p. 0.1 S.C. 1.0 i.p.

Hamsters

40-50 gm

20

1.0 i.p.

Results (dead inoculated) Exptl.

(cl (b) (b) (a) (cl Cc) (cl (a) (a) (b) (cl (b) (a) Cc) (b) (4 (a) (a) (b) (4 (a) (a) (a)

Control

8/8

O/IO

27/27 2/50 O/25 2125 6/25 o/12

o/25 o/50 O/25 O/25 IO/25 o/13

6/6 O/6 O/8 l/8

O/6

8/12 o/12 o/12

o/12 o/12 o/12

j/jb O/3

2/2 o/2

O/8 o/10

O/4

O/6 O/7

2/7

o/10

O/3

o/2

8/g o/2 2/2

O/4

o/15

o/2 o/2

O/5 a i.p., intraperitoneal injection; i.cut, intracutaneous injection; iv., intravenous injection; icer, intracerebra1 injection; i.n., nasal instillation; s.c., subcutaneous injection. b Clinical signs and symptoms and autopsy results indicated the cause of death to be feline distemper in both experimental and control animals.

TRANSMISSION

Eject

of Splenectomy, Adrenalectomy,

Animal/treatment

Weight (pm)

OF BOVINE

TABLE II and X-Radiation on Susceptibility Anaplasma marginale

No. used

2 5-30

Rats/splenectomy

150

50 8

0.5 ml i.p.

Rats/splenectomy

200

12

1.0 ml i.p.

Guinea pigs/ splenectomy

400

15

2.0 ml i.p.

10

2.0 ml i.p.

5

6.0 ml i.v.

2 kg 2 kg

Rabbits/splenectow

0.1 ml i.v.

Cats/splenectomy

3-4 kg

11

2.0 ml i.v.

Rats/adrenalectomy

250

24

1.0 ml i.v.

18-20

50

0.5 mI i.p.

Mice/(X-radiation) a All splenectomized

of Animals

Amount, route of administration, and method of preparation of inoculum

Mice/splenectomy

Rabbitslsplenectomy

59

ANAPLASMOSTS

(b) (a) (4 (b) (b) (a) (a) (b) (b)

to Infection

with

Results (dead/inoculated) Exptl.

Control

o/25

o/25

4/4a 6/6a

4/4

5/10

o/s

6/6

017

O/3

O/3

o/2

O/7 2118

O/4

4125

6/25

O/6

rats died of bartonellosis.

ication was carried out at 9 kc for 30 minutes at a cup temperature of 4°-100.3 Such treatment of A. marginale does not alter its infectivity for cattle (Bedell and Dimopoullos, 1963). These types of preparations were used to challenge experimental animals, and will be referred to in the tables as (a), (b) , and (c) , respectively. Uninfected bovine erythrocytes prepared in a similar manner were employed as the control inoculum in all experiments. Several species of experimental animals were utilized. These were (1) white mice (Webster), (2) white rats (Wistar), (3) white rabbits, (4) guinea pigs, (5) gerbils, (6) hamsters (Syrian), and (7) cats. Normal Animals Initially a series of experiments was conducted to ascertain the susceptibility of normal animals to infection with A. nwrrgilzale. These groups were classified according to weight, number utilized, amount and route of 2 Raytheon Sonic Oscillator 25OW, lOKC, mddel DFlOl; Raytheon Mfg. Co., Waltham, Massachusetts. 3 Haake thermostat type F circulation pump; Gebruder-Haake K. G., Berlin-Steglitz, West Germany.

inoculum, method of preparation of inoculum, and the result of challenge in terms of number surviving at the termination of the experiment (Table I ) . Surgically Altered Animals Splenectomy, prior to challenge with A. marginale, was performed on mice, rats, guinea pigs, rabbits, and cats. Adrenalectomy was performed only on rats. These procedures were carried out under aseptic conditions with Nembutal anesthesia supplemented when necessary by ether. A period of one week was allowed for recovery. Splenectomized animals received no special postsurgical care. Adrenalectomized rats were maintained in good condition by the addition of 1.0% sodium chloride to their drinking water (Gordon and Katsh, 1949). Completeness of organ removal was carefully checked at the time animals were autopsied. X-Irradiated

Animals

Exposure to X-radiation was carried out only on mice. These received 320 r total-body X-radiation as a single dose and were challenged with A. marginale 3 days later (Table II).

60

SUMMERS

AND

TABLE The Effect of Administration

GONZALEZ

III

of Various Resistance-Reducing

Agents on the Susceptibility

of Animals

to

-4naplasma marginale

Animal

Weight (gm)

-

No. used

Amount, route of administration, and method of preparation of inoculum

Experimental treatment employed

Results (dead/inoculated) Exptl.

Control

0.2 m1i.p.

(4

Inoculum mixed with 5.0% hog gastric mucin in ratio of 1: 1 prior to injection. Single injection

o/10

O/IO

20

0.05 ml iv.

Same as above

O/IO

O/IO

20

0.05 ml i.cer.

Same as above

z/10

l/10

Same as above

O/7

O/8

Same as above

O/5

O/5

Injected i.p. 2 hours prior to challenge with 5.0 mg of zymosan

8/25

4/25

Mice

25-30

20

Mice

25-30

Mice

25-30

Mice

IS-20

50

0.05 ml i.v.

(b) (cl (a) (a) Cc)

Mice

18-20

50

0.25 ml i.v.

(b)

Injected i.p. 2 hours, prior to challenge with 3.0 mgof zymosan

O/25

o/25

Mice

2%35

50

0.5 m1i.p.

(b)

Cortisone 2.5 mg S.C.daily for 4 days

O/23

O/25

Mice

18-20

40

0.:

m1i.p.

(b)

Bovine serum 1.0 ml i.p. for 20 days beginning 5 days before challenge

o/20

o/20

4

0.5 m1i.v.

(b)

25 mg cortisone S.C. daily for 5 days

o/2

o/2

Rats

23’0

15

0.5 m1i.p.

Guinea pigs

300

10

0.5 m1i.p.

Rabbits

Animals

4 kg

Treated by Injection of InfectionEnhancing Agents

A third series of experiments was conducted in which animals received injections of socalled infection-enhancing materials as follows: (a) Mice, rats, and guinea pigs received anaplasma-containing inoculum which had been mixed with one part of 5.0% hog gastric mucin (Olitzki, 1948); (b) mice were challenged with A. marginale 2 hours after

having been given a 3%5mg dose of zymosan intraperitoneally (Kiser et al., 1956); (c) mice were challenged while on a course of daily injections of 2.5 mg of cortisone; rabbits were similarly challenged while receiving daily amounts of 25 mg of cortisone; (d) mice were challenged during the course of prolonged administration of normal bovine serum given by the intraperitoneal route (Desowitz and Watson, 1952) (Table III).

TRANSMISSION

Effect of Riboflavin-Deficiency

OF BOVINE

TABLE IV and Lowered Blood Glucose on Susceptibility Anaplasma marginale Amount, route of administration, and method of preparation of inoculum

Weight km)

No. used

Rats

60

25

0.5 ml i.p.

(b)

Rats

90

20

0.5 ml i.p.

(4

Animal

Nutritionally

Deficient Animals

In a fourth series of experiments, the effect of the low blood glucose upon susceptibility to A. marginale was tested in rats. Chlorpropamide4 incorporated in the feed at a concentration of 4-572 caused a reduction of blood glucose to 45-60 mg%. During the initial period of low glucose, animals were challenged with A. marginale. Thereafter, blood glucose was maintained at approximately these levels for a period of 30 days by continuously supplying chlorpropamide in the diet. Glucose was determined by the method of Nelson and Somogyi.5 Riboflavin-deficient rats were obtained by placing 60-gm animals on a purified riboflavin-deficient diet6 for 30 days prior to 4 Supplied through the courtesy of Chas. Pfizer & Co., Inc., New York. 5 A micromethod modification of the NelsonSomogyi (true glucose) method as presented in Labtrol Manual, Dade Reagents, Inc., Miami, Florida. 6 General Biochemicals Inc., 690 Laboratory Park, Chagrin Falls, Ohio.

61

ANAPLASMOSIS

Experimental treatment employed

of Rats to Infection

with

Results (dead/inoculated) Exptl.

Control

Animals fed a riboflavindeficient diet for 30 days prior to challenge. Controls fed purified diet with riboflavin.

l/12

o/13

Animals fed regular diet to which was added chlorpropamide at 4.5% concentration. Controls received no drug.

O/IO

o/10

challenge (Pinkerton and Bessey, 1939). Evidence of riboflavin deficiency was noted by cessation of growth, loss of hair, and a scaliness of the skin (Table IV). Methods of Observation Close daily observation of all animals was maintained throughout in order to detect alterations in feeding habits, signs of anemia, loss of weight, inactivity, and time of death. Evidence of acquired infection was sought primarily by microscopic examination of blood cells and tissues. Peripheral blood smears, bone marrow impressions, and sliced organ impressions were stained with Giemsa. Peripheral blood smears were examined daily on several animals’ in each experimental group. At the termination of each experiment some animals from each group were sacrificed. Liver, adrenals, spleen, and lung were removed and fixed in Zenker-formalin. Fixed tissues were embedded in paraffin, sectioned, and stained with hematoxylin-eosin.

62

SUMMERS

AND GONZALEZ

RESULTS

In this study we have endeavored to transmit bovine anaplasmosis to various species of laboratory animals. The results indicate, for the most part, that normal healthy representatives of the species tested were refractory, could not be detected in since A. marginale any of the tissues examined. The same may be said to be true of those animals in which an attempt was made to break down resistance to infection. However, in certain experiments results were obtained which seem worthy of mention and which suggest the need for further investigation. Thus, new-born and lo-gm mice died following the administration of minute amounts of anaplasma inoculum. Deaths began to occur after a period of 7 days and continued until all animals had succumbed. No abnormalities of behavior were noted, and autopsy did not reveal the cause of death. There was no evidence of peritonitis. The circumstances of a very small inoculum, an incubation period of at least 7 days, and death suggested the possibility of an infectious process in these animals. Another finding which seemed noteworthy was the occurrence of death among young rats and gerbils following the intraperitoneal inoculation of defibrinated infected blood. Such animals began to die about 4 hours after inoculation while some survived as long as 18 hours. At the time of death each animal had assumed an attitude of spinal rigidity with posterior extension of the fore- and hindlegs. There was no indication that A. marginale was present in the tissues or blood stream. The rapidity with which death occurred suggested a toxic effect upon the central nervous system rather than an infectious process. Severe toxic effects have been observed in cattle inoculated with heavily infected anaplasma blood (Mott, 1957). Litter mates of both rats and gerbils in this experiment showed no such signs and remained in good health following inoculation of normal bovine blood. Further studies with rats and gerbils

by using various fractions of heavily infected anaplasma blood may aid in the isolation and identification of substances whose presence in infected cattle may help explain the pathogenesis of this disease. Among the groups of animals in which an attempt was made to alter resistance to infection the occurrence of deaths in splenectomized guinea pigs was unique. Here five of ten guinea pigs died following intraperitoneal injection of A. marginale. Deaths occurred between the eighteenth and thirtieth days. Autopsy revealed only enlargement of the adrenals. Tissue sections revealed engorgement and fatty degeneration of the liver and marked hemorrhage in the adrenal glands, particularly in the zona reticularis and medulla. No evidence of the presence of A. marginale was found. The long period which elapsed between challenge and death again suggests the possibility of an infectious process. Other splenectomized experimental and control animals in this group remained in good condition. Autopsy and microscopic examination of their tissues revealed no abnormalities. In attempting to evaluate the significance of the findings presented here, we do not wish to give the impression that we have grown A. marginale in a nonruminant host. The meager findings are encouraging enough, however, to require additional investigation. In his recent monograph Ristic (1960) stated, “the criterion for successful in vitro need not necespropagation of A. mar&ale sarily be the demonstration of classic marginal inclusions.” Such may also be the case in certain nonruminant animals following challenge. If true, the primary problem would be one of detection of the organism in the challenged host tissues from the moment of its entry by injection until it had reached some point where growth began. The application of fluorescent antibody techniques may provide valuable data concerning the fate of A. marginale in such animals as newborn, very young mice, and splenectomized guinea pigs, and also in cattle.

TRANSMISSION

OF BOVINE

CONCLUSION

Following the administration of challenging doses of A. marginale to various species of nonruminant laboratory animals, no microscopic evidence of infection was obtained. Deaths among certain experimental animals suggested an infectious process and the need for further study and for the application of more precise methods of detection of A. marginale in the tissues of both experimental and natural hosts. REFERENCES BEDELL, DAVID M., AND DIMOPOULLOS, GEORGE T. 1962. Biologic properties and characteristics of Anaplasma marginale. I. Effects of temperature on infectivity of whole blood preparations. American Journal of Veterinary Research 23, 618-625. BEDELL, DAVID M., AND D~OPOULLOS, GEORGE T. 1963. Biologic properties and characteristics of Anaplasma marginale. II. The effects of sonic energy on the infectivity of whole blood preparations. American Journal of Veterinary Research 24, 278-282. DE FARIA, G. J. 1928. Estudios sobre la Tristeza de 10s bovinos. Rev&a de1 Znstituto Bacteriologic0 Malbran 4, 429-469. DESOWITZ, R. S., AND WATSON, H. J. C. 1952. Studies on Trypanosoma vivax. III. Observations on the maintenance of a strain in white rats. Annals of Tropical Medicine and Parasitology 46, 92-100. DIKMANS, G. 1933a. Anaplasmosis. II. A short review and a preliminary demonstration of its identity in Louisiana. Journal of the American Veterinary Medical Association 62, 741-748. DIKMANS, G. 1933b. Anaplasmosis. VI. The morphology of Anaplasma. Journal of the American Veterinary Medical Association 63, 203-213. DYKSTRA, R. R., LIENHARDT, H. F., PYLE, C. A., AND FARLEY, H. 1938. Studies in anaplasmosis. Kansas Agriculture Experiment Station Report 1, l-32.

ANAPLASMOSIS

63

DYKSTRA, R. R., RODERICK, L. M., FARLEY, H., McMAHAN, V. K., AND SPLITTER, E. J. 1948. anaplasmosis. II. (1938-1948). Studies in Kansas Agriculture Experiment Station Technical Bulletin 66, l-24. D~OPOULLOS, G. T. 1959. Private communication. Louisiana State University, Veterinary Science Department, Baton Rouge, Louisiana, as quoted by RISTIC, M. 1960. Anaplasmosis. Advances in Veterinary Science 6, 111-192. GORDON, ALBERT A., AND KATSH, GRACE, F. 1949. The relation of the adrenal cortex to the structure and phagocytic activity of the macrophagic system. Annals of the New York Academy of Science 52, l-30. KISER, J. S., LINDH, H., AND DE MELLO, G. C. 1956. The effect of various substances on resistance to experimental infections. Annals of the New York Academy of Science 66, 312-328. MOTT, LAWRENCE 0. 1957. The nature of anaplasmosis. Proceedings of Third National Research Conference (“Anaplasmosis in Cattle”) Manhattan, Kansas. Pp. l-9. OLITZXI, L. 1948. Mucin as a resistance-lowering substance. Bacteriological Reviews 12, 149-172. PINKERTON, H., AND BESSEY, 0. A. 1939. The loss of resistance to Murine typhus infection resulting from riboflavin deficiency in rats. Science 69, 36&370. RISTIC, M. 1960. Anaplasmosis. Advances in Veterinary Science 6, 111-192. ROSSI, P., AND TRIOZON, F. 1953. Essais de culture d’une souche bovine francaise d’iinaplasma marginale. Bulletin de la Societie de Pathologie Exotipue 46, 312315. SMITH, T., AND KILBORNE, F. L. 1893. Investigations into the nature, causation and prevention of Texas or Southern cattle fever. U. S. Department of Agriculture, Bureau of Animal Zndustry Bulletin 1, l-301. STILES, G. W. 1929. Investigations on anaplasmosis in cattle. Journal of the American Veterinary Medical Association 74, 704-722. VEGLIA, F. 1915. The cultivation of Anaplasma marginale in vitro. Third and 4th Reports, Director Veterinary Research, Union of South Africa. Pp. 527-532.