REDUCTION IN VlRULENCE OF ~A~~~~A BUYlS DUE TO RAPID PASSAGE IN SPLENECTOMIZED CATTLE L. L. CALLOW, L. T. MELLORS and W. MCGREGOR Department

of Primary Industries, Animal Research Institute, Yeeron gpilly, Queensland 4105, Australia (Received 30 August 1978)

Abstract-Cai,r,ow L. L., MELLORSL. T. and MCGREGORW. 1979. Reduction in virulence of Bubesia bovis due to rapid passage in splenectomized cattle. International Journal for Parasitology 9: 333-338. A marked loss of virulence of Bubesiu bovis for normal cattle was observed during its rapid serial, blood passage in splenectomized calves. In 2 strains studied closely, responses to infection in cattle inoculated with parasites collected from the 1lth passages were minimal, although the splenectomized donors were severely affected. The change was reversed by passaging in intact hosts, and in one experiment the parasites had become very virulent at passage 5. The finding has proved useful in the preparation of safe, Living vaccines to control babesiosis. Selection either for immunogenic antigens, against immunosuppressive ones, or a combination of these effects may cause the decrease in virulence.

INDEX

KEY WORDS:

&be&

bovis; blood;

avirulence of strains of Bubesia bovis (previously known as B. argentina) used in vaccine against babesiosis in Australia has been mentioned in reviews (Callow, 1971; 1976; 1977). Five separate isolates of B. bovis lost virulence for nonspfenecto-

THE

REL.4nvE

mized (‘intact’) cattle after rapid passage in young, splenectomized calves, and were subsequently used in vaccine. Because of current interest in vaccines for diseases caused by protozoan parasites we are now presenting our evidence for a relationship between the virulence of B. bovis and the presence or absence of a spleen in animals used for passaging. MATERIALS AND METHODS Animals. The cattle used were 30s tuurus. Susceptible

calves, I-lo-weeks and steers I-Zyears of age were purchased from an area 160 km west of Brisbane where the tick vector of babesiosis, Boophihs microplus, does not occur, and were kept under tick free conditions at the laboratory. Valuable purebred bulls Z-3-years old, from tick free areas also served as ‘experimental’ animals. For many years the laboratory has infected the bulls with virulent B. bovis and B. bigemina, under close veterinary supervision, to immunize them prior to their transfer to the enzootic area for babesiosis (Seddon, 1952). These animals were suitable subjects in experiments designed to test for reduced virulence. Strains. Strains of 8. bovis, designated B, C and D, were used in the work. All were isolated in Queensland during outbreaks of babesiosis in widely separated districts between 1957 and 1967. Before being used in experiments and later in vaccine, the strains had been transmitted by B. microplus under laboratory conditions, and also passaged with blood. Additional history for each strain is provided in Experiments and Results. 333

cattle;

passage;

splenectomy;

vaccine;

virulence.

Procedures involving parasites. Parasitized blood was collected from the jugular vein or carotid artery (Callow & Mellors, 1966) into either sodium citrate solution or heparin, usually on day 3 or 4 of patent parasitaemia. If not used immediately it was chilled at 2-4”C usually for l-4 days, but sometimes for longer (17 days on 1 occasion), or cryopreserved (Dalgliesh, 1971) until required. The parasite concentration of blood from different donors varied considerably, particularly between splenectomized and intact donors. In experiments in which comparisons were being made, equal numbers of parasites from different donors were obtained by adjusting the volumes of the inocula. Procedures involving recipients. Cattle used during passaging were infected intravenously, as were all groups used to test virulence except once when 8. bovis was given subcutaneously to simulate vaccination (Callow, 1977). Infections were monitored by examining thin or thick blood films made from tail tip blood and stained with Giemsa’s stain, and by recording rectal temperatures once per day for laboratory cattle and twice per day for the purebred bulls. The severity of infections, taken as a measure of the virulence of the inocula, was quantified in intact steers by methods described by Callow & Pepper (1974): their scoring system was used to derive cumulative parasitaemia scores for each animal; values for the total temperature rise, that is, the total fever occurring during active infection, were obtained by adding the daily difference between a normal rectal temperature derived from controls and that of each experimentally infected steer. In addition, the number of days on which parasites could be detected microscopically (patent period) was used to measure the severity of an infection. Analysis for the significance of the differences between groups was by Student’s t test. Babesicidal treatment of acutely affected steers was avoided whenever possible. Experience with B. bovis infections has, however, taught

334

L. L.

that rectal temperatures

>2,5”C

CALLOW,

above

normal

L. T.

MELLORS

for 3 or

more days and parasitaemias of > 1 ‘A in intact cattle indicate a probable fatal outcome unless treatment is given. One group of cattle, in particular, developed these signs, and to prevent heavy losses most were treated at least once with 5 or 10 mg/kg amicarbalide (‘Diampron’, M & B) an effective babesicide. No risks could be taken with the purebred bulls, and treatment was given if the average rectal temperature (based on recordings at 0800 and 1400 h) for the day reached 40.5”C or if parasitaemia of > 0.3 ‘/” was recorded. Need for treatment was a major criterion in determining the virulence of inocula tested in the bulls. Although treatment interfered with the development of responses, it was possible to perform certain analyses of values for parasitaemia and rectal temperature. EXPERIMENTS AND RESULTS Preliminary observations were made with the B strain of B. bovis which had been held at the laboratory for approx 7 years. During most of this time it had been used in tick infectivity experiments (O’Sullivan & Callow, 1966), being inoculated in blood occasionally in connection with these experiments. For the 7 months prior to being passaged in splenectomized calves, the strain was passaged 2-3 times/month in intact cattle. Inocula prepared from these animals were virulent. The rapid passage in splenectomized calves was performed every 4-7 days. Uncontrolled observations suggested that changes occurred after about 9 passages, the strain becoming less virulent for intact cattle, but producing higher parasitaemias in the calves. Some groups of intact cattle, inoculated fro.m the splenectomized calf

and W.

MCGREGOR

I.J.P. VOL. 9. 1979

series, however, developed infections considered sufficiently severe to warrant treatment, and it was not clear until passage 30 that virulence was, in fact, less than before the strain was introduced to splenectomized animals. Experiments were then performed using appropriate controls, standardized procedures and employing 2 additional strains (a) to confirm the effect of splenectomy in reducing virulence, (b) to establish how many passages were required to change the character of a strain and (c) to determine if a return to passaging in intact cattle reversed the effect. Comparison of passaging in splenectomized and intact calves The D strain was kindly provided by Dr. D. F. Mahoney. He had isolated it 3 years earlier, and used it particularly for antigen production (Mahoney, 1962). It has been called ‘C strain’ in other work at our laboratory (Callow & Tammemagi, 1967), but is different in origin from the C strain (see below) used in the present work. The D strain was passaged with blood in intact cattle approx 12 times per year for 5 years before the experiment began. Commencing with blood from an intact calf in which patent parasitaemia had ended 24 weeks earlier, and using inocula containing approx IO8 B. bovis parasites, 2 series of 11 passages were done. One of these was in splenectomized calves (Pass S), the other in intact calves (Pass I). To compare virulence, blood containing 10” parasites obtained from passages 1, 4, 6, 8 and 11 of each series was inoculated into groups of

TABLE ~-VIRULENCE DECREASE IN D STRAIN Babesia bovis AFTER SERIAL PASSAGE IN SPLENECTOMIZED CALVES, SHOWN BY INFECTING GROUPS OF 6 INTACT STEERS AND COMPARING RESPONSESWITH THOSE PRODUCED AFTER STRAIN PASSAGED IN INTACT CALVES

Response

Passage series

Means (x) and least significant differences (LSD) for responses at uassages 1 4 6 8. -*11 -

??, LSD(PzO.05)

Splenectomized (S) 11.67 Total fever (“C)

1 I.73 5.17

Intact (1) 14.5 S

Parasitaemia duration (days)

7, LSD(P=O,05)

8.2

5.83

S

11.67

Parasitaemia intensity (score) 1

14.33

8.48

7.83 15

2.22

*Differences between means of all responses significant, P< 0.01.

2 3.01

2.45 6 ____2.83

10.5 13.14

16.68 23.17

7.98 15.27

4.83

12.33 8.83

23.5

7.50

5

5.83

x LSD(P= 0.01) 3.07

14.51

5.17 2.00

6.91

7.93

Il.75

667 1.33

I

8.45

15.3

5

k, LSD(P= 0.05) 2, LSD(P=O.05)

15.67

19.05 22.67

I.J.P. VOL.

9. 1979

Virulence change in Bubesiu bovis

~-DECREASE IN VIRULENCE OF C STRAIN Babesia WITH PASSAGINGINSPLENECTOM~ZEDCALVES,SHOWN BY DECREASING NEED FOR BABESICIDAL TREATMENT OF GROUPS OF BULLS INFECTED FROM PASSAGES BETWEEN 6

TABLE

bovis

AND11

Passage number 3 6 7 8 9 10 11

Bulls inoculated 9 10 10 9 10 10 12

Bulls treated

Bulls not treated

6 10 5 6 3 4 0

3 0 5 3 7 6 12

6 steers. Comparisons made between the 2 series at each stage of passaging were at the same time. From Table 1, Pass S parasites appeared to produce lower responses than Pass I parasites from the outset, and the difference became significant at passage 11 (P < 0.01). Virulence of Pass I parasites was high to start with, and varied little during the 11 passages. Confirmation of major virulence decrease by passage 11 using another strain The C strain of B. bovis was taken from the 3rd serially infected, intact steer following its isolation 15 months earlier. The 2nd and 3rd of these passages had been effected with ticks, the 3rd occurring 9 months prior to the commencement of passaging in splenectomized calves, The dose for passages 9, 10 and 11 in splenectomized calves was raised from lo8 to lo9 parasites. To synchronize the testing for virulence of parasites from these 3 passages and from 4 earlier passages (3, 6, 7 and S), cryopreserved material from passages 2, 5 and 6 and chilled blood from passage 7 were inoculated into splenectomized calves with the object of having all donors experiencing high parasitaemias at approximately the same time. As a result the 7 inocula were ready within a

335

period of 4 days. A dose of lOa parasites was given to each aniinal in the 7 groups comprising 9-12 purebred bulls, on the same day. From Table 2, the number of cattle fulfilling treatment criteria was highest (10 of 10) in the group inoculated with B. bovis from passage 6. The need for treatment became less for inocula prepared at subsequent passages until no animal in the group of 12 inoculated required treatment at passage 11. Statistical analysis of data from all 7 groups was not done because of the difficulty in allowing for the effect of treatment. To emphasize the marked decrease in virulence between passages 6 and 11, however, values from recipients of parasites at these 2 stages of passaging were compared by Student’s t test without allowing for the heavy treatment of passage 6 recipients. Despite bias against such a result, responses were significantly less in recipients of passage 11 than in those of passage 6 (Table 3). FuII virulence restored by passage in intact cattle In a preliminary experiment B strain B. bovis at splenectomized passage 34 reverted to full virulence after 1 passage in intact cattle. A group of 7 was inoculated, and parasites collected from the 1 animal showing moderate clinical effects produced severe reactions in a further group of intact animals. Subsequently, virulence was apparently restored to both the D and C strains by rapid serial passage through 3 or 4 intact calves. In a controlled experiment, B strain, after 92 passages in splenectomized calves, was used. Two series of 5 rapid passages, starting with an inoculum prepared from passage 92, were performed, one in splenectomized (Pass S), the other in intact calves (Pass I). The passage series were completed and inocula ready for testing after 38 days. Pass S infections developed very quickly, and parasites were stored for periods during the passaging so that inocula from passages 5 would be ready at the same time. Two groups of 7 steers were then inoculated

TABLE ~-VIRULENCE DIFFERENCEIN C STRAIN Babesia bovis COLLECTED AFTER PASSAGE 6 OR 11 IN SPLENECTOMIZED CALVES, SHOWN BY INFECTING GROUPS OF BULLS FROM THOSE PASSAGES

Response tMax T” reached (“C) Parasitaemia duration (days) Parasitaemia intensity (score)

Means for responses at passage

p = 0.05

P = 0.01

0.34

0.46

P

Reduction in virulence of Babesia bovis due to rapid passage in splenectomized cattle.

REDUCTION IN VlRULENCE OF ~A~~~~A BUYlS DUE TO RAPID PASSAGE IN SPLENECTOMIZED CATTLE L. L. CALLOW, L. T. MELLORS and W. MCGREGOR Department of Prima...
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