Effect of timing and intensity of challenge following immunization against East Coast fever

Veterinary Parasitology, 26 (1987) 29-41 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

29

Effect of T i m i n g and Intensity of Challenge F o l l o w i n g I m m u n i z a t i o n against East Coast Fever S P. MORZARIA, A.D. IRVIN*, W.P. VOIGT and E.L.N. TARACHA** International Laboratory [or Research on Animal D~seases ( ILRAD ), P 0 Box 30709, Natrobi (Kenya) (Accepted for publication 5 November 1986)

ABSTRACT Morzana, S.P., Irvm, A.D., Volgt, W.P. and Taracha, E.L.N., 1987. Effect of timing and intensity of challenge following immunization against East Coast fever. Vet Parasltol., 26:29-41 When groups of Thederia parva parva Muguga-immunized cattle were given a homologous lethal challenge at different times after immumzation, it was found that 4/6, 5/6, 6/6 and 6/6 animals survived when challenged on Days 5, 10, 20 and 30, respectively, post-immunization.With a heterologous challenge ( T p parva Mankebum), 2/6, 5/6, 4/6, 4/6 and 5/6 cattle survived when challenged on Days 5, 10, 20 and 30, respectively, after immunization. All controls, except one, died of East Coast fever (ECF). The survivor underwent severe ECF and recovered after a prolonged convalescence. When two T p parva Muguga-immunized animals were each given homologous challenge by application of 1000 infected ticks (infection rate of 20 infected acini ( i.a. ) per tick), both survived a mdd ECF reaction. When groups of T p.parva Muguga- or T p parva Muguga/Mamkebuni-immumzed cattle were challenged with different doses of T p parva Muguga sporozoites (equivalent of 140, 1400 and 14000 i.a. per ammal), 28/29 cattle survived. All controls dmd of ECF. It was concluded that cattle could be safely exposed to tick challenge 1 week after immunization by infection and treatment using appropriate immumzing stock (s). Massive homologous challenge did not break through the immunity induced by the lmmumzation procedure.

INTRODUCTION

East Coast fever (ECF) is a disease of cattle of great economic importance in East and Central Africa (MacGillivray, 1967). The disease is a syndrome caused by one or more sub-species of the protozoan parasite Theileria parva. The parasites are transmitted by the tick Rhipicephalus appendiculatus. *Present address: Overseas Development Administration, Eland House, Stagg Place, London, Gt. Britain. **Seconded from Veterinary Research Laboratory, Ministry of Agriculture and Livestock Development, Kabete, Kenya.

0304-4017/87/$03.50

© 1987 Elsevier Science Publishers B.V.

30 A method of immunization called "infection and treatment" or "chemoprophylaxis", developed in Kenya (Radley, 1981 ), has been successfully used on an experimental basis and in several field trials carried out in various parts of Africa (Robson et al., 1977; Uilenberg et al., 1977; Paling and Geysen, 1981; Dolan, 1985; Morzaria et al., 1985; Young, 1985 ). Prompted by these successes, many countries have recently agreed to use the method on a wider scale ( Irvin, 1985). Antibody responses to the macroschizont and piroplasm stages of the parasite develop 3-4 weeks following immunization and although not protective (Burridge et al., 1972), their presence provides a good indicator of immunity. On the basis of the timing of the development of these specific antibody responses, cattle have been kept tick free for 4 weeks following immunization before tick challenge has been allowed. However, if widespread use of the infection and treatment method is to be adopted, it is important to know more precisely how soon after immunization the cattle can be safely exposed to challenge. Another important point which needs clarification is whether or not massive challenge with the homologous parasite strain can break through immunity. This possibility cannot be discounted when it is well known that the severity of ECF reaction is dose dependent (Jarrett et al., 1969; Radley et al., 1974). Of the several questions which need to be answered with regard to the widescale use of the infection and treatment method of immunization (Irvin and Gill, 1981; Radley, 1981 ), we have attempted to assess the effect of timing and of massive T. parva sporozoite challenges on immunity and to test the generation of strain-specific immunity following ECF immunization. MATERIALSAND METHODS

Cattle Ninety-nine Boran (Bos indicus) steers between 8 and 12 months of age were used in these studies. They were obtained from Kapiti Plains Estates Limited, which maintains an ECF-free ranch. They were transported to the International Laboratory for Research on Animal Diseases (ILRAD) at 8 months of age and thereafter maintained tick free by twice weekly acaricidal spraying. Prior to experimentation, all animals were negative for antibodies to T. parva in the indirect fluorescent antibody test (IFA) ( Goddeeris et al., 1982 ). However, 37.5 and 56.3% had antibodies to Anaplasma spp. and Babesia bigemina, respectively, in the enzyme-linked immunosorbent assay (J. Katende, in preparation ).

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Parasite Two stocks, T. parva parva Muguga (Brocklesby et al., 1961 ) and Marikebuni (Irvin et al., 1983), were used. The parasites were maintained either as cryopreserved tick stabilates (Cunningham et al., 1973) or in adult R. appendiculatus ticks infected as nymphs while feeding on ECF-infected cattle (Bailey, 1960).

Immunization protocol The infection and t r e a t m e n t method ( Radley et al., 1975 ) of immunization against ECF was used. Cattle were inoculated with a long-acting formulation of oxytetracycline [Terramycin LA (Pfizer Ltd., Sandwich, Kent, U.K.) injectable solution 200 mg m l - 1] intramuscularly ( i.m. ) at a dose of 20 mg k g - 1 body weight (b.w.) and immediately afterwards inoculated subcutaneously (s.c.) over the parotid lymph node with a potentially lethal dose of a chosen stabilate of T.p.parva.

Challenge of cattle Immunized and unimmunized cattle were challenged with parasites by one of the following methods: (i) Stabilate challenge. The cattle were given a lethal challenge of either T.p.parva Muguga (Stabilate 836) or Marikebuni (Stabilate 1581 ). The stabilates were thawed at 37 °C in a water bath and within 30 min, 0.5 ml was injected s.c. below the ear into each animal. One millilitre of the Muguga and Marikebuni stabilates contained sporozoites derived from ~ 125 and 75 infected acini (i.a.), respectively. (ii) Sporozoite challenge. Fresh T.p.parva Muguga sporozoite suspension was prepared from infected ticks essentially according to the method described by Purnell et al. (1973). Briefly, 17 000 R. appendiculatus adults, with an infection rate of 14 i.a. per tick as determined by the method of Irvin et al. (1981) and fed for 4 days on rabbits, were homogenized. The homogenate was centrifuged (50 x g for 5 min ) and the supernatant fluid was diluted to give a concentration equivalent to 10 ticks m l - 1. Cattle were challenged by s.c. inoculation within 1 h of the preparation of sporozoite suspension. Injection was either below the ear in front of the parotid lymph node or over the shoulder in front of the pre-scapular lymph node. Three levels of challenge were used; 1 ml (140 i.a.), 10 ml (1400 i.a.) or 100 ml (14000 i.a.) per animal. (iii) Tick challenge. T.p.parva Muguga-infected R. appendiculatus ticks, with an infection rate of 20 i.a. per tick were applied on cattle ears and allowed to feed to engorgement to allow parasite transmission. Two levels of challenge

32

were used: 100 tick challenge (equivalent (equivalent to 20 000 i.a. ) per animal.

to 2000 i.a.) or 1000 tick challenge

Observations

During the experimental period, rectal temperatures were taken daily and the superficial lymph nodes nearest to the site of parasite inoculation were palpated. When the temperature of an animal was found to be 2 39.5 ‘C ( regarded as pyrexia) , a biopsy smear of the local drainage lymph node was taken. When the presence of macroschizonts was recorded in the local drainage lymph node, contralateral lymph node biopsy and blood smears were also taken. All smears were fixed in methyl alcohol, stained with Giemsa’s stain and examined for Theileria parasites. ECF reactions in the experimental cattle were classified as follows: mild reaction if the duration of detectable macroschizonts andpyrexia was < 4 days, moderate reaction if the duration of macroschizonts and pyrexia was between 4 and 8 days and severe reaction if the duration of the macroschizonts and pyrexia was > 8 days. Blood was collected from all cattle immediately prior to the experiment and weekly thereafter and sera were screened for antibodies to T. parva in the IFA test. Experimental

design

Timing of challenge

Cattle were divided into eight groups (A-H) of eight animals. Six animals in each group were immunized against T.p.parva Muguga by infection and treatment and the remaining two animals were retained as susceptible controls which were treated only with Terramycin LA. The cattle in Groups A, B, C and D were subsequently challenged on Days 5, 10, 20 and 30, respectively, post-immunization, with a lethal T.p.parva Muguga (homologous) stabilate challenge. The cattle in Groups E, F, G and H were similarly challenged with a heterologous lethal challenge of T.p.parva Marikebuni. Intensity

of challenge

In the first experiment, three animals were used. Two animals were immunized against T.p.parva Muguga and were each given a lethal homologous stabilate challenge 34 days later. Ninety-seven days thereafter, each of the immunized animals was given a massive homologous challenge by application of 1000 T.p.parva Muguga-infected adult R. appendiculatus ticks with an infection rate of 20 i.a. per tick. A control animal was challenged at the same time with 100 ticks derived from the same batch as above. In the second experiment, cattle immunized against T.p.parva Muguga and against both T.p.parva Muguga and Marikebuni were given different levels of

33 CHALLENGE Parasite

Groups

CATTLE Days post-

immunlza~on

Immun#zed

Contro~

A

S

00@@@@

00

S

lo

O

00

c

20

000000

00

D

3o

~ 0 0 0

00

T. p. parva

E

S

e e e e ~

e~

Marikebuni (Heterologous)

F

lo

O

ee

G

20

o e ~ ~

ee

H

30

e

e•

T. p. parva

Muguga (Homologous)

ECFreactions

O none (~ mild • death O death

~

~

~

~

~

~

(]~ moderate (~

severe

complicated by babaslOSm

Fig. 1. Susceptibility of Tpparva Muguga-immunized cattle to homologous and heterologous challenges at different intervals after immunization.

T.p.parva Muguga sporozoite challenge. T h e 15 T.p.parva Muguga-immunized cattle were divided into three groups ( I, II and III) each comprising five cattle. The 14 T.p.parva Muguga/Marikebuni cattle were divided into three groups (IV, V and VI); Groups IV and V comprising five cattle each and Group VI four cattle. The control groups (VII, VIII and IX) each consisted of two cattle. Each of the groups was given a different lethal dose of T.p.parva Muguga sporozoites 195 days post-immunization. Table IV shows the details of the experimental design. RESULTS

Timing of challenge The summarized results of the effect of timing of homologous and heterologous challenges following immunization are presented in Fig. 1. In Group A, challenged 5 days post-immunization, 2/6 died of acute ECF. The E C F reaction in the animals which died was comparable to those of the controls, with mean time to death of 18 days. All the survivors had ECF, one with a severe reaction, two with moderate reactions and one with a mild reac-

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TABLE I Effect of t~mmg of challenge following immunizatmn: homologous challenge Group

A

Day of challenge postimmunization

No. of ammals

ECF reaction TT a

TM b

DT c

DM d

DD e

Immumzed Controls

5 -

6 2

6.7 (6) 5.5 ( 2 )

6.8(6) 6.5(2)

8.8(6) 13 5 ( 2 )

8.5(6) 13.5(2)

18.0(2) 18.5(2)

B.

Immumzed Controls

10 -

6 2

6 0 (3 ) 6 0(2)

8.7(4) 6.0(2)

4 0(3) 20 0 ( 2 )

4.0(4) 20.5(2)

15 0(1) f 25.5(2)

C

Immumzed Controls

20 -

6 2

8.0 (2)

8.5(2)

15 0 ( 2 )

14.5(2)

22.5(2)

Immunized Controls

30 -

5 2

9.0 (1) 8 0 (2)

11.0(2) 7.0(2)

1 0(1) 14 5 ( 2 )

1.0(2) 15.5(2)

21.5(2)

D

Figures m parentheses represent the number of reacting animals. ~Mean time m days to first pyrexia bMean time in days to first macroschizonts. CMean duration m days of pyrexla. dMean duration in days of macroschizonts. ~Mean days to death. ~Died due to mixed refections of Tp parva and B blgemtna.

tion. In Group B, challenged 10 days post-immunization, 5/6 survived the challenge, all the survivors underwent mild ECF reactions. The one which died had a mixed infection of B. bigemina and T.p.parva. At the time of death, the animal was anaemic and severely jaundiced. All the cattle given a homologous challenge on either Day 20 or 30 were immune. The detailed results of ECF reactions in various groups are shown in Table I. In the groups of animals given a heterologous challenge (T.p.parva Marikebuni), the ECF reactions were variable. The details of the ECF reactions in these groups are given in Table II. In Group E, challenged 5 days post-immunization, 4/6 died of acute ECF. Two experienced severe ECF and recovered. In Group F, challenged 10 days post-immunization, all six showed ECF reactions, five with moderate reaction and recovery and one with severe reaction and death. This animal at the time of death (15 days post-challenge) had a mixed infection of T.p.parva and B.bigemina. In Group G, challenged 20 days post-immunization, all six showed ECF reactions, one with mild reaction and recovery, two with moderate reaction and recovery and one with a severe reaction and recovery, two with a severe reaction and death. One of the animals, at the time of death, had a mixed infection of T.parva and B.bigemina. In Group H, all six animals showed ECF reactions, three with mild reaction

35 TABLE II Effect of timing of challenge following immunization: heterologous challenge Group

E F. G. H.

Immunized Controls Immunized Controls Immunized Controls Immunized Controls

Day of challenge postimmunization

No. of animals

5 10 20 30 -

6 2 6 2 6 2 6 2

ECF reaction TT ~

TM b

DT c

DM d

DD ~

7.0 (6) 10.0 (2) 10.2 ( 6 ) 6.5 (2) 10 8 (5) 7.5 ( 2 ) 10 7 (6) 7.5(2)

7.2(6) 7.0(2) 8.8{6) 7.0(2) 11.0(6) 8.0(2) 10.7(6) 7.5(2)

14.2(6) 13.0(2} 8.7(6) 11.5(2) 12.0(6} 9.5(2) 62(6) 8.5(2)

16.3(6) 18.5(2) 8.8(6) 11.0(2) 12.0(6) 10.0(2) 9.2(6) 10.0(2)

22.4(4) 22.0(1) 18.0(1) f 17.0(2) 64.5 (2) g 17.0{2) 20.0(1) 16.5(2)

Figures m parentheses represent the number of reacting ammals. aMean ttme in days to first pyrexia. bMean time in days to first macroschizonts. CMean duration m days of pyrexia. dMean duration in days of macroschizonts eMean days to death fDied due to mixed infections of T p parva and B btgemtna. ~One of the animals dmd due to mixed infections of T p parva and B btgemtna.

and recovery, two with moderate reaction and recovery and one with a severe reaction terminating in death on Day 20 following challenge. All the control animals in Groups F, G and H died of acute ECF. Both controls in Group E underwent severe ECF reactions, one died and the other recovered after a prolonged convalescence.

Intensity of challenge The results of the first experiment are presented in Table III. Both immunized animals survived the massive tick/parasite challenge, undergoing mild ECF reactions. No pyrexia associated with the ECF reaction was recorded. However, both animals showed pyrexia between Days 2 and 13 post-tick application. The control animal died of acute ECF 16 days after tick application. The results of the second experiment are shown in Table IV. All but one of the immunized animals given massive T.p.parva Muguga sporozoites survived the challenge, undergoing mild ECF reactions. The one animal which died of ECF was immunized against T.p.parva Muguga and belonged to the group which received the highest sporozoite challenge (100 ml of sporozoite suspension, equivalent to 1400 i.a. ). This animal also developed an abscess at the site of the inoculation and showed Anaplasma marginale for 4 days prior to death.

36 T A B L E III E f f e c t of m a s s i v e h o m o l o g o u s c h a l l e n g e by tick f e e d i n g o n T p parva M u g u g a - i m m u n i z e d cattle A n i m a l No.

C369 C370 C283

Immune status

Immune Immune Control

Challenge ( N o . ticks/i.a. )

ECF reaction

1000/20 000 1000/20 000 100/2000

TT a

TM b

DM c

TD ~

10

14 12 9

1 1 8

16

~ T i m e in d a y s to first p y r e x i a . b T i m e in d a y s to first m a c r o s c h i z o n t s . CDuration o f m a c r o s c h l z o n t s . d T l m e in d a y s to d e a t h .

Two other animals (one in the T.p.parva Muguga group and one in the T.p.parva Muguga/Marikebuni group) which received 100 ml (14 000 i.a. ) of the sporozoite challenge showed abscess formation at the site of the inoculum and subsequently died of septicaemia. All the controls underwent severe ECF and died. TABLE IV Effect of massive Tp parva Muguga sporozolte challenge on Tpparva Muguga- and Tpparva Muguga/ Marlkebum-,mmumzed cattle Group

I II III IV V VI VII VIII IX

Immumzmg stock ( s )

Muguga Muguga Muguga Muguga/Mankebum Muguga/Mamkebuni Muguga/Marlkebum Controls Controls Controls

Sporozmte challenge (Vol ml/1 a )

No of cattle

ECF reaction TT"

TM b

DT c

DM d

1/140 10/1400 100/14000 1/140 10/1400 100/14000 1/140 10/1400 100/14000

5 5 5 5 5 4 2 2 2

5.8(5) 5.7(3) 53(4) 70(4) 5 8(4) 3 0 (2) 6.0(2) 6.0(2) 30(2)

63(3) 8 0(2) 6.0(4) 80(2) 7 0(2)

4.0(5) 3 7(3) 73(4) 2.0(4) 2 5(4) 2 0 (2) 95(2) 7 5(2) 8.5(2)

3.0(3) 2 0(2) 73(4) 3.0(2) 2 0(2) 95(2) 7 5(2) 9.0(2)

Figures m parentheses represent the number of reacting ammals ~Mean time m days to first pyrexla bMean time in days to first macroschlzonts. CMean duratmn in days of pyrexia. dMean duration m days of macroschlzonts. eMean days to death. fDied due to mixed infections of Tp parva and A margtnale

60(2) 6.5(2) 3.5(2)

TD e

190(1)' 150(2) 13.0(2) 120(2)

37 DISCUSSION In the studies investigating the timing of lethal homologous challenge following immunization, it was found that all but one of the immunized cattle had developed protective immunity by Day 10. For the protective immunity to develop, it appears necessary that the infection is established in the cells of the recipient cattle (Wagner et al., 1974). In the infection and treatment method, live sporozoites are used, which establish transiently as macroschizonts in the recipient lymphocytes before being eliminated. The immunity appears to be cell mediated ( Pearson et al., 1979; Emery, 1981; Morrison et al., 1986) and it has been demonstrated that the animals immunized in such a manner generate genetically restricted cytotoxic T-lymphocytes (CTL) which are restricted by products of the major histocompatibility complex (Morrison et al., 1986). These cells are detected in peripheral blood leucocytes ( P B L ) , coincidental with the elimination of the infection (Eugui and Emery, 1981; Emery et al.,. 1981). This suggests that the CTL response is important in protection. In the present experiments, immunization with a T.p.parva Muguga stock produced transient parasitosis between Days 12 and 15, while the mean prepatent period in susceptible control cattle was 7.6 days. Emery et al. (1981) showed that in cattle, immunized by infection and treatment, the cytotoxic activity in the PBL was first detected from 14 days post-immunization. Thus, all the immunized cattle challenged between Days 10 and 30 would have generated specific CTL by the time one would expect to detect macroschizonts in the local drainage lymph node. The one immunized animal that died following Day 10 challenge was an exception and this may be due to the fact that its immune system might have been compromised by concurrent infection with

B.bigemina. In the immunized group challenged on Day 5, evidence of specific immunity was present (4/6 survived the challenge). These cattle, receiving sporozoite challenge on Day 5, would have developed detectable macroschizonts around Day 7, i.e., 12 days post-immunization. Although this time is too early for the detection of the effector cells in P B L following primary immunization (Emery et al., 1981), it is possible that the recovered animals had already generated specific CTL which were localized in lymph nodes at sites of immunization and challenge. The purpose of using a heterologous challenge was to investigate the possible presence of protective immunity, unrelated to the establishment of macroschizonts in the host, in the few days (5-10) following immunization and to confirm the development of strain-specific immunity between Days 10 and 30 post-immunization. The selection of T.p.parva Marikebuni as an heterologous stock was based on the finding that this stock broke through T.p.parva. Mu-

38 guga-immunized animals, producing severe ECF reaction in 4/6 and deaths in 2/6 cattle (Irvin et al., 1983). The variable ECF reactions and the small numbers of animals used precluded the drawing of any firm conclusion with regard to the development of protective immunity in the group challenged 5 days after immunization. However, there was evidence that specific immunity had developed in the groups of cattle challenged on Days 10, 20 and 30. The total mortality in these groups due to the challenge was 22% (4/18). Two of these deaths were complicated by the recrudescence of B.bigemina infection. These results are comparable to those obtained by Irvin et al. (1983), who gave late (1 month post-immunization) heterologous challenge to ECF-immunized cattle using similar stocks of T.p.parva to the ones used in this study. The three cattle which died of mixed infection of B.bigemina and T.parva in these experiments were serologically positive to B. bigemina before the start of the experiment and thus, almost certainly, carriers. The recrudescence of B.bigemina during an ECF reaction indicates that these animals were immunologically compromised. The studies on the effect of timing of challenge showed that cattle can be safely exposed to tick challenge 1 week after immunization. The earliest possible sporozoite challenge is then likely to occur 4 days after the first attachment of ticks and the first appearance of macroschizonts 5 days later. Thus,. such immunized animals will see the first macroschizonts ~ 16 days post-immunization, when they would have presumably generated sufficient specific CTLs to provide protection. In the studies to investigate the effect of massive homologous challenge, the first experiment showed that the two immunized cattle withstood massive parasite challenge through tick feeding, while the susceptible control animal died of severe ECF with 10 times fewer parasites in the challenge. The purpose of using challenge through tick feeding was to ensure maximum transfer of viable sporozoites into the recipients. In the second experiment using titrated sporozoite challenges and a larger number of animals, all but one immunized animal survived the ECF challenge. This substantiated the results of the first experiment, showing that the massive sporozoite challenge (equivalent to 14 000 i.a.) did not break through the immunity induced by infection and treatment. The animal that died had a massive abscess at the site of the inoculum and also a concurrent Anaplasma infection. Again, these mixed infections might have played a role in its death. There has been a report that massive challenge can break through the immunity to ECF (Barnett, 1957). However, no experimental confirmation has ever been provided. Our studies showed that massive homologous challenge of sporozoites, as high as 20 000 i.a. equivalent, given 195 days post-immunization did not break through the ECF immunity. This is between 6 X l0 s and 1 X 109 sporozoites (Fawcett et al., 1985) per animal. Such massive challenge

39 is unlikely to occur in the field. However, in the field where tick control is minimal or absent, the sporozoite challenge occurs over a period of several days. Such natural conditions are difficult to mimic in the laboratory and the consequences of such challenges are not predictable. The two control cattle given a sporozoite challenge equivalent to 1400 i.a. showed pre-patent periods prior to macroschizont development of 3 and 4 days, respectively. Radley et al. (1974), challenged cattle with similar massive doses of stabilate, but were unable to detect macroschizonts before 5 days. However, a 4-day pre-patent period has been reported ( Purnell, 1977 ). The level of challenge we subjected these cattle to has never been reported before and it is likely that the sporozoites could have developed to macroschizonts even earlier than detected. Such an early pre-patent period further substantiates the in vitro studies of Stagg et al. (1981) and Fawcett et al. (1982) which showed that sporozoites, when exposed to susceptible lymphocytes, enter and develop into schizonts within a few hours. It seems very unlikely, therefore, that a preschizontal stage as proposed by Radley et al. (1974) exists. Antigenically different strains exist in ECF ( Snodgrass et al., 1972; Young et al., 1973; Radley et al., 1975; Dolan et al., 1980). Such strain differences were identified using cross-immunity tests in cattle. However, some doubt exists on the validity of such tests because comparable quantified sporozoite challenges have not been used. The existence of solid immunity despite massive challenge, as shown in these studies, provides indirect evidence that breakdowns in immunity are due to strain differences and substantiates the results of earlier in vivo cross-immunity tests carried out by other workers. Thus, cross-immunity tests, although costly, are an important way to differentiate T.p.parva strains. In conclusion, the studies described here provide practical evidence that protective immunity to ECF develops sufficiently early following immunization by infection and treatment, to allow immunized animals to be safely exposed to tick challenge 1 week post-immunization. The studies also showed that the protection induced following immunization was strain specific and was not broken down by unnaturally high homologous challenge. ACKNOWLEDGEMENTS Drs. Sam Black and Cynthia Baldwin are thanked for their helpful discussion during this study. The technical assistance of Mr. J. Kiarie is gratefully appreciated. This is ILRAD Publication No. 458.

REFERENCES Bailey,K.P., 1960.Noteson the rearingof Rh~picephalus appendiculatus and their infectionwith Thederia parva for experimentaltransmission. Bull. Epizoot. Dis. Aft., 8: 33-43.

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