Trap. Anim. Hlth Prod. (1979) 11, 87-90

INFECTIVITY OF PREPARED FROM

H Y A L O M M A

G R O U N D - U P T I C K S U P E R N A T E S T H E I L E R I A A N N U L A TA I N F E C T E D A N A T O L I C U M AlVA T O L I C U M

D. K : SINGH, 1 S. JAGDISH, 2 0 . P. GAUTAM and S. DHAR

Department of Veterinary Medicine, Haryana Agricultural University, Hissar, India

SUMMARY

Groups ofTheileria annulata infected Hyalomma anatolicum anatolicum adults prefed on a calf for 1 to 6 days were separately ground in tissue culture medium- 199, supplemented with bovine albumin powder, Fraction-V. Supernatant fluid was collected, made up with additional medium, so that each mtThTitre represented material from 25 ticks, and was injected subcutaneously into groups of cross-bred male cah, es. The results indicated that ground-up tick supernate (GUTS) prepared from unfed ticks was not infective, whereas that prepared from 1 to 6 days prefed ticks was infective. GUTS prepared from 3 days prefed ticks appeared to contain highest infectivity and I ml of it induced fatal theileriosis.

INTRODUCTION

In routine tick transmission of theileriosis neither infective dose nor time of infection is determined (Wilde, 1975). However subcutaneous injections of ground-up tick supernates prepared from Theileria parva infected and prefed Rhipicephalus appendiculatus have been used by several workers for experimental transmission of East Coast fever (Cunningham, Brown, Burridge, Irvin, Purnell and Radley, 1973; Radley, Brown, Burridge, Cunningham, Kirmi, Purnell and Young, 1975). Such material cryopreserved as stabilates makes uniform production of the disease possible for extended periods of time and simplifies the experimental procedure.

As regards bovine tropical theileriosis (T. annulata infection), reports are available, regarding the. necessity for prefeeding the ticks as a prelude to preparing infective ground-up tick supernates (GUTS) from some of its vectors such as, Hyalomma dromedarii, H. excavatum and H. detritum (Mazlum, 1969; Samish and Pipano, 1976). Some of these have given varying views on this aspect of the infectivity of species of Hyalomma (Bhattacharyulu, Chaudhry and Gill, 1975a; Shrivastava and Sharma, 1978). The present report summarises the results of studies carried out with GUTS prepared from T. annulata infected H. a. anatolicum adults prefed for variable periods of time.

MATERIALS AND METHODS Animals

Fourteen calves divided into 7 pairs were used for the present study. The calves were crosses of Hariana with European breeds (8os indicus x Bos taurus). At the time of experimental infection they were approximately 2 months old, reared under tick-free conditions since birth and free from Theileria infection microscopically as well as serologically.

Present addresses: i Department of Veterinary Medicine, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, India. 2 Department of Pharmacology, Bombay Veterinary College, Pard, Bombay, India.

87

88 SINGH, JAGDISH, GAUTAM AND DHAR

Parasite A virulent strain of T. annulata isolated locally and maintained cyclically in H. a.

anatolicum nymphs and adults and susceptible calves was used in the present study.

Preparation of ground-up tick supernates Hungry nymphs from clean colonies of H. a. anatolicum, were allowed to engorge

on a splenectomised T. annulata positive calf having 4 ~o infected erythrocytes. Follow- ing moulting, nearly 400 adult ticks were attached for prefeeding in ear bags on a T. annulata negative calf. Later, batches of 50 ticks each (equal numbers of males and females) were detached manually at 24 h intervals and were kept for a few minutes at 4°C followed by trituration with glasswool powder in a precooled mortar with 5 ml of medium-199 s supplemented with 3-5 ~ bovine albumin powder (Armour Fraction V) and antibiotics (benzyl penicillin, 100 iu/ml) and streptomycin sulphate (100/~g/ml). After making a fine paste, the material was transferred into a test tube and the mortar and pestle were rinsed thoroughly with additional 5 ml of medium. The tube was allowed to stand for I h at 4°C. Two millilitres of supernatant fluid were separated and 1 ml (equivalent to the material from 25 ticks) was injected subcutaneously in the right mid-neck region of the calves. GUTS were also prepared from T. annulata infected but unfed ticks in the same way. Results were thus obtained from GUTS made from 1 to 6 days prefed 14. a. anatolicum adults and from infected but unfed ticks injected into calves.

Parameters The inoculated calves were observed for time and duration of enlargement of

prescapular lymph nodes, rectal temperature and percentage parasitaemia. Necropsy was conducted in all animals which died. Antibody response of the surviving calves was assessed on day 40 by the indirect fluorescent antibody (IFA) test as described by Burridge (1971) and Dhar and Gautam (1977). Later they were challenged on the 48th day post-inoculation by the attachment of 30 presumed infective H. a. anatolicum adults.

RESULTS

The course of infection in different groups of calves is summarised in Table I. All the calves showed enlargement of the right prescapular lymph node in 3 to

6 days. The enlargement was slight and subsided in 2 days in calves injected with GUTS prepared from infected but unfed ticks, whereas it was marked and persisted for average periods of 15-5, 16-5, 17"5, 12.5 and 6 days in the calves injected with GUTS prepared from ticks prefed for 1, 2, 3, 4, 5 and 6 days, respectively.

The calves injected with GUTS prepared from 1 to 5 days prefed ticks exhibited a febrile reaction, which persisted for variable periods of time. Highest parasitaemias (8 and 28 ~) were observed in the calves injected with GILTS prepared from 3 days prefed ticks and the lowest (< 1 ~o) in the calves injected with the GUTS prepared from 6 days prefed ticks. No piroplasms were observed in the calves injected with the GUTS prepared from unfed ticks. Both the calves injected with the GUTS prepared from 3 days prefed ticks and 1 out of 2 injected with GUTS prepared from 2 days prefed ticks died of theileriosis. On post-mortem examination of these calves typical lesions of theileriosis were seen. All the surviving calves of groups injected with GUTS prepared from 1 to 6 days prefed ticks showed IFA titres of 1:640 to

a M/s Bios, Bombay, India.

INFECTIVITY OF GUTS SUPERNATES 89

TABLE I

Reaction of cross-bred calves inoculated with Theiteria annulata infected ground-up tick supernate (GUTS) prepared from ticks prefed for varying periods of time

Duration of prefeeding of ticks on a calf (days)

Parameters Unfed 1 2 3 4 5 6

Mean max. temperature (°C) Mean duration of temperature (days) Max. parasitaemia (%) Mean max. parasitaemia (%) Mean time to lymph node

enlargement (days) Mean duration of appearance of

piroplasms (days) Mean duration of lymph node

enlargement (days) Number died Mean IFA titre (40 days

post-infection) Result of infective tick challenge

38-8 39.8 40-4 41.3 40.6 39"9 39-1 0"0 5"0 10.5 12"5 8"0 2"5 0 .0 0 .0 1"5-<1 3-4 8-28 2-5 1-3 Rare 0 .0 < 1 . 0 3-5 18.0 3.5 2.0 Rare 6-0 3"5 4.5 4"5 5"5 5"5 6.0

- v e 13"5 12-5 11 "0 12"0 14"5 15.0

2"0 15.5 16.5 19"0 17"5 12"5 6"0

0/2 0/2 1/2 2/2 0/2 0/2 0/2 --ve 1:640 1:1280 - - 1:960 1:960 1:960

Suscep- Immune Immune tible I

Immune Immune Immune

1 Both the calves died on challenge due to typical theileriosis.

1:1280 and were found immune to 30-tick challenge, whereas calves inoculated with GUTS of unfed ticks did not show any IFA titre and died on challenge showing all the symptoms indicative of theileriosis. The calf used for prefeeding of ticks also developed typical theileriosis and died on 18th day post-infection.

DISCUSSION

From the results it appears possible, within the limits set by the small numbers of calves used, that the GUTS prepared from T. annulata infected but unfed H. a. anatolicum was not infective to calves. The slight lymph node enlargement in these calves might have been due to tick tissue material and not to Theileria infection as the swelling subsided within 2 days. This suggests that, for the development of the infective stages of T. annulata, prefeeding of ticks is necessary. This finding is in agreement with the morphological studies made by Bhattacharyutu, Chaudhry and Gill (1975b) who failed to observe stages of 7". annulata in the salivary glands of infected but unfed adults of H. a. anatolicum. Transmission studies carded out by Samish and Pipano (1976) with the crushed preparations obtained from T. annulata infected H. detritum and H. excavatum also agree with the present findings. However, this observation is contrary to that of Mazlum (1969) and Shrivastava and Sharma (1978) and others who claimed to transmit T. annulata successfully through unfed H. drome- darii and H. a. anatolicum respectively. Their claims call for further work since Samish (1977) is of the opinion that though infective stages of T. annulata in H. excavatum can develop without blood meal the ticks have to be incubated for 6 days at 37°C and 95% rh. It is possible that infected ticks in the experiments of Mazlum (1969) and Shrivastava and Sharma 0978) might have been exposed inadvertently to a higher temperature resulting in the development of infective stages of T. annulata though from their protocols this seems unlikely. However it seems possible that the process of feeding or of incubating at a higher temperature might provide some stimulus which enables the parasite in its vector to develop into infective stages.

90 SINGH, JAGDISH, GAUTAM AND DHAR

The results also suggest that the opt imum period o f prefeeding the ticks in the conditions pertaining in this experiment is 3 days in order to prepare the G U T S o f highest infectivity since both the calves inoculated with 3 days prefed G U T S died o f acute theileriosis (Table I). The other pairs were less severely affected. This was probably because in ticks prefed for the shorter duration, only a few parasites might have attained maturi ty and in ticks removed after 3 days o f prefeeding some o f the matured parasites might have escaped into the calf used for prefeeding as this calf died o f acute theileriosis on day 18.

I t is felt that subcutaneous injection o f G U T S prepared f rom 72 h prefed T. annulata infected H. a. anatolicum is to be preferred over usual tick a t tachment for experimental transmission o f tropical theileriosis. The method provides a degree of precision o f time and infection dose and it also reduces the necessity for regular monitoring o f the experimental animals.

Accepted for publication September 1978

REFERENCES

BHATTACHARYULU, Y., CHAUDHRY, R. P. & GILL, B. S. (1975a). Parasitology, 71, 1-7. BH^TrACHAItYULU, Y., CHAUDHRY, R. P, & GILL, B. S. (1975b). Annales de Parasitologie, 50, 397-408. BURRIDGE, M. J. (1971). Research in Veterinary Science, 12, 338. CUNNINOHAM, M. P., BROWN, C. G. D., BUR~UOOE, M. J., IRVIN, A. D., PURNELt., R. E. & R^DLEY,

D. E. (1973). Research in Veterinary Science, 15, 263-265. DHAR, S. & GAUTAM, O. P. (1977). Indian Journal of Animal Science, 47, 720-723. MAZLUM, Z. (1969). Parasitology, $9, 597-600. RADLEY, D. E., BROWN, C. G. D., BURRIIX;E, M. J., CUNNINGHAM, M. P., KntMl, I. M., PURNELL, R. E.

& YOUNG, A. S. (1975). Veterinary Parasitology, I, 35-41. SAMtSH, M. (1977). Nature, 270, 51-52. SAMISH, M. & PZPANO, E. (1976). Transmission of Theileria anmdata by two host and three host ticks

of genus Hyalomma. PrOc. Int. Conf. Tick-borne Diseases and Their Vectors, Centre Trop. Vet. Med., Univ. of Edinburgh, 1976.

SHRIVASTAVA, P. S, & SHARMA, N. N. (1978). Veterinary Parasitology, 4, 83-89. WILDE, J. K. H. (1975). Theileria and Theileriasis in East Africa and Mediterranean area. Prec. 29th

World Vet. Congr., 1975. Thessaloniki, Greece, I, pp. 481--486.

INFECTIOSITE DU SURNAGEANT D'UNE CULTURE PREPAREE A PARTIR DE THEILERIA ANNULATA INFECTEES PAR H YALOMMA ANATOLICUM ANATOLICUM

Rqhmm~,--Des tiques Hyalomma anatoiicum anatolicum adultes infect6es par Theilerla anmdata et pr~nourries de 1 6 6 jours sur un veau ont ~t~ broy6:s s~par~ment en milieu de culture de tissu 199 additionn~ avcc de la poudre d'albumine bovine (Fraction V).

Le surnageant a ~t6 r~olt~, compl6t~ avec du milieu de fac~on que chaque ml repr~ente le materiel issu de 25 tiques et inject6 par vole sous-cutan~/~ des groupes de veaux males m~tis. Les r~sultats ont montr6 que le surnageant du broyat (GUTS) pr~par6 avec des tiques/t jeun (non nourries) n'~tait pas infcctieux alors que celui pr~par6 avec les tiques nourries entre 1 et 6 jours ~tait infectieux. Le GUTS pr~par6 avec des tiques nourries pendant 3 jours est apparu etre le plus hautement infectieux, 1 ml provoquant une theileriose mortelle.

INFECTIVIDAD DE MACERADOS DE GARRAPATAS ADULTAS H YALOMMA ANATOLICUM ANATOLICUM INFECTADAS CON THEILERIA ANNULATA

ResumenwGrupos de garrapatas adult.as Hyalomma anatolicum anatolicum infeetadas con Theileria annulata y prealimentadas pot perlodos de uno a seis dias, se maceraron separadamente en cultivo de c~lulas medio-199, suplementado con albfimina bovina en polvo y Fracci6n V. El liquido sobrenadante se colect6 adieionandole rruts medio, de manera que cada mL representara material de 25 garrapatas y se inyect6 subcutaneamente en grupos de terneros mestizos. Los resultados indicaron que el sobrenadante proveniente de garrapatas sin alimentar fue in6cuo, mientras que el preparado de garrapatas prealimentadas pot periodos de uno a seis dias fue infectivo. Los sobrenadantes preparados de garrapatas prealimentadas pot periodos de tres dias fueron les m~ infectivos; un ml. indt~jo teileriosis fatal.