Veterinary Parasitology, 4 (1978) 83--89 83 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

STUDIES ON THE I N F E C T I V I T Y OF T H E I L E R I A A N N U L A T A I N F E C T E D NYMPHS, A D U L T S AND G R O U N D TISSUES O F THE TICK H Y A L O M M A A N A T O L I C U M t

P.S. SRIVASTAVA* and N.N. SHARMA

Division of Parasitology, Indian Veterinary Research Institute, Izatnagar -243122 (India)

tSupported by a grant from the Department of Atomic Energy, Government of India and carried out while the senior author was a research fellow.

*Present address: Department of Parasitology, College of Veterinary Sciences, Govind Ballabh Pant University of Agriculture and Technology, Pantnager-263145 (India).

(Received 24 November 1976)

ABSTRACT

Srivastava, P.S. and Sharma, N.N., 1978. Studies on the infectivity of Theileria annulata infected nymphs, adults and ground tissues of the tick Hyalomma anatolicum. Vet. Parasitol., 4: 83--89.

The infectivity of unfed nymphs, adults and ground tissue suspensions of the cattle tick, Hyalomma anatolicura, infected with Theileria annulata was investigated using sus- ceptible calves. Sporozoites of T. annulata introduced into the host by the above mentioned tick stages were able to induce clinical theileriasis in calves. The nature of the disease was the same, whether it was induced by sporozoites injected via 200 infected nymphs or 20 adults (both sexes in equal numbers). This suggests a direct correlation between the infectivity of the ticks and the volume of T. annulata-infected blood sucked by them during their preceding instars.

INTRODUCTION

The catt le t ick, H y a l o m m a ( H y a l o m m a ) anato l icum ana to l i cum Koch, 1844, is considered to be the principal vec tor of bovine theileriasis (Theileria annulata infect ion) in India (Bha t t acha ryu lu et al., 1975). However , there is di f ference o f op in ion on the compara t ive infect ivi ty o f sporozoi tes injected into the cat t le t h rough the n y m p h a l and adult t icks and t ick tissue suspen- sions o f infected, unfed adults. Accord ing to R e i c h e n o w (1940) , n y m p h a l t icks derived f rom larvae fed on an infected beast ha rbour more theilerial particles o f T. parva than unfed adults ob ta ined f rom n y m p h s engorged on the same animal. This hypo thes i s was later disproved by histological evidence in T. parva- in fec ted Rh ip icepha lus appendicu la tus (Purnell et al., 1971) . Excep t fo r the repor t of Hadani et al. (1963) , the major i ty o f the investigators opined tha t bo th n y m p h a l and adul t H. ana to l icum t icks (Delpy, 1949;

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Daubney and Sami Said, 1951; Bhattacharyulu et al., 1975), and crushed infected unfed adults of H. dromedarii (Mazlum, 1969), were equally capable of transmitting T. annulata infection in cattle. The present report describes the results of our preliminary experiments on the infectivity of T. annulata-infected intact tick stages and ground tick tissues in susceptible crossbred calves.

MATERIALS AND METHODS

Experimental animals

The cattle used were high grade crossbred male calves, aged 1- -8 weeks, procured from the Indian Veterinary Research Institute, Izatnagar and the Military dairy farm, Bareilly, where strict tick-control measures were prac- tised. The experimental calves were kept under tick-free conditions. Labora- tory-bred, healthy, New Zealand white rabbits, preferably virgin females and young males were used for tick feeding, as described by Srivastava and Sharma (1976a, b; 1977a).

The strain o f T. annulata

The strain of T. annulata used was a locally isolated, virulent strain, main- tained through alternate passages in calves, and larval/nymphal stages of the tick, H. anatolicum, for 1 year (Srivastava and Sharma, 1976c).

The ticks

The ticks used for infectivity trials were from a laboratory-bred colony of unfed nymphal /adul t H. anatolicum. During their preceding stages (larvae/ nymphs), these ticks had been allowed to engorge on T. annulata-infected cattle showing erythrocytic parasitaemia in excess of 20%. In the laboratory, the tick stages were maintained at a temperature of 28 + 2°C and 80 + 2% relative humidi ty (Srivastava and Sharma, 1976b).

Preparation of infected ground tick tissues

One hundred nymphs of H. anatolicum were applied to a T. annulata-in- fected calf showing erythrocyt ic parasitaemia from 20 to 25%. The engorged nymphs were collected and kept in the laboratory until they moul ted into adults. Out of these ticks, three groups of 10 pairs each (both sexes in equal number) were made. These were thoroughly crushed in three separate, sterilized, glass pestles and mortars in 10 ml of sterile, phosphate-buffered, saline (PBS), pH 7.4. The suspension was then sieved through fine muslin cloth. The final volume of the sieved suspension was adjusted to 15 ml,

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by addition of PBS. After addition of penicillin (50 units/ml) and streptomy- cin (5 pg]ml ) to prevent secondary bacterial growth, the suspension was stored at 4°C in rubber-stoppered glass vials (Srivastava and Sharma, 1977b). This material was injected subcutaneously, behind the base of the pinnae of the right ears of susceptible calves, within 4 hours of its preparation.

Experimental design

A total of 10, crossbred, male calves was divided into four groups (T1, T2, T3 and T4 ). Each animal of group T~ (Nos 2 and 101) and T2 (Nos 70 and 135) was exposed to 50 and 200 infected nymphs, respectively, while those of group T3 (Nos 31, 33 and 35) were exposed to 10 pairs of unfed infected adult ticks. Similarly, calves of group T4 (Nos 242, 243 and 246) were each injected with a crushed tissue suspension of 20 ticks (10 males and 10 females).

Clinical and parasitological observations

Following introduction of infection, morning rectal temperature was recorded daily. Biopsy smears from pre-scapular lymph nodes, as well as smears from peripheral blood, were prepared daily for 5 weeks, from the 3rd day onwards. These were stained with Giemsa, and examined for the pre- sence of schizonts and erythrocytic stages of T. annulata. Clinical reactions, such as enlargement of parotid and pre-scapular lymph nodes, digestive disturbances and respiratory distress were also recorded when observed.

The criteria for the establishment of infection were the detection of schi- zonts in lymph node biopsy materials and in peripheral blood smears, the degree of clinical response and mortalities in experimental calves.

RESULTS

The results of the experiments are summarized in Table I. The calves of group T1 (Nos 2 and 101), in which the infection (sporozoites of T. annu- lata) was introduced by applying 50 infected, unfed nymphs, showed the presence of schizonts in the peripheral blood on the 5th and 12th days, and erythrocytic stages (piroplasms) on the 18th and 15th days, respectively, after exposure to infected ticks. The peak parasitaemia in these calves did not exceed 7% of schizonts and 18% of piroplasms. During the initial reac- tion period, i.e. 6th--15th day after application of ticks, both these calves exhibited an elevated body temperature (max. 41.0°C) and lacrimation from both eyes. After a brief reaction period of 10--15 days, their body tempera- tures returned to 39°C (normal) and the parasitaemia declined to less than 1%, resulting in clinical recovery of both calves.

Subsequently, another group (T2) of calves (Nos 70 and 135) were ex- posed to 200 infected, unfed nymphs. Calf No. 135 exhibited a moderate

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type of reaction, showing highest lymphocytic and erythrocytic parasitaemia of 10 and 23%, respectively, followed by clinical recovery. The reactions in the other calf (No. 70) were acute, resulting in death, after an illness lasting over a month. The maximum parasitaemia recorded in calf No. 70 was 18% of schizonts and 25% of piroplasms on the 15th and 24th days, respectively, after the application of infected nymphs. The three clinically recovered cal- ves (Nos. 2, 101 and 135) continued to harbour a low-level erythrocytic parasitaemia (less than 1%), with the occasional appearance of schizonts in their blood smears.

The calves of group T3 (Nos. 31, 33 and 35), exposed to 20 infected, un- fed adults reacted very severely, showing peak parasitaemia of 12--18% of schizonts between the 9th and 12th days, and 27--40% of erythrocytic piro- plasms between the 17th and 21st days, after the application of ticks, respectively. All the calves exhibited acute febrile reactions, i.e. profuse lacrimation and salivation, and a steep rise in body temperature (maximum 41.7°C) resulting in their deaths. Similarly, calves of group T4 (Nos. 242, 243 and 246), inoculated with infected, crushed tick tissue suspensions, also exhibited acute febrile reactions, with maximum body temperatures varying from 41.2 to 41.8°C. Three to 4 days prior to this peak thermal reaction (be- tween 20--25 days after inoculation), the maximum parasitaemia of 10% of schizonts and 24---33% of erythrocytic piroplasms was observed in their peri- pheral blood smears. All the calves died between 26 and 38 days after inocu- lation of infected tick tissue suspensions as a result of severe respiratory dis- tress and pneumonia. On autopsy, marked characteristic ulcerations in abomasal and intestinal mucosa, together with enlargement of parotid and pre-scapular lymph nodes and spleen, were observed.

DISCUSSION

The results of the present experiments demonstrate that T. annulata-in- fected, unfed nymphs and adults, as well as crushed tick tissue suspensions, are capable of inducing clinical theileriasis in calves. However, the degree of clinical reactions produced by the application of 50 infected, unfed nymphs (group T1) to calves was comparatively milder than that seen in calves ex- posed to 20 (10 males and 10 females) infected, adults ticks (group T3). The clinical reactions of calves in group T3 were similar to those in group T2 (exposed to 200 unfed nymphs). The present results agree with those of Sergent et al. (1936a, b) for T. annulata, and Purnell and Joyner (1967, 1968) and Purnell et al. (1973) for T. parva infections. These authors con- sidered that a comparatively greater number of nymphs was required for the production of an identical disease to that induced by adult ticks. This also confirms the observations of Delpy (1949) and Daubney and Sami Said (1951), who reported variable degrees of reaction in calves, after their exposures to T. annulata-infected nymphs and adults. However, the present observations differ from those reported by Hadani et al. (1963), who were unable to transmit T. annulata infections to calves through nymphs derived from infected larvae.

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The results obtained with crushed infected tick tissues (group T4 ) were similar to those observed after exposure to infected, unfed adults. However, the prolongation of incubation and reaction periods in infections induced by sporozoites injected through tick tissue suspensions indicates that perhaps some of the sporozoites were damaged during preparation of the suspension. The present results are in agreement with those of Mazlum (1969}, who suc- cessfully transmitted T. annulata to cattle through inoculation of unfed, crushed, infected tick tissues of H. dromedarii.

ACKNOWLEDGEMENTS

We are thankful to the Director, Indian Veterinary Research Institute, Izatnagar and the Head of the Division of Parasitology, for the facilities pro- vided. We are also grateful to Dr H.C. Tewari, Officer-in-charge, Irradiated Vaccine laboratory.

REFERENCES

Bhattacharyulu, Y., Chaudhri, R.P. and Gill, B.S., 1975. Transstadial transmission of Theileria annulata through common ixodid ticks infesting Indian cattle. Parasitology, 71: 1--7.

Daubney, R. and Sami Said, M., 1951. Egyptian fever of cattle: the transmission of Theileria annulata (Dschunskowsky and Luhs, 1904) by Hyalomma excavatum Koch, 1844. Parasitology, 41 : 249--260.

Delpy, L.P., 1949. Essai critique de synonymic du genre Hyalomma C.L. Koch, 1844. Ann. Parasitol Hum. Comp., 24: 464--494.

Hadani, A., Tsur, I., Pipano, E. and Senft, Z., 1963. Studies on the transmission of Theileria annulata by ticks (Ixodoidea, Ixodidae). I. Hyalomma excavatum. J. Proto- zool., 10 (Suppl): 35.

Mazlum, Z., 1969. Transmission of Theileria annulata by the crushed, infected, unfed Hyalomma dromedarii. Parasitology, 50: 597--600.

Purnell, R.E. and Joyner, L.P., 1967. Artifical feeding technique for Rhipicephalus appendiculatus and the transmission of Theileria parva from the salivary secretion. Nature (London), 216 : 494--485.

Purnell, R.E. and Joyner, L.P., 1968. The development of Theileria parva in the salivary glands of the tick, Rhipicephalus appendiculatus. Parasitology, 58: 725--732.

Purnell, R.E., Boater, C.D.H. and Peirce, M.A., 1971. Theileria parva: Comparative infec- tion rates of adult and nymphal Rhipicephalus appendiculatus. Parasitology, 62 : 349--353.

Purnell, R.E., Brown, C.G.D., Cunningham, M.P., Burridge, M.J., Kirimi, I.M. and Ledger, M.A., 1973. East Coast fever: correlation between the morphology and infectivity of Theileria parva developing in its tick vector. Parasitology, 66 : 539--544.

Reichenow, E., 1940. Der Entwicklungsgang des Kustenfiebererregers im Rinde und in der Ubertragenden Zecke. Arch. Protistenkd., 94: 1--56.

Sergent, E., Donatien, A., Parrot, L. and Lestoquard, F., 1936 a. Etude morpholgique due cycle ecolutif de Theileria dispar chez le boeuf et chez la tique. Ann. Inst. Pasteur, Paris, 57: 30--35.

Sergent, E., Donation, A., Parrot, L. and Lestoquard, F., 1936 b. Cycle evolutif de Theileria dispar du boeuf chez la tique Hyalomma mauritanicum. Arch. Inst Pasteur Alger, 14: 259--294.

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Srivastava, P.S. and Sharma, N.N., 1976 a. Effects of 6°Co irradiation on the early developmental stages of an ixodid tick, Hyalomma anatolicum. Int. J. Radiat. Biol., 29: 159--168.

Srivastava, P.S. and Sharma, N.N., 1976 b. On the rearing of Hyalomma (Hyalomma) anatolicum anatolicum Koch, 1844 and their infection with Theileria annulata (Dschunskowsky and Luhs, 1904). Indian J. Anim. Res., 10: 21--26.

Srivastava, P.S. and Sharma, N.N., 1976 c. Characteristics of a tick transmitted virulent strain of Theileria annulata (Dschunskowsky and Luhs, 1904) in crossbred calves. Pantnagar J. Res., 1: 83--88.

Srivastava, P.S. and Sharma, N.N., 1977 a. A simple, economical and improved plastic collar for restraining rabbits during tick feeding. Lab. Anita, 11: 59.

Srivastava, P.S. and Sharma, N.N., 1977 b. Potential of immunoprophylaxis using 6°Co irradiated Theileria annulata in salivary gland suspensions of the tick Hyalomma ana- tolicum. Vet. Parasitol., 3: 183--188.

RESUME

Srivastava, P.S. et Sharma, N.N., 1978. Etudes sur l'infectivit~ des nymphes, adultes et suspensions du tissu de la tique Hyalornma anatolicum infect~e par Theileria annulata. Vet. Parasitol., 4 :83- -89 (en anglais).

L'infectivit4 des nymphes affam~s et adultes affam~s et suspensions du tissu des tiques bovines, Hyalomma anatolicum, infect~es par Theileria annulata etait investig~e par usage des veaux susceptibles. Sporozoites des T. annulata inocul~s dans l 'host par stades diff~rantes des tiques induient la maladie theileriose en form clinicale dans les veaux. La nature identique de la maladie induit par sporozoites inocul~s par 200 nymphes infect~es ou 20 adultes (m~le et femelle, les deux en ~galit~) sugg~t la correlation directe entre l'infectivit~ des tiques et la volume du sang infect~ par T. annulata suc~ par eux dans leurs stades precedents.

KURZFASSUNG

Srivastava, P.S. und Sharma, N.N., 1978. Zur Ansteckungskeit von mit Theileria annulata infizierten Nymphen, Erwachsenen und zermahlte Gewebe der Zecken Hyalomma anatolicum. Vet. Parasitol., 4:83- -89 (in Englisch).

Die Ansteckungskeit der ungefutterten Nymphen, Erwachsenen und zermahlten Gewebesuspensionen der mit Theileria annulata infizierten Viehzecken Hyalomma ana- tolicum wurde unter Beniitzung der empf~nglichen K~lber untersucht. Sporozoiten yon T. annulata in den Wirt durch die obenerw~hnten Zeckenstufen eingefiihrt, kSnnten in der K~flber klinischen Theileriasis induzieren. Die identische Art der Krankheit induziert dutch Sporozoiten eingespritz durch 200 infizierten Nymphen oder 20 Erwachsenen (beide Geschlechter in gleicher Zahl) deutet eine direkte Korrelation zwischen der Anstechungskeit den Zecken und dem Volumen yon T. annulata infizierte Blut geschliickt von ihnen w~'hrend ihren fr~heren Entwichlungsstufen an.