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Transmission of a Trypanosoma sp. to cattle by the tick Hyalomma anatolicum anatolicum
Veterinary Parasitology, 19 (1986) 1 3 - 2 1 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
13
T R A N S M I S S I O N OF A T R Y P A N O S O M A SP. TO CATTLE BY THE TICK HYALOMMA ANA TOLICUM ANA TOLICUM
S.P. M O R Z A R I A a, A.A. L A T I F b, F. JONGEJAN c and A.R. WALKER d
FAO Tick and Tick-borne Diseases Control Project', P.O. Box 1117, Khartoum (Sudan) (Accepted for publication 28 February 1985)
ABSTRACT Morzaria, S.P., Latif, A.A., Jongejan, F. and Walker, A.R., 1986. Transmission of a Trypanosorna sp. to cattle by the tick Hyalomma anatolicum anatolicum. Vet. Parasitol., 19: 13--21.
Hyalomma anatolicum anatolicum engorged nymphs and flat adults were collected from two areas in northern Sudan. Various developing stages of Trypanosoma theilerilike flagellates were observed in the engorged nymphs, freshly moulted adults and mature adults partially engorged on rabbits. When these ticks were applied to two calves, one calf became infected with the trypanosome. The parasites were observed for one day in the enlarged lymph node nearest to the tick-feeding site 5 days after the tick application. Subsequently the trypanosomes were re-isolated in vitro from the infected calf. Inoculation of a ground-up tick supernatant suspension from the infected batch of ticks containing 104 trypanosomes into a calf did not produce a patent infection.
INTRODUCTION The chief vectors of the trypanosomes pathogenic for cattle are tsetse flies. I n a r e a s w h e r e t s e t s e f l i e s d o n o t o c c u r , b i t i n g f l i e s , m a i n l y s t o m o x i d s and tabanids are considered to be responsible for transmission and maintenance of infection. Hard ticks, which are vectors of economically important sporozoan blood p a r a s i t e s o f c a t t l e , h a v e n e v e r b e e n i n c r i m i n a t e d as v e c t o r s o f c a t t l e t r y p a n o somes (Hoare, 1972), although many reports of the presence of developing forms of Trypanosoma spp. have been published (O'Farrell, 1913; Carpano, 1 9 3 2; A r i f d z h a n o v a n d N i k i t i n a , 1 9 6 1 ; B u r g d o r f e r e t al., 1 9 7 3 ; K r i n s k y a n d apermanent address for reprints: International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya. bInternational Centre for Insect Physiology and Ecology, Nairobi, Kenya. CUNDP/FAO Project ZAM/77/002, Animal Diseases Centre, Lusaka, Zambia. dCentre for Tropical Veterinary Medicine, Roslin, Midlothian, Gt. Britain. 1Project GCP/SUD/024/DEN, supported by the Danish International Development Agency and executed by the F o o d and Agriculture Organisation of the United Nations.
0304-4017/86/$03.50
© 1986 Elsevier Science Publishers B.V.
14 Burgdorfer, 1976; Kirmse and Taylor-Lewis, 1976; Lopez et al., 1979). Recently Shastri and Deshpande (1981) have described experiments which strongly suggest that the tick Hyalomma anatolicum anatolicum may be a vector of T. theiIeri. During studies on the epidemiology of tick-borne diseases in the Sudan between 1979 and 1982 large numbers of H.a. anatolicum were examined for the presence of theilerial parasites. During the course of this work many ticks were found to be heavily infected with various developing stages of trypanosomes. The regularity and the intensity of the trypanosome infection led to the suspicion that this might n o t be an u n c o m m o n phenomenon, and subsequently further studies were initiated. This paper describes the morphology of the trypanosome and its successful transmission to a calf by H.a. anatolicum. MATERIALS AND METHODS Tick collection and examination Ticks were collected from two farms located in the semi-desert of Sudan: the University of Khartoum, Shambat Farm (15°40'N, 32°35'E), 10 km northeast of Khartoum, and the University of Gezira, Nisheishiba Farm (14°28'N, 33°30'E), 200 km southeast of Khartoum. Engorged nymphs, and occasionally adult ticks, were collected from cattle pens. The ticks were mostly found in crevices between bricks on the drinking and feeding troughs, or trapped in cobwebs f o u n d in large numbers within the hollow metal tubular frames supporting the pens. Occasionally ticks were found underneath dried cattle dung. Engorged n y m p h s were maintained in the laboratory at 28°C and 75% relative h u m i d i t y (RH) until moulted and then identified and transferred to 20°C and 75% RH together with unfed adults collected in the field. In the experiments described below only H.a. anatolicum were used. After collection, and at variable periods during and after moulting, engorged nymphs were punctured with a fine needle and the haemolymph, occasionally with the gut extruding from the wound, was smeared on to a microscope slide. The smears were air-dried, fixed in methanol and stained in Giemsa for microscopic examination. Experimental cattle Young male Friesian and Zebu crossbred calves (1--3 days old) were purchased from a farm where strict acaricidal control of ticks is practised. The calves were reared in the laboratory in a tick-free environment and were used in experiments only when t h e y were found to be free of blood parasites and negative for Theileria annulata and Babesia bigemina antibodies by the indirect fluorescent antibody test (Morzaria et al., 1981).
15 During the experiments, blood smears were taken daily from calves and superficial lymph nodes were palpated. Whenever an enlarged lymph node was f o u n d a biopsy was taken and smeared on a microscope slide for Giemsa staining and examination of parasites.
Preparation o f trypanosome suspension from field-collected ticks A sample of H.a. anatolicum adults from Nisheishiba Farm was fed on a rabbit. Unattached ticks were discarded on day 1. On day 3 after application the ticks were removed and counted. Twenty females and 17 males were then processed to prepare suspensions of ground-up ticks in a tissue culture medium containing RPMI 1640, 20 mM HEPES (Flow Laboratories, Gt. Britain), bovine plasma albumin (Sigma, Gt. Britain) at 3.5% w/v, 200 i.u. ml -~ penicillin, 200 pgm1-1 streptomycin and 5 t~gml -~ fungizone. The procedure used was essentially similar to that described by Brown (1979) for the preparation of sterile Theileria sp. sporozoites except that filtration was through a 25 mm diameter Swinnex holder with a glass microfiber pre-filter and an 8 pm pore diameter cellulose ester high porosity membrane filter (Millipore type AP 25 and MF, respectively, Millipore Corporation, U.S.A.). The filtrate volume was adjusted to contain the equivalent of 4 ground-up ticks ml- 1 Wet preparations on a h a e m o c y t o m e t e r (Improved Neubauer) and Giemsastained cytospin smears (Cytospin 2, Shandon, at 550 r.p.m, for 10 min) of 50 pl samples of the suspension were examined for parasites. Trypanosomes at a concentration of 104m1-1 and Theileria sporozoites (32 per 40 microscope fields of 0.175 mm diameter with X 100 oil immersion objective) were seen. This suspension was used for infecting a susceptible bovine calf.
Infection o f cattle Attempts to induce infection in the experimental calves were carried out by either inoculation of the trypanosome suspension or by application of infected H.a. anatolicum adults. One calf (A19) was used for testing infectivity o f the suspension by inoculation of the suspension which contained 104 trypanosomes m1-1 (0.5 ml intravenously and 0.5 ml subcutaneously). Infectivity of the field ticks from Shambat and Nisheishiba, respectively, were tested by applying 12 H.a. anatolicum (six females and six males from each location) separately in earbags to each o f two calves (A47 and A48).
In vitro isolation o f trypanosomes from infected calf Using aseptic techniques, biopsies were taken from an infected lymph node using 38-mm disposable needles and immediately transferred to a growth medium (GM) containing 20 mM HEPES buffered RPMI 1640, 20%
16 heat-inactivated foetal calf serum penicillin (200 i.u. m1-1 ), streptomycin (200 ~ g m l -~) and fungizone (5 ~gml-~). The biopsy material was vigorously agitated in the medium with a pipette to break down large pieces of tissue and the resultant cell suspension was centrifuged at 400 g for 10 min. The pellet was then resuspended in GM and cells were dispensed into T-25 plastic tissue culture flasks, each containing 10 ml of 107 cells. The cultures were incubated at 37°C and 48 h later 2 ml of the GM was added to each culture. Thereafter cultures were replenished twice weekly: half the medium was removed and centrifuged (400 g for 10 min), the supernate was discarded and the pellet resuspended in fresh GM and returned to the culture flasks. All cultures were examined daily and Giemsa-stained cytospin smears from each of them were made twice weekly. RESULTS Tick examination
Ticks collected from both farms showed the presence of flagellates. In the first collection 1/49 of the adult ticks from Shambat and 2/55 from Nisheishiba were infected with flagellates. In the second collection 3/45 and 2/30 adult ticks from Shambat and Nisheishiba, respectively, were found to be infected. The origin of the flagellates in the cuticular-puncture smears could not be determined as the smears were often found to be contaminated with the contents of tick-gut and Malpighian tubules. Four different morphological forms were seen but the predominant forms were long, slender vacuolated epimastigotes with pointed ends, a free flagellum, a central nucleus and a club-shaped kinetoplast. They measured 20.1 pm in length (minimum 15.1 ~m and m a x i m u m 23.2 pm). Other forms seen were: (i) sphaeromastigotes (10.1 pm in length, minimum 7.1 ~m and maximum 13.5 pm); (ii) transition forms from epimastigotes to trypomastigotes with the kinetoptast in juxtaposition to the nucleus and measuring 25.1 pm (minimum 20.8 pm and maximum 28.1 pm); and (iii) trypomastigotes of 26.0 ~m in length (18.5 pm minimum and 32.0 pm maximum). Several dividing stages of the sphaeromastigotes and epimastigotes with two kinetoplasts, nuclei and flagellae were observed. Figures 1, 2 and 3 show various forms of the flagellate observed in the ticks examined. Intracellular forms were not detected. As selective tick organ smears were not prepared other locations of the flagellate development could n o t be determined. It was interesting to note that although the epimastigote stages predominated other stages were f o u n d simultaneously in the same smear. It was not possible to determine the species of the trypanosomes seen but the morphology appeared to be similar to that of T. theileri (Wells, 1971).
17
Q
O
Ib
Fig. 1. Epimastigote forms: the most c o m m o n l y found stage o f the flagellate in H.a. anat o l i e u m . A r r o w indicates a dividing form.
Fig. 2. Sphaeromastigotes in the tick h a e m o l y m p h . Fig. 3. T w o different stages: (a) a transition f o r m b e t w e e n epimastigote and trypomastigote stages and (b) a t r y p o m a s t i g o t e , seen in a h a e m o l y m p h smear o f an adult H.a. anatolicurn.
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Infection o f cattle Of the 3 calves used in the experiment (A19 inoculated with the trypanosome suspension, A47 and A48 infested with H.a. anatolicum from Shambat and Nisheishiba, respectively) only A48 showed trypanosomes. The parasites were seen in the enlarged lymph node nearest to the tickfeeding site on day 5 after tick application. The trypanosomes were only observed on one day. They were not detected in blood smears or in any other lymph nodes.
Figs. 4 a n d 5. L y m p h n o d e b i o p s y s m e a r o f a n i n f e c t e d calf s h o w i n g t r y p a n o s o m e s w i t h a l o n g free flagellum a n d an u n d u l a t i n g m e m b r a n e . N o t e close a s s o c i a t i o n o f t h e k i n e t o plast w i t h t h e parasite nucleus.
19 The size of the trypanosomes seen in the lymph node of A48 varied from 32.0 gm to 54.0 pm. All were slender trypomastigote forms with the kinetoplast invariably adjacent and posterior to the nucleus. The kinetoplast was difficult to detect in m a n y trypanosomes. The flagellum was long and free with a well-developed undulating membrane. It was not associated with the kinetoplast and appeared to emerge from the posterior end of the trypomastigote (Figs. 4 and 5). Approximately two trypanosomes were observed per 100 lymphocytes. Clean H.a. anatolicum nymphs were applied on two calves (A19 and A48) between days 9 and 13 after the start of the experiment. The resultant adult ticks (120) were examined for trypanosomes but were found to be negative. Long-term monitoring of the 3 experimental calves could n o t be carried out as all of them died of T. annulata infection 14--15 days after the start of the infection.
In vitro isolation o f the trypanosomes One day after the parasites were observed in the lymph node of A48, biopsy cultures were set up. Sixteen days later large numbers of organisms, predominantly amastigote forms of the flagellates, were observed. DISCUSSION Our observations confirm that various developing stages of T. theileri-like parasites are regularly found in adult H.a. anatolicum collected as engorged n y m p h s from the field. The experiments also indicate that the moulted ticks, when applied on a susceptible calf, can transmit the flagellates. This appears to be the first demonstration of biological transmission of a trypanosome by a tick. Several authors have reported the presence of various Trypanosoma spp. in hard as well as soft ticks. O'Farrell (1913) found crithidia-like flagellates in the cattle tick H. aegyptium which were considered by Wenyon (1926) to be forms of T. theileri and this was supported later by Carpano (1932) when he discovered an adult H.a. anatolicum to be infected with a similar flagellate. Arifdzhanov and Nikitina ( 1 9 6 1 ) r e p o r t e d that 25 out of 110 adult H.a. anatolicum collected from a cattle barn in Tashkent were infected with this parasite, indicating a high susceptibility of this tick to T. theileri. Burgdorfer et al. (1973) reported the presence of T. theileri in Rhipicephalus pulchellus and Boophilus decoloratus collected in Ethiopia. Krinsky and Burgdorfer (1976) reported ~T. theileri-like forms in A m b l y o m m a americanum collected from deer in the U.S.A. In a systematic study, Kirmse and Taylor-Lewis (1976) showed limited development of T. lewisi, T. musculi, T. congolense and T. vivax in hard and soft ticks for a variable period of time. Lopez et al. ( 1 9 7 9 ) i n South America demonstrated T. vivax in B. decoloratus. However, none of the above workers were able to successfully transmit trypanosomes to cattle using hard ticks as vectors.
20 A successful transstadial transmission of a T. thylacis by a hard tick, Ixodes tasmani, to an Australian marsupial, the short-nosed bandicoot, has been reported by Weilgama (1980), who found four developmental stages, amastigotes, sphaeromastigotes, epimastigotes and trypomastigotes, in the tick tissues. More recently, experiments by Shastri and Deshpande (1981) showed that infection with T. theileri could be established with H.a. anatolicum when a suspension of infected ticks was inoculated together with the application of infected ticks on susceptible calves. Although this does not confirm tick transmission the infectivity of tick-derived T. theileri for cattle is confirmed. Shashtri and Deshpande were n o t able to directly demonstrate trypanosomes in the infected calves but the transmission was confirmed by xenodiagnosis. The experiments in our study confirmed tick-transmission of Trypanosoma sp. by demonstration of the flagellate in a susceptible bovine host after application of a batch of ticks previously identified as infected. From these experiments it appears that the ticks acquire infections as nymphs from chronically infected cattle and transmit the parasite to susceptible calves as adults. Thus transstadial transmission appears to occur. Some workers have shown massive development of T. theileri in the ovaries of infected ticks (O'Farrell, 1913; Arifdzhanov and Nikitina, 1961; Burgdorfer et al., 1973; Krinsky and Burgdorfer, 1976) and therefore the possibility of transovarian transmission cannot be discounted. In the present study the flagellates were re-isolated in vitro after transmission by infected ticks to a calf. No trypanosomes were observed in the blood smears of the calf during a short period of examination. It was unfortunate that the experimental animals in the study died due to contaminating T. annulata infection. However, this was an expected outcome as Hoa. anatolicurn collected from the field are usually heavily infected with T. annulata {Walker et al., 1983). It was not possible to determine the species of trypanosome in the tick and the calf. The morphology of the flagellate in the tick appeared to be similar to that of the T. theileri-like parasite described by Burgdorfer et al. {1973) in A. americanum. The forms seen in the lymph node of the calf after application of the ticks do n o t conform to the typical forms of T. theileri described from the peripheral blood of the bovine host. However, these stages detected in the lymph node may represent the early developing forms and therefore may n o t be typical of T. theileri. At present there is no serological test available to detect T. theileri infection in a bovine host, and hence it was n o t possible to be absolutely certain that the experimental calves used in the present study were free of T. theileri before the experiment. The evidence of tick transmission must, therefore, be treated with caution and is based on the appearance of trypanosomes transiently for one day in a hyperplastic lymph node following application of a batch of ticks previously identified to be infected with the flagellates. Further studies are necessary to determine the identity of the flagellate described and its infectivity and pathogenicity to cattle through H.a. anatolicum.
21 ACKNOWLEDGEMENTS Thanks are due to the Ministry of Agriculture and Animal Resources (MAAR), Khartoum and Central Veterinary Research Laboratory, Soba, S u d a n f o r t h e f a c i l i t i e s p r o v i d e d . Dr. R . J . T a t c h e l l , D r , A . M . O s m a n , P r o f . G. U i l e n b u r g , D r . J.P. O v e r d u l v e a n d D r . P. G a r d i n e r a r e t h a n k e d f o r t h e i r h e l p f u l s u g g e s t i o n s . T h e p a p e r is p u b l i s h e d b y t h e k i n d p e r m i s s i o n o f t h e Under Secretary of the MAAR, Khartoum.
REFERENCES Arifdzhanov, K.A. and Nikitina, R.E., 1961. Detection of Crithidia hyalomma (O'Farrell 1913) in Hyalomma a. anatolicum (Koch 1944). Zool. Zh., 4 0 : 2 0 - - 2 4 (in Russian). Brown, C.G.D., 1979. Propagation of Theileria. In: R. Maramorosch and H. Hirumi (Editors), Practical Tissue Culture Applications. Academic Press, New York, pp. 223--254. Burgdorfer, W., Schmidt, M.L. and Hoogstraal, H., 1973. Detection of Trypanosoma theileri in Ethiopian cattle ticks. Acta Trop., 30: 340--346. Carpano, M., 1932. Localisations du Trypanosoma theileri dans les organes internes des bovins, son cycle 6volutif. Ann. Parasitol. Hum. Comp., 10: 305--322. Hoare, C.A., 1972. The Trypanosomes of Mammals, a Zoological Monograph. Blackwell Scientific Publications, Oxford/Edinburgh, pp. 30--56. Kirmse, P. and Taylor-Lewis, G., 1976. Ticks as possible vectors of trypanosomes. In: J.K.H. Wilde (Editor), Tick-borne Diseases and their Vectors. University of Edinburgh, pp. 177--180. Krinsky, W.L. and Burgdorfer, W., 1976. Trypanosomes in A m b l y o m m a americanum from Oklahoma. J. Parasitol., 62: 824--825. Lopez, V.G., Thompson, K.C. and Bazalar, H., 1979. Transmission experimentale de Trypanosoma vivax par la garrapata Boophilus microplus. Rev. Inst. Colomb. Agropec., 14 : 93--96. Morzaria, S.P., Mustafa Um E1 Hassan, Shawgi, M.H., Pedersen, V. and Osman, A.M., 1981. Isolation, identification and transmission of Theileria mutans in Northern Sudan. In: A.D. Irvin, M.P. Cunningham and A.S. Young (Editors), Advances in the Control of Theileriosis. Martinus Nijhoff Publishers, The Hague/Boston/London, pp. 166--168. O'Farrell, W.R., 1913. Hereditary infection with special reference to its occurrence in Hyalomma aegyptium infected with Crithidia hyalomma. Ann. Trop. Med. Parasitol., 1: 545--556. Shastri, U.V. and Deshpande, P.D., 1981. Hyalomma anatolicurn anatolicurn (Koch, 1844) as a possible vector for transmission of Trypanosoma theileri, Laveran, 1902 in cattle. Vet. Parasitol., 9 : 151--155. Walker, A.R., Latif, A.A., Morzaria, S.P. and Jongejan, F., 1983. Natural infection rates o f Hyalomma anatolicum anatolicum with Theileria in Sudan. Res. Vet. Sci., 35: 87--90. Weilgama, D.J., 1980. Cyclical development of trypanosomes in the tick and their transmission. In: O.P. Gautam, R.D. Sharma and S. Dhar (Editors), Haemoprotozoon Diseases of Domestic Animals. Proceedings of a Seminar, 27 October--1 November 1980, at Hissar, India, pp. 187--197. Wells, E.A., 1971. Studies on Trypanosoma theileri-like trypanosomes of cattle. Br. Vet. J., 127: 466--475. Wenyon, C.M., 1926. Protozoology. A Manual for Medical Men, Veterinarians and Zoologists. Bailli~re, Tindall and Cox, London, pp. 358--361.
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T R A N S M I S S I O N OF A T R Y P A N O S O M A SP. TO CATTLE BY THE TICK HYALOMMA ANA TOLICUM ANA TOLICUM
S.P. M O R Z A R I A a, A.A. L A T I F b, F. JONGEJAN c and A.R. WALKER d
FAO Tick and Tick-borne Diseases Control Project', P.O. Box 1117, Khartoum (Sudan) (Accepted for publication 28 February 1985)
ABSTRACT Morzaria, S.P., Latif, A.A., Jongejan, F. and Walker, A.R., 1986. Transmission of a Trypanosorna sp. to cattle by the tick Hyalomma anatolicum anatolicum. Vet. Parasitol., 19: 13--21.
Hyalomma anatolicum anatolicum engorged nymphs and flat adults were collected from two areas in northern Sudan. Various developing stages of Trypanosoma theilerilike flagellates were observed in the engorged nymphs, freshly moulted adults and mature adults partially engorged on rabbits. When these ticks were applied to two calves, one calf became infected with the trypanosome. The parasites were observed for one day in the enlarged lymph node nearest to the tick-feeding site 5 days after the tick application. Subsequently the trypanosomes were re-isolated in vitro from the infected calf. Inoculation of a ground-up tick supernatant suspension from the infected batch of ticks containing 104 trypanosomes into a calf did not produce a patent infection.
INTRODUCTION The chief vectors of the trypanosomes pathogenic for cattle are tsetse flies. I n a r e a s w h e r e t s e t s e f l i e s d o n o t o c c u r , b i t i n g f l i e s , m a i n l y s t o m o x i d s and tabanids are considered to be responsible for transmission and maintenance of infection. Hard ticks, which are vectors of economically important sporozoan blood p a r a s i t e s o f c a t t l e , h a v e n e v e r b e e n i n c r i m i n a t e d as v e c t o r s o f c a t t l e t r y p a n o somes (Hoare, 1972), although many reports of the presence of developing forms of Trypanosoma spp. have been published (O'Farrell, 1913; Carpano, 1 9 3 2; A r i f d z h a n o v a n d N i k i t i n a , 1 9 6 1 ; B u r g d o r f e r e t al., 1 9 7 3 ; K r i n s k y a n d apermanent address for reprints: International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya. bInternational Centre for Insect Physiology and Ecology, Nairobi, Kenya. CUNDP/FAO Project ZAM/77/002, Animal Diseases Centre, Lusaka, Zambia. dCentre for Tropical Veterinary Medicine, Roslin, Midlothian, Gt. Britain. 1Project GCP/SUD/024/DEN, supported by the Danish International Development Agency and executed by the F o o d and Agriculture Organisation of the United Nations.
0304-4017/86/$03.50
© 1986 Elsevier Science Publishers B.V.
14 Burgdorfer, 1976; Kirmse and Taylor-Lewis, 1976; Lopez et al., 1979). Recently Shastri and Deshpande (1981) have described experiments which strongly suggest that the tick Hyalomma anatolicum anatolicum may be a vector of T. theiIeri. During studies on the epidemiology of tick-borne diseases in the Sudan between 1979 and 1982 large numbers of H.a. anatolicum were examined for the presence of theilerial parasites. During the course of this work many ticks were found to be heavily infected with various developing stages of trypanosomes. The regularity and the intensity of the trypanosome infection led to the suspicion that this might n o t be an u n c o m m o n phenomenon, and subsequently further studies were initiated. This paper describes the morphology of the trypanosome and its successful transmission to a calf by H.a. anatolicum. MATERIALS AND METHODS Tick collection and examination Ticks were collected from two farms located in the semi-desert of Sudan: the University of Khartoum, Shambat Farm (15°40'N, 32°35'E), 10 km northeast of Khartoum, and the University of Gezira, Nisheishiba Farm (14°28'N, 33°30'E), 200 km southeast of Khartoum. Engorged nymphs, and occasionally adult ticks, were collected from cattle pens. The ticks were mostly found in crevices between bricks on the drinking and feeding troughs, or trapped in cobwebs f o u n d in large numbers within the hollow metal tubular frames supporting the pens. Occasionally ticks were found underneath dried cattle dung. Engorged n y m p h s were maintained in the laboratory at 28°C and 75% relative h u m i d i t y (RH) until moulted and then identified and transferred to 20°C and 75% RH together with unfed adults collected in the field. In the experiments described below only H.a. anatolicum were used. After collection, and at variable periods during and after moulting, engorged nymphs were punctured with a fine needle and the haemolymph, occasionally with the gut extruding from the wound, was smeared on to a microscope slide. The smears were air-dried, fixed in methanol and stained in Giemsa for microscopic examination. Experimental cattle Young male Friesian and Zebu crossbred calves (1--3 days old) were purchased from a farm where strict acaricidal control of ticks is practised. The calves were reared in the laboratory in a tick-free environment and were used in experiments only when t h e y were found to be free of blood parasites and negative for Theileria annulata and Babesia bigemina antibodies by the indirect fluorescent antibody test (Morzaria et al., 1981).
15 During the experiments, blood smears were taken daily from calves and superficial lymph nodes were palpated. Whenever an enlarged lymph node was f o u n d a biopsy was taken and smeared on a microscope slide for Giemsa staining and examination of parasites.
Preparation o f trypanosome suspension from field-collected ticks A sample of H.a. anatolicum adults from Nisheishiba Farm was fed on a rabbit. Unattached ticks were discarded on day 1. On day 3 after application the ticks were removed and counted. Twenty females and 17 males were then processed to prepare suspensions of ground-up ticks in a tissue culture medium containing RPMI 1640, 20 mM HEPES (Flow Laboratories, Gt. Britain), bovine plasma albumin (Sigma, Gt. Britain) at 3.5% w/v, 200 i.u. ml -~ penicillin, 200 pgm1-1 streptomycin and 5 t~gml -~ fungizone. The procedure used was essentially similar to that described by Brown (1979) for the preparation of sterile Theileria sp. sporozoites except that filtration was through a 25 mm diameter Swinnex holder with a glass microfiber pre-filter and an 8 pm pore diameter cellulose ester high porosity membrane filter (Millipore type AP 25 and MF, respectively, Millipore Corporation, U.S.A.). The filtrate volume was adjusted to contain the equivalent of 4 ground-up ticks ml- 1 Wet preparations on a h a e m o c y t o m e t e r (Improved Neubauer) and Giemsastained cytospin smears (Cytospin 2, Shandon, at 550 r.p.m, for 10 min) of 50 pl samples of the suspension were examined for parasites. Trypanosomes at a concentration of 104m1-1 and Theileria sporozoites (32 per 40 microscope fields of 0.175 mm diameter with X 100 oil immersion objective) were seen. This suspension was used for infecting a susceptible bovine calf.
Infection o f cattle Attempts to induce infection in the experimental calves were carried out by either inoculation of the trypanosome suspension or by application of infected H.a. anatolicum adults. One calf (A19) was used for testing infectivity o f the suspension by inoculation of the suspension which contained 104 trypanosomes m1-1 (0.5 ml intravenously and 0.5 ml subcutaneously). Infectivity of the field ticks from Shambat and Nisheishiba, respectively, were tested by applying 12 H.a. anatolicum (six females and six males from each location) separately in earbags to each o f two calves (A47 and A48).
In vitro isolation o f trypanosomes from infected calf Using aseptic techniques, biopsies were taken from an infected lymph node using 38-mm disposable needles and immediately transferred to a growth medium (GM) containing 20 mM HEPES buffered RPMI 1640, 20%
16 heat-inactivated foetal calf serum penicillin (200 i.u. m1-1 ), streptomycin (200 ~ g m l -~) and fungizone (5 ~gml-~). The biopsy material was vigorously agitated in the medium with a pipette to break down large pieces of tissue and the resultant cell suspension was centrifuged at 400 g for 10 min. The pellet was then resuspended in GM and cells were dispensed into T-25 plastic tissue culture flasks, each containing 10 ml of 107 cells. The cultures were incubated at 37°C and 48 h later 2 ml of the GM was added to each culture. Thereafter cultures were replenished twice weekly: half the medium was removed and centrifuged (400 g for 10 min), the supernate was discarded and the pellet resuspended in fresh GM and returned to the culture flasks. All cultures were examined daily and Giemsa-stained cytospin smears from each of them were made twice weekly. RESULTS Tick examination
Ticks collected from both farms showed the presence of flagellates. In the first collection 1/49 of the adult ticks from Shambat and 2/55 from Nisheishiba were infected with flagellates. In the second collection 3/45 and 2/30 adult ticks from Shambat and Nisheishiba, respectively, were found to be infected. The origin of the flagellates in the cuticular-puncture smears could not be determined as the smears were often found to be contaminated with the contents of tick-gut and Malpighian tubules. Four different morphological forms were seen but the predominant forms were long, slender vacuolated epimastigotes with pointed ends, a free flagellum, a central nucleus and a club-shaped kinetoplast. They measured 20.1 pm in length (minimum 15.1 ~m and m a x i m u m 23.2 pm). Other forms seen were: (i) sphaeromastigotes (10.1 pm in length, minimum 7.1 ~m and maximum 13.5 pm); (ii) transition forms from epimastigotes to trypomastigotes with the kinetoptast in juxtaposition to the nucleus and measuring 25.1 pm (minimum 20.8 pm and maximum 28.1 pm); and (iii) trypomastigotes of 26.0 ~m in length (18.5 pm minimum and 32.0 pm maximum). Several dividing stages of the sphaeromastigotes and epimastigotes with two kinetoplasts, nuclei and flagellae were observed. Figures 1, 2 and 3 show various forms of the flagellate observed in the ticks examined. Intracellular forms were not detected. As selective tick organ smears were not prepared other locations of the flagellate development could n o t be determined. It was interesting to note that although the epimastigote stages predominated other stages were f o u n d simultaneously in the same smear. It was not possible to determine the species of the trypanosomes seen but the morphology appeared to be similar to that of T. theileri (Wells, 1971).
17
Q
O
Ib
Fig. 1. Epimastigote forms: the most c o m m o n l y found stage o f the flagellate in H.a. anat o l i e u m . A r r o w indicates a dividing form.
Fig. 2. Sphaeromastigotes in the tick h a e m o l y m p h . Fig. 3. T w o different stages: (a) a transition f o r m b e t w e e n epimastigote and trypomastigote stages and (b) a t r y p o m a s t i g o t e , seen in a h a e m o l y m p h smear o f an adult H.a. anatolicurn.
18
Infection o f cattle Of the 3 calves used in the experiment (A19 inoculated with the trypanosome suspension, A47 and A48 infested with H.a. anatolicum from Shambat and Nisheishiba, respectively) only A48 showed trypanosomes. The parasites were seen in the enlarged lymph node nearest to the tickfeeding site on day 5 after tick application. The trypanosomes were only observed on one day. They were not detected in blood smears or in any other lymph nodes.
Figs. 4 a n d 5. L y m p h n o d e b i o p s y s m e a r o f a n i n f e c t e d calf s h o w i n g t r y p a n o s o m e s w i t h a l o n g free flagellum a n d an u n d u l a t i n g m e m b r a n e . N o t e close a s s o c i a t i o n o f t h e k i n e t o plast w i t h t h e parasite nucleus.
19 The size of the trypanosomes seen in the lymph node of A48 varied from 32.0 gm to 54.0 pm. All were slender trypomastigote forms with the kinetoplast invariably adjacent and posterior to the nucleus. The kinetoplast was difficult to detect in m a n y trypanosomes. The flagellum was long and free with a well-developed undulating membrane. It was not associated with the kinetoplast and appeared to emerge from the posterior end of the trypomastigote (Figs. 4 and 5). Approximately two trypanosomes were observed per 100 lymphocytes. Clean H.a. anatolicum nymphs were applied on two calves (A19 and A48) between days 9 and 13 after the start of the experiment. The resultant adult ticks (120) were examined for trypanosomes but were found to be negative. Long-term monitoring of the 3 experimental calves could n o t be carried out as all of them died of T. annulata infection 14--15 days after the start of the infection.
In vitro isolation o f the trypanosomes One day after the parasites were observed in the lymph node of A48, biopsy cultures were set up. Sixteen days later large numbers of organisms, predominantly amastigote forms of the flagellates, were observed. DISCUSSION Our observations confirm that various developing stages of T. theileri-like parasites are regularly found in adult H.a. anatolicum collected as engorged n y m p h s from the field. The experiments also indicate that the moulted ticks, when applied on a susceptible calf, can transmit the flagellates. This appears to be the first demonstration of biological transmission of a trypanosome by a tick. Several authors have reported the presence of various Trypanosoma spp. in hard as well as soft ticks. O'Farrell (1913) found crithidia-like flagellates in the cattle tick H. aegyptium which were considered by Wenyon (1926) to be forms of T. theileri and this was supported later by Carpano (1932) when he discovered an adult H.a. anatolicum to be infected with a similar flagellate. Arifdzhanov and Nikitina ( 1 9 6 1 ) r e p o r t e d that 25 out of 110 adult H.a. anatolicum collected from a cattle barn in Tashkent were infected with this parasite, indicating a high susceptibility of this tick to T. theileri. Burgdorfer et al. (1973) reported the presence of T. theileri in Rhipicephalus pulchellus and Boophilus decoloratus collected in Ethiopia. Krinsky and Burgdorfer (1976) reported ~T. theileri-like forms in A m b l y o m m a americanum collected from deer in the U.S.A. In a systematic study, Kirmse and Taylor-Lewis (1976) showed limited development of T. lewisi, T. musculi, T. congolense and T. vivax in hard and soft ticks for a variable period of time. Lopez et al. ( 1 9 7 9 ) i n South America demonstrated T. vivax in B. decoloratus. However, none of the above workers were able to successfully transmit trypanosomes to cattle using hard ticks as vectors.
20 A successful transstadial transmission of a T. thylacis by a hard tick, Ixodes tasmani, to an Australian marsupial, the short-nosed bandicoot, has been reported by Weilgama (1980), who found four developmental stages, amastigotes, sphaeromastigotes, epimastigotes and trypomastigotes, in the tick tissues. More recently, experiments by Shastri and Deshpande (1981) showed that infection with T. theileri could be established with H.a. anatolicum when a suspension of infected ticks was inoculated together with the application of infected ticks on susceptible calves. Although this does not confirm tick transmission the infectivity of tick-derived T. theileri for cattle is confirmed. Shashtri and Deshpande were n o t able to directly demonstrate trypanosomes in the infected calves but the transmission was confirmed by xenodiagnosis. The experiments in our study confirmed tick-transmission of Trypanosoma sp. by demonstration of the flagellate in a susceptible bovine host after application of a batch of ticks previously identified as infected. From these experiments it appears that the ticks acquire infections as nymphs from chronically infected cattle and transmit the parasite to susceptible calves as adults. Thus transstadial transmission appears to occur. Some workers have shown massive development of T. theileri in the ovaries of infected ticks (O'Farrell, 1913; Arifdzhanov and Nikitina, 1961; Burgdorfer et al., 1973; Krinsky and Burgdorfer, 1976) and therefore the possibility of transovarian transmission cannot be discounted. In the present study the flagellates were re-isolated in vitro after transmission by infected ticks to a calf. No trypanosomes were observed in the blood smears of the calf during a short period of examination. It was unfortunate that the experimental animals in the study died due to contaminating T. annulata infection. However, this was an expected outcome as Hoa. anatolicurn collected from the field are usually heavily infected with T. annulata {Walker et al., 1983). It was not possible to determine the species of trypanosome in the tick and the calf. The morphology of the flagellate in the tick appeared to be similar to that of the T. theileri-like parasite described by Burgdorfer et al. {1973) in A. americanum. The forms seen in the lymph node of the calf after application of the ticks do n o t conform to the typical forms of T. theileri described from the peripheral blood of the bovine host. However, these stages detected in the lymph node may represent the early developing forms and therefore may n o t be typical of T. theileri. At present there is no serological test available to detect T. theileri infection in a bovine host, and hence it was n o t possible to be absolutely certain that the experimental calves used in the present study were free of T. theileri before the experiment. The evidence of tick transmission must, therefore, be treated with caution and is based on the appearance of trypanosomes transiently for one day in a hyperplastic lymph node following application of a batch of ticks previously identified to be infected with the flagellates. Further studies are necessary to determine the identity of the flagellate described and its infectivity and pathogenicity to cattle through H.a. anatolicum.
21 ACKNOWLEDGEMENTS Thanks are due to the Ministry of Agriculture and Animal Resources (MAAR), Khartoum and Central Veterinary Research Laboratory, Soba, S u d a n f o r t h e f a c i l i t i e s p r o v i d e d . Dr. R . J . T a t c h e l l , D r , A . M . O s m a n , P r o f . G. U i l e n b u r g , D r . J.P. O v e r d u l v e a n d D r . P. G a r d i n e r a r e t h a n k e d f o r t h e i r h e l p f u l s u g g e s t i o n s . T h e p a p e r is p u b l i s h e d b y t h e k i n d p e r m i s s i o n o f t h e Under Secretary of the MAAR, Khartoum.
REFERENCES Arifdzhanov, K.A. and Nikitina, R.E., 1961. Detection of Crithidia hyalomma (O'Farrell 1913) in Hyalomma a. anatolicum (Koch 1944). Zool. Zh., 4 0 : 2 0 - - 2 4 (in Russian). Brown, C.G.D., 1979. Propagation of Theileria. In: R. Maramorosch and H. Hirumi (Editors), Practical Tissue Culture Applications. Academic Press, New York, pp. 223--254. Burgdorfer, W., Schmidt, M.L. and Hoogstraal, H., 1973. Detection of Trypanosoma theileri in Ethiopian cattle ticks. Acta Trop., 30: 340--346. Carpano, M., 1932. Localisations du Trypanosoma theileri dans les organes internes des bovins, son cycle 6volutif. Ann. Parasitol. Hum. Comp., 10: 305--322. Hoare, C.A., 1972. The Trypanosomes of Mammals, a Zoological Monograph. Blackwell Scientific Publications, Oxford/Edinburgh, pp. 30--56. Kirmse, P. and Taylor-Lewis, G., 1976. Ticks as possible vectors of trypanosomes. In: J.K.H. Wilde (Editor), Tick-borne Diseases and their Vectors. University of Edinburgh, pp. 177--180. Krinsky, W.L. and Burgdorfer, W., 1976. Trypanosomes in A m b l y o m m a americanum from Oklahoma. J. Parasitol., 62: 824--825. Lopez, V.G., Thompson, K.C. and Bazalar, H., 1979. Transmission experimentale de Trypanosoma vivax par la garrapata Boophilus microplus. Rev. Inst. Colomb. Agropec., 14 : 93--96. Morzaria, S.P., Mustafa Um E1 Hassan, Shawgi, M.H., Pedersen, V. and Osman, A.M., 1981. Isolation, identification and transmission of Theileria mutans in Northern Sudan. In: A.D. Irvin, M.P. Cunningham and A.S. Young (Editors), Advances in the Control of Theileriosis. Martinus Nijhoff Publishers, The Hague/Boston/London, pp. 166--168. O'Farrell, W.R., 1913. Hereditary infection with special reference to its occurrence in Hyalomma aegyptium infected with Crithidia hyalomma. Ann. Trop. Med. Parasitol., 1: 545--556. Shastri, U.V. and Deshpande, P.D., 1981. Hyalomma anatolicurn anatolicurn (Koch, 1844) as a possible vector for transmission of Trypanosoma theileri, Laveran, 1902 in cattle. Vet. Parasitol., 9 : 151--155. Walker, A.R., Latif, A.A., Morzaria, S.P. and Jongejan, F., 1983. Natural infection rates o f Hyalomma anatolicum anatolicum with Theileria in Sudan. Res. Vet. Sci., 35: 87--90. Weilgama, D.J., 1980. Cyclical development of trypanosomes in the tick and their transmission. In: O.P. Gautam, R.D. Sharma and S. Dhar (Editors), Haemoprotozoon Diseases of Domestic Animals. Proceedings of a Seminar, 27 October--1 November 1980, at Hissar, India, pp. 187--197. Wells, E.A., 1971. Studies on Trypanosoma theileri-like trypanosomes of cattle. Br. Vet. J., 127: 466--475. Wenyon, C.M., 1926. Protozoology. A Manual for Medical Men, Veterinarians and Zoologists. Bailli~re, Tindall and Cox, London, pp. 358--361.