Hereditary transmission of Theileria annulata infection in the tick, Hyalomma aegyptium neum

93 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 44. No. 1. July, 1950.

H E R E D I T A R Y T R A N S M I S S I O N OF THEILERIA ANNULAT.4 INFECTION IN THE TICK, HYALOMMA AEG YPTIUM NEUM.* BY

H. N. RAY,

Officer-in-charge, Section of Parasitology, Indian Veterinary Research Institute, Mukteswar, Kumaun, U.P. India.

SERGENT and his collaborators (1936), FOTHERINGHAM and LEWIS (1937), and TZELISHCHEVA (1940) found that theileria parasites did not persist in the egg stage of Hyalomma; while according to COWDRY and HAM (1932), and LEWIS et al. (1946), ticks of the genus Rhipicephalus also behaved in the same way. On the other hand, the hereditary transmission of Theileria annulata in Hyalomma has been recorded on the basis of the findings of the present writer in the Annual Report of the M u k t e s w a r Institute (1940-41), a n d in a p a p e r b y RAGHAVACHARI et al. (1944), whilst KORIENKO and SHMUIREVA (1944), in Russia, also observed this to be the case with the same genus of tick. I n this article detailed evidence is produced in confirmation of the earlier observations of the writer, namely, that theileriasis (T. annulata infection) is t r a n s m i t t e d to animals b y the adult, but not b y the larval or n y m p h a l progeny of infected ticks, H. aegyptium. MATERIAL AND METHOD. In June, 1940, and September, 1945, numerous adult specimens of H. aegyptium were collected from a cattle farm in the Multan District (West Punjab), where acute theileriasis amongst calves was prevalent.]- They were brought to Mukteswar (altitude 7,500 feet) and kept in an incubator at 27 ° to 28 ° C., the engorged adults being kept * The author is deeply indebted to the following who worked under him from tinle to time and rendered valuable help in the capacity of Research Assistants : K. RAGHAVACHARI,AZIZAHMAD, S. N. SAPRE, C. V. G. CHAUDHURY, MANMOHANSINGHand HARBANS SINGH. The author also wishes to place on record his grateful thanks to Dr. S. DATTA, Director, Indian Veterinary Research Institute, for his keen interest and helpful suggestions during the initial stage of this work. t Accordingto DUTOIT'S(1931) scheme of classificationof Theileria, the species concerned in these cases is referable to T. annulata.

94

HEREDITARY TRANSMISSION OF THEILERIA ANNULATA

singly, and the unfed ones in groups of 30 to 50 in test tubes plugged with cotton wool. Within each tube was placed a crumpled piece of filter paper to prevent accumulation of moisture. Ticks in all stages were fed on calves and hill cattle according to the method described by SEN 0 9 3 3 ) and SAPRE (1943). Briefly, the method is as follows : T h e hair is clipped close at the site--preferably the upper portion of the h i p - - w h e r e the ticks are to be applied (Fig. 1). A circular piece of cloth, measuring 1½ to 2 inches in diameter, is glued to the area along the margin by means of celloidin dissolved in a mixture of alcohol and ether, a small portion being left unglued for the introduction of ticks from a small glass tube (Fig. 2). After the adhesive has hardened and become free from the smell of the solvent, the contents of the tube are emptied through the opening and the latter closed up with the celloidin. A metallic cup with a flat edge and with perforations for admitting air is fixed to the skin by means of Chatterton's compound ~ so as to cover the circular piece of cloth (Figs. 3 and 4). After 6 to 10 days the metallic cup was first removed by cutting the hairs adjoining its rim. T h e piece of cloth was then partially detached and the ticks collected in a test tube. Fed larvae or nymphs were usually kept together in large numbers in small specimen tubes, and engorged females were kept singly in test tubes. In these experiments only 3 to 9 months old calves, all born and bred at the Institute, were used, excepting calf No. " X , " which belonged to the Jahangirabad Cattle Farm, Jahangirabad, District Multan. Four to 6 days prior to the application of the ticks, their superficial lymphatic glands were palpated, temperature recorded, and blood examined microscopically to make sure that they were free from T . annulata. HISTORICAL.

Very little is known about the role of ticks of the genus Hyalomma in the transmission of animal diseases. BRUMPT (1920) mentions CARPANO (1915) as having incriminated H. aegyptium as the vector of Theileria parva and T, mutans. In European Russia, Nuttallia equi is reported by BELLITZER(1925) to be transmitted by H. aegyptium, but WARBURTON (1923) stated that there is no definite proof of H. aegyptium being pathogenic, although there is a strong suspicion that it is a transmitter of N. equi to horses. In India, COOPER (1926) says-evidently on the basis of circumstantial evidence--that T. mutans infection in Indian cattle is transmitted by H. aegyptium, while according to K/~ASHNIKOV, cited by FOTHERINGHAMand LEWIS (1937), piroplasmosis in Astrakhan is transmitted by the same species of tick. North African theileriasis caused by T. dispar was shown by SERGENT and his collaborators (1928) to be transmitted by the adults of H. mauritanicum. With regard to this finding, DU TOIT (1931) observes : " It should be mentioned that this is the first time that a species of Hyalomrna has been found capable of transmitting a disease." Outbreaks of T. annulata infection in Jordan valley was believed by ROSEN (1931) to be due to the introduction of H. aegyptium and Boophilus sp. into this area. GALUZO (1935, 1936), from his experimental and field observations from 1928 to 1929, states that T. annulata in Central Asia is transmitted by H. dromedarii anaticum, t * Chatterton's compound is a proprietary preparation, and is obtainable from suppliers of electrical goods, viz., Messrs. Sieman's, Ltd. (India), Calcutta. It has a low melting point, and on setting proves to be an excellent adhesive. 1"DELPHY (as cited by FOTHERINGHAM and LEWIS, 1937) considers this v~ariety to be a synonym of H, dromedarii.

It. N. RAY

95

FIG. 1.--ApIJlication of the metallic tick cup.

FIG. 2. FIG. 3.

..............

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FIG. 4. FIG. 2 . ~ M u s l i n b a g glued all r o u n d except at one end t h r o u g h w h i c h a small test tube c o n t a i n i n g ticks is e m p t i e d into t h e bag. FIG. & - - T o p view of the metallic tick c u p - - a c t u a l size. FIG. 4 . - - T h e cup in section.

96

HEREDITARY TRANSMISSION OF THEILER1A ANNULATA

SERCENT and his collaborators (1936), working with T. dispar, found that sp0rogony of this protozoan occurs in H. mauritanicum, and that the larva and nymph feed on the same animal. The engorged nymphs, after detachment, hibernate for 6 to 8 months, the adults appearing the following summer to attack another animal. The protozoa, according to these workers, do not pass through the ova of an infected tick. SraGENT (I940) failed to transmit the disease through Anopheles. It is noteworthy that FOTItERINGrtAM and LEWIS (1937), working in Kenya, found that T. parva was not only transmissible, as recorded by previous workers, through the agency of Rhipicephalus appendiculatus, R. evertsi and R. simus, but that Hyalomma impressum near planum also is capable of transmitting East Coast Fever in the nymphal and adult stages after the larvae and nymphs respectively have fed on an infected animal. The parasite does not pass through the egg. These observations would appear to necessitate a modification of the statement made by DV TOIT (1931) that the chief difference between T. parva and T. dispar is that the former is carried by Rhipicephalus spp. and not by Hyalomma. In Southern Kazakstan, T. annulata was shown by TZELISHCHEVA (1949) to be transmitted by H. marginatum, besides 1t. detritum, H. savignyi and H. dromedrii, which had already been known as the vectors of the disease in the Russian Union. Larval or nymphal progeny of infected 11. savignyi and H. marginatum did not, however, transmit the infection. WACNEa (1941) reports transmission of 7". parva infection to young bovines at Frankport through the bite of infected nymphs of R. appendiculatus obtained from South Africa. The nymphs were infective after fasting for 6 months but not after fasting for 12 months. GALUZO(1943), while dealing with natural vectors of Theileria in Kazakstan, emphasizes the fact that an experimental vector cannot be a natural one if its immature stages do not feed on cattle in nature, since the species of Theileria do not persist in ticks from one generation to the other. This is not clear. Even if the immature stages do not feed on cattle the adult progeny of infected ticks may be infected. KORIENKO and SHMUIREVA (1944), however, indicated the possibility of the transmission of theileria parasites to their progeny by ticks of the species H. turkmeniense. The abstract of their article is as follows : " I n 1939 females of Hyalomma turkmeniense Olen., taken in Central Asia on cattle infected with Theileria, were allowed to oviposit in the laboratory, and their offspring were reared by feeding the larvae and nymphs on rabbits in September. Fifty of the resulting adults were placed on a healthy call on which they engorged in 5 to 11 days. The calf did not show infection in autumn of 1939 but did so in the spring of 1940. To obtain a more definite result, a further 30 of the ticks were sent to Omsk, where the disease does not occur. There they engorged in 8 to 13 days on a healthy bullock, and it became infected 11 days later, Theileria bodies being present in its blood for 5 to 6 days."

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It is pointed out that the results of this experiment differ from those of previous workers, who concluded that infection with Theileria is not passed through the eggs in ticks of the genus Hyalomma. The results of transmission experiments carried out by the present writer are essentially in accord with those of KORIENKO and SHMUIREVA. SECTION A. TRANSMISSION EXPERIMEN'i'S WITII TICKS COLLECTED IN JUNE,

1940.

Series I. Experiments with the Progeny of First Generation. In September, 1940, a batch of larvae representing the progeny of ticks collected in June, 1940, from the Jahangirabad Cattle Farm (W. Punjab) were put on a clean calf at Mukteswar. They became engorged in 10 days when they were removed from the animal and the latter kept under observation for nearly 2 months with negative result. Nymphs that emerged from these larvae were put on another clean calf, but this time, too, the result was negative. In the following November the nymphs were kept in the incubator at 27 ° C. and allowed to moult into adults. In January, 1941, a batch of 12 of these adults were put on a 29-day-old calf, on which they became engorged in 9 days. On the 7th day after removal of the ticks, the calf showed a temperature of 105.4 ° F., with theileria parasites in the circulating blood. The number of parasites in the blood kept on increasing concurrently with a rise in temperature until it reached the maximum of 107.0 ° F. on the 12th day (Fig. 1). Thereafter, the temperature fell gradually and the calf succumbed to acute theileriasis on the 26th day after the application of the ticks. Fro. l.--Calf No. 15. Age 29 days. t07 t06 I05 104 103 t02 101 tOO 99 DAY5

A--Application of ticks. B Removal of ticks. C ~ T h e first appearance of theilcria parasites in the peripheral circulation. D ~ D e a t h of the animal. O---Disappearance of theileria parasites from the peripheral circulation.

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A month later, 10 adult ticks of the same batch were put on another calf aged/3~ months. The ticks became engorged in 9 days and 4 days later G

98

HEREDITARY TRANSMISSION OF THEILERIA ANNULATA

theileria parasites were first seen in tile peripheral blood, where they persisted for the next 10 days. On the 9th day after the removal of ticks, schizonts (Koch's bodies) appeared in the blood, and the animal showed a temperature of 105"0 ° F. (Fig. 2). This condition lasted for 2 days, during which period, and also on the 2 following days, the animal was dull and showed marked lack of appetite. During the next 14 days the temperature fluctuated between 101.6 ° and 102"8 ° F., with a progressive decrease in the number of parasites in the peripheral circulation until they completely disappeared at the end of this period. The animal's condition gradually returned to normal and it was discontinued from experiments after another week. FIG. 2.--Calf No. 453. to6 104

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Series 11. Experiments with the Progeny of Second, Third, Fourth, Fifth and Sixth Generations. Second and third generation adults derived from the ticks collected in 1940 failed to produce infection in calves at Mukteswar. In February, 1942, a batch of 30 adults of the third generation were taken over to Jahangirabad Cattle Farm and put on a calf aged 3 weeks. This animal had been weaned at birth and reared in a tick-free paddock, and microscopical examination of its blood for 4 days prior to application of ticks had given negative results for the presence of protozoan parasites. Fully engorged ticks were recovered from the animal on the 10th day. A day previous to the removal of tieks slight swelling of the superficial glands was noticed, and its peripheral blood showed theileria parasites. It gradually recovered after showing the parasites in its blood for 13 days, the maximum rise of temperature (104.6 ° F.) being registered on the 5th day after removal of the ticks (Fig. 3). Fourth, fifth and sixth generation adults proved non-infective for calves at Mukteswar. It is possible that this was due to the ticks being depleted of their store of parasites as a result of repeated feeding in all their stages, through several generations, on clean animals. Further experiments were suspended

H. ~. RAY

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until September, 1945, when a fresh consignment of unfed specimens of adult H. aegypthtm was obtained from Jahangirabad. SECTION B. TRANSMISSION EXPERIMENTS WITH TICKS COLLECTED IN SEPTEMBER,

1945.

About 40 adult ticks referred to in the preceding section were put on a young calf, and out of these only six ticks (five females and one male) were recovered after engorgement on the 9th day; the others were found dead. Nineteen days after the application of ticks the animal showed a body temperature of 105"6 ° F. (Fig. 4), and 9 days later it died of theileriasis. The following series of experiments relate to the descendants of the five females mentioned above, the individuals immediately descended from these females being Fu;. 3 . - - C a l l No. " X."

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designated as first generation, the progeny of the latter as second generation, and so on, The number of adult ticks put at a time on an animal varied from 30 to 50, unless otherwise mentioned.

Series I.

Experiments with Ticks of the First Generation.

The first generation larvae and nymphs, when put on clean animals, failed to produce theileriasis, but in February, 1946, when the adults were allowed to feed on a young calf aged 8 months, it died of acute theileriasis on the 15th day after showing the maximum body temperature of 106.2 ° F. on the 14th day (Fig. 5). In March another batch of 100 first generation adults were applied to the calf aged 9 months. This animal likewise succumbed to acute theileriasis on the 14th day, after showing a maximum temperature of 107.2 ° F. on the 1 lth day (Fig. 6). t08.

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Series II. Experiments with Ticks of the Second Generation. In August, 1946, some second-generation adults were put on a bull calf aged 3 months and also on two adult animals. The bull calf reacted but

H. No RAY

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recovered, while the two adult animals remained unaffected. In October, 1946, the experiment was repeated on two more bull calves aged about 40 days ; both developed acute theileriasis and died within 18 and 25 days respectively.

Series IlL

Experiments with Ticks o f the Third Generation.

Between November, 1946, and January, 1947, some third generation adults were put on four calves Of which only one r&cted and developed the disease. Between 30 and 50 ticks were used in each case.

Series IV.

Experiments with Ticks of the Fourth Generation.

During April and May, 1947, 50 and 65 fourth generation adults respectively were applied to two calves; one of these showed the parasite in the blood on the 15th day, with a rise of temperature to 105.4 ° F. The parasites persisted for the following 29 days, after which the animal recovered and was discontinued from experiment. The second animal showed the parasites and the maximum elevation of temperature (105.0 ° F.) on the same day as the first one, but in this case the parasites persisted in tile blood for only 14 days. The animal died about 3 months later, of causes other than theileriasis. Twelve partially fed adults were examined for the presence of theileria parasites in their salivary glands, and nine showed the parasites in their acini, almost every acinus being infected in the majority of cases. In a third experiment, two fourth generation adults (one male and one female) were put on a calf aged about 2½ months. These ticks had moulted into adults about 7 months before. The calf died 20 days after the application of the ticks without showing a rise in its body temperature or the symptoms of the disease (Fig. 7). On postmortem examination, however, it showed icterus, slight enlargement of FIG. 7.--Caff No. 73. ....

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the liver and spleen, and petechiae in the abomasum. Smears made from the liver, spleen and the bone marrow showed schizonts and a few endoglobular forms. The absence of a thermal reaction in this case is contrary to experience.

102

HEREDITARY TRANSMISSION OF THEILERIA ANNULATA

According to NEITZ (1946), the causal agent of East Coast Fever--Thdleria parva--is associated with a symbiotic virus, and the thermal reaction on the part of the vertebrate host is due to the viral accompaniment and not to the protozoan. It is possible that T. annulata has a similar viral accompaniment and, if so, it must have been absent in the present instance.

Series V. Experiments with the Ticks of the Fifth Generation. During September and October, 1947, fifth generation adults were found to fail in transmitting theileriasis to calves, although the salivary glands of two out of five partially fed females revealed the presence of parasites in them. The number of acini found infected in the two positive cases was, however, very small as compared with that of the fourth generation adults.

Series VI.

Experiments with Ticks of the Sixth Generation.

In February and March, 1948, 30 sixth generation adults were put on each of two young calves. These ticks, like those of the previous generation, proved non-infective to the calves, although two out of four partially fed female ticks revealed the presence of theileria parasites in the acini of their salivary glands. The number of infected aeini in this case also was very small.

Series VII.

Experiments with the Ticks of the Seventh Generation.

During May, 1948, 14 seventh generation adults were put on a young calf, and this time, too, the result was negative. As only three females and two male ticks succeeded in engorging themselves, none of these was dissected for the purpose of examining its salivary glands. No further experiments could be made after July, 1948, when all attempts to feed the eighth generation larvae on cattle or rabbits met with failure. Presumably, continued rearing under artificial conditions had some untoward effect on the general metabolism of the ticks. SECTION C. EXPERIMENTS W I T H F I F T H GENERATION ADULTS THAT HAD THEIR LARVAL AND NYMPHAL FEED ON RABBITS.

Two series of experiments were carried out with a brood of adults that had been fed on rabbits in their larval and nymphal stages. These experiments are briefly described below. in August, 1947, 22 adult ticks were put on a young calf aged about 3 months. Six days later two females were removed and the salivary glands of one of them revealed developing stages of theileria parasites while those of the other were totally free from them. T h i r t y ticks were put on the same calf about a week later, and in this lot three partially fed females removed after 96 hours' feed were found to be free from the parasites in their salivary glands.

H. N. RAY

103

A few days later another batch of 35 adults were put on this calf. The salivary glands of six of these ticks after 24 hours' feed, and of six others after 36 hours' feed, were found to be negative for theileria parasites ; but those of one fully engorged female removed after a feed of 6 days showed numerous parasites in the acini. The calf died, 16 days after the first application of ticks, of causes other than theileriasis. In early September, 1947, 25 ticks were put on another calf aged about 2½ months. Six partially fed females removed 6 days later, and their salivary glands examined for theileria parasites, showed developing parasites in the salivary glands. A month after the application of the ticks this calf died of causes other than theileriasis.* DISCUSSION.

The possibility of transmitting theileria parasites to the progeny of infected ticks through thcir eggs has been investigated only by a very small number of workers, and in the majority of cases the infectivity of ticks has not been tested beyond their nymphal stagc. Thus the negative finding recorded by FOTHERINGHAM and LEWIS (/.c.) in the case of T. parva is based on the result of a trial made with the larvae only. SERGENTand his collaborators (I.e.) and TZELISHCHEVA (/.C.) likewise experimented with the larvae and nymphs only, with the same results. The same remarks apply t o COWDRY and HAM (l.c.) and LEWIS et al. (l.c.). The positive results recorded by KORIENKO and SHMUIREVA (l.c.) with the adult progeny of infected ticks are open to the criticism that they disprove the capability of larvae and nymphs for transmission as they were not fed on bovines but on rabbits. The results of a fairly large number of trials with H. aegyptium carried out by the present writer are claimed to have shown conclusively that only the adult, and not the larval and nymphal progeny of infected ticks, is capable of transmitting T. annulata to susceptible bovines. As for the objection that the ticks in the course of their larval and nymphal feed might have acquired the parasites from infected bovines, it may be pointed out: (1) that natural T. annulata infection is not known to occur in calves born at the Institute; (2) that H. aegyptium has not been recorded from the locality (altitude 7,500 feet) where the experiments were carried out; and (3) that in the case of all experimental animals their lymphatic glands were palpated and their blood carefully examined for the presence of theileria parasites before submitting them to the bites of the ticks. SUMMARY.

1. Hyalomma aegyptium is the intermediate host of Theileria annulata in India. * In these cases it is not possible to declare definitely that death was not due to some

latent forms of ThefTeria, though the parasites could not be found in the peripheral blood or in organ smears,

104

HEREDITARY TRANSMISSIONOF TIIF.ILERIA ANNULATA

2. Progeny of the infected Hyalomma aegyptium in their adult stage only are capable of transmitting Theileria annulata to susceptible bovines. 3. Positive results were obtained up to third generation adults i n the first series of observations (1940-42) and up to fourth generation adults in the second series (1945-48). 4. T h e salivary glands of a few of the fifth and sixth generation adults revealed the presence of theileria parasites in their acini but the n u m b e r of infected acini was not so high as that of the fourth generation adults. 5. T h e association with T. annulata of a symbiont virus was suspected in one instance. REFERENCES. Ann. Rep. Indian Vet. Res. Inst. (1940-41). BELLITZER,A. (1925). Zbl. Bakt., 94, 51. BRUMPT, E. (1920). Bull. Soc. Path. exot., 13, 416. COOPER, H. (1926). Agric. ft. India, 12, 95. COWDRY, E. V. & HAM, A . W . (1932). Parasitology, 24, 49. DAUBNEY, R. (1936). Rep. Dep. Agric. Kenya, 2, 55. DU TOIT, P. J. (1931). Eleventh Int. vet. Congr., London, 1930, 3, 539° FOTHERINGHAM,W. & LEWIS, E. A. (1937). Parasitology, 29, 504. GALUZO, I. G. (1935). Rev. appl. Ent., 23, 1. (1936). Ibid., 24, 148. (1943). Izv. Kazakh. Fil. Akad. Nauk. (Set. Zool.). No. 2, 41. KORIENKO, Z. P. • SHMUIREVA, M. K. (1944). Veterinarya, Moscow, 21, 24 (in Russian). LEWIS, E. A., PIEREY, S. E. 8c WILEY, A . J . (1946). Parasitology, 37, 60. NEITZ, W. O. (1946). ft. S.Afr. Vet. Med. Ass., 17, 92. RAGHAVACHARI,K., SHAH, A. & RAY, H. N. (1944). Indian ft. vet. Sci., 15, 149. ROSEN, G. (1931). Arch. Schiffs-u. Tropenhyg., 35, 373. SAP1RE, S. N. (1943). Indian ft. vet. Sci., 13, 175. SEN, S . K . (1933). Ibid., 3, 356. SERGEN'r, E. (1940). Arch. Inst. Pasteur, Algdr., 18, 237. - - , DONATIEN, A., PARROT, L. & LESTOQUARD, F. (1928). C.R. Acad. Sci., Paris, 187, 433. , , & . (1936). Ibid., 202, 809. TZELISHCHEVA,L.~ M. (1940). Sovetsk, Vet., Moscow, 17, 31. WAGNER, O. (1941). Dtsch. tropen med., 45, 97. WARBURTON, C. (1923). In G. H. Woodridge's Ticks. Encyclopaedia of Veterinary Medicine, Surgery and Obstetrics, London : Henry Frowde & Hodder & Stoughton, 1, 488.