Preliminary Observations on a Theilerial Species Pathogenic to Cattle Isolated from Buffalo (Syncerus Caffer) in Tanzania

Br. vet.

J.

(1973), 129, 382

PRELIMINARY OBSERVATIONS ON A THEILERIAL SPECIES PATHOGENIC TO CATTLE ISOLATED FROM BUFFALO (SYNGERUS GAFFER) IN TANZANIA By

M.

J.

A. S.

YOUNG·,

BURRIDGE,

M.

D.

BRANAGAN·*, C.

P. CUNNINGHAM, AND

G. D. R. E.

BROWN, PURNELL··*

Immunological Research on Tick-borne Cattle Diseases and Tick Control Project, East African Veterinary Research Organization, Muguga, P.O. Kabete, Kenya

SUMMARY

A theilerial species, pathogenic to cattle, and resembling Theileria lawrencei, was isolated from two buffalo captured in the Serengeti National Park, Tanzania. When nymphal Rhicephalus appendiculatus ticks were fed on the buffalo, on four out of six occasions the resultant adults were shown to be capable of causing highly pathogenic T. lawrencei-type infections in cattle. Aspects of the life cycle of the parasite in both ticks and cattle were studied. Its development in the tick was apparently similar to that of T. parva, and, in cattle, only differed from T. parva by causing the host's death when relatively few lymphocytes were infected with macroschizonts and when neither microschizonts nor piroplasms were detectable. The parasite was designated T. lawrencei (Serengeti). INTRODUCTION

Although buffalo are considered to be involved in the epizootiology of a theileriosis pathogenic for cattle in many areas of Africa (Brocklesby & Barnett, Ig66a), isolations from buffalo have been reported only from South Africa (Neitz, 1955) and Kenya (Brocklesby & Barnett, 1966b). Neitz (1955, 1957) considered the parasite from buffalo to be distinct from Theileria parva, the causative organism of East Coast fever (ECF) of cattle, as fatal infections in cattle involved only small numbers of macroschizonts, and microschizonts and piroplasms were scanty or absent. Tick transmission of the parasite was thus difficult (Neitz, 1958). These differences from T. parva led Neitz (1955) to name the buffalo-derived parasite T. lawrencei. Barnett & Brocklesby (1966) isolated a similar parasite from buffalo and called it T. lawrencei (Kenya). Mter tick transmission through captive buffalo and cattle, the parasite appeared in some cases to behave like T. parva. Furthermore, they found that behavioural transformation to T. parva occurred when

* Wellcome Research Fellow seconded from Nuffield Institute of Comparative Medicine, London, attached to the East Mrican Veterinary Research Organization and the Serengeti Research Institute. ** Present address : The Royal (Dick) School of Veterinary Studies, Centre for Tropical Veterinary Medicine, Easter Bush, Roslin, Midlothian, Scotland. *•• On ODA secondment from the ARC Institute for R esearch on Animal Diseases, Compton, near Newbury, Berkshire, U.K. (ODA Research Project 2396) .

THEILERIAL SPECIES PATHOGENIC TO CATTLE

T. lawrencei (South Mrica) was serially passaged through cattle (Barnett & Brocklesby, 1969). This paper reports the isolation of a similar theilerial parasite from buffalo in Tanzania and describes its pathogenicity for cattle. MATERIALS AND METHODS

The buffalo, one of each sex, came from herds in the Serengeti National Park, which had had no observed contact with cattle since 1961 (Sinclair, personal communication). They were captured in 1967 when less than three weeks of age, and housed thereafter at Banagi in the Serengeti National Park, where again no cattle contact was possible. The cattle were of Bas taurus type, purchased in Kenya from farms where strict acaricidal regimes are practised. The rabbits used were chinchilla crosses from the E.A.V.R.O. Small Animal Unit. The ticks, Rhipicephalus appendiculatus, were reared and maintained according to the methods described by Bailey (1960).

Isolation of Theileria from buffalo Thin blood smears were taken from the buffalo at regular intervals, stained with Giesma and examined for theilerial parasites. Attempts to isolate the parasite in ticks were made by applying, to the ears of the buffalo, either 500 nymphs or 2000 larvae in earbags. Engorged nymphs or larvae were collected and returned to E.A.V.R.O. to moult. Either two or seven months after moulting, 50 or 100 adult ticks (equal numbers of each sex) were applied to cattle. On one occasion nymphs, fed as larvae on the male buffalo, were applied to cattle (Table I). A series of observations on the reactions of the cattle was made. The rectal temperature of each animal was recorded daily, and the appropriate parotid and contralateral prescapular lymph nodes were palpated. When these nodes became enlarged, biopsy smears were taken from them, stained with Giemsa, and examined for macroschizonts. Once macroschizonts were detected, blood smears were taken daily, stained with Giemsa, and examined for theilerial piroplasms. Macroschizont indices (MS I) (Jarrett, Crighton & Pirie, 1969) in prescapular lymph nodes were determined by counting the number of macroschizonts associated with 10,000 lymphoid cells in biopsy smears. Whenever an MSI was above I per cent, the number of nuclei per macroschizont was determined for at least 100 macroschizonts. The prepatent period was taken as the interval in days between tick application and initial detection of macroschizonts in the local drainage lymph node. An animal was considered to be undergoing a febrile response when its rectal temperature was 103 °F or more. Attempts to transmit the parasite between cattle When a theilerial infection was d etected in an animal,

500

R. appendiculatus

BRITISH VETERINARY JOURNAL, 129, 4

nymphs were applied to each of its ears. The infectivity for cattle of the resultant batches of adult ticks was tested by applying 100-400 of them to the ears of susceptible calves two months later.

Detection of theilerial parasites in R. appendiculatus Batches of adult ticks which had fed as nymphs on the buffalo were applied to rabbits' ears. Approximately equal numbers of each sex were removed three, four and five days after attachment. Their salivary glands were then dissected out, processed according to the methods of Martin, Barnett & Vidler (1964), and examined for parasites.

RESULTS

Isolation of buffalo Theileria Four of the six batches of ticks fed on the captive buffalo produced infections in cattle. Details of the attempted isolations are shown in Table I. In transmission attempts, peak piroplasm parasitaemias in the buffalo varied from 0'9 per cent to 1'4 per cent. Ticks were shown to remain infective for cattle for at least seven months, and buffalo to be carriers of tick-transmissible piroplasms for at least three months. The resultant infection in cattle Some of the characteristics of the theilerial syndrome in cattle are shown in Table II. All infections were fatal, and the syndromes produced by ticks fed on both buffalo were similar. Little variation was seen in the prepatent period (mean 10'2 days), the time to fever (mean 12'4 days), or the time to death (mean 17.6 days). All calves died when their MSI's were below 5 per cent (mean 4'4 per cent), and the number of nuclei per macroschizont in the terminal part of the infection shows little variation (mean 4'0). No microschizonts or piroplasms were observed. The mean peak temperature was I06·0°F. The MSI's during the course of infection are shown in Table III. In two of the calves the MSI decreased before death. MSI's were determined in various lymphoid tissues taken during postmortem examination of calves C 492 and C 493 (Table IV). There was little variation between MSI's observed in different lymphoid tissues. The mean number of nuclei per macroschizont was 4'0 (range 1-19) compared to 6'7 (range 1-75) for T. lawrencei Kenya (Barnett & Brocklesby, 1966) and 8'0 (range 1-85) for T. parva (Muguga) (Barnett, Brocklesby & Vidler, 1961 ) . Post-mortem findings in infected cattle Relatively few macroscopic lesions were detected. All lymph nodes examined showed hyperplasia, and in many instances were extremely oedematous. Pulmonary oedema was not marked, but extensive frothy exudate was seen in the trachea and bronchi.

TABLE I ATTEMPTED TRANSMISSION OF A Theileria FROM BUFFALO TO CATTLE, USING R, appendiculatus TICKS AS THE VECTOR

B,/female

Reference No , Buffalo

Peak piroplasm parasitaemia during tick feeding

0'9 %

Batch No,

1'3%

2

3

1'5%

0'9%

1'4%

6

5

4 Oct. '68 Nymphs 50 adults 2

Jan, '69 Nymphs 100 adults 2

Mar, '69 Nymphs 50 adults 2

C54

C 217

C 213

C238

C 492C 493

C654

o

+

+

+ +

+

rJl

T, lawrencei (Kenya)·

Time to febrile response in days

Time to death in days

Mean dairy no, of nuclei per macroschizont

20 16 16 16 20 17 ,6

3'7 3'7 4'1 3'9 4'7 4 '0

4'0 4'5 4,8 4'4 4'3 4 '4

21

6' 7

2'0··

15

10 10 10 10 10'2

II

12'4

106'0 106'4 105'4 105'8 106'4 106'0

Not known

14

106'2

14 II

II

• According to Brocklesby (1964), bovine 3600, 1St passage from buffalo, 10 tick challenge, *'" Number of macroschizonts per 1000 lymphoid cells ,

t""

rJl

~

tt:I

~o ~ o

Maximum temperature of,

II

::0

;;

~

SOME CHARACTERISTICS OF THE THEILERIOSIS IN CATTLE

C54 C 217 C49 2 C493 C6S4 Average

~

May'68 Larvae 200 nymphs 7

Infections produced in cattle

Prepatent period in days

tt:I .....

May'68 Nymphs 50 adults 7

TABLE II

Bovine ref. no,

1-3

::t:

May'68 Nymphs 50 adults 2

Reference No, Cattle

1' 1%

1

Date of feed on buffalo Stage fed on buffalo No, of ticks applied to cattle Months since moulting

Ticks

B,2male

Maximum MSI

1-3

o

~

tt:I

<.»

co

(.J1

386

BRITISH VETERINARY JOURNAL, 129,4 TABLE III

MACROSCHIZONT INDEX AND NUCLEAR NUMBER DURING BUFFALO

Days after application o/ticks

Theileria

INFECTIONS OF CATTLE

Macroschh,ont index with mean macroschizont nuclear ,lumber in parentheses CS4

C 217

C492

C493

C654

+ +

+ + +

+ + + +

+ + + + +

g

10

+ + + + +

II

12 13 14 15 16 17 18

1'3 (3'4) 2'5 (3 '2) 3,8 (3,6) 4'0 (3'7) 1' 1 (3'5)*

Ig

20

1,6 4'5 2'7 1'4

+

(2'3) (3'5 ) (3'7) (3'9) *

1'0 (3 '5) I'g (3'7) 4'2 (3'9) 4,8 (4'1 ) *

1'4 (3' 7) 2'1 (3'9) 4'4 (3'7)*

+

1' 1 (3,6 ) (3,8) 2,8 (4'0) 4'3 (4'7) 4,6 (4'4)* I 'g

Mean

0'5 1'5 1'8 2,6 2'2 3'3 4'1 2,8

(3'4) (3,6) (3,8) (3'7 ) (3'5) (3,8) (4'2) (3'9)

* Day of death, TABLE IV MACROSCHIZONT INDEX FROM BOVINE L YMPHOID ORGANS AT DEATH ( TICKS APPLIED TO ANIMALS' LEFT EARS) .

Buffalo Theileria Lymph nodes and tissues MSI Bovine C 492 Left parotid Right parotid Left prescapular Right prescapular Left retropharyngeal Left atlantal Bronchial Mesenteric Spleen Right precrurals Left cervicals Mean Standard error

MSI Bovine C 493 3,6 5'4

4'4

4,6 11'2 6'2 3'5 5' 1 3'4 4'0 NT 5'15 ± o'66

NT = Not taken,

In one calf, extensive petechiation was noted in the cortex of the kidneys, but no kidney lesions were found in other animals. Epicardial and endocardial petechiation was present in two animals and petechiation of the serosa of the rumen was observed in three animals.

Transmission between cattle All attempts to transmit buffalo Theileria between cattle were unsuccessful. Development of the parasite in R. appendiculatus Twenty-three salivary glands, 10 from male ticks and 13 from female ticks, were examined from tick batch 2 (see Table I). Only one female tick, fed for

THEILERIAL SPECIES PATHOGENIC TO CATTLE

five days, had infected salivary glands but 2 I of the acini were infected and they contained stages corresponding to both intermediate and mature forms of T. parva (Purnell & Joyner, 1968). Thirty-nine salivary glands, 19 from male ticks and 20 from female ticks, were examined from tick batch 4. Only two female ticks, fed for four and five days, had infected salivary glands. Intermediate stage parasites were detected in no less than 140 salivary acini of the four-day fed tick. Only one lobe of the salivary gland was infected. The other tick had only one infected acinus, containing mature parasites. Thus, only three ticks of the 62 examined contained parasites in their salivary glands, but two of them were heavily infected. DISCUSSION

Our results show that buffalo in Tanzania harbour a theilerial parasite pathogenic for cattle. Buffalo and cattle come into frequent contact in East Mrica, and there is considerable circumstantial evidence that buffalo are important in the epizootiology of bovine theileriosis in many areas. In Tanzania, Milne (1956), McCulloch (1964) and Branagan (personal communication) have observed a theilerial syndrome in cattle similar to that recorded in this paper. The parasite caused infections in which death of the cattle ensued when MSI's were generally about 5 per cent. The disease thus resembled those caused by T. lawrencei (South Mrica), described by Neitz (1955, 1957) and by T. lawrencei (Kenya) described by Barnett & Brocklesby (1966), rather than that caused by T. parva, where MSI's have generally approached 100 per cent terminally (Neitz, 1957; Jarrett, Crighton & Pirie, 1969; Radley, 1971). Despite the facts that neither microschizonts nor piroplasms were detected, and that transmission between cattle was not successful, transmission from buffalo to cattle appeared to be fairly efficient since four out of six tick batches transmitted fatal infections to cattle. Examination of the ticks' salivary glands revealed a low infection rate in the ticks, but a high level of infection in the individual infected ticks. This contrasts with the results obtained by Purnell & Joyner (1968) for T. parva (Muguga), where the exact reverse was observed. The Theileria sp. causing the disease syndrome described in this paper is designated T. lawrencei (Serengeti). This is based on its similarities to T. lawrencei (South Mrica) (Neitz, 1955), namely, that it was transmitted from buffalo to cattle through the tick R. appendiculatus and that it induced a fatal disease in cattle characterized by the presence of few macroschizonts and no detectable piroplasms. The captive buffalo used in the present study had no opportunity to come into contact with cattle, so T. lawrencei (Serengeti) must be maintained by game animals in the Serengeti National Park. The majority (95 per cent) of 120 Serengeti buffalo examined harboured piroplasms morphologically indistinguishable both from those of the captive buffalo and from T. parva (Young, unpublished data). Further studies on T. lawrencei (Serengeti) will involve continued attempts to passage the parasite through cattle to see if we can confirm the behavioural

BRITISH VETERINARY JOURNAL, 129,4

transformations to T. parva recorded by Barnett & Brocklesby (1966, 1969) for T. lawrencei (Kenya) and T. lawrencei (South Africa). ACKNOWLEDGEMENTS

This work was done when one of us (ASY) was in tenure of a grant from the Well come Trust, London. The facilities provided by the Directors of Tanzania National Parks, of the Serengeti Research Institute (SRI) and of the East Mrican Veterinary Research Organization, are gratefully acknowledged. The assistance of Dr B. Schiemann, Dr A Sinclair, Dr H. Lamprey, and Mr J. Martin Moshe of S.R.I. was greatly appreciated, as was the advice of Dr L. G. Goodwin of the Nuffield Institute of Comparative Medicine, London. The Immunological Research on Tick-borne Diseases and Tick Control Project is supported by the United Nations Development Programme/Special Fund, with the Food and Agriculture Organization of the United Nations as the Executing Agency, in co-operation with the East Mrican Community. The Project is also assisted by the Overseas Development Administration of the United Kingdom (Research Projects 2396 and 2494), the United States Department of Agriculture, the Rockefeller Foundation, the International Atomic Energy Agency, and the Pfizer Corporation. REFERENCES

BAILEY, K. P. (1960). Bull. epizoot. Dis. Afr. 8, 33. BARNETT, S. F. & BRoCKLESBY, D. W. (1966). Br. vet. J. 122,396. BARNETT, S. F. & BROCKLESBY, D. W. (1969). In Diseases in Free-living Wild Animals, Symp. zool. Soc. Lond. 24, 147. BARNETT, S. F., BROCKLESBY, D. W. & VIDLER, B. 0 . (1961). Res. vet. Sci., 2, II. BROCKLESBY, D . W. (1964). D.V.M. thesis, University of Zurich. BRoCKLESBY, D. W. & BARNETT, S. F. (1966a). Br. vet. J. 122,371. BRoCKLESBY, D. W. & BARNETT, S. F. (1966b). Br. vet. J. 122,387. JARRETT, W. F. H., CRICHTON, G. W. & PIRIE, H. W. (1969). Expl. Parasit. 240 9. MCCULLOCH, B. (1964). Bull. epizoot. Dis. Afr. 12,63. MARTIN, H. M., BARNETT, S. F. & VIDLER, B. 0. (1964). Expl. Parasit. 15,527. MILNE, A. M. (1956). Ann. Rep. vet. Dep. Tanganyika 1954,2, 18. NEITZ, W. O. (1955). Bull. epizoot. Dis. Afr. 3, 12I. NEITZ, W. O . (1957). Onderstepoort J. vet. Res. 27, 275. NEITZ, W. O. (1958). Bull. epizoot. Dis. Afr. 6, 151. fuRNELL, R. E. & JOYNER, L. P. (1968). Parasitology, 58, 725. RADLEY, D. E. (1971). Ph.D. Thesis, University of Edinburgh. (Accepted for publication 12 December 1972)

Observations prelirnjnajres sur une espece de Theileria pathogene pour Ie betaU, isolee de la bu1Be (Syncerus caffer), en Tanzanie (Young et al.) Reswne. Une espece de Theileria, pathogene pour Ie betail, et ressemblant a Theileria lawrencei a ete isolee de deux bufHes capturees au Parc National Serengeti, en Tanzanie. Quand les tiques de Rhipicephalus appendiculatus a l'etat de nymphes, etaient places sur les bufHes, dans quatre sur six occasions, les adultes qui en resulterent ont ete prouves capables de causer des infections tres pathogeniques, du type T. lawrencei, dans de betail.

THEILERIAL SPECIES PATHOGENIC TO CATTLE Les aspects du cycle d'evolutioll du aprasite, tant chez les tiques que chez Ie betail, ont ete etudies. Son developpement dans Ie tique etait apparemment semblable a celui de T . parva, et dans Ie betail, differait de T. parva seulement parce qu'il causait la mort de l'h6te, quand relativement peu de lymphocytes etaient infectes par les macroschizontes, et quand ni les microschizontes, ni les piroplasmes, n'etaient detectables. Le parasite a ete denomme T. lawrencei (Serengeti). VorIaufige Mitteilung iiber einen Theileria Stanun, der fiir Rinder pathogen ist und von tanzanischen Biiffeln (Syncerus caffer) isoliert wurde (Young et al.) ZusaDlDlenfassung. Eine fUr Rinder pathogener Theileria Stamm, der Theileria lawrencei iihnelt, wurde von zwei Btiffeln isoliert, die in Tanzanias Serengeti Naturschutzpark gefangen worden waren. Wenn Rhipicephalus appendiculatus Zecken als Larven and Buffel verftittert wurden, zdgte sich bei vier von sechs Versuchen, dass die sich entwickelten erwachsenen Parasiten imstande waren bei Rindern hOchst pathogene Infektionen des T. lawrencei Typs zu verursachen. Der Entwicklungszyklus der Parasiten wurde in den Zecken und den Rindern studiert. Bei Zecken ahnelte er offenbar dem der T. parva und beim Rind unterschied er sich nur insofern davon, dass der Wirt verendete, wenn relativ wenige Lymphozyten mit Makroschizonten infiziert waren, und wenn weder Mikroschizonten noch Piroplasmen beobachtet wurden, Der Parasit wurde T. lawrencei (Serengeti) bennant. Observaciones prellininares sobre una especie Theileria patogenica al ganado que se aisl6 del bufalo (Syncerus caffer) en Tanzania (Young et al.) ResUD1en, Se ha aislado una especie teileriana, patogenica al ganado y que se parece a Theileria lawrencei, de dos bUfalos que se capturaron en el Parque Nacional de Serengeti, Tanzania. Cuando se aliment6 a las ninfas de las garrapatas Rhipicephalus appendiculatus en los bUfalos, se demostr6 que en cuatro de seis casos los adultos resultantes eran capaces de causar unas infecciones altamente patogenicas del tipo de T. lawrencei en el ganado. Se estudiaron aspectos del ciclo de vida del parasito tanto en las garrapatas como en el ganado. Su desarrollo en la garrapata se asemeja aparentemente al de T. parva, y, en el ganado, solamente se diferenci6 de T. parva en que caus6 la muerto del huesped cuando relativamente pocos linfocitos fueron infectados por macrosquizontes y cuando no se podian descubrir ni microsquizontes ni piroplasmas, El parasito fue llamado T. lawrencei (Serengeti).