# Hæmatoxenus separatus sp. n. (Sporozoa, Theileriidae),
a new blood parasite of domestic sheep
in Tanzania

by G. UILENBERG (*) and M. P. ANDREASEN (**)

## RESUME

Haematoxenus separatus sp.n. (Sporozoa, Theileriidae),
nouvel bématozoaire du mouton domestique en Tanzanie,
transmissible par tiques

Après une revue du genre *Haematoxenus*, connu chez le bovin, le buffle africain et des antilopes africaines, les auteurs rapportent la découverte d'une nouvelle espèce de ce genre chez le mouton domestique en Tanzanie, *H. separatus* sp. n. Le parasite, apparu chez un mouton après splénectomie, a pu être transmis à un second mouton par inoculation de sang infecté. Le parasite diffère morphologiquement des *Theileriidae* déjà connues chez le mouton par la présence d'un voile, particularité du genre *Haematoxenus*; de plus, ses dimensions sont plus grandes que celles de *Theileria ovis* de Madagascar et de la République Centrafricaine. On ne sait pas encore si des éléments sans voile, apparus en même temps que les formes à voile, appartiennent à l'espèce *H. separatus* ou s'ils doivent être attribués à une infection mixte. Le voile est séparé du parasite, contreirement à celui d'*H. veliferus*. Le voile semble provenir du cytoplasme de l'érythrocyte infesté. La division d'*H. separatus* dans les globules rouges se fait en quatre, comme chez les autres *Theileriidae*. Des stades exoérythrocytaires n'ont pas encore été observés. Une modification de la définition du genre *Haematoxenus* est proposée.

## INTRODUCTION

The genus *Haematoxenus* was created for an intracellular blood parasite of cattle in Madagascar, *H. veliferus* Uilenberg, 1964. The only stages in the life cycle of this parasite which have been recognized until now are the erythrocytic forms, morphologically similar to

*Theileriae*, apart from the fact that many have a rectangular « veil » arising from the side of the organism. Division in four is also similar to the *Theileriae* (11).

*H. veliferus* has been found in cattle (both *Bos indicus* and *B. taurus*) in Madagascar (10, 11, 12, 13), in Nigeria (4, 3), in Kenya (2, 17), in Uganda (2, 8, 9), in the Central African Republic, the Chad and Mali (14). We have also seen it in a bloodsmear from a cow taken in the Ivory Coast in 1972 by our colleague Dr M. Clair, and recently in several cattle in Tanzania. The parasite is therefore widely spread in tropical Africa, and is quite common

<footnote>(*) Tanzania F.A.O./U.N.D.P. Project "Improve-
ment of Tick Control", c/o Central Veterinary
Laboratory, P.O. Box 9254, Dar-es-Salaam, Tanzania.
(**) The Royal Veterinary and Agricultural Uni-
versity, Department of Veterinary Virology and Im-
munology, 13 Bülowsvej, 1870 Copenhagen V, Den-
mark.</footnote>
in Madagascar (11, 12, 13), in Uganda (9, UILENBERG and ROBSON, unpublished observations, 1973), in Mali (15) and in Tanzania (UILENBERG, unpublished observations, 1973, 1974).

A morphologically identical parasite occurs in wild African buffaloes (*Syncerus caffer*) in the Central African Republic (14) and in Uganda (17).

*Haematoxenus* also occurs in African antelopes from impala (*Aepyceros melampus*) in Tanzania (5), and YOUNG (personal communication, 1973), has found it in hartebeest (*Alcelaphus buselaphus*) in Kenya. The morphology of the veil of the parasites in antelopes seems to be somewhat different from that of *H. veliferus*, being less regularly rectangular.

In this paper, we describe a new pathogenic parasite of domestic sheep in Tanzania, for which we propose the name *Haematoxenus separatus* sp. n., the "separated" *Haematoxenus*.

## MATERIAL AND METHOD

A lamb (nº 1549) of the local breed of sheep, approximately 6 months old, born at Dar-es-Salaam, was splenectomized. Scanty theilerial parasites had been seen on one occasion in its blood prior to the operation, and it was hoped to obtain a high parasitaemia of *Theileria ovis* Rodhain, 1916, for transmission experiments with ticks. A second lamb, black-headed Persian, approximately one month of age, born at Dar-es-Salaam, was also splenectomized; it was hoped that this young animal, born in a flock where some tick control was practised, would still be free from *T. ovis*, in order to serve for transmission experiments.

## OBSERVATIONS ON THE SHEEP

### Lamb n° 1549

Scanty theilerial organisms appeared in its blood 3 days after splenectomy. They increased in number from day 6 after the operation, until on day 17 over 5 p. 100 of the erythrocytes were infested; the parasitaemia decreased after this maximum and the parasites were again scanty on day 21.

Theilerial organisms associated with veils were first noted on day 6. Their number increased at the same time as that of the normal theilerial organisms, until on day 17 a maximum of at least 1 p. 100 of erythrocytes infested was reached; their decrease also paralleled that of the normal organisms. During this period, the proportion of organisms associated with a veil to the normal ones appeared to remain relatively stable, at any one time some 10 to 15 p. 100 of the total number of parasites being associated with a veil. Both types fluctuated in number after day 21, from very scanty to fairly numerous (never exceeding 0,2 p. 100 of cells infested), and both could be found for at least fourteen months after splenectomy. The ratio of veiled to unveiled parasites appeared far less constant during this period of low parasitaemia.

Lamb 1549 also showed *Anaplasma ovis* Lestoquard, 1924 after the operation. The first anaplasms appeared on day 10, and they had become so numerous, associated with marked anaemic changes in the blood picture, on day 20, that the animal was treated with a tetracycline (Reverin®) at 10 mg/kg. The anaplasms then disappeared within a few days and have not been seen again.

Fever was noted on days 11 to 13, with a maximum of 41,0° C on day 12, associated with a rapid rise in number of both types of theilerial organisms, while *A. ovis* was still scanty. Anaemia was noted from day 19 onward, while the theilerial parasites were decreasing in number, associated with a rapid increase in parasitaemia by *A. ovis*; anaemic changes in the blood picture were most pronounced on days 21 to 23.

### Persian lamb

No blood parasites appeared following splenectomy. On day 40 after the operation, it was injected subcutaneously with 5 ml of blood taken from lamb 1549. This was 58 days after the splenectomy of lamb 1549; both types of theilerial parasites were fairly numerous in its blood that day.

Very scanty theilerial organisms were seen in the blood of the Persian lamb 18 days after this injection. The type associated with a veil was observed with certainty 21 days after the injection. Unfortunately, the animal, which had
lost its mother and had become very weak, died 22 days after the injection.

## DESCRIPTION OF HAEMATOXENUS SEPARATUS SP. N.

### Morphology

(methanol fixation, Giemsa staining)
(fig. 1, 2, 3, microphoto)

*Theileria-like* organisms in the red blood cells, associated with a veil that does not arise from the parasite as in *H. veliferus*, but is separated from it. The long axis of the veil does not seem to have any particular orientation in relation to the parasite. No difference in colour or intensity of staining between the veil and the cytoplasm of the red cell has been observed, except in erythrocytes in the process of lysis, when the veil retains its original colour, while the normal cell cytoplasm becomes faint. The veil is most commonly situated outside the red cell, opposite a corresponding gap in the cell, and it appears to originate from erythrocyte cytoplasm. The veil can also be situated on or in the cell, in which case it is surrounded by an unstained halo. [A similar gap around the veil of *H. veliferus* is commonly seen, see UILENBERG (13, p. 432)]. The organism and its associated veil are sometimes seen lying free between the red cells, presumably a result of lysis of the host cell. The shape of the veil is on the whole rectangular, but the angles are commonly rounded, and ellipsoid and crescent-shaped veils occur. The dimensions of the veils vary from slightly more than 1 µ to over 3 µ in length, with a width of approximately 0,5 µ to more than 1 µ.

The organisms are typically *Theileria*-like. Round and oval parasites predominate, narrow rod-shaped ones also occur, but are less frequent. The largest parasites are well over 2 µ in length, the smallest are approximately 1,5 µ.

Dividing forms, associated with a veil, are present. Division is as in *Theileria*, with the formation of a Maltese cross, resulting in four small parasites.

Some or all of the unveiled theilerial organisms present at the same time as typical *H. separatus*, may belong to this species; small theilerial organisms without veils also occur in *H. veliferus* (11). Their average dimensions

are less than those of typical *H. separatus*; many are only 1 µ by less than 0,5 µ.

### Life cycle

Unknown, apart from multiplication in four in the red cells.

### Host

Domestic sheep.

### Transmission

Artificial transmission is possible by subcutaneous injection of infected blood.

### Pathogenicity

The fever in lamb 1549, although the temperature was normal at the maximum of the theilerial parasitaemia, may have been caused by the (possibly mixed) theilerial parasites, as *A. ovis* was still scanty at that time. The anaemia in lamb 1549 may well have been due mainly to *A. ovis*.

### Differential diagnosis

*H. separatus* differs from *T. ovis* and *Theileria hirci* Dschunkowsky and Urodschevich, 1924 in the presence of the veil. The veil is such a striking feature that it is inconceivable that the different authors who have observed sheep *Theileriae* could have overlooked it. *T. hirci* has only been reported from northern Africa, southern Europe and Asia Minor, while *T. ovis*, the only other known *Theileria* of sheep, has been reported from many parts of Africa, including Tanzania [see for instance NEITZ (7)]. We have compared *H. separatus* to what we consider to be *T. ovis* from splenectomized sheep in Madagascar and the Central African Republic. The biggest forms of *T. ovis* do not reach the size of some almost *Babesia*-like parasites of well over 2 µ that are found in *H. separatus*. No veils like those of *H. separatus* were seen in smears from Malagasy and Central African sheep. Within some of the *T. ovis* infested red cells a darker patch like an ill-defined veil was seen, as well in smears from Madagascar as from the Central African Republic; this has been reported before in Malagasy sheep (13). Such darker patches, which are quite different from the clearly defined veils of *H. separatus* or *H. veliferus*,
<figure><img src="image_1.png" /><figcaption>Fig. 1. — *Haematoxenus separatus* dans les erythrocytes de mouton.</figcaption></figure>

<figure><img src="image_2.png" /><figcaption>Fig. 2. — *Haematoxenus separatus* comprenant un parasite libre avec voile et un parasite dans un globule rouge en voile de lyse, le voile gardant sa couleur originale.</figcaption></figure>

<caption>Fig. 2. — *Haematoxenus separatus*, including one free-lying parasite with veil, and one parasite in lysing red cell, the veil retaining its original colour.</caption>
<figure><img src="image_3.png" /><figcaption>Fig. 3. — *Haematoxenus separatus* comprenant un parasite libre avec voile, trois formes en division dans des globules rouges, et un parasite libre qui vient apparemment de terminer sa division en 4, avec son voile.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 4. — Microphotographie d'*Haematoxenus separatus* dans les erythrocytes de mouton.</figcaption></figure>

<caption>Fig. 4. — Microphotograph of *Haematoxenus separatus* in sheep erythrocyte.</caption>
have not been seen in red cells of lamb 1549
infested with unveiled or veiled organisms.

The position of the veil in relation to the
parasite is quite different in *H. separatus* from
that in *H. veliferus*. One single attempt at
transmission of *H. veliferus* from cattle to a
splenectomized sheep has given a negative
result (11).

DISCUSSION

The definition of the genus *Haematoxenus*
by BARNETT (1) and LEVINE (6) should be
amended as follows :

*Theileriidae* in which the erythrocytic forms
are associated with a clearly defined more or
less rectangular veil, separated from the orga-
nism or appearing to arise from it.

As noted before (15), even in *H. veliferus*
the veil probably does not arise directly from
the organism, but seems to be separated from
it by a narrow band of erythrocytic cytoplasm,
visible with the electron microscope (unpu-
blished electron microscopic photographs made
by Dr H. M. D. HOYTE, Brisbane, 1965). It

now appears that the veil originates from ery-
throcytic cytoplasm. Its mode of origin and
its function remain quite unknown.

Veil-like structures may be more wide-spread
amongst the *Theileriidae* than is realized at
present. As noted above, darker patches of
red cell cytoplasm are associated with some
forms of *T. ovis* in Madagascar and the Central
African Republic. They also occur in infec-
tions with the British cattle *Theileria* (16),
which may not be *T. mutans* after all, as such
structures have not been seen with African
or Malagasy *T. mutans*. Intermediate species
between typical representatives of *Theileria*
and *Haematoxenus* might thus occur.

Specimen slides of *H. separatus* have been
deposited in the collections of both authors.

Acknowledgements

We are grateful to the Director of the
Livestock Development of the Ministry of
Agriculture and to the Director of the Central
Veterinary Laboratory in Dar-es-Salaam for
permission to publish this paper. We also wish
to thank Mr D. S. M. Kadaso, Assistant Field
Officer, for his assistance.

SUMMARY

The authors describe a new species of the *Theileriidae, Haematoxenus separatus* sp. n., in domestic sheep in Tanzania. The parasite appeared in the blood after splenectomy of a carrier, transmission by injection of infected blood was successful. The veil of this species is separated from the parasite, not appearing to arise from it as in *H. veliferus* of cattle. An amendment for the definition of the genus *Haematoxenus* is proposed.

RESUMEN

Haematoxenus separatus sp. n. (Sporozoa, Theileriidae) nuevo
hematozoario de la oveja doméstica en Tanzania, trasmisible
por garrapatas

Los autores pasan en revista los conocimientos actuales del genero *Haematoxenus*, conocido en el bovino, el búfalo africano y antílopes africanos. Después, notan el descubrimiento de una nueva especie de dicho genero en la oveja doméstica en Tanzania, *H. separatus* sp.n. Se pudo trasmitir el parásito, encontrado en una oveja después de esplenectomia, a una segunda oveja por inoculación de sangre infectada.

El parásito difiere morfologicamente de las *Theileriidae* ya conocidas en la oveja por la presencia de una vela, particularidad del genero *Haematoxenus*. Además sus dimensiones son más grandes que las de *Theileria ovis* de Madagascar y de República Centroafricana. No se sabe todavía si los elementos sin vela, aparecidos al mismo tiempo que las formas con vela, pertenecen a la especie *H. separatus* o si deben de ser atribuidos a una infección mixta. La vela es separada del parásito, al contrario de la de *H. veliferus*. Parece provenir del citoplasmo del eritrocito infestado. *H. separatus*, como los otros *Theileriidae*, se divide en cuatro en los glóbulos rojos. No se observaron todavía estados exeritrocitarios. Se propone una modificación de la definición del genero *Haematoxenus*.
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