Diagnosis of Theileria annulata infection of cattle in Tunisia: comparison of serology and blood smears

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Diagnosis of Theileria annulata infection of cattle in Tunisia: comparison of serology and blood smears

Mea Darghouth, A Bouattour, L Ben Miled, L Sassi

To cite this version:

Mea Darghouth, A Bouattour, L Ben Miled, L Sassi. Diagnosis of Theileria annulata infection of

cattle in Tunisia: comparison of serology and blood smears. Veterinary Research, BioMed Central,

1996, 27 (6), pp.613-621. �hal-00902453�

Original ^article

Diagnosis ^{of Theileria annulata} infection of cattle in Tunisia: comparison ^of serology ^{and blood smears}

MEA Darghouth A Bouattour L Ben Miled 2 L Sassi

1 Laboratoire de parasitologie, École nationale de médecine vétérinaire, ^{2020 Sidi} Thabet;

2 Institut Pasteur de Tunis, ^Tunis Belvédère, Tunisia

(Received ²⁰ February 1996; accepted ³⁰ May 1996)

Summary ― The immunofluorescent antibody ^test (I FAT) using schizont and piroplasm antigens ^was compared ^{with the} microscopic examination of Giemsa-stained blood smears for the diagnosis ^of

Theileria annulata infection in experimentally ^and naturally infected cattle. The results obtained on 100 naive cattle showed that non-specific fluorescence disappeared ^{at serum dilution levels of 1/40}

and 1/160 for the piroplasm and schizont antigens, respectively. ^{These levels were therefore retained}

as the starting dilutions for this study. ^On day ³⁰ post-infection, ¹⁶ experimentally ^{infected calves}

were shown to be serologically positive for schizont and piroplasm IFAT, while 13 of them were pos- itive for blood smears. A total of 109 cattle from an endemic region ^of tropical theileriosis were sampled

before the onset of the disease season in April ^{and later in} September. Globally ^{the IFAT results}

revealed more cattle exposed ^to T annulata infection than the blood ^smear examination. The piro- plasm IFAT and the blood smears were less reliable than the schizont IFAT. The latter appeared ^{to be}

the best test for detecting exposure ^to T annulata.

cattle I immunofluorescent antibody ^test I Theileria annulata I Tunisia

Résumé ― Diagnostic ^de l’infection des bovins par Theileria annulata. Étude comparative ^de

la sérologie ^{et de l’examen de frottis} sanguins. Le test d’immunofluorescence indirecte (7F/! ^utili-

sant l’antigène ^schizonte (IFI schizonte) ^et l’antigène piroplasme (IFI piroplasmes) ^{a été} comparé ^{à l’exa-}

men microscopique de frottis sanguins pour le dépistage d’infections expérimentales ^et naturelles de bovins parTheileria annulata. Les résultats obtenus sur 100 bovins indemnes ont montré la disparition

de la fluorescence non spécifique ^{aux dilutions} sérologiques ^{de 1/40 e et de 11160e} respectivement pour l’IFI schizonte et l’IFI piroplasme. Ces dilutions ont de ce fait été retenues comme dilutions test pour le diagnostic sérologique. Un groupe de ^{i6 veaux} infectés expérimentalement ^{ont tous} présenté ^une sérologie positive ^au 30 e jour de l’infection alors que ^{seuls 13} d’entre eux ont montré un frottis sanguin positif. ^Des prélèvements ^{obtenus avant et} après ^la saison de theilériose sur un échantillon de i09 bovins

en zone d’endémie de theilériose ont permis de constater que /’/FI était globalement plus ^sensible

*

Correspondence ^and reprints

que l’examen de frottis ^de sang pour ^le dépistage ^de l’exposition à l’infection parT annulata. Par ailleurs l’IFI schizonte apparaît ^{comme la} technique ^de dépistage ^la plus ^fiable par comparaison ^à

/’IF/ piroplasme ^{et au frottis de} sang.

bovins limmunofluorescence indirecte /Theileria annulata 1 Tunisie

INTRODUCTION

Tropical theileriosis is a parasitic ^disease

caused by ^the protozoan ^{Theileria annu-} lata. This disease represents ^a major ^threat

to cattle and in particular exotic pure breeds, in a large ^zone extending from the Mediter-

ranean littoral, up ^to the eastern part ^of

China (Purnell, 1978). ^In Tunisia, during ^the period from June to August tropical ^theile-

riosis represents ^{one of the} major ^diseases affecting ^cattle, particularly ^for improved dairy ^breeds (Darghouth, 1991 ).

The immunofluorescent antibody ^test (IFAT) ^{can detect serum} antibodies in cattle

recovering from Theileria infections

(Schindler ^{and Wokatsch,} 1965). ^{This test}

was successfully ^{used for} detecting ^anti-

bodies against schizont and piroplasm ^anti- gens in cattle infected with T annulata.

(Pipano ^and Cahana, 1969; Burridge ^{et al,} 1974; Goldman and Pipano, 1976; Dhar and

Gautam, 1977). Despite the fact that the IFAT has been in use for a long ^{time and}

may be somewhat dated, and also the absence of a more convenient operational ELISA, ^{it still} represents the reference test

currently ^{used in} investigations ^of tropical

theileriosis. Some studies have reported

that the IFAT is globally ^{more sensitive than} blood smear examination for the detection of T annulata piroplasms ^{in the field} (Dhar ^and Gautam, 1977; Jongejan ^{et al, 1995;}

Kachani et ai, 1995; Sayin ^{et al,} 1995). ^How-

ever, the level of superiority of IFAT rela- tive to blood smears appeared ^to vary ^con-

siderably depending ^{on the authors,} region

and season. Furthermore, ^{these sudies did}

not present clear data concerning ^{the rela-} tionship between antibodies and piroplasms

in animals exposed ^to T annulata natural infection. This paper reports the results of a

comparative study ^{conducted on} experi- mentally ^and naturally ^{infected cattle, in}

order to establish the relationship ^between

the antibodies in schizont and piroplasm

IFAT and piroplasms, ^{in cattle} exposed ^{to a}

T annulata infection. This was a preliminary step ^towards identifying ^{the most reliable} diagnotic ^{test for use under} Tunisian field conditions.

MATERIALS AND METHODS

Comparative study of IFA T and Giemsa blood smears in the diagnosis

of experimental ^infection

Animals

The IFAT using both schizont and piroplasm ^anti-

gens ^was evaluated on two groups ^of Friesian-Holstein cattle coming from two state farms free of tropical theileriosis. The animals were divided in two groups, Group I consisted ^of

84 animals (36 ^{calves of more than 3} months, ^{13 3} heifers and 35 Friesian-Holstein cows in lacta-

tion). Group II contained 16 male calves of 3 to 5 months old and who were born during ^{the win-}

ter.

IFAT serology

The IFAT was performed using schizont and piro- plasm antigens according ^{to the} protocol

described by Burridge and Kimber (1973); ^and Burridge ^{et al} (1974). The schizont antigen ^was prepared ^{from a T} annulata cell line at low pas- sage ^levels (< ²⁰ passages). Piroplasm antigen

was prepared from two naive calves showing ^a parasitaemia ^{of 25 and 30% after an} experimen-

tal infection with a tick stabilate. Rabbit anti-bovine

IgG fluorochrome conjugate (Nordic ^Laborato- ries) ^{was used, after} previous titration, ^{at a dilution} of 1/80 in PBS. The slides were observed in a Leitz Dialux SM 100 microscope equipped ^{for flu-}

orescence. For each test known positive and neg- ative sera were used ^as controls.

Determination of non-specific

fluorescence in naive cattle

Sera samples ^were collected from Group ^{I cat-}

tle and Group II calves before the experimental

infection. The serology ^was performed ^{with the}

two antigens using ^serum dilutions of 1/10, 1/40

and 1/160.

Detection of antibodies and piroplasms

in calves experimentally ^infected

with T annulata

The 16 calves of Group II were infected with four Tunisian T annulata cell lines at low passage ^of culture (< ¹⁰ passages) isolated from natural clin- ical cases of tropical theileriosis and recorded as cell lines A, B, ^{C and} D, respectively. ^{The ani-}

mals were inoculated subcutaneously ^{with 2 x 10 6}

cells from fresh culture. They ^{were monitored}

over a period ^{of 30} days ^for clinical and para-

sitological reactions to the infection. On day ³⁰

blood samples ^{were collected from each calf and} Giemsa-stained blood smears and serum sam-

ples ^were prepared. ^The presence of piroplasms

of T annulata was investigated ^{on 50} microscope

fields. Schizont and piroplasm ^{IFAT were} per- formed using ^{serum dilutions of} 1/10, 1/40, 1/160, 1/640 and 1/2 560.

Comparison ^between IFAT serology

and Giemsa-stained blood smears to determine the exposure ^{of cattle} to T annulata in ^an endemic region

Farms and animals

Six farms from the endemic region ^{of Sidi Thabet}

with a previous history ^of tropical theileriosis

were included in this study. ^{These farms con-}

tained 109 cross-bred and Friesian cattle. The animals were individually identified using ^labelled ear tags.

Sampling ^{of animals}

The six farms were visited twice, ^{first in} April

before the theileriosis season and then in Septem-

ber after the theileriosis season. On each of these visits blood samples ^{were collected from all ani-}

mals and individual serum samples and Giemsa- stained blood smears were prepared. ^{On the sec-}

ond visit, in September, three out of the 109 cat- tle sampled ⁱⁿ April ^{were not} found. Calves which

were born between the two visits were excluded from the sampling.

IFAT serology and examination of blood smears

The IFAT was performed ^{as described} previously.

Individual serum collected on each of the two vis- its were screened at a serum dilution of 1/40 and 1/160 for the piroplasm IFAT and the schizont

IFAT, respectively. The presence of T annulata

piroplasms ^{was assessed on each Giemsa-}

stained blood smear by observing ^{of 20 micro-}

scope fields (10 3 magnification). ^{For each of the}

two visits the mean number of piroplasms

recorded in ^a positive ^{blood smear was calcu-}

lated.

The kappa coefficient was calculated for eval-

uating ^the agreement between blood smears and

serology (Toma ^{et al,} 1991). The ^chi square ^test

was used for distribution analysis. ^{In case of small}

numbers this test was used with the Yates cor- rection. The Student’s t test was used for ^com-

parison ^{of mean values} (Schwartz, 1983).

RESULTS

Comparative study ^{of IFAT}

and blood smears in the diagnosis of experimental ^infection

Determination of non-specific

fluorescence on naive cattle Schizont IFAT

Only ^a single ^cow among the 84 animals from Group ^{I was} serologically positive ^at

the serum dilution of 1/40 in schizont IFAT

and showed a weak fluorescence. All the

other cattle of Groups ^{I and // were sero-} logically negative ^at the three dilutions tested

(1/10, 1/40 ^and 1/160). ^{Some sera} gave, ^at the dilution of 1/10 and 1/40, ^a fluorescence of the whole leucocyte without any particu-

lar fluorescence of the schizont. The level of this fluorescence was moderate at the dilu- tion of 1/10 and weak at the next dilution of 1/40. However, ^at the 1/160 dilution this flu-

orescence was totally eliminated in all the

sera tested resulting ^{in a} specificity ^{of 100%.}

Piroplasm ^IFAT

At a dilution of 1/10 and before any infec- tion, ^{some sera} _gave ^a weak diffuse fluo-

rescence of both infected and uninfected cells. At the next dilution of 1/40 all the sera

from naive calves were negative, giving ^a specificity ^{of 100%.}

Detection of antibodies and piroplasms

in calves experimentally ^infected

with T annulata

The results obtained on day ³⁰ post-infec-

tion from the 16 experimentally ^infected

calves using IFAT and blood smears for

detecting anti-schizont and anti-piroplasm

antibodies and piroplasms of T annulata are

shown in table I. All the infected calves were

serologically positive ^{at or above the serum}

dilutions of 1/40 and 1/160 in piroplasm ^and

schizont IFAT respectively. At the dilution of 1/10 and 1/40 respectively the tested sera were positive but all the infected cells

appeared fluorescent without there being ^a

clear contrast between the parasite ^{and the} cytoplasm of the host cell. Piroplasms ^were

detected on day ^{30 in 13 out of the 16 6}

infected calves (81.3%) ^The piroplasm-pos-

itive calves all showed a parasitaemia ^below

0.1%.

Comparison of the IFA T serology

with blood smears examination for the detection of natural exposure to Tannulata infection

The results of the comparison ^between

blood smears and piroplasm IFAT and blood

smears and schizont IFAT are shown in

table II. Before the theileriosis season

(beginning ⁱⁿ April), ^the prevalence ^{of cat-}

tle giving positive ^{blood smears} (31.19%)

was not significantly different from the results given by schizont IFAT (37.62%)

and by piroplasm ^IFAT (25.68%). ^{After the}

theileriosis season (in September), ^{the two} serological ^{tests detected} significantly ^more

cattle exposed ^to T annulata infection

(55.66 and 46.23% for schizont and piro- plasm ^IFAT, respectively) than blood

smears (20.75%) (test Chi two, ^P < 0.01 ).

The sensitivity ^of serology ^evaluated by refering ^{to blood} smears was 70.6 and 82.4% in April and 90.9 and 95.5% in

September ^{for the} piroplasm and schizont IFAT respectively.

The proportion ^of positive ^{sera to schizont}

and piroplasm ^{IFAT are} shown in table Ill.

Almost all the serologicaly positive ^cattle (to

both schizont and piroplasm IFAT) ^were

detected with schizont IFAT, 100 and 98.3%

in April ^and September, respectively. During

the corresponding ^visits piroplasm ^IFAT

detected 68.3 and 81.7% of the serologi- caly positive animals. For the two periods

of sampling ^the difference between the two

serological ^{tests was} significant (test ^Chi-

square, P ^< 0.001 for April, ^{and P < 0.01 in} September).

The levels of agreement between blood

smears and piroplasm IFAT and blood

smears and schizont IFAT were calculated

using ^the kappa ^test. The values obtained for the kappa coefficient were 0.7 and 0.62 in April and 0.39 and 0.31 in September,

for piroplasm IFAT and schizont IFAT

respectively. ^In April, ^cattle positive ^to piro- plasm IFAT alone or to both piroplasm ^and

schizont IFAT presented ^a significantly higher prevalence ^of piroplasms ^{than those}

who were positive only ^to the schizont IFAT, respectively ^{23/28 and 5/13} (test chi-square,

P < 0.01 ). In September, this difference was not significant.

The prevalence ^of piroplasm ^{in cattle} sig- nificantly ^{decreased from} April ^to September (test chi-square, ^P < 0.001 ). In addition the average parasitemia ^{estimated on a total}

count of 20 fields in piroplasm-positive ^cat-

tle showed a similar pattern: 8.4 and 2.2

piroplasms ⁱⁿ April ^and September respec-

tively (test ^{t, P} < 0.001 ). Finally the preva-

lence of piroplasm ^{in cattle} testing positive

in piroplasm and schizont IFAT was signif- icantly higher ⁱⁿ April (68.3%) ^{than in} September (35.6%) (P < 0.001 ).

DISCUSSION

Because of the high specificity ^{noted on the}

100 naive cattle, and also the high ^sensi- tivity obtained in the experimentally ^infected calves, ^the dilutions of 1/40 and 1/160 were

retained as the screening ^test dilution levels in piroplasm I FAT and schizont IFAT respec-

tively, ^for assessing ^the serological ^{state of}

cattle in the field. It could, however, _{be sug-} gested ^{that the} single ^{cow from the} Group ^I

of naive cattle, that showed a weak posi-

tive serology ^{at the 1/40} dilution in schizont IFAT might ^{have been} previously ^infected

with T annulata. However, the fact that the infection of this animal with a mild T annulata cell line resulted in a clinical and serological

reaction similar to the other naive serologi- cally negative ^cattle (Darghouth, unpub-

lished data), strongly ^supports the view that this mild case of seropositivity ^was non-spe- cific.

At the April ^visit piroplasm IFAT and sch- izont IFAT had, globally, ^a good ^{level of} agreement with the blood smears with val-

ues of kappa coefficient between 0.61 and 0.8 (kappa coefficient of 0.7 and 0.62

respectively ^for piroplasm ^{IFAT and}

schizont IFAT). These results tend to confirm that the antibodies detected are specific ^to

T annulata. The good ^{level of} agreement

between I FAT and blood smears, and their similar levels of sensitivity, might ^indicate

that during April (before ^the theileriosis sea-

son), serology ^{and blood smears gave a}

similar reliability ^for detecting infected cattle.

Negative serological ^{reactions were}

observed during April ^{in ten and six out of}

the 34 cattle with positive ^{blood smears, in}

schizont and piroplasm ^IFAT, respectively.

These results indicate that antibodies to T annulata in some cattle could have a

shorter duration than the infection itself.

Similar observations were also made by

Dolan et al (1986); ^with T parva ^infected

animals and by Jongejan ^{et al} (1995); ^{for T}

annulata using PCR-specific primers. ^Fur- thermore, it is also possible ^{that in} April

some cases of latent infection were not detected by serology ^and by ^{blood smears,}

since such negative serological ^reactions

might have occurred in infected cattle in which, because of very low parasitemia levels, piroplasms ^{were not detected in}

blood films. In Turkey, Sayin ^{et al} (1995);

found that before the theileriosis season the IFAT was significantly ^more sensitive than blood smears. The difference from the results of this study ^can probably ^be

attributed to differences in the screening ^of

the sera dilutions and in the number of fields examined for the detection of piroplasms.

Shortly after the end of the theileriosis sea- son, during ^the September ^{visit, the two}

IFAT became significantly ^more reliable than the blood smears (P ^< 0.001 ). A ^similar

observation was also reported by ^{Dhar and}

Gautam (1977). ^The higher reliability ^of serology ^was also observed in this work on

calves recovering ^from experimental ^infec-

tion. On day ³⁰ post-infection, piroplasm

and schizont IFAT detected all the infected animals while blood smears only ^detected

81.25% of them (table I). ^It would have been

interesting ^to have followed these calves

longer ^{than 30} days ^{in order to assess the} sensitivity ^{of the} diagnostic ^{tests a few}

months afterwards. This _was, unfortunately,

not possible ^{in the} present study ^because

the animals were used in another experi-

ment. However a subsequent study ^{on naive}

calves exposed ^to natural T annulata pri- mary infection confirmed that IFAT serol- ogy is ^more sensitive than blood smears

few months after the occurrence of the infec- tion (Soudani, 1995). ^{Indeed 3 to 4 months} after their infection all the calves were pos- itive in schizont IFAT at 1/160 dilution while

only ^73.3% of them showed positive ^blood

smears.

This superiority ^{of the} serology ^{to the}

blood _smears, after the theileriosis _season,

was related to the fact that the level of sero-

prevalence ^{and the} sensitivity of the serol- ogy increased significantly ^while, in contrast with what might normally ^be expected, ^the prevalence ^of piroplasms ^decreased (table II). ^This explains ^also why ^the serology ^and

blood smears had a lower level of agree- ment during ^the September ^visit (kappa

coefficient below 0.4 for both schizont and

piroplasm IFAT). Furthermore in the sero-

logically positive animals the prevalence ^of piroplasms ^was significantly reduced from

April ^to September. It could therefore be

suggested that the stimulation of the immune system throughout ^{the summer} by

natural infections resulted in an increase in the antibody levels, and also in an efficient immune control of the parasite replication,

which probably limited the appearance of

piroplasms in the infected cattle even after the end of the disease season in September,

as noted in this study. This view is strongly supported by the fact that during ^the September ^{visit, the mean} parasitemia ⁱⁿ piroplasm-positive ^{cattle was} only ^a quar- ter of the previous ^{level in} April. Similarly,

in Morocco, the study of Flach and Ouhelli

(1992) showed that in September, the par- asitemia level in cattle was half as much as

in April. Interestingly, ^{the same} sample ^of

cattle followed in this study between the visit of September ^{to the next of} April, ^{showed a} higher prevalence ^of piroplasm-positive ^cat-

tle (data ^not shown). This observation could

possibly ^be explained, in the absence of natural infections which would have increased the immunity, by ^{a relative}

decrease in the ability of the immune system

to control the parasite replication ^{in chronic}

infection. Early ^{cases of mild} tropical ^thei-

leriosis in the spring ⁱⁿ Algeria, ^{which were}

described by Sergent ^{et al} (1945), ^{are also}

observed in Tunisia before the beginning ^of

the activity period ^{of the vector tick} (Dargh- outh, unpublished data). ^{It is} possible ^that

these mild cases of disease may correspond

to a relapse that follows the replication ^of

the parasite in infected cattle. More inten- sive field studies are required ^{to assess this} hypothesis.

The schizont IFAT appeared significantly

better than the piroplasm ^{IFAT at} detecting

animals exposed ^to T annulata infection

(table 111). ^{Almost all, 100 and 98.3%, of} seropositive ^{cattle were detected} by

schizont IFAT during ^the spring ^{and autumn}

visit respectively, ^while only 68.3 and 81.7%

of these cattle were positive ⁱⁿ piroplasm

IFAT. Furthermore, in cattle which were pos- itive to blood smears, the schizont IFAT gave ^a higher sensitivity ^than piroplasm IFAT, particularly ^{in the} spring visit where 82.4% of cattle carrying piroplasm ^were pos- itive for this test against ^{70.6% with} piro- plasm ^IFAT. However, the piroplasm ^IFAT

correlated better with the blood smear

results since it gave the highest ^{values of}

the kappa coefficient. This was also illus- trated by the fact that cattle positive ^to piro- plasm ^{IFAT showed a} higher prevalence

of piroplasm than animals positive ^to

schizont IFAT only (table II). Furthermore, since the prevalence ^of anti-piroplasm-anti-

bodies in piroplasm-positive ^{cattle was} higher during ^{the autumn visit} (90.9%) shortly after the theileriosis _season, in com-

parison ^{to the} spring ^visit (70.7%), it could be

suggested ^{that the} occurrence of detectable levels of anti-piroplasm antibodies in IFAT is

more dependent ^on the exposure ^{to new} infections during ^{the summer than on the}

presence of piroplasms ^from persistent

infections. This hypothesis ^could possibly explain the results reported by ^{Kachani et al} (1995) ^{in a} preliminary evaluation of an

ELISA test using ^{crude sonicated} piroplasm antigen of T annulata. These authors observed that before the disease _season, blood smear examination was more sensi- tive than ELISA, ^while after the disease sea- son the ELISA became more sensitive but less specific.

The higher reliability of the schizont IFAT

was also reported ^for T parva (Burridge ^and Kimber, 1972, 1973). ^{This is} probably ^{due to}

a longer duration of detectable titre of anti- schizont antibodies compared ^to anti-piro- plasm antibodies (Anonyme, 1984) ^{as is}

illustated in this study by the difference in

sensitivity between schizont and piroplasm

IFAT in the spring, ^{8 months at} least after the previous theileriosis season. According

to Bishop ^{et al} (1992); schizont antibodies could persist ^even longer ^{than the infection}

itself. Using ^the polymerase chain reaction for revealing T parva ^{carrier animals, these}

authors reported that the schizont IFAT- detected cattle that were no longer ^infected

with the parasite. However, ^{as described} earlier, the antibodies to T annulata could have a shorter duration than the infection itself in some cattle.

In experimental T annulata infections inducing ^a morbidity of 100% and a mortal-

ity ^of 50%, ^{it was} observed that over a

period of 66 weeks a higher titre of anti- bodies were detected in piroplasm ^IFAT

than with schizont IFAT (Darghouth, unpub-

lished results). Similar results were also

reported by Perry (1993). ^{It is} possible ^that

in this study, ^the single animal from the field

sample positive ^to piroplasm ^{IFAT, was} showing ^a similar humoral profile ^{with anti-}

schizont antibodies absent or present ^{at lev-}

els undetectable in IFAT. However with the

exception ^{of this animal,} the results of the

antibody ^{kinetics obtained under} experi-

mental situations may ^not be able to be

extrapolated ^{to cattle} exposed ^{to a natural}

infection. In these animals the anti-schizont antibodies appear ^to persist longer. ^Differ-

ences in the extent of infection and in the

parasite ^{stocks, and} continuous stimulation of the immune system by repeated ^natural challenges ^could explain this observation.

In conclusion, this work confirmed that

serology using ^{IFAT is more} sensitive than the detection of T annulata piroplasm ⁱⁿ

blood _smears, and showed that the majority

of cattle carrying piroplasm, ^{82.4 to 95.5%}

depending ^{on the} sampling period, ^could

be detected serologically. ^{In addition, this} study showed that the schizont IFAT at a

screening ^sera dilution of 1/160 as com-

pared ^to piroplasm ^IFAT appeared ^more

appropriate ^{to use for} detecting ^cattle

exposed ^to T annulata infection.

ACKNOWLEDGMENTS

We are grateful ^{to F Ben Salem} (Circonscription

v6t6rinaire de Kalaat El Andaleus), K Ben Ammar and S Zouaoui (Circonscription v6t6rinaire de Sidi Thabet) ^{for their} help ^{in the field work. This}

work was a part ^{of a} project ^funded by ^{the EU} (contract TS2-0260-UK).

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