Evaluation of the Acridine Orange Fluorescence Technique and the Indirect Fluorescent Antibody as Diagnostic Tests for Tropical Theileriosis

This study was carried out to evaluate the use of acridine orange fluorescence technique on blood slides as a rapid diagnostic test for tropical theileriosis in comparison with the Giemsa-stained thin blood film technique. Also the indirect fluorescent antibody test has been employed for the serodiagnosis of tropical theileriosis. The study was carried out on 62 young and 48 adult Friesian cattle suffering from clinical tropical theileriosis in Qassim Region, Central Saudi Arabia, during the period from August 2006 to July 2008. For control, blood samples were also obtained from 25 young and 25 adult, clinically healthy, Friesian cattle, selected at random from different dairy farms in Qassim Region. Thin blood films were fixed with methanol and stained with Giemsa and acridine orange and were examined by two independent microbiologists. There was 100% correlation in the interpretation of slides stained with Giemsa and acridine orange both with respect to positivity and negativity, between the two microbiologists. It is concluded that if facilities are available acridine orange is a valuable alternative for screening tropical theileriosis. The method may also have potential value in the diagnosis of Theileria parva, which causes East Coast fever, and also other Theileria species. Results of the present study also showed that IFA test was not found sufficiently sensitive and specific as has been reported earlier.


Introduction
An accurate diagnosis is a prerequisite for an effective disease management of tropical theileriosis.Tropical theileriosis or Mediterranean Coast Diagnosis of tropical theileriosis is based on clinical fever is a disease of cattle caused by the protozoan signs, knowledge of disease and vector distribution parasite Theileria annulata (Dschunkowsky and and examination of Giemsa-stained blood and lymph Luhs, 1904) and transmitted by species of ixodid ticks node smears (OIE, 2008). of the genus Hyalomma, principally Hyalomma Acridine orange (AO), a fluorescent dye, binds anatolicum anatolicum (Robinson, 1982).Domestic with nucleic acids and fluoresces.The fluorescence cattle and the water buffalo (Bubalus bubalis) are can easily be observed in contrast to the dark susceptible, although the latter is apparently more background (Kong and Chung, 1995).The acridine resistant than cattle (Dhar et al., 1973).The exotic orange fluorescence technique has been used breeds and cross-bred cattle are particularly extensively for diagnosis of malaria (Hänscheid, susceptible with mortality rates of 40-60% (Brown, 1999) and showed a good diagnostic performance 1990) reaching up to 80% in some areas (Gill et al., when compared with Giemsa's or other staining 1977; Hashemi-Fesharki, 1991).

Theileria annulata is known to occur in northern
The indirect fluorescent antibody (IFA) test has Africa, along the valley of the River Nile to at least the been widely used for serodiagnosis of theileriosis in th 13 parallel in the Sudan, Eritrea, the Middle and Near cattle (Mehlhorn et al., 1994).It is sensitive and East, Southern Europe, the Indian subcontinent, requires little standardization (OIE, 2008).However, Central Asia and the Far East (Purnell, 1978; the major disadvantage of the test is that it relies on subjective observation of the degree of fluorescence Uilenberg, 1981).(Norval et al., 1992).Its inconvenience for the screening blood smears of acridine orange were fixed with of large numbers of sera limits its use in epidemio-methanol and air dried.A drop of acridine orange stain logical surveys (Kachani et al., 1992).
(pH 7.2) was placed on a clean cover slip, which was Fluorochromes have never been used for then placed on the film.Films were examined at 1000 diagnosis of theileriosis.Hence, the present study was magnification using a fluorescence microscope carried out to assess the applicability of acridine (Olympus BX51, Olympus Optical Co, Tokyo, Japan) orange fluorescence technique as a rapid diagnostic fitted with a 100-Watt high pressure mercury burner test for tropical theileriosis.In this report we also and equipped with DP12 camera unit.All stained describe the comparison of the Giemsa-stained thin slides (Giemsa and fluorescence) were coded in a blood film technique with the IFA test for diagnosis of blind manner and examined by two independent tropical theileriosis microbiologists.

Materials and methods
was carried out according to the method of Burridge Animals: and Kimber (1972) and OIE (2008).The conjugate, The current study was carried out on 62 Bos taurus fluorescein isothiocyanate conjugated rabbit antibovine young (< 1 year) and 48 adult (> 1 year) IgG (RAB-FITC), was obtained from Sigma (Sigma Friesian cattle of both sexes suffering from clinical Chemical Company, USA).The reference positive tropical theileriosis.The case definition was based on serum was obtained from a chronically infected calf.overt clinical signs (fever > 39° C and enlarged lymph The reference negative serum was obtained from a nodes), detectable parasitaemia and parasitosis.These recently imported cow which was confirmed non-animals were presented to the Veterinary Teaching infected by blood smear and lymph node examination.Hospital, College of Agriculture and Veterinary The slides were examined as described above.Pilot Medicine, Qassim University during the period from studies showed disappearance of non-specific August 2006 to July 2008.For comparison, blood fluorescence at serum dilution level of 1/40.This level samples were also obtained from 25 young and 25 was therefore, used as the starting dilution throughout adult, clinically healthy, Friesian cattle, selected at the study.random from different dairy farms in Qassim Region.

Results
very low parasitemia were not included.Blood samples: Blood samples were collected by There was 100% correlation in the interpretation venipuncture into plain and EDTA-containing vacutainer of slides stained with Giemsa and acridine orange, tubes from adult and young Friesian cattle.Thin blood both with respect to positivity and negativity, between smears were prepared from the ear veins of all animals.the two microbiologists.Piroplasms, schizonts and Lymph node aspirates were prepared from suspected merozoites of Theileria in the cytoplasm of cases of tropical theileriosis.mononuclear cells and leukocytes fluoresce green Methods: The thin blood film and lymph node (Fig. 1A, 1B).impression smears were stained with Giemsa.Thin Out of 110 young and adult Friesian cattle with detectable parasitaemia 95 (86.4%) were found studies (Darghouth et al., 1996).The low specificity positive by the indirect fluorescent antibody test.The (44%) recorded in the present investigation was seropositivity was higher in adults (87.5%) than in obviously due to the falsehood of using absence of T. young (85.5%) (Table 1).Of 50 examined known annulata.Piroplasms as a criterion (gold standard) for negative sera, 28 were found positive by the indirect determining true negative animals.PCR technology fluorescent antibody test (Table 2).Using presence of for the detection of parasite DNA sequences in blood T. annulata piroplasms as a gold standard, the IFA test samples should be used as a gold standard instead of has a sensitivity of 86.4% and specificity of 44%. the insensitive blood smear examination technique.

Discussion
of the IFA test in the immunodiagnosis of acute T. annulata infections.These results came in line with Theileria-infected young and adult exotic and earlier reports that the IFA test is not always cross-bred cattle suffered from severe and often fatal Theileria sufficiently sensitive to detect all infected individuals disease (Brown, 1990).Rapid detection of is (Darghouth et al., 1996).Further studies are needed to a prerequisite for an effective treatment and thus in delineate the dynamics of antibody response during reducing the morbidity and mortality due to the the course of T. annulata infections as the differences disease.Giemsa-stained blood smear examination, obtained could be due to different immunoglobulin which is the cornerstone in the laboratory diagnosis of

Figure
Figure.1a & 1b.A micrograph showing piroplasm of Theileria annulata in the RBCs (a) and merozoites or schizonts of Theileria annulata in the cytoplasm of a mononuclear cell (b) in acridine orange-stained blood smear (bar scale = 20 µm).