doi:10.5455/vetworld.2013.76-82 Epidemiology, diagnosis and therapy of Theileria equi infection in Giza, Egypt 1

Aim: To study the prevalence of Theileria equi among horses in different age groups, both sexes, months and seasons of the year, and regions of Giza governourate. Studying the changes in the blood picture, blood chemistries, liver enzymes associate with T.equi infections in horses. Evaluating IFA and CFT at different dilutions in the serodiagnosis of T.equi infections in horses. Evaluating four anti-Theileria medication regimens (diminazine aceturate, imidiocarb 7%, buparvaquone and a combination of imidiocarb 7% and buparvaquone) in treatment of T.equi infections in horses. 
 
Materials and Methods: Total of 149 horses were examined by clinical signs and blood smears. Fortey whole blood samples from T.equi infected horses were examined to measure haemoglobin, total RBCs count and PCV. Fortey serum samples from T.equi infected horses were examined to measure total bilirubin, direct bilirubin, ALT and AST enzymes. Serum samples from T.equi infected (40) and non infected (14) horses were tested by indirect fluorescent antibody test (IFA) and complement fixation test (CFT) at different dilutions. Four groups of T.equiinfected horses (A,B,C,D), each group was represented by 10 horses and was separately treated with diminazine aceturate, imidiocarb 7%, buparvaquone and a combination of imidiocarb 7% and buparvaquone respectively. 
 
Results: the prevalence of T.equi was 41.61% in totally examined horses. The prevalence was higher in males than females. The highest prevalence was among age group ranged from 5-10 years as (22.81%). The highest prevalence was in July and was recorded as (25.81%) and the disease was more prevalent in summer than winter. The highest prevalence was recorded in Nazlet-alsamman as (51.61%). Equine theileriosis was clinically characterized by fever, haemoglobinuria, oedema, anaemia and icterus. The best dilution for IFA was 1/160 where sensitivity, specificity and accuracy were the highest for this test as (98%), (92.86%) and (97.44%) respectively. The best dilution for CFT was 1/32 where sensitivity, specificity and accuracy were collectively the best as (90%), (92.86%) and (90.74%) respectively. 
 
Conclusion: It was concluded that T.equi is prevalent among horses in Giza governourate, its prevalence is varied according to the age, sex of horses, months, seasons and regions. T.equiinfections in horses are accompanied with changes in blood pictures, blood chemistries and liver enzymes. Both IFA and CFT could be used for the serodiagnosis of T.equi . The used four anti-Theileria medication regimens have the same ability to eradicate T.equi from the infected horses.


Introduction
in Netherland [4], in Venezuela [12] and in Repbublic of Korea [19].The polymerase chain reaction was used Equine theileriosis , an OIE list disease, caused by to diagnosis of T.equi prevalence in Israel [21], in tick borne Theileria equi, is responsible for important North-Eastern Mongolia [20] and in Poland [2].The economic losses in the equine industry.The disease is diagnosis of equine theileriosis depends on the endemic in tropical and subtropical regions of the detection of Theileria equi in the blood cells in acute world, including Southern Europe, Africa, The Middle cases.Screening antibodies specific to Theileria equi East, Asia, South America and Central America.
in the sera of horses is very important to detected Disease can occur in peracute, acute and chronic forms.chronic cases of equine theileriosis.The treatment of In animals there are fever, anemia, jaundice, haemoequine theileriosis includes both specific and supportive globinuria and in some cases death can occur [14].lines of therapy.The specific line of treatment depends Donkeys usually remain asymptomatic carriers with on using anti-Theileria equi drugs with control of ticks positive antibody titre throughout life.The clinical while supportive line was carried out on fluid therapy, form of the disease is diagnosed by peripheral blood antipyretics, blood tonics and stomachics.smear examination, but in carrier donkeys it is very The aim of this study was to record the clinical difficult to demonstrate the parasite in stained blood signs of T.equi infection in horses, to determine the smears as the parasitaemia is extremely low.The prevalence of T.equi in horses in Giza governourate, to Seroprevalence of the disease was studied in Saudi record the changes in blood picture and blood Arabia [3], in Jordan [1] , in Mexico [5], in Brazil [11], chemistry associated with equine theileriosis before the other slide in angle of fourty fife touched the drop of and after treatment, to evaluate complement fixation blood by the end of the slanted slide so the blood run in space beneath it.The slanted slide was drawn quicly test and indirect fluorescent antibody technique for the and the blood was pulled behind.The blood smear was diagnosis of equine theileriosis and to evaluate died in air, fixed by absolute methyl alcohol and was different drugs for the treatment of equine theileriosis.
stained with Giemsa 10% for 20 minutes in neutral

Materials and Methods
phosphate buffer saline then was washed and dried.Animals: A total of 149 horses showed signs of fever, b-Thick drop test: Blood samples were collected from emaciation and exhaustion were examined for ear veins and small blood droplets were spread detection of blood parasites.The blood smears, whole moderately in a clean glass slide and mixed with tooth blood and serum samples were collected from each picks, dried on incubator 37°C and stained for twenty animal.The Theileria infected horses were classified minutes with Giemsa stain in neutral phosphate buffer into groups and each group was treated separately.The then wash and dry and examined microscopically as blood smears, whole blood and serum samples were carried out [24].collected post-treatment to evaluate the efficacy of anti-theileria medications.The serum samples were Whole blood examination: Total red blood cell (RBC) used for complement fixation test and indirect counts were made by a haemocytometer.The interfluorescent antibody technique to evaluate both them national microhaematocrit method was employed for in comparison the gold standard (presence of T.equi in packed cell volume (PCV) determination; the haemoblood smears).The horse was considered Theileria free globin concentration (Hb) was obtained using a after three negative successive blood smears.Animal spectrophotometric method [6].

respiration rates. Inspection of mucous membranes
Group-B was treated with Imidiocarb (Imidox, parnel and lymph nodes was also done [16].The presence of laporatories (aust) 7% at dose rate of 120 mg /100kg ticks and urine colour were also taken in consideration body weight by intramuscular injection.to diagnose equine theileriosis.

Parasitological examination:
Schering-plough animal health 50mg/ml) at dose rate a-Blood smear: As previously mentioned [24], Blood of 50 mg/20 kg body weight by intramuscular samples were collected from ear veins and small drop injection. of fresh blood were placed at the end of one slide and by Group-D was treated with a combination of Imidiocarb (Imidox, parnel laporatories (aust))7% at dose rate of 41C°), Jaundice, red urine, oedema, anaemia and ticks 120 mg/100kg by intramuscular injection and buparvaquone infestation.(Butalex, Schering-plough animal health 50mg/ml) at The prevalence: Prevalence of Theileria equi was (62 dose rate of 25mg/ 20 kg body weight by intramuscular out of 149 horses),(41.61%) in examined horses.It was injection.
higher in males (54) than females (8), it was recorded All horses were examined before and after treatment as (36.24%) and (5.36%) respectively.The prevalence by blood smear, blood picture, blood chemistry, IFA was the highest in age group ranged from 5-10 years and CFT.Each anti-theileria medication was evaluated where 34 horses (29 males and 5 females) were infected clinically by disappearance of disease symptoms; with T.equi as shown in table-2.The distribution of haematologically by examinations of blood smears, T.equi infected horses according to months were blood pictures and blood chemistries; and serologically illustrated in table-3, the highest number of infected by results of indirect fluorescent antibody test and horses was in July (16, 25.21%).The distribution of complement fixation tests before and after treatments.
T.equi infected horses according to regions were Supportive treatment was given to all Theileria illustrated in table-4 the highest number of infected infected horses.It was included Metamizol (Chemical horses was in Nazlet-alsamman (32 51.61%).Industries Development (CID) at dose rate of 10-20 ml per horse by intravenous injection).
The evaluation of IFA and CFT: The results of statistical Poly-Minerals mixture as blood tonics was added evaluation of IFA and CFT are presented in tables (5 on animal ration.Cafosal (Arabcomed) at dose rate of and 6).Statistically, the best dilutions of IFA and CFT 5.0 -25.0 ml according to body weight by intravenous for diagnosis of T.equi were 1/160 and 1/32 respectively.injection).Glucose 5% (El-nasr company) at dose of Therapy: The four anti-Theileria medications have the one litre for 100 kg b.w. by intravenous infusion).
same ability to eradicate T.equi from horse blood and Doramectin (Dectomax, Pfizer) at dose rate of improve clinical signs, blood pictures, blood chemistry 1ml/50 kg body weight by intramuscular injection to and also lower antibody titres in sera of horses that control ticks.
were very clear at one month post-treatment as reservoir for infection in Africa.The economic prevalent in summer than in `winter as previously importance of this disease are concerned in weakness described in table-3.That could be explained by the and inability of horse to work, cost of treatment and deaths.
summer season is the suitable time for ticks multipli-In our study, the prevalence of Theileria equi was cations accordingly spread the infestations of T.equi to recorded as 41.61% (62 out of 149 horses) in examined ticks infested horses.That was proved [18], they horses by using blood films examinations.Piroplasms confirmed transmission of T. equi from tick to horses of T. equi are small measuring 2.0 x 1.0 µm, circular, by using indirect immunofluorescence antibody test (IFA).but mostly piriform and in the latter shape usually The highest prevalence was recorded in Nazletpresented as a Maltese cross [13], that means it is in a alsamman as (51.61%).This could be explained by group of 4 merozoites united by their top ends in the presence of heavy ticks infestation among horses in shape of a cross that found in a single red blood cell.
Nazlet-alsman region.The role of ticks in transmission Another stage found in lymphocyte of infected horses; of T.equi was reported [13], who mentioned that T. equi which is Schizont of Theileria equi as presented in Figure -3.are transmitted by ticks, which become infected when The prevalence was higher in males (54) than they ingest parasites in the blood of infected equids.females (8), it was recorded as (36.24%) and (5.36%)Approximately 14 species of ticks in the genera respectively.The sex of horses could play a role in Dermacentor, Hyalomma and Rhipicephalus can be infestation of T.equi according to the system of vectors for these organisms.breeding and working of horses, most of females used The variation in the prevalence of T.equi according for breeding while most of males used for working to age, sex, months of year and regions is recorded in consequently working horses are more exposed to our study as presented in tables (2,3 and 4), that is in stress and immune suppression.While prevalence of concordance with that reported [10], they stated that Theileria equi was previously studied [23], they found the effects of the variation of the prevalence has been that out of the 111 samples, 38 (34%) and 36 (32%) observed with different categories and breeding systems samples were sero-positive for B. equi infection and B.
in different regions.The management of the horses caballi infection, respectively.In addition, 14 (12%) appears to be an important factor for the prevalence of samples were sero-positive for both B. equi and B.
T. equi infections.Among horses raised with access to caballi as a mixed infection.
pasture there was a significant difference in the seropositive The highest prevalence was among age group percentage of reactors (15.05%) compared with horses ranged from 5-10 years as (22.81%) (34 included 29 without access to pasture (55%).male and 5 female).The age may play a role in the Equine babesiosis was clinically characterized by infestation of horses with T.equi as old animals oedema (Figure -2) that could be explained by decrease especially if working hard are under stress and immune in the mean total serum protein due to haemolytic suppressed for that they are susceptible to infestation anaemia which lower the blood osmotic pressure.with T.equi.
Anaemia was also recorded in the infested horses that The highest prevalence was in July and was resulted from infection of the RBCs by the Theilleria recorded as (25.81%) and the disease was more Merozites which disrtucted the cell wall of the RBCs  recorded where the number of RBCs decreases due to observed in Theileria equi infested horses that could be it's destruction by piroplasmoses causing anemia.The happened due to increase in serum concentration of haemoglobin decreased due to destruction of RBCs.bilirubin which discoloured tissues including mucous Bilirubin was increased as a result of pre-heapatic membranes and body fluid.[13,16].Deaths from T.
jaunedice caused by haemolytic anaemia [13,16].equi infection among horses was not recorded in our When RBCs were destructed; haemoglobin released study because it usually happen in peracute form of the then it was destructed to haem and globin.The Haem disease.It is caused by severe haemolytic anaemia and part was converted into biliverdine by oxygenase destruction of RBCs that released toxins and enzyme and consequently biliverdine was converted disseminated intervascular coagulation in the into bilirubin by reductase enzyme.Liver enzymes vascularity of the brain [6,13].
were elevated due to the increased amount of unconjugated The changes in blood and chemistry were bilirubin, at the same time the liver has certain capacity  to conjugate bilirubin so with continous release of as the test of choice for diagnosis of Theileria equi haemoglobin and formation of unconjugated bilirubin infection in several countries, allowing detection of the level of liver enzymes increased [16].
acute cases and chronic carriers, becoming negative As previously illustrated in table-5, the best soon after treatment.IFA is even more sensitive and dilution for IFA was 1/160 where sensitivity, specificity recommended in combination with CFT [13].and accuracy were the highest for this test as (98%), Although the four anti-theileria medications (92.86%) and (97.44%) respectively.The same results regimens have different medications, doses, routes of were recorded [15], who mentioned that the sensitivity administrations and modes of actions they have the and specificity of IFA were 89.2% and 99.0% same ability to eradicate T.equi from horse blood and respectively.While for CFT, the best dilution was 1/32 improve clinical signs, blood pictures, blood biowhere sensitivity, specificity and accuracy were chemistry and also lower antibody titres in sera of collectively the best as (90%), (92.86%) and (90.74%) infected horses that were very clear at one month postrespectively.That disagree with that recorded [15], treatment.they found that the CFT sensitivity and specificity for Imidocarb dipropionate is a derivative of the diagnosis of T. equi were 63.1% and 96.4%, carbanilide antiprotozoals.Although the exact mode of respectively.At the comparison of IFA 1/160 and CFT action of the drug is not known, a possible mechanism 1/32 we found that IFA has the higher sensitivity, is interference with cellular repair and replication specificity and accuracy.So it is preferable to use IFA at through binding with DNA.Imidocarb is highly dilution 1/160 for diagnosis of equine babesiosis than effective against infections caused by Theileria.

CFT. That agrees with that reported [13], indirect
Our results disagree with other authors reported fluorescent antibody test is the primary tests used for that although there are different anti-theileria drugs, qualifying horses for the importation.The test has been imidocarb dipropionate is the drug of choice for equine proved more effective in detecting long-term infected theileriosis.It has a widespread distribution in body animals and animals treated with antiparasitic drugs fluids and tissue.Plasma concentration of imidocarb but these animals may be CFT negative but still be was not still detectable twelve hours after administration.infected.
It could be administered intramuscularly or subcutan-The false positive results of IFA and CFT could be eously, but not intravenously.The precise mode of explained by firstly, treatment with protozoal drug action against piroplasmosis is not yet clearly elemenate the protozoan from RBCs but the antibodies understood.It is thought to involve the compound's is found in serum.And secondly, by infection of combination with nucleic acids, causing partial Equines with T. equi appeared to remain permanent.
uncoiling and denaturation of the DNA double helix of Parasitemia was often absent in carriers, but can the parasite.Imidocarb also inhibits the entry of reoccur after immuno-suppression or strenuous inositol into the parasitised erythrocyte and therefore exercise.T. equi can be passed to the foal in utero, and leads to "starvation" of the parasite [9].some foals can be healthy carriers.
Diminazene aceturite has effect on the Theileria, The false negative results of IFA and CFT could it appears to relate to interference with aerobic be explained by the early infection by T.equi where glycolysis, as well as with synthesis of DNA in the they were found inside RBCs but the antibodies were parasite.not formed yet or the immune system was suppressed We also observed that the diminazene diaceturate and did not react to form antibodies that occurred in too causes swelling and necrosis at the injection sites that agrees with that reported [9].In addition to toxic doses young or too old equines.
of diminazene diaceturate may result in respiratory The kappa value (the degree of agreement) of the distress, depression and other signs of intoxication [9].IFA and CFT as illustrated in table (6) showed that the In our study, the dose of imidocarb was 120mg/ best value was at IFA 1/320 and CFT 1/128 as (1) that 100kg body weight, it eliminate T.equi, improve means complete agreement while the worst value was clinical signs and leave undetectable traces of the at IFA 1/320 and CFT 1/8 as (0.02) that means slight parasite that stimulate immune system cause horse agreement.Complement fixation test has been selected C.A= Complete Agreement = 1, F.A.= Fair Agreement from 0.21-0.40,A.P.A.= Almost Perfect Agreement >0.81, S.A. = Slight Agreement from 0 -0.20,S.A. = Substantial Agreement from 0.61 -0.80, P.A. = Poor Agreement = 0, M.A. = Moderate Agreement from 0.41-0.60causing haemolytic anaemia.Icterus (Figure-1) was

Figure- 1 .Figure- 2 .Figure- 3 .
Figure-1.Icteric mucous membrane in Theileria equi infested horse ethics committee permit us to do this research work on Serum chemistry: Estimations of total and direct horses.All horses were cared according to animal bilirubin, Alanine aminotransferase(ALT), aspirate ethics committee instructions.
aminotransferase (AST), were carried out [8].Samples: Blood smears were collected from ear vein of

Table - 2
. Age and sex of T.equi infected horses equi).Both organisms belong to the phylum Apicomplexa after three negative successive blood smears.andorderPiroplasmida.They can infect horsesResultsconcurrently. Other related protozoa such as Babesia The clinical signs observed in Theileria infected bovis (the organism that causes bovine babesiosis) horses are illustrated in table-1.havebeen reported rarely in horses.It affects horses, The clinical signs recorded were fever (39.5mules, donkeys and zebras.Zebras are an important

Table - 3
. Temporal distribution of T.equi infected horses.Table-4.Spatial distribution of T.equi infected horses.

Table - 5
. Results of statistical evaluation of IFA and CFT at different dilutions.

Table - 6
. Results of Kappa values for IFA and CFT at different dilutions

Table - 7
. Assessment of anti-Theileria equi drugs for treatment of T.equi infection by blood smear, clinicalimprovement and serological tests.