Serological prevalence of Babesia caballi and Theileria equi in camels and donkeys from Karamoja sub-region , North-eastern Uganda

Equine piroplasmosis is a severe disease of horses caused by the intra-erythrocyte protozoan, Theileria equi and Babesia caballi. T. equi and B. caballi infections were assessed in serum from camels and donkeys using competitiveenzyme-linked immunosorbent assay (cELISA) assay. A total 110 animals were studied including 25 donkeys and 85 camels from two districts viz. Moroto and Amudat in Karamoja sub-region, North-eastern Uganda. All the (100%) donkeys tested were positive for Babesia/T. equi while none of the camels had been exposed to the infection. All animals were negative to B. caballi cELISA. Our findings indicated that all donkeys sampled in Karamoja sub-region have been exposed to T. equi and this could be prevalent in equine population in Uganda. No exposure status to B. caballi was reported. This study represents the first report on the status of T. equi and B. caballi infection in Uganda.

In Uganda, camels and donkeys are kept in Karamoja and Sebei sub-regions, North-eastern Uganda.They are kept by peasant farmers, pastoralists in this semi-arid region.They are kept for meat, milk, dowry, prestige, and carriage.These animals receive little or no veterinary care.Nakayima et al., (2017) detected helminth parasitosis in these animals in the absence of veterinary intervention.No studies have been undertaken on equine piroplasmosis in Uganda.Therefore, the aims of this work were to determine the infection rate of EP in donkeys and possibly in camels given the fact that they occupy the same ecological setting and could therefore act as accidental hosts.Information from this study will help update current knowledge on the health of camels and donkeys in Uganda for their improved production and productivity.

MATERIALS AND METHODS
Serum samples were collected from Karamoja sub-region in two districts namely: Moroto: N 2° 31' 41.604", E 34° 39' 28.794" and Amudat: N 1° 47' 29.841", E 34° 54' 23.583" districts, Uganda.The study was conducted in March 2016.The camels and donkeys were classified as: Infant, juvenile, sub-adult and adult.Both sexes were sampled (Tables 1 and 2 and Figure 1).Blood was collected from the jugular vein of both camels and donkeys following restraint.5 ml of blood was collected, 2.5 ml blood was put in anticoagulant ethylene diamine tetra acetic acid (EDTA) vaccutainers for parasitological and DNA analysis while 2.5 ml blood was put in serum tubes.The serum was collected into plain vaccutainer tubes without anticoagulant, serum was separated from blood cells by centrifugation at 2500 rpm for 15 min and stored at -20ºC until use in a competitive-ELISA (cELISA) (Kouam et al., 2010) for both T. equi and B. caballi.The total number of donkeys was 25, while camels were 85 giving a total of 110 animal samples collected.The sample size determination was based on purposive sampling based on the availability of the animals.The age and sex were recorded for each animal.The samples were transferred to the laboratory and stored at -20ºC until use.The serum from clinically healthy animals was examined for T. equi and B. caballi antibodies by two separate cELISA.

ELISA
Commercial cELISA kits were used to analyze sera from donkeys and camels for the presence of antibodies to T. equi and B. caballi as described by the manufacturer (VMRD Inc., Pullman, WA, USA).The cut-off values for positive infections was 40% of inhibition for both tests, as indicated by the manufacturer (VMRD Inc., Pullman, WA, USA) (Shkap et al., 1998;Kappmeyer et al.,1999).Thus samples with %I above 40 are considered as positive, and below 40, considered as negative.The results were expressed as a value of the percent inhibition (%I) according to the following formula:  location and size of parasite in an infected erythrocyte in Giemsastained blood smears (Sadeghi Dehkordi et al., 2010).Morphological detection of the parasites could be described as single round, double round, single pyriform and double pyriform with obtuse or acute angle.Microscopically, B. caballi is a larger paired pyriform parasite, while T. equi is a smaller paired pyriform, rounded and tetrad or Maltese cross arrangement of merozoites (Kuttler, 1988;Levine, 1971).

Statistical analysis
Data were analyzed using SPSS (Version 16).A value of p < 0.05 was considered as statistically significant.

RESULTS
110 samples were analyzed with 25 donkeys and 85 camels.All donkeys were positive for T. equi.Donkey no.22 was not done, the sample had dried out.All camels were negative for Babesia/ T. equi ELISA.Corrected ODs were calculated from sample ODs and blank ODs (Table 3).Sample Id represents animal species, age, sex and Nakayima et al. 139 sample number.
All the serum samples from donkeys and camels were negative to Babesia caballi Competitive-ELISA.This suggests no history of exposure to this parasite.110 samples were analyzed with 25 donkeys and 85 camels.Donkey no.22 was not done, the sample had dried out.Corrected ODs were calculated from sample ODs and blank ODs (Table 4).Sample Id represents animal species, age, sex and sample number.

DISCUSSION
Diagnosis of piroplasmosis can be achieved by clinical observation and confirmed by microscopy (Irwin, 2010).However, given the low sensitivity of parasitological diagnosis, there is need to combine parasitological diagnosis with molecular diagnostics (Abedi et al., 2014;Salib et al., 2013;Baptista et al., 2013;Rosales et al., 2013).
Camels were sampled alongside donkeys.Much as camels are not equines, they could serve as accidental hosts or reservoirs of the parasites since they are in the same ecological setting.However, all camels tested negative to B. equi cELISA (Table 3).The serological prevalence of B. equi cELISA was 100% in donkeys and 0% in camels (Table 3).Apparently, equine piroplasmids are enzootic in Uganda and their distribution pattern is likely affected by the presence and densities of suitable hosts rather than by ecological conditions.There is a significant correlation between host species and age with the distribution of EP.Horses are more susceptible to the infection than donkeys.Adult animals have a higher risk of infection.All the serum samples from camels and donkeys tested negative to B. caballi cELISA (Table 4).This suggests no history of exposure of these animals to this parasite or the fact that the infection is cleared in 1 to 4 years.All the 25 donkey serum samples (100%) were found positive for T. equi antibodies (Table 3), B. caballi was not detected in the study area (Table 4) while all the 85 camels tested negative to equine piroplasmosis (Tables 3 and 4).A study of equine piroplasmosis in horses in Sudan by Salim et al. (2008) recorded 100% prevalence of T. equi in Khartoum North (100%) and Atbara (100%); a low prevalence for B. caballi was reported and 0% prevalence was detected in Khartoum, Khartoum North, and Kosti areas.The infection rate of EP varies in different countries.Several factors account for this namely: disease management practices, tick vector abundance and climate.Climatic factors influence the habitat of tick vectors like rainfall, humidity and temperature (Oncel et al., 2007).In endemic countries, equines adopt to infection possibly because of the phenomenon of endemic stability.However, stress and immune-suppression could revert otherwise sub-clinical infection to overt disease.T. equi infection results into life-long carrier status (Brüning, 1996) while B. caballi could persist in subclinical form for at least 1 to 4 years Treatment strategy for piroplasmosis in equines depends on the endemic status of the country.So it is either parasite elimination in disease free countries or resolution of clinical disease in endemic countries.It is not recommended to eliminate the parasite from equids in endemic countries because the animals need endemic stability due to constant exposure to the parasite at low levels (Donnellan and Marais, 2009).Several drugs can be used to treat EP.Generally, B. equi has been reported to be more refractory to babesiacidal drugs than B. caballi.More common drugs include imidocarb and diminazene.However, donkeys are more susceptible to imidocarb toxicity than horses (Donnellan and Marais, 2009).
Imidocarb causes a dose-dependent hepatotoxicity and nephrotoxicity (Donnellan and Marais, 2009).Other drugs could include artesunate, arteether, buparvaquone.Equine piroplasmosis is endemic in Karamoja sub-region and possibly Uganda at large due to the distribution of equine species and the tick vectors.

Conclusion
All donkeys tested positive to Babesia / T. equi cELISA while all camels were negative.This exposure status indicates that this piroplasm could be endemic in Karamoja sub-region and in the equine population in Uganda in the absence of veterinary intervention.No exposure status to B. caballi was reported.Camels are not accidental hosts or reservoirs of equine piroplasmosis.The results of this study will help inform policy on the improvement of the health and welfare of these animals through veterinary intervention.To our knowledge, this is the first report on epidemiology of equine piroplasms in Uganda.Molecular studies for accurate diagnosis of equine piroplasmosis are essential for providing baseline information about its epidemiology, distribution, and prevalence in the affected equine population and for effective control measures.

Table 1 .
Infection of camels (Total = 85; all negative for both infections).

Table 4 .
B. caballi competitive-ELISA of camel and donkey serum.the serum samples from donkeys and camels were negative to Babesia caballi Competitive-ELISA.This suggests no history of exposure to this parasite.110 samples were analyzed with 25 donkeys and 85 camels.Donkey no.22 was not done, the sample had dried out.Corrected ODs were calculated from sample ODs and blank ODs.Sample Id represents animal species, age, sex and sample number. All