Molecular detection and phylogenetic analysis of Babesia caballi in domestic donkeys (Equus asinus) in Baghdad Zoo, Iraq

1. Phylogenetic analysis and molecular detection of Babesia caballi in donkeys (Equus asinus) in Baghdad, Iraq.
2. Equine piroplasmosis is considered a severe haem-protozoan infection of, which has not been molecularly reported in donkeys in Iraq.
3. The molecular diagnosis was based on partial amplification about 540 bp of the 18S rRNA gene.
4. The study concludes that donkeys are carriers of piroplasmosis and can act as reservoirs. These animals may be a major source of infection for other equids.

Introduction

Tick-borne disease was thought to be one of the most common blood-protozoan intra-erythrocytic parasites that affected a lot of different types of equids (horses, donkeys, mules, and zebras), caused by ticks transmitting two protozoa (Theileria equi and Babesia caballi) inside the erythrocytes (1,2). Ticks transmit both pathogens belonging to Hyalomma, Rhipicephalus, and Dermacentor species (3-5). The presence of transmitter vectors in tropical and subtropical areas makes piroplasmosis endemic. The disease spreads worldwide, causing economic loss and affecting the movement of horses across borders (2,6,7). Equine piroplasmosis has been studied in Iraq, and previous articles studied piroplasmosis using molecular and serological methods in horses and serological detection in donkeys (8-11). Molecular methods have been used as sensitive techniques for identifying and characterizing B. caballi in horses and donkeys (12-15). For example, Sunday Idoko et al. (12) in Nigeria used conventional PCR for detecting rap-1 of B. caballi in horses and donkeys, and this study suggested that donkeys act as reservoirs of these parasites. Bashiruddin et al. developed a PCR technique that amplified targets of 659 bp regions of 16S rRNA genes to detect B. caballi (13). Abedi et al. (14) modified the multiplex PCR of 18S rRNA to detect T. equi and another B. caballi designed by Alhassan et al. (15). The donkeys with piroplasmosis were asymptomatic. Still, they could act as carriers (14). Salinas-Estrella et al. used a duplex qPCR method and nested PCR test for amplification of the rap1 gene of B. caballi; this study suggested the duplex qPCR was more sensitive than nested PCR (16). Mshelia et al. (17) used nested PCR to screen the 18S rRNA of B. caballi and T. equi, and they used nested PCR to identify specific species of the B. caballi rap1 gene. The comparison between nested and duplex real-time PCR (nPCR) and duplex PCR showed that nPCR can detect B. caballi with more sensitivity and specificity than duplex real-time PCR. In contrast, duplex real-time PCR can readily diagnose B. caballi infection in equines, but this test has low sensitivity (18).

The present study used the conventional polymerase chain reaction (PCR) method to detect B. caballi in the blood of 70 donkeys domesticated in the Baghdad Zoo of the Al Zawra’a Gardens area, Baghdad, Iraq, from April to September 2018. In addition, the 18S rRNA gene of 5 isolates was sequenced, and phylogenetic variations of B. caballi sequences were compared with other countries.

Materials and methods

Ethical approve

All animals and procedures in this research were approved by the Committee of University of Baghdad, committee number 16 in the 6 June/2022.

Blood collection and hematological analysis

Blood samples (n = 70; 59 females and 11 males) were collected from domestic donkeys from April to September 2018 in the Baghdad Zoo of the Al Zawra’a Gardens area, Baghdad, Iraq. These donkeys were prepared to be food for carnivores in the zoo. A total of 2.5 mL of blood samples were collected from the jugular vein using sterile tubes containing Ethylene Diamine Tetra Acetic Acid (EDTA) anticoagulant. The haematologic parameters of all blood samples were investigated, and from each sample, 25 µl blood was analyzed using blood haemolyze (HumaCount-30TS-Veterinary Mode, Germany). The blood samples were kept at -20°C for further molecular analysis in the Department of Internal and Preventive Veterinary Medicine at the College of Veterinary Medicine at the University of Baghdad. Blood samples.

Extraction of genomic DNA and PCR

The DNAs were extracted from all blood samples using the Blood gDNA Miniprep kit (Promega, USA) according to the manufacturer's instructions for amplification of the 18S rRNA gene of B. caballi, the forward primer UFP (5-TCG AAG ACG ATC AGA TAC CGT CG-3) and the reverse primer CabR (5-CTC GTT CAT GAT TTA GAA TTG C-3) (15) (Microgen Bioproducts, UK) were used. The PCR reaction was 25 μl consisting of 12.5 μl Green Master Mix (Promega, USA), 1 μl of each primer, 8.5 μl nucleus free water, and 2 μl genomic DNA.

The PCR reaction used Thermal Cycler (TC9610, Inc. USA) to amplify the 18S rRNA gene according to the following conditions: initial denaturation for 10 min. At 96 °C, followed by 36 cycles, including a first step of denaturation at 96 °C for 60 s, an annealing step at 55.9 °C for 60 s, and an extension step at 72 °C for 60 s, while the final extension was done at 72 °C for 10 min. For each PCR, a negative control was added using deionized pathogen-free distilled water to the mixture instead of the DNA template. Afterward, the PCR products were loaded at 5 μl volume on a 1.2% agarose gel prepared in 1X TBE buffer, stained with Ethidium Bromide, and electrophoresed for 45 minutes at 90 volts, 110 Ampere. A ladder of 100 bp was used as a size marker. The DNA bands on the agarose gel were visualized by a UV transilluminator and photographed by a digital camera. The annealing temperature 55.9°C was optimized using a clinically positive sample with severe clinical signs of piroplasmosis (bloody urine, icterus, and intraerythrocytic parasites).

Sequence analysis

The PCR products of five samples were analysed and sequenced for the 18S rRNA gene according to the manufacturer's instructions (Macrogen, Korea). The nucleotide sequences were aligned with the NCBI data. The software Molecular Evolutionary Genetics Analysis version (MEGA 6.0) generated phylogenetic trees and the sequence analysis.

Statistical analysis

SPSS was used for the statistical analysis (version 20.0, USA). To calculate the risk factors of sex with B. caballi, the odd ratio was used. To compare means at the level of statistical probability at P<0.05, analysis of variance (ANOVA) was used in this study.

Results

The infection rates and hematological features

In the present study, the B. caballi-positive cases were 65 out of 70 donkeys, representing an infection rate of 92.8%. All 65 DNA samples were positive for B. caballi and showed a band of about 540 bp (Figure 1).

Figure 1: Partial fragments 540 bp of the 18S rRNA gene of B. caballi in donkeys. Lanes 1-12 represent positive cases, and lane 13 is the negative control.

The results revealed a non-significant increase in the risk factor (1.10 odds ratio) for males compared to females, where the infection rate between females was 91.5% compared to males at 100% (Table 1).

Table 1: The infection rates of B. caballi in donkeys

*Refer to non-significant difference at (P<0.05) (1.10 odds ratio).

The changes in blood values in animals infected with B. caballi, compared with uninfected animals, showed non-significant differences in mean±SE (Table 2) despite an apparent decrease in hemogram values, but it is insignificant. Infected donkeys showed severe emaciation, signs of a lack of nutrition, and pale mucous membranes because of poor management. Babesia caballi infection did not have a significant effect on blood values.

Table 2: The hematological values of donkeys infected with B. caballi

Differences in letters horizontally refer to significant differences at P<0.05.

Phylogenetic analysis of B. caballi

A phylogenetic tree of B. caballi 18S rRNA sequences of five DNA samples was analyzed and submitted in the NCBI (accession numbers: MN723600.1, MN723592.1, MN723851.1, MN723855.1, and MN723849.1) (Figure 2). The sequences were grouped into five individual clades: the first clade involved MN723600 and has 81% resemblance with global isolates from Sereba, Bosnia and Herzegovina, Tobago, Brazil, China, and other countries with high heterogeneity of this clade, while the second clade included MN723592 with a matching percentage of 99% with isolates from one of the neighboring countries of Iraq (Turkey, MN481269, unpublished but data of sequence available in NCBI website). The other three clades of B. caballi infecting donkeys in this study (MN723851.1, MN723855.1, and MN723849.1) were similar to each other at 98-99% and also to the Indian isolate (India, MF384422.1, unpublished but data of sequence available in NCBI website).

Figure 2: Molecular phylogenetic tree of partial 18S rRNA gene sequences of Babesia caballi infecting domestic donkeys in Baghdad Zoo and other sequences obtained from NCBI database. Phylogenetic analysis was carried out using the MEGA6 program illustrated by five Clades.

Discussion

In donkeys, a few studies on B. caballi are available. Most epidemiological studies show that B. caballi is less widespread than Thaleria equi (19). However, a high rate of B. caballi infection was seen in Egypt, and the infection rate in donkeys was 15.7% (20). In another recent study, an infection rate of B. caballi of 8.8% in Nigeria was recorded in donkeys, and no case was diagnosed in horses (12). Most of the DNA samples amplified by PCR were positive for B. caballi with bands between about 540 bp; the band size is similar to that obtained by the researchers who designed the primers (15), who mentioned that the expected positive result was about 540 bp where they used the multiplex method. Therefore, conventional optimization of the annealing temperature for diagnosing B. caballi is recommended due to its high sensitivity.

 These donkeys live in crowded environments and are neglected in the Baghdad Zoo, where the donkeys gather from most areas of Iraq. This increases the possibility of infection among them. The donkeys were suffering from severe emaciation, signs of nutritional deficiency, and pallor of the mucous membranes due to poor management conditions and neglect. These donkeys were prepared to be food for carnivores in the zoo. For these reasons, the infection rate in the present study was higher than that in other research papers performed by other researchers in Iran (14,15). The Iranian researchers (14) noticed, when using the same type of forward and reverse primers by multiplex PCR, that no infection with B. caballi was recorded in donkeys. In contrast, a high infection rate with Thaleria equi was observed 50.9%. Alhassan et al. (15) were the first to use primers (UFP and CabR) to diagnose B. caballi with a 17.9% infection rate.

 This study suggests that donkeys are more infected than other Equidae species due to the long hair of donkeys and the lack of grooming hair. In our opinion, the skin of the donkeys became good hideouts for the ticks that transmit Babesia. Since the donkeys received no anti-babesia treatments, these animals became reservoirs for Babesia. Using PCR assay, the most recent studies in Israel found that 9.7% of horses had been infected with B. caballi, and the prevalence of infection depended on breed, tick infestation, and geographical area (21). According to case history, the donkeys in Baghdad Zoo do not receive adequate veterinary care, unlike horses. 

According to Abedi (14), both infected and uninfected donkeys had low hemoglobin levels; this means that donkeys do not get enough food and are neglected, making it hard to find control donkeys living in the same conditions for comparison. The blood tests for the group infected with B. caballi showed that their hemoglobin was 10.48 g/dl, and their HCT was 30.15%. These results are similar to donkeys living in similar conditions in Egypt (20), where the hemoglobin levels were 10.37 g/dl and the HCT was 29%. They recorded a significant decrease in these values compared to the control group. This reinforces the result that the animals in Iraq generally suffer from anemia. In Iraq, one study estimated the normal hematological values of donkeys in Iraq, where these values ranged from PCV 32.4-48.7%, Hb 9.6-14.3 g/dl and RBC 4.69-7.6× 10 ⁶/μ L (22). This study indicated that the hematological values in donkeys were low under normal conditions.

Davitkov et al. (23) found a very low incidence of B. caballi infection 2.1%. While the annealing temperature of the Sant et al. (24) isolate Tobago, KU289101.1 was reduced to 51.3, this study also revealed a low infection rate of B. caballi in horses of 3.6%. The common infection rates in these studies, when compared to the infection rate reported in this study, might be because of a difference in the geographical distribution among countries, animal species, or differences in the annealing temperature of PCR.

The present study first investigated the prevalence of B. caballi in donkeys in Baghdad/Iraq by molecular approach. Al-Mola et al. (25) were clinical and pathological studies of equine babesiosis in Mosul/ Iraq in draught horses. Alsaad et al. (10) was seroprevalence reported of B. caballi in Mosul, Iraq, in both horses and donkeys. Alsaad et al. (26) detected equine babesiosis in horses in Basrah/ Iraq by hematological and clinical assay. Another study was designed to detect equine piroplasmosis by molecular technique in the tick activities season (27). The importance of molecular investigation of protozoa ticks gives additional information about the life cycle of the protozoa (28). The hematological studies and investigation of protozoa were conducted on donkeys in Iraq, but this species required further investigations on internal and blood parasites (22,29,30).

From what was presented in the discussion, it is necessary to determine the optimal conditions for PCR to use the primers designed by Al-Hassan (15) and delete the variable nucleotide base (T) that is at the end of the reverse primer (cabR) to detect B. caballi and conventional PCR should be used instead of multiplex PCR. All of these procedures give more accurate results and make the PCR more sensitive as well.

In the phylogenetic tree of B. caballi, the heterogeneity of the sequence of one isolate was 19% compared to other countries, which may be due to inadequate sequencing techniques. In contrast, the other four isolates had a high identity of 99%, especially with neighbouring countries. For this reason, we sent five isolates to be sequenced. Some B. caballi isolates had a high matching rate of 98-99% with other isolates from nearby countries, such as the Turkish isolate in the horse MN481269.1 and the Indian isolate in the horse MF384422.1, both of which have not been published but have information available in NCBI. The sequences of isolates in the results matched each other at a high rate, except for two isolates (MN723600.1 and MN723592.1). There may have been more Turkish horses coming into and out of Iraq, or there may have been more free movement of animals between Iraq and Turkey.

Conclusion

The study concluded that donkeys are carriers of B. caballi and act as reservoirs. These donkeys may be a major source of infection for other equids. This study is the first to use molecular diagnosis to confirm the presence of B. caballi in donkeys in Iraq.

Acknowledgments

The authors thank the Baghdad Zoo, the Baghdad Veterinary Hospital, and the College of Veterinary Medicine at the University of Baghdad for assisting in this study.

Conflict of interest

The authors declare that there is no conflict of interest.