Prevalence of camel babesiosis in southeast of Iran

Abstract Babesiosis is a globally distributed zoonotic parasitic disease in a broad range of vertebrates with great importance in the veterinary field. The standard diagnostic test for Babesiosis in animals is microscopic identification of the parasite in a venous blood smear stained with Giemsa combined with assessment of clinical manifestations throughout the acute phase of the disease. The present study was planned to determine the presence of Babesia species in camels from the southeastern regions of Iran. A total of 140 blood samples of camels were randomly collected in four selected cities including Qaen, Nehbandan, Iranshahr, and Zahedan from March to August 2019. Blood smears of each case were also examined by the Giemsa staining method and extracted DNA samples were subjected to internal transcribed spacers (ITS1) polymerase chain reaction (PCR) amplification. The prevalence rates using microscopically and molecular examinations were 10% and 19.28%, respectively. The prevalence rates significantly vary between the selected regions (p = 0.003). PCR technique showed higher sensitivity than microscopy. We found that all infected camels were positive for Babesia caballi. The rate of infection with Babesia among the camel in Zahedan is remarkable. Early diagnosis and early treatment can prevent further spread of the disease in this area.

examination that can be the cause of false-negative results (Terkawi et al., 2012). Since various parasite species can infect animals, the morphology-based diagnosis of babesiosis is difficult (Bilgiç et al., 2013). Therefore, the application of molecular diagnostic tests can bring potential benefits such as more accurate identification of the Babesia species and differentiation from Theilera parasites, especially in the case of low parasitemia in a time and cost-effective method (Alvarez et al. 2019;Bahrami et al. 2014;Motevalli Haghi et al. 2014).
Molecular-based techniques target internal transcribed spacers (ITS) among nuclear genomes located in the ribosomal region. ITS region is characterized as highly conserved sequences within species but variable between different species, so it is explicitly used as gene targets for identifying and discriminating different Babesia species (Liu et al., 2014). A majority of studies on the detection of babesiosis in infected animals have been performed on sheep and cattle in Iran, while there are limited studies on other species such as camels that could be infected by Babesia (Kalani et al., 2012;Sazmand & Joachim, 2017). Camel babesiosis with B. caballi as one the most important species of Babesia have been reported in a different part of the world (Abd-Elmaleck et al., 2014;Ibrahim et al. 2017;Jasim et al., 2015;Khamesipour et al., 2015). During the acute stage of the disease, it causes fever, anemia, jaundice, and edema in the infected camels. It sometimes results in death which induces significant economic losses in the camel industry (Hosseini et al., 2017).
Camels are well known for surviving in harsh conditions in arid and semi-arid regions and play an essential role in human life in Iran (Khalkhali-Evrigh et al., 2018). Iranian camel population is around 150,000 in desert areas, particularly in Eastern regions of Iran. Ticks can transmit many different pathogenic organisms such as protozoan pathogens that cause piroplasmosis (Bitaraf Sani et al., 2020).
In the recent decade, consumption of camel dairy products and meat increased among Iranians, and therefore the control of infectious diseases is essential for food safety of camel products (Mohammadpour et al., 2020). Considering the inadequacy of data on the distribution of Babesiosis and genetic diversity of this parasite among camels in southeastern Iran, we aimed to investigate the prevalence and distribution of various Babesia species by polymerase chain reaction (PCR) method to address the research gap in this particular field.

DNA extraction
According to the manufacturer's instructions, genomic DNA was extracted utilizing a Takapozist DNA extraction kit (Tehran, Iran). For each sample, 10 micro-punches (1.2 mm) of blood-spotted filter paper were placed into a 1.5-ml microcentrifuge tube and centrifuged for 10 min at 12,000 rpm and the supernatant gently poured off. Two milliliters of 70% ethanol was added to the pellet, mixed by vortexing for 3-5 s, centrifuged at 12,000 rpm for 5 min, and the residual DNA was transferred into a new tube. Next, DNA concentration was determined in A260/A280 ratio by a NonoDrop spectrophotometer (Thermo Scientific, U.S.) and stored at -20 • C until used.

Statistical analysis
Data analysis was carried out using SPSS software version 18 (IBM SPSS Statics 18, USA). Descriptive statistics, including frequency distribution, central and dispersion indexes together with chi-square were applied for analysis and interpretation of research findings. All statistics were considered significant at p < 0.05. Also, the kappa coefficient (κ) was used to measure agreement between the two screening diagnostic methods for babesiosis detection, indicating a relatively strong agreement between Giemsa staining microscopy and PCR methods.
During the past decades, much more information has become available on Babesiosis in a wide variety of vertebrate hosts; while it has been addressed in several investigations in Iran, there have been few scientific investigations on Babesia infection in Iranian camels.
In a study conducted in Zabol in 2008, the presence of Babesia was observed in 3.54% of camels detected by microscopic examination of blood smears (Ranjbar & Afshari, 2009). In another survey performed in Ahvaz, Babesia infections have been identified in eight out of 122 camels without determining the type of species (Khamesipour et al., 2015). Genome sequencing results revealed that the infection with B.
Certain geographical and strategical specifications of the studied area and probably close communication of native camels with imported camels of neighboring countries including Afghanistan and Pakistan, and the distribution of vector ticks are factors that increase infection in this region. Therefore, this situation emphasizes the importance of border monitoring and quarantine, and control of vector ticks. However, research conducted on the prevalence of Babesia infection among camels was 5%-18.43% in Egypt (El-Naga & Barghash, 2016;Mazyad & Khalaf, 2002;Nassar, 1992) and 74.5% in Sudan (Ibrahim et al., 2017).
Comparison of the findings with previous studies reveals the increased prevalence of babesiosis in camels across the world and particularly in Iran over the past few years. A possible reason for the low prevalence of B. caballi could be associated with the earlier removal of the parasite after a short term of infection (Ibrahim et al., 2017). Hence, utilizing an appropriate diagnostic method for early detection of infection is required to break the chain of transmission among vertebrate hosts, which may avoid the spread of Babesia species in larger populations.
Although most of the infected cases remain asymptomatic, the clinical presentations of babesiosis are relatively similar to theileriosis.
Therefore, generally, the detection of babesiosis is confirmed with the microscopic examination of blood smears.
Indeed, the most common and cost-effective method for diagnosing babesiosis in vertebrates is a traditional microscopic examination of thin and thick blood smears obtained from superficial and deep vessels of live ruminants or heart, bone marrow, and kidney, or spleen regard-

CONFLICT OF INTEREST
We declare that all authors listed on the manuscript are employed by Iran's government as university researchers and faculty staff who do not have any primary function or representative other than research and/or education.

ETHICAL APPROVAL
All the procedures in this investigation have been reviewed and approved by the Ethics Committee of Zahedan University of Medical Sciences (IR.Zaums.REC.1396.17).

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/vms3.666