An epidemiological survey of bovine Babesia and Theileria parasites in cattle, buffaloes, and sheep in Egypt

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Highlights

  • Babesia and Theileria were surveyed in Egyptian cattle, buffaloes, and sheep.

  • Cattle were PCR-positive for both genera with T. annulata the commonest species.

  • B. bovis RAP-1 and B. bigemina AMA-1 were conserved, T. annulata Tams1 was diversed.

  • This is the first report of T. orientalis in Egypt and MPSP type 2 in buffaloes.

  • We highlight the potential risks of hemoprotozoan infections in Egyptian livestock.

Abstract

Cattle, buffaloes, and sheep are the main sources of meat and milk in Egypt, but their productivity is thought to be greatly reduced by hemoprotozoan parasitic diseases. In this study, we analyzed the infection rates of Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis, using parasite-specific PCR assays in blood–DNA samples sourced from cattle (n = 439), buffaloes (n = 50), and sheep (n = 105) reared in Menoufia, Behera, Giza, and Sohag provinces of Egypt. In cattle, the positive rates of B. bovis, B. bigemina, T. annulata, and T. orientalis were 3.18%, 7.97%, 9.56%, and 0.68%, respectively. On the other hand, B. bovis and T. orientalis were the only parasites detected in buffaloes and each of these parasites was only found in two individual DNA samples (both 2%), while one (0.95%) and two (1.90%) of the sheep samples were positive for B. bovis and B. bigemina, respectively. Sequence analysis showed that the B. bovis Rhoptry Associated Protein-1 and the B. bigemina Apical Membrane Antigen-1 genes were highly conserved among the samples, with 99.3–100% and 95.3–100% sequence identity values, respectively. In contrast, the Egyptian T. annulata merozoite surface antigen-1 gene sequences were relatively diverse (87.8–100% identity values), dispersing themselves across several clades in the phylogenetic tree containing sequences from other countries. Additionally, the T. orientalis Major Piroplasm Surface Protein (MPSP) gene sequences were classified as types 1 and 2. This is the first report of T. orientalis in Egypt, and of type 2 MPSP in buffaloes. Detection of MPSP type 2, which is considered a relatively virulent genotype, suggests that T. orientalis infection may have veterinary and economic significance in Egypt. In conclusion, the present study, which analyzed multiple species of Babesia and Theileria parasites in different livestock animals, may shed an additional light on the epidemiology of hemoprotozoan parasites in Egypt.

Introduction

Piroplasmosis caused by different species of Babesia and Theileria in various wild and domestic animals affects the health status of the infected hosts [1]. Disease outbreaks in livestock animals related to infections with these parasites are of great economic significance. Among the Babesia species associated with Bovine babesiosis, Babesia bovis, Babesia bigemina, and Babesia divergens are considered the most virulent [2]. While B. bovis and B. bigemina are found in tropical and subtropical regions of the world, B. divergens, which is also defined as a zoonotic agent, is common in Europe [2], [3]. Babesia sporozoites released from infected ticks during blood feeding infect the host's red blood cells (RBCs), where they transform into merozoites [4]. The asexual multiplication of merozoites within the RBCs results in hemolysis of the cells, leading to anemia and jaundice in a host animal [2]. The clinical picture associated with B. bovis infection includes nervous and respiratory symptoms caused by the sequestration of infected RBCs in the capillary beds of vital internal organs [5].

In cattle, Theileria parva and Theileria annulata are the main etiological agents of severe clinical theileriosis [6], but Theileria orientalis, a benign Theileria parasite, has also caused outbreaks of theileriosis in several countries [7], [8]. In contrast to most Babesia species, Theileria sporozoites infect the host leukocytes, where they undergo schizogony and merogony [4], [6]. Because T. parva and T. annulata schizonts induce rapid proliferation of leukocytes, these species are classified as transforming Theileria parasites [9]. In contrast, T. orientalis does not induce leukocyte proliferation and is therefore referred to as a non-transforming Theileria parasite [6]. Merozoites released upon schizont lysis are infective to RBCs [6]. While T. annulata and T. orientalis merozoites efficiently multiply in RBCs, merogony in RBCs is less pronounced in T. parva [6], [10]. While T. parva is endemic in eastern, central, and southern Africa, T. annulata is common in north Africa, southern Europe, and Asia [11], and T. orientalis has a worldwide distribution [7], [8], [12], [13], [14], [15], [16], [17], [18].

Most of the animals that recover from the clinical diseases caused by Babesia and Theileria parasites remain carriers of these diseases [19], [20]. Subclinical infections may also be common among animals that are resistant to clinical piroplasmosis [2]. Detection of carriers and subclinical infections is essential for estimating the level of risk posed by Babesia and Theileria parasites. Therefore, data from epidemiological surveys could be useful for gauging the efficacy of the parasite control programs implemented in the past. Based on the findings of such surveys, parasite control strategies could be modified where needed. Microscopic examination of Giemsa-stained blood smears is a simple and common method for identifying blood parasites. However, because of low parasitemias at the carrier stage, microscopy may not be an effective diagnostic tool as it lacks sensitivity and specificity [21], [22]. Currently, DNA detection techniques, such as PCR assays are preferable for epidemiological investigations, because these methods are specific, sensitive, and capable of detecting active infections [23].

In Egypt, cattle, buffalo, and sheep are the main source of meat, milk, and their related products. Clinical diseases caused by Theileria and Babesia species are common among the cattle and buffaloes in this country [24], [25], [26]. Disease outbreaks often lead to economic losses from reduced productivity, require costly veterinary treatment, and can result in the death of affected animals. Previously, a number of epidemiological studies of Babesia and Theileria parasites have been conducted in Egypt. B. bovis and B. bigemina have been reported in cattle, buffaloes, and ticks [27], [28]. However, the study areas in these investigations were usually limited to one or two provinces of the country. Furthermore, most of the past epidemiological surveys have focused on either Babesia or Theileria, but studies aimed at simultaneous detection of both parasites have not been conducted. Additionally, T. orientalis, which has been reported in several countries, has never been studied in Egypt. In the present study, therefore, we conducted an epidemiological survey of B. bovis, B. bigemina, T. annulata, and T. orientalis, using blood–DNA samples collected from cattle and buffaloes reared in four Egyptian provinces. Sheep populations were also surveyed to investigate the possible infections with these parasite species.

Section snippets

Blood sampling and DNA extraction

A total of 594 blood samples were collected from cattle, buffaloes, and sheep during a period from August to October, 2013. In detail cattle (n = 439) were sampled in four different Egyptian provinces (Menoufia, Behera, Giza, and Sohag), while blood samples were collected from buffaloes (n = 50) reared in the same provinces, except for Giza (Fig. 1, Table 1). Similarly, sheep (n = 105) were sampled in the same provinces, except for Sohag. In Egypt, livestock animals are maintained under three major

Results

The species-specific PCR assays detected all parasite species surveyed (i.e., B. bovis, B. bigemina, T. annulata, and T. orientalis) in the cattle populations (Table 1). Among the DNA samples sourced from cattle (n = 439), 86 (19.6%) were positive for at least one parasite species. In cattle, the most common parasite was T. annulata (9.56%), followed by B. bigemina (7.97%), B. bovis (3.18%), and T. orientalis (0.68%). Among the provinces surveyed (Fig. 1), Behera had the highest positive rates

Discussion

Bovine piroplasmosis caused by Babesia and Theileria parasites has a worldwide distribution and inflicts a severe economic burden on farming communities [1]. In this study, cattle, buffalo, and sheep populations, which were bred in different geographical locations in Egypt, were surveyed for bovine parasites. Subsequently, the genetic diversity of each parasite species, based on the gene sequences determined from the PCR amplicons generated by species-specific primers, was studied.

Our findings

Acknowledgments

We thank Dr. Amer Hamada (Faculty of Veterinary Medicine, Sadat City University) and Ms Hiroko Yamamoto (National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary medicine) for their excellent technical assistance. We also thank the owners and staff of the study farms in Egypt. This study was supported by grants from the Scientific Technique Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry (25034B), from the Japan Society

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