Original article
Molecular and serological detection of Theileria equi and Babesia caballi infection in horses and ixodid ticks in Iran

https://doi.org/10.1016/j.ttbdis.2013.11.008Get rights and content

Abstract

Equine piroplasmosis is a hemoprotozoan tick-borne disease with worldwide distribution that is caused by Theileria equi and Babesia caballi. However, the geographical distribution of equine piroplasmosis in Iran is unknown. The aim of the current study was to determine the causative agents and vector ticks of equine piroplasmosis in horses in the North Khorasan Province. In the year 2011, 100 horses were randomly selected from 14 villages. Blood samples and ixodid ticks were collected and examined using microscopical, molecular, and serological methods. Theileria equi infection was microscopically detected in 5 (5%) of the blood smears with low parasitemia, while serum samples were tested by the indirect immunofluorescent antibody test (IFAT). Antibodies against T. equi, B. caballi, and a mixed infection were detected in 48 (48%), 2 (2%), and 3 (3%) of the serum samples, respectively. A multiplex PCR was used to detect T. equi and B. caballi DNA in blood samples. No B. caballi infections could be found, but Theileria equi DNA was detected in 45 (45%) of the blood samples, and a BLAST analysis of the sequenced samples indicated a 99% similarity with T. equi 18S rRNA gene sequences in GenBank. Both molecular and serological results did not identify any significant association between T. equi infection and risk factors. A comparision of the results of 3 diagnostic methods demonstrated a poor agreement between microscopical examination with IFAT and PCR and a moderate agreement between IFAT and PCR. Thirty-seven adult ticks (20 females and 17 males) were collected from 15 horses. The most common tick was Hyalomma marginatum marginatum (n = 19), followed by Hyalomma anatolicum excavatum (n = 10), Rhipicephalus bursa (n = 4), Hyalomma marginatum turanicum (n = 3), and Hyalomma anatolicum anatolicum (n = 1). The salivary glands and ovaries were also examined using PCR. The genomic DNA samples of the salivary glands of 3 ticks, H. a. excavatum (n = 2) and R. bursa (n = 1), had a positive reaction for T. equi, but no tick contained B. caballi DNA. Thus, our results indicate that T. equi occurs more frequently than B. caballi in the investigated geographical region.

Introduction

Equine piroplasmosis is an important tick-borne disease of equids (horses, donkeys, zebras, and mules) that is caused by Theileria equi and Babesia caballi (Mehlhorn and Schein, 1998, Rothschild and Knowles, 2007). The disease has a worldwide distribution (De Waal, 1992).

The clinical signs of equine piroplasmosis are categorized as peracute, acute, subacute, and chronic. The peracute form is observed in neonatal foals following infection in utero. The clinical signs for acute piroplasmosis, which occurs frequently, include fever, anemia, anorexia, limb edema, icterus, and hemoglobinuria. The subacute form is characterized by anorexia, intermittent pyrexia, anemia, weight loss, limb edema, lethargy, and poor performance. The clinical signs of the chronic cases are ambiguous and display in appetence, poor body condition, and a poor performance. Anemia may be absent or minimal in the chronic form (Friedhoff and Soule, 1996, Rothschild and Knowles, 2007).

In endemic areas, infected horses are apparently healthy without any clinical signs, although stress and excessive exercise results in subclinical manifestations of the disease in those horses that suffer from a chronic infection (Hailat et al., 1997, Takeet et al., 2009) (Fig. 1).

Several laboratory methods have been used for the diagnosis of infection. Microscopical examination is reasonable only in case of clinical disease because parasitemia is low in the latent phase, and serodiagnosis is suitable for epidemiological studies. Three serological tests such as complement fixation test (CFT), indirect fluorescence antibody test (IFAT), and enzyme-linked immunosorbent assay (ELISA) have been used in epidemiological studies of equine piroplasmosis. The CFT is the primary test used for horses traveling between countries. Because the CFT may not identify all infected animals, especially not those that have been treated, and because of anti-complementary reactions produced by some sera, the IFAT is used as a supplementary test. A strongly positive IFAT reaction is simply recognized, but detecting weak and negative reactions requires considerable experience in interpretation (Taylor et al., 2007). Molecular methods are more sensitive than other diagnostic techniques to detect carrier animals (Böse et al., 1995). Several PCR methods based on ribosomal 18S RNA sequence, EMA-1 gene, Rhoptry-associated protein 1, and 16S-rRNA have been developed to detect piroplasms in horses (Bahiruddin et al., 1999, Nicolaiewsky et al., 2001, Battsetseg et al., 2001, Rampersad et al., 2003). Recently, a multiplex PCR and multiplex real-time PCR were used for the B. caballi and T. equi detection (Alhassan et al., 2005, Heim et al., 2007a, Heim et al., 2007b).

Equine piroplasmosis is endemic throughout Asia except Siberia and Japan, and the infection rates in the Middle East are high (Friedhoff et al., 1990, Ruegg et al., 2007).

In Mediterranean countries, which are endemic for piroplasmosis, symptomatic infections can occur all the year round with specific or nonspecific signs (Zobba et al., 2008). Although Iran is located in the endemic areas and B. caballi and T. equi infections have been reported in horses (Aslani, 2000, Seifi et al., 2000), there is no epidemiological information about equine piroplasmosis available for Iran.

The aim of our current study was to determine the relative frequency of T. equi and B. caballi infections in horses in the North Khorasan Province of Iran. In addition, tick infestation of horses and piroplasm infection in ticks were investigated.

Section snippets

Field study area

The study was conducted in the North Khorasan Province from June to August 2011. The North Khorasan Province, which covers an area of more than 28,400 km2, is located next to the north-eastern border of Iran (36°37′–38°17′N and 55°53′–58°20′E). The average annual rainfall in the province is approximately 250 mm. The province is a breeding center for Turkoman horses, an oriental breed from the steppes of Central Asia.

Sampling

One hundred horses from 14 villages were randomly selected. First, the data of

Results

Theileria spp. infections were microscopically detected in 5 (5%) of the blood smears with low parasitemia (approximately 0.001–0.003%). Fifty-three horses (53%) showed seropositive reaction in IFAT. Antibodies against T. equi, B. caballi or mixed infections were detected in 51 (51%), 2 (2%), and 3 (3%) horses, respectively (p < 0.05) (Table 1).

In multiplex PCR, 45 horses (45%) were positive for T. equi, and none had a positive reaction for B. caballi. The nucleotide sequences were assembled and

Discussion

In this study, T. equi was microscopically detected in only a small number of blood smears of horses, which could be due to the low sensitivity of microscopic examination. Although microscopy is the method of choice to detect piroplasms in infected horses with clinical signs (Böse et al., 1995), it is recommended that more sensitive diagnostic tests, such as serological and molecular methods, should be used to detect infections in carrier horses (De Waal, 1992). CFT has been found to provide

Acknowledgements

We are very grateful to Dr. Abdoljalil Ghiadi, the manager of the Turkoman horse breeding company (THBC) for his hospitality. We also thank Mohammad Abedi and Dr. Ali Sarani for their help with sampling, and Mr. H. Eshrati and Mr. G.A. Azari for their technical assistance. This study was supported by the grant VPRTFM-22992.2 from The Vice President Research and Technology of Ferdowsi University of Mashhad, Iran.

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