DIAGNOSTIC AND GENETIC STUDIES OF THEILERIA ANNULATA WITH SPECIAL REFERENCE TO GENETIC POLYMORPHISM OF THEILERIA ANNULATA MEROZOITE SURFACE (TAMS-1) ANTIGEN

DIAGNOSTIC AND GENETIC STUDIES OF THEILERIA ANNULATA WITH SPECIAL REFERENCE TO GENETIC POLYMORPHISM OF THEILERIA ANNULATA MEROZOITE SURFACE (TAMS-1) ANTIGEN

AMIRA A.T. AL-HOSARY^*; LAILA S. AHMED^* and ULRIKE SEITZER^**

^* Department of Animal Medicine (Infectious diseases), Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt, PO Box 71526.

^** Division of Veterinary Infection Biology and Immunology, Research Center Borstel, parkallee 22, 23845, Borstel, Germany.

Email: amiraelhosary@ yahoo.com

INTRODUCTION

Theileriosis is the most economically important disease of cattle; it causes severe losses in livestock production in Egypt Al–Gaabary, (1995) and AL-Hosary, (2009). (Tams-1) is an antigen of    T. annulata, it is one of the merozoite piroplasm surface antigen (MPSA) (Katzer et al., 2002).Tams-1, polypeptide characterized by variable molecular mass it is between 30 to 32 kDa (Katzer et al., 1998).Thisgene encoded for protein of 281 amino acids (aa) containing a putative hydrophobic N-terminal signal peptide, which is highly variable. Several studies reported using of (Tams-1) target based PCR for sensitive and specific amplification of T. annulata DNA from blood of cattleand ticks D'Oliveira et al. (1995), Gubbels et al. (2000), Shiels et al. (1994, 2000) and Kirvar et al. (2000). The present study aimed to compare between blood films and (Tams-1) target based PCR as a diagnostic tools for diagnosis of bovine theileriosis. Genetic polymorphism of Tams-1 gene in different isolates of T. annulata from different localities in Upper Egypt was monitored.

MATERIALS and METHODS

Since August 2010 till August 2013, a total number of 210 cattle belong to different localities in Upper Egypt were randomly selected to this study. Out of them, 11 positive samples were subjected to cloning, sequencing, amino acids translation and multiple sequences alignment. Two types of samples were collected from each animal; one blood sample was collected directly from the ear vein and used for preparation of blood films, another whole blood sample was collected on Ethylene Diamine Tetra-Acetic acid (EDTA) as anticoagulant (1mg/1ml) by direct puncture of the jugular vein and marked with numbered labels then stored at (-20°c) till use in DNA–extraction Charles, (2002).

Blood film:

Thin blood films were prepared immediately post ear vein puncture then fixed and stained by Giemsa stain 8%. Three thin blood films from each animal were examined for detection of piroplasms or trophozoite (Coles, 1986).

Polymerase chain reaction (PCR):

(Tams-1) target based polymerase chain reaction was used for molecular diagnosis of T. annulata infection in examined blood samples.

DNA Extraction and polymerase chain reaction:

DNA extraction from whole blood was carried out according to manufacturer's instructions of commercial kits (according to QIA amp blood kit, Qiagen, Ltd, UK)

DNA amplification by polymerase chain reaction (PCR) using Tams-1 target-based primer:

For the standard PCR, primer Tams1 F (5\ ATG CTG CAA ATG AGG AT) and Tspms1 R (5\GGA CTG ATG AGA AGA CGA TGA G), Amplifying a (785 bp) fragment of T. annulata 30 KDa major merozoite surface antigen gene Kirvar et al. (2000), Murat et al. (2008) and AL-Hosary, (2009).                                  

Cycling conditions: 94°C for 5 min., followed by 37 cycles consisting of 1 min at 94°C, 1 min. at 55°C, 2 min. at 72°C and final extension step at 72°C for 10 min. longer then, the samples were stored at 4°C until use. The cycling condition carried out in thermocycler TECHNE TC–312. In addition to the samples positive control sample contain "DNA from T. annulata infected lymph" and negative control sample (DNase free water), then all were included in the amplification step.

Gel Electrophoresis: The chamber was connected to 75 volt power supply for 90 min., 10µl of each PCR product were separated by electrophoresis on 1.8% agarose gel (GX 040.90, Gen AGarose, L.E., Standard DNA /RNA agarose, Molecular Biology Grade, Inno–Train Diagnostic, D–61476, Kronberg/Taunus) Containing Ethidium bromide as 1 µl /ml electrophoresis buffer. The result obtained through high performance ultraviolet transilluminator, the specific band were detected by using DOC–It ® LS, Image acquisition–software, (UVP, INC, UK) at 785 bp.

Cloning of PCR product, DNA sequencing and data analysis

Cloning:

Ligation of the PCR product was carried out using pDrive Cloning vector according to manufacturer's instructions of QIAGEN Kit (Cat. NO.231224), Qiagen, Ltd, UK. Transformation by using QIAGEN Kit was carried out according to the manufacturer's instructions of Qiagen, Ltd, UK then Platting/Culture and the selection of clones was depended on (Blue / White Colony Selection).

Preparation of the samples for sequencing: The samples measured for detection the amount of dDNA by using eppendorf-Biophotometer, NO.6131 then adjust the sample concentration to 1µg/µl in total volume 15 µl in two tubes one for forward primer and the second for reverse primer.

Sequencing and data analysis: The amplicons were sequenced in both directions in the research center, Borstel, Germany by using Sanger 3730xl, Applied Biosystems (Life Technologies) as previously described by Holman, et al. (2005) and Schoelkopf   et al. (2005).Tams-1 sequences were determined from alignments of 3 to 12 clones from each isolate and then the sequence results were subjected to Basic Local Alignment Search Tool (BLAST) similarity searches according to Altschul et al. (1990).

Sequences alignment and translation:

All the sequences were alignment together by using Clustal W, also translated into its amino acids (aa) by using Expasy.translate software and then alignment the obtained amino acids (aa) together by the same alignment program.

RESULTS

In the current study 210 cattle were randomly selected and subjected to examination for diagnosis of T. annulata infection.

Blood film and PCR:

The results revealed that the infection rate was 28.57% while the infection rate according Tams-1 target based PCR was 46.19% (Table 1 and Fig. 1, 2).

Table 1: Infection rate by using blood film and Tams-1 target based PCR

Sequences analysis:

The obtained results revealed that the local isolates of T. annulata are characterized by the polymorphism of Tams–1 gene. This finding obtained through alignment of the different obtained sequences together and with those in the gene bank by using (Clustal W)(Fig. 3). This was also confirmed after translation of the each sequence to its amino acids (aa) by using Expasy.translate software and then alignment together (Fig. 4). The obtained sequences and their translated amino acids were deposited in the GenBankTM databases and available under accession numbers as following (GenBank^TM: KJ021626, GenBank^TM: KJ021627, GenBank^TM: KJ021628, GenBank^TM: KJ021629).

Figure 3: Sequences’ alignment for T.  annulata Tams-1 gene.

Figure 4:The alignment of translated (aa) of T. annulata Tams-1 gene

DISCUSSION

The infection rate with T. annulata of the examined cattle was 28.57% by thin blood film and 46.19% by Tams-1 target based PCR. These obtained results revealed that molecular technique was more sensitive it was in agreement withD'Oliveira et al. (1995), Aktas et al. (2002, 2006), Tabidi et al. (2006), Azizi et al. (2008), Durrani and Kamal, (2008) and AL-Hosary, (2009). They reported that blood smear is a quick and cheap for diagnosis of T. annulata infection in cattle, and PCR test was more sensitive and specific test.                                                                   

Regarding to the genetic polymorphism of (Tams-1) gene that responsible for T. annulata Merozoite piroplasm surface antigen, the results revealed that Tams-1 gene from different T. annulata isolates are differ from each other. This agree with Manuja et al. (2006)who reported that (Tams-1) is a polymorphic gene and it was studied amongst four T. annulata isolates collected from three different parts of India. The four isolates of T. annulata were different from each other and may be express different antigenic determinants on their cell surface. Furthermore, Altay et al. (2007) and Esmaelizad et al. (2001)reported that the polypeptide (Tams1) is a highly variable immunodominant major merozoite piroplasm surface antigen of the protozoan parasite T. annulata and its associated with the evasion of the bovine immune system. Another possible explanation for the high levels of genetic diversity in Tams-1 gene among T. annulata isolates was reported by Gubbels et al. (2000) who concluded that it may be due to random mutation of nucleotides during asexual reproduction. This makes it difficult to use in vaccination or serological diagnostic assays. It concluded that Tams-1 gene merozoite surface antigens in T. annulata characterized by high level of genetic polymorphism making it unfit for serological diagnostic tools or vaccine production.

ACKNOWLEDGMENT

We would like to express sincere thanks for DFG project “Molecular epidemiology network for promotion and support of delivery of life vaccines against Theileria parva and Theileria annulata infection in Eastern and Northern Africa” (DFG AH 41/7-1) for supporting this work.

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