Molecular identification of Anaplasma platys in cattle by nested PCR

Abstract

Background and objectives: Anaplasmosis is a zoonotic disease caused by Gram-negative bacterium from Anaplasmataceae family. Anaplasma causes high economic losses worldwide. 16S rRNA analysis was used to diagnose Anaplasma platys in Cattle. Phylogenetic tree and estimation of evolutionary divergence between A. platys isolates were performed.

Materials and methods: A total of 60 blood samples were collected from a cattle farm in AL-Diwaniyah province. 16S rRNA gene was identified using nested PCR. Overall, 40% of cattle that were chosen to collect the blood were identified to be infected with A. platys.

Results: The results have shown presence of targeting partial region of 16S rRNA gene in 24 samples out of 60. Sequencing results of 10 samples have revealed that the phylogenetic tree was divided in to two separate clades. Five isolates of A. platys-Iraq (accession no. OP646782, OP646783, OP646784, OP646790, and OP646791) were located in one clade with the A. platys-China (accession no. MN193068.1). While, five isolates (accession no. OP646785, OP646786, OP646787, OP646788, OP646789) were in different clade with two isolates of A. platys-Africa and A. platys-Zambia in distinct branches, close to the Rickettsiales.

Conclusion: The phylogenetic study of A. platys sequences indicated that the isolates were collected from a cattle farm in Al-Dewaniyah were similar and close related to A. platys-China, A. platys-Zambia and A. platys-Africa). This study suggests that cattle can be considered a reservoir of A. platys.

Keywords: 16S rRNA; Anaplasma platys; Cattle diseases; Tick-borne diseases.

Fig. 1.

Gel electrophoresis image (1.5%) agarose shows the PCR product (–24) of Anaplasma platys targeting partial region within 16S rRNA gene (size = 926 bp). M: is molecular marker (Promega, USA); C: is control negative in which similar PCR components were used except H O was added instead of template DNA.

Fig. 2.

Phylogram of the identified sequences targeting partial region within 16S rRNA gene of Anaplasma platys. this was carried out by MEGA X using Bootstrap analysis with 1000 replicates and rooted with Rickettsiales bacterium (20).