Molecular identification of Babesia canis canis genotype A in a dog from Iran

Abstract Background Canine babesiosis is a common and clinically significant tick‐borne disease caused by obligate haematozoan parasites of the genus Babesia. Purpose To report Babesia canis canis genotype A infection in a dog. Methods A 2‐year‐old female Shih Tzu dog was submitted with the history of anorexia and depression for one week and no prior surgery. Fever, anorexia, depression and vomiting as well as mucosal pallor were noticed on physical examination. Microscopic examination of the Giemsa‐stained blood smear disclosed large form of Babesia, and single to four pear‐shaped merozoites within erythrocytes (RBCs). The specific primers were used for detecting Babesia canis. Results The result of PCR was confirmed by 18S rRNA gene sequence analyzing and has been registered in GenBank under following accession numbers for Babesia canis canis (MW199108). The sequences were compared to those in GenBank, and alignments showed that the B. canis canis isolate belonged to genotype A. Conclusions This is the first description of B. canis canis genotype A in dog from Iran.


INTRODUCTION
Canine babesiosis is a common and clinically significant tick-borne disease caused by obligate haematozoan parasites of the genus Babesia.
Detection of the pathogen in Giemsa-stained peripheral blood samples under a light microscope has long been established for the diagnosis of Babesia and other haematozoans. However, this morphologybased approach is labour and time consuming because of its low sensitivity. Particularly, the efficiency of the method is undermined in cases of mixed infection or low parasitaemia which results in a false negative diagnosis (Oyamada et al., 2005). To circumvent these pitfalls, novel This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd. methods including molecular assays, such as polymerase chain reaction (PCR) have been developed. PCR and sequence analysis offer a remarkably higher sensitivity and specificity enabling the differentiation of haematozoan parasites (Shabani et al., 2020).
Based on morphology, the causative agents of canine babesiosis have been divided into two distinct groups, namely the large Babesia measuring 3-5 µm B. canis which mainly appears to be pyriform, and the smaller counterpart with the size of 1-3 µm, known as B. gibsoni which mainly appears to be signet ring form (Laha et al., 2015 (Zahler et al., 2000).

B. canis canis infection is distributed in Europe and
Asia, which is a moderate virulent-subspecies, potentially causing a wide range of clinical signs in dogs (Boozer & Macintire, 2003). Rhipicephalus sanguineus has been implicated in the transmission of this parasite in Iran. Little is known about molecular identification of B. canis spp in Iran (Habibi et al., 2020). The present study presents the first description of molecular and phylogenetic analysis of B. canis subspecies canis genotype A in blood sample collected from a dog.

CASE HISTORY
In for molecular analysis. Giemsa-stained blood smear was examined for the presence of the canine piroplasms and other haemopathogens (Furlanello et al., 2005). Then, based on the results obtained from the microscopic examination, molecular assays were performed as below.
The DNA was extracted from blood using a DNA extraction kit  (Birkenheuer et al., 2003). were also used for construction of the phylogenetic trees using maximum parsimony and neighbour-jointed methods. All positions containing gaps and missing data were eliminated (Tamura et al., 2007). Furthermore, to assess B. canis canis genotypes, the sequences obtained in this study were compared with those of group A (AY703072) and group B (AY649326) (Adaszek et al., 2009;Hornok et al., 2016). Netherland and Poland. Moreover, the sequences were grouped in two major genotypes A and B (Figure 2).

F I G U R E 3
Comparison of a part of B. canis canis sequence (Tehran isolate) obtained in this study with those of genotypes A and B Following the comparison of the obtained nucleotide sequence with those of genotypes A and B, the isolate shared 100% similarity with genotype A and the main difference was observed in positions 194 and 195. The differences of the two genotypes in the row of adenine and guanine nucleotides are in genotype A as GA and AG in genotype B (Figure 3). The parasitaemia was estimated to be 2.4%.
The first study reporting canine babesiosis in Iran was published in 1973. In that study, 155 dogs and one fox were included from the north of Iran. Eighty-six dogs were splenectomized and the blood was examined at two-day intervals. Blood smears from one splenectomized dog were found to contain B. canis. The fox was also identified to be infected with B. gibsoni (Niak et al., 1973).  Iran.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

ETHICS STATEMENT
The authors would like to thank the Office of the Vice Chancellor for Research of Urmia University for financial support of this study. This paper is part of DVM thesis of Milad Ghasemzade, numbered 3927, under supervision of Drs. Bijan Esmaeilnejad, Siamak Asri-Rezaei.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study is openly available in GenBank at https://www.ncbi.nlm.nih.gov/nuccore/, reference number MW199108.