Neobalantidium coli: First molecular identification from the Eurasian wild boar, Sus Scrofa in Bushehr Province, Southwestern Iran

Abstract Balantidium coli is a common parasite of pig and wild boars (Sus scrofa) which can infect humans and several species of mammals. This study aimed to determine the genotype of Balantidium isolated from Eurasian wild boars in Bushehr province, Southwestern Iran. Twenty‐five faecal samples, originating from 25 wild boars captivated in our previous study, were processed. DNA was extracted from the faecal samples and PCR‐amplified, targeting an ITS1–5.8s‐rRNA–ITS2 region of Balantidium genome. PCR product was purified from the gel, and sequenced. BLAST analysis was performed in order to compare our isolates with other previously reported ones. A phylogenetic tree was constructed, using MegaX software, to find out the phylogenetic diversity of the isolates. With PCR it was possible to detect Balantidium DNA in the faecal samples of 13 out of 25 (52%) of the wild boars. BLAST analysis of seven isolates revealed that the isolates belong to the newly introduced genus Neobalantidium coli. Sequences of three isolates were deposited in the GenBank. Moreover, molecular analysis revealed six areas of nucleotide differences within the isolates and nine areas of difference between the sequences obtained in this study and those available in the GenBank. Phylogenetic analysis revealed that the sequences of isolates of this study have up to 2.2% dissimilarity from those published in the GenBank. The findings of this study, for the first time, revealed that some of the isolates of Balantidium originating from wild boars in Southwestern Iran belonged to the N. coli.

reported that 67% of the Eurasian wild boars, S. scrofa, in Lorestan Province, western Iran, were infected with B. coli (Solaymani-Mohammadi et al., 2004). Human infection has been reported from Khuzestan and Hormozgan province in the south of Iran (Solaymani-Mohammadi et al., 2005). Swine balantidiasis is often asymptomatic, yet these asymptomatic pigs serving as the major reservoir hosts of the parasite. Wild boars in rural Western Iran are considered as a reservoir for human balantidiasis.
Due to the pleomorphism of B. coli and also the range of its hosts, the Balantidium taxonomy is rather controversial (Pomajbikova et al., 2013). A previous study advised that morphological features and host species are insufficient for the identification of Balantidium species (Ponce-Gordo, Jimenez-Ruiz, & Martinez-Diaz, 2008). Considering the morphological differences or the host species, a number of Balantidium species have been described from various wild and domestic mammals.
It has been proposed that B. coli be transferred to a new genus, Balantioides (Mathison & Pritt, 2019). Recently, based on genetic analysis of Balantidium isolated from different hosts, Pomajbíková et al. (2013) proposed a new genus, Neobalantidium, to accommodate B. coli and other Balantidium species of warm-blooded hosts (Pomajbikova et al., 2013). Neobalantidium is considered to be a junior synonym of Balantioides (Chistyakova, Kostygov, Kornilova, & Yurchenko, 2014;Pomajbikova et al., 2013). As B. coli lack mitochondria, the only available genetic data for B. coli are from the nuclear small subunit rRNA gene (SSU-rDNA) and the internal transcribed spacer (ITS1-5.8S rD-NAITS2) regions. For differentiating among narrowly related subtypes, analysis of the ITS region, and particularly the ITS2 fragment, is considered the best possibility.
Given the worldwide distribution of Balantidium in different hosts and its unknown epidemiology in human populations, the need for further studies on the genetic diversity of this pathogen is evident (Pomajbikova et al., 2013). So far, there has been no molecular study to identify the genotypes of Balantidium in domestic or wild boars in Iran.
Therefore, this study, for the first time, aimed to determine the genotypes of Balantidium isolated from wild boars in the southwest of Iran.

| Ethics
The study was approved by and carried out under the guidelines of the Ethical Committee of the Shiraz University of Medical Sciences.

| Study area
Bushehr province is located at 28.9184° N; 50.8382° E and Dilam Port (where the samples were collected) is located at the north of the province. The climate of the Dilam Port is warm and humid and is mainly covered with massive forests and pasture plants.

| Sample collection
In this study, stool samples collected from the gastrointestinal tracts of 25 wild boars in our previous study, from the northern regions of Bushehr Province were evaluated .

| Microscopical identification of Balantidium
Temporary staining of stool samples with Lugol's solution was performed. Moreover, stool smears were prepared and stained with both trichrome and Ziehl-Neelsen stains (Yaghoobi et al., 2016).

| DNA extraction and PCR
Total genomic DNA was extracted from the wild boars' faecal samples, using the Nucleic Acid Extraction Kits (Vivantis), following the manufacturer' instructions. The ITS1-5.8s-rRNA-ITS2 region of Balantidium was amplified, using the forward 144 | NOORPISHEH GHADIMI Et Al.
PCR was carried out in a 25 μl reaction volume, containing 2 μl of template DNA (50 ng), 0.5 μl of each primer (10 pmol/μL), 12.5 μl of master mix (Qiagen) and 9.5 μl of ddH 2 O. PCR was performed with the following strict temperature profile; initial denaturation of 94°C, Sequence results were analysed by the Geneious software (www. genei ous.com) and compared with sequences available in GenBank, using the BLAST system (http://www.ncbi.nlm.nih.gov/). The phylogenetic relationships were constructed using sequences obtained in this study and reference sequences deposited in GenBank, using the maximum-likelihood method, based on the Kimura two-parameter model, using Mega-X software. Bootstrap analyses (using 1,000 replicates) were carried out to determine the robustness of the finding.

| D ISCUSS I ON
Balantidium is a common parasite among pigs that can infect humans and many other species of mammals (Schuster & Ramirez-Avila, 2008). Balantidiasis is considered as an often-neglected disease which has not received much attention. Human reported cases have been associated with contact with domesticated pigs.

However, there is still controversy about the epidemiology of
Balantidium infection in humans. Several cases of Balantidium infection have been reported in humans who have no contact with pigs, such as those reported in Muslim countries (Maleky, 1998). In contrast, residents of endemic areas with a high prevalence of infection in pigs are often reported as having negative or an asymptomatic Balantidium infection (Schuster & Ramirez-Avila, 2008).
Cases of human balantidiasis have been previously reported from the Persian Gulf region in Iran (Maleky, 1998) In a study on wild boars in Lorestan province, western Iran, 25% of wild boars were found to be infected with B. coli (Solaymani-Mohammadi et al., 2004). These animals were considered as the reservoir of human balantidiasis in this area of the country. In this study, F I G U R E 3 Phylogenetic tree (using maximum likelihood method) of ITS1-5.8s -ITS2 region of Neobalantidium coli isolates obtained in this study and references sequences retrieved from GenBank To date, no studies on molecular analysis of B. coli isolated in Iran have been reported and this is the first study which looked at the molecular features of Balantidium isolated from wild boars in Iran.

| CON CLUS ION
Overall, the findings of this study revealed that some of the isolates of Balantidium originating from wild boars from the Southwest of Iran belonged to the newly introduced genus known as N. coli.
Further studies in this field, especially in other areas of Iran, may explore other aspects of this protozoan in the country.

ACK N OWLED G EM ENTS
This study was funded by the Vice-Chancellor of Research of Shiraz University of Medical Sciences (Grant No. 10436). The study was extracted from Dr. Mohammadreza Abedini MD dissertation.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

E TH I C A L S TATEM ENT
The study was approved by and carried out under the guidelines of the Ethical Committee of Shiraz University of Medical Sciences (SUMS).