J Arthropod-Borne Dis, March 2021, 15(1): 97–107 M Khanmohammadi et al.: Molecular Identification and … 97 http://jad.tums.ac.ir Published Online: March 31, 2021 Original Article Molecular Identification and Genotyping of Babesia canis in Dogs from Meshkin Shahr County, Northwestern Iran *Majid Khanmohammadi1; Reza Zolfaghari-Emameh2; Mehdi Arshadi3; Elham Razmjou4; Poorya Karimi4 1Department of Laboratory Sciences, Marand Branch, Islamic Azad University, Marand, Iran 2Department of Energy and Environmental Biotechnology, Division of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran 3Al-Zahra Hospital, Tabriz University of Medical Sciences, Tabriz, Iran 4Department of Medical Parasitology and Mycology, Iran University of Medical Sciences, Tehran, Iran *Corresponding author: Dr Majid Khanmohammadi, E-mail: Majid593@gmail.com (Received 12 Oct 2019; accepted 13 Mar 2021) Abstract Background: Canine babesiosis is one of the mainly worldwide-distributed tick-borne haemoprotozoan parasitic dis- eases in dogs. Methods: A total of 43 blood samples were randomly collected from naturally infected dogs in seven villages from different geographical areas of Meshkin Shahr, Ardabil Province, Iran. The presence of Babesia species detected with standard methods including parasitological and gene sequencing techniques targeting the 18S rRNA gene. Results: Our results revealed that four dogs 9.3% (4/43) including one female and three male dogs were infected with Babesia. All four Babesia-infected dogs were confirmed B. canis by the molecular-based method. Sequence alignments comparison of the B. canis genotypes A and B, it was revealed that all B. canis isolates belonged to genotype B. Conclusion: This study provides essential data for subsequently define the critical importance of the molecular studies in management and prevention of the canine babesiosis in Iran. Keywords: Babesia canis; Babesiosis; Dogs; Genotyping; RNA, ribosomal, 18S; Iran Introduction Canine babesiosis is a tick-borne parasitic dis- ease with worldwide importance and caused by intra-erythrocytic Babesia species. The identi- fication of each Babesia species routinely is based on the host specificity and the morpho- logical characteristics of piroplasmids (1). The differences in geographical distribution, vector specificity, antigenic properties, genetic char- acteristics and severity of the clinical manifes- tations sub divided the former species into the three subspecies, namely B. canis canis (3–5μm) is transmitted by Dermacentor reticulatus in Eu- rope, B. canis vogeli transmitted by Rhipiceph- alus sanguineus sensu lato in tropical and sub- tropical regions, and B. canis rossi transmitted by Haemaphysalis leachi in South Africa (1, 2). Babesia gibsoni (1.5–2.5μm) is present in Asia, North America, Africa, Australia and Eu- rope (3-6). The geographical distributions of both species of D. marginatus and D. reticula- tus in Europe range from Portugal to Ukraine (continue to the east of Kazakhstan), Turkey and probably to the northern parts of Iran (7- 10). The first report of Dermacentor ticks in Iran was documented in 1971 by Mazlum (11). This study performed among the 30 provinces and the results defined that, Dermacentor ticks were found only in six provinces (Semnan, Khorasan, Kurdistan, Ardabil, East Azerbaijan, and Zanjan) with the highest rate of distribution in Ardabil in which ticks was found to be re- stricted to four species; D. niveus, D. margina- Copyright © 2021 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2021, 15(1): 97–107 M Khanmohammadi et al.: Molecular Identification and … 98 http://jad.tums.ac.ir Published Online: March 31, 2021 tus, D. raskemensis and D. daghestanicus (12- 17). In a study 200 adult Dermacentor ticks (139 D. niveus and 61 D. marginatus) were col- lected from a sheep babesiosis infected in the Ardabil region of Iran, B. ovis was detected in ticks by semi-nested PCR. Based on the results obtained D. niveus and D. marginatus, which are distributed in Ardabil region of Iran, might play a crucial role in the transmission of Babe- sia infection to domestic animals (12). To date, there are no reports of D. reticulatus in Iran. Dermacentor ticks usually occurred in the moun- tainous area with cold climatic conditions and high altitude (15). A cross sectional study in southeastern Iran, among tick-infested dogs, three dogs were infected with B. gibsoni. In this study, ticks were identified and belonged to R. sanguineus sensu lato and no Babesia DNA was detected. This study first record of B. gibsoni in dogs in Iran (18). It seems that B. canis and B. gibsoni were the major species infecting dogs and causing various clinical symptoms in Iran (19-21). Based on the morphological features of the infection, it has documented that tick spe- cies including R. bursa, R. sanguineus, R. tura- nicus and D. marginatus have distributed in dif- ferent regions of Iran. R. bursa and R. san- guineus may play the major roles as the vector of the parasite respectively in the case of ani- mal babesiosis in Iran (13). The babesiosis in- fection was detected with molecular and sero- logical methods in dogs and other wild canine (22-27). The molecular-based techniques ena- ble differentiation of morphologically undistin- guishable Babesia species (28). The molecular diagnostic methods (for example PCR) are cost- effective with high sensitivity and specificity and the most reliable techniques for Babesia DNA detection in blood (29-31). Easy applica- tion and accessibility of databases and growing amount of annotated genomic sequences in databanks caused an improvement in the phy- logenetic studies on B. canis (31). Limited in- formation on the canine babesiosis has been documented, while a high number of suspicious clinical cases reported in dogs from Iran. This study provides essential and valuable data to insight into the prevalence and distribution of canine babesiosis in Iran. Thereby, regarding to raise of knowledge on this parasite, the de- tection and characterization of the Babesia spe- cies and subspecies from canine babesiosis in Meshkin shahr has a great importance through application of PCR and sequencing of 18S rRNA gene sequences. Materials and Methods Study area, blood and spleen samples From July 2017 to February 2018, a total of 43 blood samples were randomly collected from shepherd dogs (Canis familiaris) (32 males and 11 females, 9 months to 7 years old) in Mesh- kin Shahr, Ardabil Province, Northwest of Iran. Blood samples were collected into 0.001M EDTA-containing tubes, and transported in ice- boxes to the laboratory of protozoology, facul- ty of medicine, Iran University of medical sci- ences. Blood samples aliquoted, smears were prepared from EDTA-sampled, blood air-dried, and stained with Giemsa. Genomic DNA was extracted from each blood samples using a DNA extraction kit (Qiagen DNA Blood Mini- Kit, Germany). All samples were identified and followed the detection process using PCR. Molecular analysis and characterization of the isolated Babesia species DNA was extracted from whole blood sam- ples using the DNA extraction kit from blood (Qiagen, Hilden, Germany) through following manufacturer instructions as previously de- scribed (22, 31). To detect Babesia species, the gene fragment (~550bp) from 18S rRNA was amplified and sequenced using the primers BAB GF2 (5′-GYYTTGTAATTGGAATGA TGG- 3′) and BAB GR2 (5′- CCAAAGAC TTTGA TTTCTCTC-3′). All stages were performed us- ing the previously described PCR protocol (23). Generally, reactions were performed in a total of 25μl, including 2.5μl of 10X PCR buffer, 2.0μl of dNTP (2.5mM each), 1.25U of Taq http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2021, 15(1): 97–107 M Khanmohammadi et al.: Molecular Identification and … 99 http://jad.tums.ac.ir Published Online: March 31, 2021 DNA polymerase (SinaColon Co. Iran) 1.0μl of template DNA, 1.0μl of each primer (10pmol), and 16.25μl of double distilled water (Sina Co- lon Co. Iran). The PCR reaction was 95 °C (3min), [95 °C (30s), 55 °C (30s), 72 °C (90s)]× 35 cycles, 72 °C (5min). In the second round of PCR 418bp of DNA fragments were gener- ated using another pair of primers, PIRO-nest F (5′-GGATAACCGTGST AATTSTAGGGC- 3′) and PIRO-nest R (5′-GTGTGTACAAAGGG CAGGGACG-3′) (4). The amplified PCR prod- ucts were maintained at -20 °C until analyzed. The products were run on electrophoresis in a 1.5% agarose gel containing 0.2μg of safe stain/ ml in Trisacetate-EDTA buffer at 120V for 30 min and consequently transilluminated under UV light. Ethical approve This study was approved by admission with the ethics procedures and guidelines of the re- spective national the animal ethics use commit- tees of research issued by the council of the Iran University of Medical Sciences (IR. IUMS.REC. 27899.). Sequences analysis Sequences subjected to online BLAST al- gorithm and were compared with previously registered sequences in the GenBank database. To confirm the classification of the parasite, large fragments of the 18S rRNA gene were amplified from each sample that was positive for Babesia. The18S rRNA genes sequences were analyzed by standard technique using a sequencer and BioEdit software (Perkin-Elmer, USA) (32). Analysis of DNA sequences and phylogenetic relationships for B. canis isolates and the group of isolates from dogs were aligned using Clustal W software (33). A phylogenetic tree was created using alignments performed with neighbor joining (NJ) phylogenetic tree using Kimura-2-Parameter algorithm with boot- strap as the tree construction method (34). Fur- thermore, phylogenetic analysis of gene se- quences were performed with maximum like- lihood method with MEGA 7.0 software. The representative sequence was annotated in the GenBank database with accession number MN173220, MN173221, MN173222 and MN 173223 (Table 1). To assess B. canis genotypes (4, 35), the obtained sequences were compared with the members from genotype A (AY 703072) and genotype B (AY649326). Results In the direct microscopic diagnostic inves- tigation of blood smear and molecular study of blood samples revealed that four dogs 9.3% (4/43) including one female and three male dogs were infected with B. canis (Fig 1). In the clinical examinations, all four dogs had major symptoms of babesiosis and most of the infect- ed dogs had fever and splenomegaly. In addi- tion, blood parameters including hemoglobin concentration, haematocrit, RBC count, and di- rect bilirubin had increased (Table 1). Out of the total samples subjected to PCR, four dogs was Babesia-positive including one female and three males. DNA was purified from all blood samples of the collected dogs and used as the PCR template, which a 550bp band was ob- served in the analysis. The results of sequence analysis were the same as with the other pre- viously annotated sequences. The nucleotide se- quences from canine samples were identical to each other and had shown a 99.6–100% iden- tity with B. canis derived from dogs in Gen- Bank reference sequences originated from dif- ferent countries such as Turkey (KY247106 and KF499115), China (MK256974), Slovakia (DQ869307), Estonia (KT008057), Romania (HQ662634), and Croatia (AY072926). The comparison of B. canis nucleotide sequences obtained in this study with genotypes A and B revealed that all our isolates were classified as genotype B and the main difference was ob- served in positions 490 and 491. The difference of the two genotypes in the row of adenine and guanine nucleotides are in genotype B as AG and GA in genotype A (Fig. 2). The results of phylogenetic analysis revealed that the 18S http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2021, 15(1): 97–107 M Khanmohammadi et al.: Molecular Identification and … 100 http://jad.tums.ac.ir Published Online: March 31, 2021 rRNA gene sequences obtained in this study matched with B. canis and alignments showed that all B. canis isolates belonged in the cate- gory of genotype B (Fig. 3). In present study, the common ancestor of genotype A and B was obtained with confidence level 99%. Genetic confidence intervals can help to better under- stand genealogical relationships to DNA match- es. Fig. 1. Direct microscopic detection of Babesia canis in the blood of naturally infected dogs. Field-Giemsa stained thin smears showing various forms of B. canis in erythrocytes. A: Closed angle pyri form bodies of B. canis and B: Wide angles B. canis near the margin of the infected RBCs Fig. 2. Multiple sequence alignment of the partial 18S rRNA gene and the sequences of genotype A and B. In this posi- tion nucleotide changes can be seen http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2021, 15(1): 97–107 M Khanmohammadi et al.: Molecular Identification and … 101 http://jad.tums.ac.ir Published Online: March 31, 2021 Fig. 3. Neighbor-joining analysis of canine Babesia sequences obtained from samples submitted. A 550bp fragment of the 18S rRNA was aligned with representative sequences derived from GenBank. Bootstrap values (1000 replications) are shown in the phylogenetic tree. Comparison of the B. canis sequences obtained in this study with genotypes A and B. Samples sequenced in the present study are marked with red cycle (MN173220- MN173223). The tree was inferred using the neighbor joining method of MEGA7, bootstrap values are shown at each branch point http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2021, 15(1): 97–107 M Khanmohammadi et al.: Molecular Identification and … 102 http://jad.tums.ac.ir Published Online: March 31, 2021 Table 1. Principle information on the animals sampled and the Babesia species isolated Samples Pathogen Clinical symptoms Accession number (s) Blood analysis* Diagnostic investigation Location and Coordinates Breed ID of animal Age Sex Mongrel Mesh 1 1 year Female B. canis Fever, splenomegaly MN173220 RBC: 4.3 HGB: 99 HCT: 27.6% Direct Bilirubin: 1.08 PCR: positive Blood smear: positive Tissue: positive Ag bolagh 38°20′57″N 47°39′57″E Kurd Mastiff (Pshdar) Mesh 2 3 years Male B. canis Fever, vomi- tin, lethargy MN173221 RBC: 4.8 HGB: 90 HCT: 26.1% Direct Bilirubin: 1.01 PCR: positive Blood smear: positive Tissue: positive Parikhan 38°24′51″N 47°38′38″E Anato-lian Karabas Mesh 3 4 years Male B. canis Fever, icter, splenomegaly MN173222 RBC: 4.3 HGB: 91 HCT: 29.1% Direct Bilirubin: 1.15 PCR: positive Blood smear: Not tested Tissue: positive Qurt tappeh 38°25′43″N 47°37′29″E Mongrel Mesh 4 14 months Male B. canis Cough, splenomegaly MN173223 RBC: 4.1 HGB: 97 HCT: 26.6% Direct Bilirubin: 1.12 PCR: positive Blood smear: positive Tissue: positive Qara darvish 38°56′43″N 47°28′48″E *Normal ranges: Red blood cell (RBC) count, 4.6–10×109/L; Hemoglobin concentration (HGB), 93–153g/L; Haematocrit (HCT), 28–49%; Direct bilirubin, 0.15±0.01 http://jad.tums.ac.ir Published Online: March 31, 2021 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2021, 15(1): 97–107 M Khanmohammadi et al.: Molecular Identification and … 103 http://jad.tums.ac.ir Published Online: March 31, 2021 Discussion In this study due to the most availability of 18S rDNA sequences from B. canis in the Gen- Bank, the 18S rDNA was used to search for the intraspecific variability and the most available abundant B. canis sequence. Out of the 43 sam- ples subjected to PCR, 9.3% (4/43) were found to be positive for Babesia infection. This is the first study of a molecular detection and iden- tification of B. canis infection in dogs from Iran and our results revealed that B. canis was prev- alent in Meshkin Shahr, Iran. On the basis of 18S rRNA gene sequence analysis, genetic het- erogeneity of B. canis has been reported in Po- land, Croatia, Estonia, Lithuania, Hungary and china (4, 22, 35). Two genotypes of B. canis, includes A and B, have been documented so far, and have shown to have variable virulences (4, 36). The results provided principle infor- mation toward a better understanding of the ep- idemiology of canine babesiosis in Iran and pre- pared the situation for implementation of an ef- fective control planning on babesiosis. A vari- able interspecies pathogenicity of the B. canis genotypes stated by previous studies (1, 4, 25, 28). The clinical manifestations of B. canis in- fection are mild to acute, and the severity of disease has a significant relationship with the species of Babesia causing infection (4). There are few reports and studies on Babesia spp. in Iran, while a widespread distribution of the par- asite vector and suitable weather condition were observed in some areas of Iran (18, 37). There- fore, there is the probability canine babesiosis and establishment of an infection chain in some geographic areas of Iran. Niak et al. (1973) stud- ied the blood parasites of 155 dogs and one fox (Vulpes vulpes) in the north of Iran, B. canis was just found in one splenectomized dog and B. gib- soni was found from fox (21). Jalali et al. (2013) applied a PCR method in the study and docu- mented that the prevalence of canine babesiosis was 0.36% (20). In another study, Akhtardanesh et al. (2016) detected 60 tick-infested anemic dogs, among which three dogs (5%) were posi- tive through using a genus-specific PCR and all infected with B. gibsoni. None of the collect- ed ticks was positive at the Babesia specific PCR (18). In the collected blood samples of dogs from seven regions of Shiraz in south of Iran, only one positive sample was infected with B. canis (19). The results provided useful data on the distribution of B. canis genotypes in dogs from Iran, and showed the necessity to use a mo- lecular-based analysis for an accurate diagno- sis of canine babesiosis. The PCR-based analy- sis demonstrated that the molecular techniques can a highly sensitive easy to use and cost-ef- fective tools for the simultaneous detection and differentiation of B. canis genotypes. Howev- er, since a limited number of target gene se- quences are currently available for molecular detection of this parasite, any consideration on the population genetics of Babesia in the study areas would be highly scrupulously (38). Ge- netic intraspecific variability is a vital mecha- nism for piroplasm parasite survival in hosts (39). It is proven that B. canis transmitted by D. reticulatus, and the distribution area of the par- asite is directly related to the presence of this tick species. Although B. canis has been ob- served in dogs in Iran (19-21, 37), But so far we have no reports of D. reticulatus ticks in Iran. These results demonstrate that probably B. ca- nis and D. reticulatus have infested a dog’s pop- ulation, at least in the northern part of Iran. Der- macentor reticulatus, not occurring in the Medi- terranean climatic zone, is a tick of some cool regions generally in wooded areas. This ticks has a wide spread geographical overlap with D. marginatus. Preferred habitats are forests and swamps zones where it can survive for long pe- riods (8). The main clinic pathological sings in Babesia infections were a moderate to acute dis- ease haemoglobinuria and a mild to very severe normochromic normocytic haemolytic anemia but, the symptoms of the disease are classified based on clinical sings and severity of the in- fection (40). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2021, 15(1): 97–107 M Khanmohammadi et al.: Molecular Identification and … 104 http://jad.tums.ac.ir Published Online: March 31, 2021 In the present study, the main clinical signs in Babesia infected dogs were fever, splenomegaly, vomiting, cough. Haemoglobinuria and haemo- lytic anemia was not seen, which may be due to host immune system status, age and stages of infection. Based on clinical signs and mortality rates, genotype B is more virulent than geno- type A (4). Considering that, all of the positive cases of Babesiosis in this study were of geno- type B and all of them had typical clinical symp- toms of the disease, the results of this study are consistent with those of other studies (4, 36, 40). The babesiosis infection is detected with mo- lecular and serological methods in domestic dogs and other wild canine in the world. The basic method of diagnosis is the observing intracel- lular parasite, however, this method has limi- tations such as false positive, co infections and non-identification of the species (41). Serolog- ical analysis is a very useful diagnostic method, but has some limitations such as cross-reactiv- ity between different Babesia species and it can- not differentiate between acute infection and pri- or exposure with the parasites (40, 41). The mo- lecular-based techniques enable differentiation of morphologically undistinguishable Babesia species and the most reliable techniques for Babesial DNA detection in blood and tissue (36, 39, 42). Ardabil Province and especially Mesh- kin Shahr region have cold and mountainous climate with forest and swamp conditions. The presence probability of this tick, because of the proof of B. canis not far-fetched. It has prov- en that global warming and climate change will lead to a further spread of the vectors and transmitted pathogens (42). Indeed, the climate change is a global challenge, which may ex- plain not only the increase of density and scat- tering of tick vectors, but also the pattern dis- tribution their hosts, changes in periods of ac- tivity, and variations in geographical distribu- tion (42). The studies suggest a possible role of Dermacentor spp. as vectors of tick-borne path- ogens that affect human and animal health (12, 19). Fast diagnostic technique is necessary for the accurate determination of Babesia in canine that could be carried and possibly transmitted by Dermacentor or other related spp. More stud- ies are needed to increase the knowledge in the epizootiology, ecology and epidemiology of ca- nine babesiosis in Ardabil area. Epidemio-mo- lecular studies are necessary to provide important data for the development of new vaccines and effective therapies against canine babesiosis (43). Through a novel diagnostic strategy, our study could characterize B. canis infection in dogs in Meshkin Shahr, Iran. Due to the increasing num- bers of piroplasm species, infected dogs may state a drastic health position threat to dog’s pop- ulation and prevalence of animal infectious dis- ease in Iran. Conclusion Our study identified the presence of B. ca- nis in dogs in Meshkin Shahr, Iran, but further studies are needed on the prevalence of Babe- sia spp. in large sample dog populations from extended areas in Iran to understand better about the epidemiology of canine babesiosis and to promote an effective control program to deter- mine the tick species diversity in dogs in dif- ferent areas of Iran. The finding of this study provides essential data for subsequently define the critical importance of the molecular stud- ies in management and prevention of the ca- nine babesiosis in Iran. Acknowledgements The authors are pleased to thank Prof Me- hdi Mohebali and Dr Zabihollah Zareei (De- partment of Medical Parasitology and Mycol- ogy, Tehran University of Medical Sciences, Iran) and Prof Lameh Akhlaghi (Department of Medical Parasitology and Mycology, Iran University of Medical Sciences, Iran), for their technical advises and valuable assistances. 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