J Arthropod-Borne Dis, Sep 2022, 16(3): 243–250 A Azarm et al.: Molecular Identification of … 243 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Original Article Molecular Identification of Leishmania infantum kDNA in Naturally Infected Dogs and Their Fleas in an Endemic Focus of Canine Visceral Leishmaniasis in Iran Amrollah Azarm1, *Abdolhossin Dalimi1, Mehdi Mohebali2, Anita Mohammadiha1, Majid Pirestani1, Zabihollah Zarei2, *Alireza Zahraei-Ramazani3 1Department of Medical Parasitology and Entomology, Faculty of Medical Sciences of Tarbit Modares University, Tehran, Iran 2Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran *Corresponding authors: Dr Alireza Zahraei-Ramazani, E-mail: azahraei@tums.ac.ir, Dr Abdolhossin Dalimi, E-mail: dalimi_a@modares.ac.ir (Received 24 June 2020; accepted 09 Aug 2022) Abstract Background: Fleas (Insecta: Siphonaptera) are considered as highly specialized bloodsucking on mammals such as dogs. The existence of three factors, namely a vast distribution area, different hosts, and digestive system with a specific mecha - nism for digesting blood has led to species of fleas who nourish from mammals be introduced as the potential vectors of diseases. The aim of this study was to assess Leishmania infantum natural infection of dog fleas in northwest Iran in 2018. Methods: A total of 20 infested domestic dogs (Canis lupus familiaris) were randomly selected from 5 villages. Fleas were collected using brushing against dog hairs and fine forceps. Then, they were morphologically identified and pre- served in ethanol for molecular assay. The kinetoplast DNA of the parasite was used for detection of Leishmania infan- tum using a semi-nested polymerase chain reaction (PCR) assay. Results: The human flea, Pulex irritans, and the cat flea, Ctenocephalides felis were identified on 40% and 35% of dogs, respectively. The results of PCR indicated that L. infantum was found in the Ctenocephalides canis (75%) and C. felis (66.7%) collected from infected dogs. No leishmanial infection was observed in P. irritans. Conclusion: It is concluded that fleas could be infected by Leishmania infantum, but maintenance of the parasite and their vectorial competence needs to be determined. Keywords: Ctenocephalides canis; Ctenocephalides felis; Leishmania infantum; Pulex irritans Introduction Fleas (Insecta: Siphonaptera) are considered as highly specialized bloodsucking on mammals such as humans, livestock, dog, cat, rabbit, squir- rels, rats, and mice (1). They transmit agents of some diseases such as bubonic plague, murine typhus, tularemia, and listeriosis (2). One of the most important families of fleas that transmit dangerous diseases to humans is Pulicidae. Ctenocephalides canis, C. felis, Pulex irritans, Xenopsylla cheopis, and X. austria, are species of this family (3). Several studies have shown that already various species of fleas have been reported from dogs and in most of these stud- ies, three species C. canis, C. felis, and P. irri- tans have been observed (4). Ctenocephalides canis and C. felis are the known vector of many pathogens of dogs that some of them have a zoonotic role (5). Due to the biological flex- ibility of C. canis, C. felis, and the global dis- tribution of dogs and cats, these species are Copyright © 2022 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/ mailto:azahraei@tums.ac.ir mailto:dalimi_a@modares.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, Sep 2022, 16(3): 243–250 A Azarm et al.: Molecular Identification of … 244 http://jad.tums.ac.ir Published Online: Sep 30, 2022 the most common flea in the world. In addi- tion, these insects can transfer a great variety of bacterial, viral, and fungal diseases from dogs to humans and other hosts (5). Leishmaniasis is endemic in many parts of the world and considered as a major public health problem. There are two forms of this disease in Iran: cutaneous and visceral leish- maniasis. Visceral leishmaniasis is a life-threat- ening disease caused by Leishmania infantum and is one of the most important diseases trans- mitted by vectors. Leishmania parasites are trans- mitted by sand flies infected with the parasite. In the Old World, Phlebotomus (Diptera: Psy- chodidae) is the only known biological vector of Leishmania spp. parasites (6). In the Medi- terranean area, Canidae has a definite role in the transmission of visceral leishmaniasis (6). Previous studies have shown that even if there are no Phlebotomus, healthy dogs may receive Leishmania parasites from infected dogs. It shows that other species of arthropods such as fleas, ticks, mites, and lice may be involved (7). Fleas can receive many types of microor- ganisms during blood-feeding. This study was focused on the fleas gathered from healthy dogs and those infected with Leishmania par- asites and assessing natural infection of dogs to Leishmania spp. and their fleas in Meshkin- Shahr County in Ardabil Province. Materials and Methods Study area Meshkin-Shahr County locates in the Ar- dabil Province in the northwest of Iran (Fig. 1). Meshkin-Shahr County with geographical coordinates 38° 44’ N and 47° 40’ E and an altitude of 4811 meters above sea level (8). Sample population In this study, a population of 20 dogs and their fleas were examined to detect Leishmania parasites. The dogs were collected from five dif- ferent villages, named Ahmad Abad, Kojanagh, Parikhan, Ur Kandi, and Sarikhanlou in Mesh- kin-Shahr County, Ardabil Province in 2018. The dogs’ age range was between 1 and 5 years old, which was estimated by the teeth. Blood collection and DAT examination Blood samples from dogs were prepared. A blood sample (8ml) was collected from the saphenous vein of each dog. Then, each blood sample was divided into two parts. One part was used for Direct Agglutination Test (DAT) and another part was transferred to the labora- tory for molecular tests. Blood samples were stored at -20 °C until tested. The collected blood samples were centrifuged at 800g for 5–10 min and the sera were separated and stored at -20 °C until examined by DAT. Afterward, sam- ples were tested according to the protocol de- scribed by Harith et al. (9). Collection and morphological identification of fleas All fleas were collected from the body of dogs using brushing against their hair. In some cases, fleas were collected by fine forceps. Then, the collected specimens from each dog were finally conserved in 70% ethanol and identified by the standard keys. At the labora- tory, fleas were cleaned by water and im- mersed in lactophenol for 1 hour. Finally, tem- porary mounts were prepared with lactophenol to identify the collected fleas. After mounting, the slides of fleas were evaluated using a light microscope (×400) and identified by the stand- ard keys (10). Molecular study In this study, the method of Ish-Horowitz was used to extract the DNA of L. infantum from fleas (11). Also, DNA of parasites was extracted from blood samples using the Ge- nomic DNA Extraction Kit (Bioneer) and stored at -20 ºC for later use. For the detection and identification of species of Leishmania para- sites, the sequence of kinetoplast DNA (kDNA) was used. A Semi-nested PCR assay was car- ried out to detect the Leishmania infection of http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 243–250 A Azarm et al.: Molecular Identification of … 245 http://jad.tums.ac.ir Published Online: Sep 30, 2022 blood samples and collected fleas from healthy and infected dogs. The primers introduced by Aransay et al. (2000) were applied for the de- tection of leishmanial infection of blood sam- ples and fleas. The sequences of the primers were LINR4 (5'- GGG GTT GGT GTA AAA TAG GG -3') for forward and LIN17 (5'- TTT GAA CGG GATTTCTG -3') and LIN19 (5'- CAGAACGCCCCTACCCG -3') for reverse used in a semi-nested PCR technique (12). In the semi-nested PCR assay, the kDNA was am- plified using LIN primers (length 720bp). The PCR test was performed individually for each species of fleas. In the first round of the PCR, 8µl of the master mix, 1µl of each primer (LINR4, LIN17), 2µl of sterile distilled water, and 3µl of DNA were used. The amplification conditions were 94 ºC for 5min, followed by 30 cycles of denaturation at 94 ºC for 30sec; annealing at 52 ºC for 3sec and extension at 72 ºC for 40sec with a final extension step at 72 ºC for 5min. The second round of the PCR was performed with 8µl of the master mix, 1µl of each primer (LINR4, LIN19), 3µl of sterile distilled water, 2µl of the product of the first round of the PCR, in 33 cycles (94 ºC for 30sec, 58 ºC for 30sec and 72 ºC for 1min) and a final extension at 72 ºC for 10min. PCR products were visualized by UV rays in a 1.5 % agarose gel after electrophoresis with a safe stain solution. Results Twenty dogs, including seven females (35%) and 13 males (65%) were examined. Using DAT (8), a high level (titer) of antibody (above 1/ 320) was observed in 12 out of 20 dogs and eight dogs did not have a high level (titers) of antibody. A total of 974 fleas belonging to the genus Ctenocephalides and Pulex were col- lected from dogs in different localities. Three species C. canis, C. felis, and P. irritans were identified which C. canis was the most abun- dant (95.48%) followed by P. irritans (3.28 %) and C. felis (1.23%). The dog flea, C. canis was the most common flea on all dogs (100%), and P. irritans and C. felis were found on 8 (40%) and 7(35%) of dogs respectively (Fig. 2, Table 1). Out of 12 types of a blood sample which were positive by DAT, 10 samples were positive using semi-nested PCR method (Fig. 3). The molecular test was done on 192 C. canis, 12 C. felis, and 18 P. irritans that had been collected from infected dogs to identify Leishmania parasite. 160 (83.3%) of C. canis (Fig. 4) and 9(75%) of C. felis (Fig. 5) were infected with L. infantum and no infection was observed in P. irritans. By semi-nested PCR, it was found that the collected fleas from healthy dogs are not infected with L. infantum (Table 1). Table 1. The number of collected fleas from healthy and infected dogs and the results of Leishmania infantum molecu- lar tests in fleas collected from dogs using the PCR method, Meshkin-Shahr County, Ardabil Province in 2018 Species The number of collected fleas The number of tested fleas Fleas infection with Leishmania infan- tum The infected fleas with healthy host The infected fleas with infected host Canis lupus familiaris Healthy dog Infected dog Healthy dog Infected dog Number % Number % Ctenocephalides canis 72 858 72 192 0 0 160 83.4 Ctenocephalides felis 0 12 0 12 0 0 9 75 Pulex irritans 10 22 10 18 0 0 0 0 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 243–250 A Azarm et al.: Molecular Identification of … 246 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Fig. 1. Location of Meshkin-Shahr County in Ardabil Province, Iran, 2016 Fig. 2. A: Adult of Ctenocephalides felis; 1 shape of the head, 2 length of the first spine of the genal comb, and 3 One short, stout bristle in the interval between the postmedian and long apical bristles of the dorsal margin of the hind tibia, B: Adult of Ctenocephalides canis; 1 shape of the head, 2 length of the first spine of the genal comb, 3 Two short, stout bristles in the interval between the postmedian and long apical bristles of the dorsal margin of the hind tibia, C: Adult of Pulex irritans; 1 Eye hair in this type is under the eye, 2 the borderline of moral rood does not exist in this type, 3 the first part of spermatheca not distinguishing, 4 the picture of the ending part in the male gender http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 243–250 A Azarm et al.: Molecular Identification of … 247 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Fig. 3. Agarose gel (1.5%) electrophoresis of semi-nested PCR products (720bp) of DNA extracted from blood sam- ples of Leishmania-free and infected dogs with L.eishmania infantum. Lad: A 100bp ladder (SinClon, Iran); lanes 1–6, 8–10, and 12: PCR products of infected blood; lanes 7 and 11: PCR products of Leishmania-free blood samples; Neg: a negative control free of DNA template and lane +: positive control for L. infantum Fig. 4. Agarose gel (1.5%) electrophoresis of semi-nested PCR products (720bp) of DNA extracted from Cten- ocephalides canis collected from Leishmania-free and infected dogs with Leishmania infantum. Lad: A 100bp ladder (SinClon, Iran); lanes 1–4, 6–9 and 11: PCR products of infected fleas; lanes 5, 10, and 12: PCR products of Leishma- nia-free fleas; Neg: a negative control free of DNA template and lane +: positive control for L. infantum Fig. 5. Agarose gel (1.5%) electrophoresis of semi-nested PCR products (720bp) of DNA extracted from Ctenocephalides felis collected from infected dogs with Leishmania infantum. Lad: A 100bp ladder (SinClon, Iran); Neg: a negative control free of DNA template; lanes 2, 4, 5, and 8: PCR products of infected fleas; lanes 1, 3, and 7: PCR products of Leishmania-free fleas and lane +: positive control for L. infantum http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 243–250 A Azarm et al.: Molecular Identification of … 248 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Discussion Fleas are one of the most important arthro- pods in transmitting many diseases. There are many reasons that fleas may have a role in the transmission of infectious pathogens. Some of them include a long time of blood-feeding, the mode of obtaining blood, digestion, and con- tact with the host, and the frequency of host exchange. In the present study, 12 out of 20 (60%) dogs were infected with L. infantum using DAT. But PCR assay confirmed leishmanial infection in 10 out of them (Fig. 2). Three spe- cies of fleas collected including C. canis, C. felis, and P. irritans. All the caught dogs (100%) were infested with 974 fleas. The fleas are the most common ectoparasites in domestic dogs of the Meshkin-Shahr area. In recent studies, the probable role of fleas in the transmission of Leishmania parasites has been discussed. In our study, L. infantum was observed in 9/12 (75%) of C. canis (Fig. 3) and 4/6 (66.67%) of C. felis (Fig. 4) using semi-nested PCR and P. irritans fleas did not infect with the para- site. Also, no infection was observed in fleas collected from Leishmania-free dogs by PCR assay. Lainson and Shaw (1985) isolated agent of visceral leishmaniasis from 8 Lutzomyia long- ipalpis and 3 Lutzomyia antunesi on the island of Maraio (13). There are sporadic cases of visceral leishmaniasis on this island (13). In another study conducted, kDNA of L. infan- tum was isolated from salivary glands of col- lected ticks (Rhipicephalus sanguineus) from infected dogs in southern Italy. In this study Dantas-Torres et al. performed by PCR tech- nique, kDNA of L. infantum in the larvae of R. sanguinus tick was isolated after 4 months of experimental infection, which represents the possibility of transovarial transmission of Leish- mania parasites in the ticks (14). Dabaghmanesh et al. (2016) collected a total of 180 Leishma- nia-free ticks collected from fields and bred on lab rodents, were divided into eight groups, and allowed to feed on a dog for fixed periods of time. The infection rate was significantly higher in female than male ticks. The rates were higher among nymphs than adult ticks. The kDNA of L. infantum was not detected in ticks 24h post-feeding. It was, however, posi- tive among the second to fourth groups of nymphs and adult ticks. Eggs and unfed larvae recovered from the third and fourth adult groups were 100% PCR-positive. The data re- vealed the passage of L. infantum kDNA in nymphs and adults of brown dog tick follow- ing fixed time intervals post blood feeding on an infected dog (15). Coutinho and Linardi in 2007 conducted a study on the possibility of transmission of Leishmania chagasi caused by devouring collected fleas (C. felis) from infect- ed dogs in the Golden Hamsters (Mesocricetus auratus. In this study, of the fleas collected, 4/207 (1.9%) showed the presence of pro- mastigotes in smears stained by Giemsa, whilst 43/144 (29.9%) exhibited positive PCR assay for DNA of Leishmania parasites. Fourteen of the hamsters tested PCR amplification and 4 of them by indirect fluorescent antibody test (IFAT) were positive with Leishmania para- sites. In addition, out of 16 infected hamsters, 11 had been infected peritoneally and 5 orally (16). In 1932, Wenyon reviewed most existing theories about the transmission of Leishmania infection and believed that ticks have no role in the transmission of visceral leishmaniasis in the Mediterranean region (17). Prior to this study, similar cases on the likelihood of trans- mission of the disease had not been made on dogs. The results are very important because it has been shown in practice that R. sanguinus ticks have failed to transfer L. infantum from infected dogs to healthy dogs. Conclusions Visceral leishmaniasis has been increasing worldwide, principally due to a substantial rise in human and domestic animal traffic contrib- uting to spreading leishmanial infection in low or non-endemic areas (18). Female sand flies of http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 243–250 A Azarm et al.: Molecular Identification of … 249 http://jad.tums.ac.ir Published Online: Sep 30, 2022 some species of the genus Phlebotomus are the main and proven vectors of L. infantum trans- mission in humans and dog. There has long been speculation about the role of fleas as biologi- cally or mechanically vectors of L. infantum and recent studies have reinforced this hypothesis (15, 18). The human flea, P. irritans, and the cat flea, C. felis were identified on 40% and 35 % of dogs, respectively. The results of PCR in- dicated that L. infantum was in the collected C. canis (75%) and C. felis (66.7 %) from infected dogs. The result of the present study confirms that fleas can be infected by L. infantum. But it is not yet clear whether these insects as vectors are able to transmit Leishmania parasites from an infected host to healthy hosts and the vectorial competence of fleas needs to be determined. Given this, it is recommended that the Xenodi- agnoses test be carried out to determine the probable role of Leishmania spp. transmission. Acknowledgements The authors would like to thank Mrs F Baghkhani for her laboratory support and co- operation. Ethical considerations The protocols conducted in this study fol- lowed the guidelines of the institutional ethi- cal committee (Tarbiat Modares University). The protocols were approved by TMU ethical committee under registry TMU-3674. Conflict of interest statement Authors declare that there is no conflict of interest. References 1. Darvishi MM, Youssefi MR, Changizi E, Li- ma RR, Rahimi MT (2014) A new flea from Iran. Asian Pac J Trop Dis. 4: 85– 87. 2. Ratovonjato J, Rajerison M, Rahelinirina S, Boyer S (2014) Yersinia pestis in Pulex irritans fleas during plague outbreak, Mad- agascar. Emerg Infect Dis. 20(8): 1414– 1415. 3. Hamzaoui EL, Zurita B, Cutillas A, Parola C (2020) Fleas and flea-borne diseases of North Africa. Acta Trop. 211: 105627. 4. Takahashi K, Takahashi M, Hamasaka K (2016) Finding of the human flea Pulex irritans (Siphonaptera: Pulicidae) in Hok- kaido, Japan with human dermatitis caused by this flea. Med Entomol Zool. 67(4): 233–235. 5. Perez-Martinez L, Venzal JM, Gonzalez- Acuna D, Portillo A, Blanco JR, Oteo JA (2009) Bartonella rochalimae and other Bartonella spp in fleas, Chile. Emerg In- fect Dis. 15(7): 1150–1152. 6. Mohebali M (2013) Visceral leishmaniasis in Iran: a review of the epidemiological and clinical features. Iran J Parasitol. 8 (3): 348–358. 7. Ferreira MGPA, Fattori KR, Souza F, Lima VMF (2009) Potential role for dog fleas in the cycle of Leishmania spp. Vet Par- asitol. 165(1–2): 150–154. 8. Kakeh Mami A, Ghorbani A, Kayvan Beh- joo F, Mirzaei Mosivand A (2017) Com- parison of visual and digital interpreta- tion methods of land use/cover mapping in Ardabil Province. Journal of RS and GIS for Natural Resources. 8(3): 121–134 (Persion). 9. Harith A, Kolk A, Kager P, Leeuwenburg J, Muigai R, Kiugu S, Kiugu S, Laarman JJ (1986) A simple and economical direct agglutination test for serodiagnosis and seroepidemiological studies of visceral leishmaniasis. Trans R Soc Trop Med Hyg. 80(4): 583–536. 10. Kumsa B, Abiy Y, Abunna F (2019) Ecto- parasites infesting dogs and cats in Bish- oftu, central Oromia, Ethiopia. Vet Para- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 243–250 A Azarm et al.: Molecular Identification of … 250 http://jad.tums.ac.ir Published Online: Sep 30, 2022 sitol Reg Stud Reports. 15: 100263. 11. Ish-Horowicz D, Burke J (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res. 9(13): 2989–2998. 12. Aransay A, ME Scoulica, Tselentis Y (2000) Detection and identiÞcation of Leishma- nia DNA within naturally infected sand ßies by seminested PCR on minicircle ki- netoplastic DNA. Appl Environ Micro- biol. 66: 1933–1938. 13. Lainson R, Shaw J, Ryan L, Ribeiro R, Sil- veira F (1985) Leishmaniasis in Brazil. XXI. Visceral leishmaniasis in the Am- azon Region and further observations on the role of Lutzomyia longipalpis (Lutz and Neiva, 1912) as the vector. Trans R Soc Trop Med Hyg. 79(2): 223–226. 14. Dantas-Torres F, Lorusso V, Testini G, de Paiva-Cavalcanti M, Figueredo LA, Stanneck D, Mencke N, P. Brandao-Fil- ho SC, Alves L, Otranto D (2010) De- tection of Leishmania infantum in Rhip- icephalus sanguineus ticks from Brazil and Italy. Parasitol Res. 106(4): 857–860. 15. Dabaghmanesh T, Asgari Q, Moemenbel- lah-Fard MD, Soltani A, Azizi K (2016) Natural transovarial and transstadial trans- mission of Leishmania infantum by naïve Rhipicephalus sanguineus ticks blood feeding on an endemically infected dog in Shiraz, south of Iran. Trans R Soc Trop Med Hyg. 110(7): 408–413. 16. Coutinho MTZ, Linardi PM (2007) Can fleas from dogs infected with canine vis- ceral leishmaniasis transfer the infection to other mammals? Vet Parasitol. 147 (3–4): 320–325. 17. Wenyon CM (1932) The transmission of Leishmania infections: A review. Trans R Soc Trop Med Hyg. 25: 319–348. 18. Colombo FA, Odorizzi RMFN, Laurenti MD, Galati EAB, Canavez F, Pereira- Chioccola VL (2011) Detection of Leish- mania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs. Parasitol Res. 109(2): 267– 274. http://jad.tums.ac.ir/