J Arthropod-Borne Dis, Sep 2022, 16(3): 196–205 A Azarm et al.: Pulex irritans on … 196 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Original Article Pulex irritans on Dogs and Cats: Morphological and Molecular Approach Amrollah Azarm1, *Abdolhossin Dalimi1, Majied Pirestani1, Anita Mohammadiha1, Alireza Zahraei-Ramazani2, Narges Marvi-Moghaddam2, Esmaiel Amiri2 1Department of Medical Parasitology and Entomology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 2Department of Biology and Vector Control of Diseases, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran *Corresponding author: Dr Abdolhossein Dalimi, E-mail: dalimi_a@modares.ac.ir (Received 02 Jan 2022; accepted 02 May 2022) Abstract Background: The painful bite of Pulex irritans; causes wound on the host body and is a vector for Bartonella bacteria species, which can cause trench fever, Rickettsia species, Rocky Mountain spotted fever and Mediterranean spotted fe- ver. We conducted a study to find out the hosts, abundance, as well as the molecular characteristics of this flea species in Meshkin-Shahr County, Ardabil Province, northwest of Iran. Methods: After collecting fleas from different reservoir hosts and transferring them to the laboratory, identification was done using different morphological characters as well as the internal transcribed spacer1 (ITS1) molecular marker. Results: This morphological study indicated that from the 1053 fleas, which were collected from 162 different animals, including cats and dogs, 74 specimens belonged to human flea, Pulex irritans. In addition, in molecular analysis showed a high sequence similarity (99.5%) with the P. irritans counterparts from Spain country and Zanjan of Iran available in GenBank. Conclusion: Pulex irritans species is an obligatory hematophagous ectoparasite of human and animals. Therefore, con- sidering the relatively high frequency of this species on the body of cats and dogs, it is suggested to conduct more stud- ies on its distribution and the possibility of being a vector of pathogens among these animals and human. The results of these studies will be used to compile and review the control programs of this vector. Keywords: Pulex irritans; Dog; Cat; ITS1; Iran Introduction Fleas, the common name for the order Si- phonaptera, have unique properties like the lack of wings, sucking mouthparts, with a specific digestive system. Their digestive system is dif- ferent from other blood-feeding insects struc- turally and functionally. The flea body pressed laterally. It is different from other insects that have stretched bodies from each side. In addi- tion, a flea has a great leaping ability, which is the result of strong feet and the evolution of the curves of sides. It feeds from a wide range of hosts including birds and mammals. These features make this order of insect different from others (1). The results from the molecular stud- ies on DNA and morphological studies and ovary features showed that the closest relative of fleas is Boreidae from Mecoptera species (2). Today in the world, over 2500 species of fleas have been identified whose hosts are most- ly birds and mammals (3). The desire for blood feeding from warm-blooded animals has led to some of these species being vectors of some infectious diseases, and besides being the main host for some tapeworms like Dipylidium cani- num (4). The family Pulicidae of fleas has a great desire to feed blood from humans or oth- er mammals. The existence of similar morpho- logical characters among different species of 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: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): 196–205 A Azarm et al.: Pulex irritans on … 197 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Pulex, as caused most of the species of this type, to be hard to differentiate (5). One of the most famous species of this genus is Pulex ir- ritans. Despite its name, this species does not have a definite host and can suck blood from a wide range of mammals, and this factor makes it a carrier of various diseases such as Dipylid- ium caninum and trench fever. Rickettsia spe- cies, which can cause Rocky Mountain spot- ted fever and Mediterranean Spotted Fever (6). The existence of a wide range of hosts and com- patibility with different climatic areas results in finding this flea in many different areas of the world (7). Most of the studies done on this spe- cies in some areas of Iran have related to mor- phological characters and there is little data about its molecular characters. In this study, the internal transcribed spacer1 of ribosomal DNA (ITS1-rDNA) gene, was determined to provide a molecular marker for identification of the local Iranian P. irritans population. Materials and Methods Study area Ardabil Province is one of the thirty-one provinces of Iran. Meshkin-Shahr County is one of the counties located in the Province in the northwest part of Iran (Fig. 1). Meshkin- Shahr County with geographical coordinates 38° 44' N and 47° 40' E is at an altitude of 1400 meters above sea level. Morphological studies The present study was conducted during March and July 2018 in Meshkinshahr Coun- ty, Ardabil Province. The sampling was done from random captured cats (Felis catus) and dogs (Canis lupus) specimens, with two stand- ard methods of combing and separating with forceps (9). After collecting and recording the reservoir's host data; their ectoparasites were transferred to the glass tubes with lids contain- ing 70% ethanol for further identification (Fig. 2). First, the flea specimens from each host were cleared with 10% KOH individually and the permanent slides in Canada balsam were pre- pared. For the morphological identification of flea specimens, we used the standard key of Harimalala et al. (10). After morphological iden- tification and final confirmation, specimens were taken out of slides, washed, and transferred to tubes containing 90% ethanol. Molecular Studies The identified flea specimens were used for DNA extraction and molecular study. After morphological studies, a sample of each flea was placed under a binocular on a slide and its abdominal contents were completely emp- tied and its information was recorded and trans- ferred to a new microtube. The flea was then crushed into a microtube with a pistol. The mi- crotube was transferred to a nitrogen tank for 5 minutes and then the DNA of fleas was ex- tracted using DNeasy kit (Qiagen, Hilden, Ger- many) and stored at -20 ºC for later use. The ITS1 primers used of P. irritans were NC5 (5′-GTA GGT GAA CCT GCG GAA GGA TCA TT-3′) and ITS1rev (5′-GCT GCG TTC TTC ATC GAC CC-3′) as forward and reverse respectively (11). In each 15µl reaction of PCR, the 0.50–1µl of each extracted DNA, 2µl pri- mer (10pmol of each), 7.5µl 2x master mix (Taq DNA Polymerase Master Mix RED, 2mM MgCl2, Amplicon) were used. The PCR reac- tion was conducted with a thermal cycler de- vice along 33 cycles (94 degrees for 30 sec- onds, 58 degrees for 30 seconds, and 72 de- grees for 1 minute) and for the final extension, 10 minutes with 72 degrees was used. The PCR products were visualized in a 1.5% agarose gel containing a DNA-safe stain (cat no., EP5082, Sinaclon). PCR product of each sample se- quenced by the ABI3730XL sequence analyz- er (Macrogen, S. Korea). The sequences were edited and aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and compared with reference sequences from GenBank. For ITS1 sequences, the phyloge- netic tree was built with the maximum likeli- hood (ML) algorithm using molecular evolu- tionary genetics analysis (MEGA) software, in- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 196–205 A Azarm et al.: Pulex irritans on … 198 http://jad.tums.ac.ir Published Online: Sep 30, 2022 cluding sequences representative of three fam- ilies (Pulicidae, Leptopsyllidae, Ceratophylidae) of order Siphonaptera. Results The results of the morphological study in- dicated that among the 1053 flea specimens, which were isolated from two animal hosts, 47 dogs and 115 cats, 74 (7.03%) specimens be- longed to P. irritans. It was specified that from 74 specimens of P. irritans fleas, 35 (47.29%) were males and 39 (52.70%) were females (Ta- ble 1). Out of the 74 P. irritans fleas, 49 (66.21 %) were collected from dogs; 32 (43.24%) were collected from female dogs, and 17 from male dogs, and the other 25 samples were taken from male cats. In this study, P. irritans flea were not found on female cats (Table 2). The mor- phological characteristic of P. irritans is shown in Figure 3. The specific primers used in this study am- plified about 900bp of ITS1 region of rDNA gene. (Fig. 4). The analysis of the ITS1 sequence of the identified isolates approved the mor- phological identification. The sequence of the isolated from the present study is accessible under GenBank Accession No. MG745174. Af- ter trimming the sequence data, 802bp of se- quences were blasted. The sequence obtained in this study displayed 99.5% sequence ho- mology with two P. irritans specimens from Spain (GenBank ID: LT797452) and Zanjan (GenBank ID: KX822017) in west of Iran (Fig. 5). In addition to these two sequences, there were some other P. irritans ITS1 sequences in GenBank originated from Spain, different lo- cations of west of Iran (including Mahneshan Zanjan, Khoramabad, Kamyaran, Hamedan, Ba- har, GilanGharb, Kerrmanshah, Mahabad, Sana- ndaj, and Urmia), China, and India which dis- played 95.69-99.38% sequence homology with the sequence obtained in this study. To assess phylogenetic relationship among P. irritans iso- lates, multiple alignments were generated for a subset of P. irritans specimens plus the se- quence obtained in this study, and the alignment was used to construct a phylogenetic tree by Neighbor Joining (NJ) method. Phylogenetic relationships of the P. irritants using the ITS1 region of the rRNA gene produced the highest log likelihood as shown in Figure 6. The se- quence obtained in this study was strongly as- sociated with the specimen from Spain (Gen- Bank ID: LT797452). Most of the nodes in the tree had 98-99% bootstrap values indicating very strong support for the nodes created in the tree. Table 1. Geographical features of the study area, prevalence and distribution of fleas and their animal hosts in Mesh- kinshahr County, Ardabil Province, Iran, 2016-2018. collected fleas cats dogs Location Sampling Area % N % N % N East North 14.43 152 16.52 19 10.64 5 473852 382452 Parikhan 9.59 101 21.79 25 14.89 7 474054 382246 Meshkinshahr 2.66 28 15.65 18 6.37 3 473110 383105 Sarikhanloo 33.33 351 13.04 15 12.76 6 473804 382115 Urkandi 17.76 187 6.96 8 6.37 3 473336 382441 Ahmad Bigloo 0.57 6 5.22 6 12.76 6 474023 382102 Aghbalagh 19.37 204 16.52 19 17.02 8 473053 382918 Koojangh 2.28 24 4.35 5 19.14 9 473534 382200 Ahmad Abad 99.99 1053 100 115 99.95 47 …… …….. Total http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 196–205 A Azarm et al.: Pulex irritans on … 199 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Table 2. Prevalence of host’s flea infestation in Meshkinshahr County, Ardabil Province, Iran, 2016-2018. M: male flea, F: female flea Pulex No. (%) Fleas on Cats Fleas on Dogs Total Fleas Sampling Area % F % M % Total %/74 Pulex % No %/74 Pulex % No 12.1 9 8.1 6 18.9 14 4.0 3 15.9 18 14.8 11 14.2 134 152 Parikhan 2.7 2 0.0 0 2.7 2 1.3 1 19.4 22 1.3 1 8.5 79 101 Meshkinshahr 6.7 5 0.0 0 6.7 5 0.0 0 0.0 0 6.7 5 2.9 28 28 Sarikhanloo 33.7 25 16.2 12 50.0 37 8.1 6 33.6 38 41.8 31 33.3 313 351 Urkandi 2.7 2 0.0 0 2.7 2 0.0 0 4.4 5 2.7 2 19.3 182 187 Ahmad Bigloo 0.0 0 0. 0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.6 6 6 Aghbalagh 5.4 4 9.4 7 14.8 11 2.7 2 15.0 17 12.1 9 19.8 187 204 Koojangh 1.3 1 1.3 1 2.7 2 2.70 2 11.5 13 0.0 0 1.1 11 24 Ahmad Abad 64.8 48 35.1 26 100 74 18.9 14 100 113 81.0 60 100 94 1053 Total Fig. 1. Map of area study Meshkinshahr County, Ardabil Province, Iran. Fig. 2. Isolation and collection of fleas from a dog in Meshkinshahr, northwest of Iran using the standard method of combing and transferring them to tubes containing 70% ethanol (original) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 196–205 A Azarm et al.: Pulex irritans on … 200 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Fig. 3. Morphological characters of Pulex irritans. A: Eye hair is under the eye; B: The borderline of moral rood which does not exist; C: The first part of Spermatheca which is not distinguishing; D and E: end part of the female and male genus (original) Fig. 4. The ITS1-rDNA PCR products (about 900bp) on agar gel 1.5%. Ladder: 100bp DNA Ladder (SinClon, Iran), 1: Negative control (ddH2O); 2: Pulex irritans isolated from stray cats, 3 and 4: Pulex irritans isolated from stray dogs http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 196–205 A Azarm et al.: Pulex irritans on … 201 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Fig. 5. The multiple alignments of 802bp of ITS1-rDNA sequence of Pulex irritans from this study (MeshkinShahr, GenBank ID: MG745174) and two reference sequences from Znajan, Iran (GenBank ID: KX822017) and Spain (Gen- Bank ID: LT797452). There was 99.5% similarity between MeshkinShahr specimen with the Zanjan and Spain speci- mens. * indicates similar nucleotide, and gaps indicates substitution in the position http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 196–205 A Azarm et al.: Pulex irritans on … 202 http://jad.tums.ac.ir Published Online: Sep 30, 2022 Fig. 6. Phylogenetic relationships inferred from neighbor joining analyses constructed by Mega10 based on 802bp se- quences of ITS1-rDNA gene of Pulex irritans obtained in this study and a subset of Pulex irritans sequences available in GenBank. The GenBank ID of the sequences are shown in front of branches. Xenopsylla cheopis with accession number DQ 295061.1 was used as out-group. Scale bar indicates genetic distance. Our sample is marked with an arrow Discussion All human fleas collected specimens are characterized by laterally compressed bodies and large hind legs. Pulex irritans lack the combs that lend the appearance of genal and pronotal combs. It has a rounded forehead, which allows differentiation from the anteri- orly flattened head of the stick-tight flea. Be- hind the antennae, the head has only a single pair of hairs. It has no pleural rod, a feature that differentiates it from Xenopsylla cheopis (12). In terms of medical importance, P. irri- tans can play the role of the intermediate host of the dog tapeworm (for example D. cani- num). In addition, P. irritans may be a vector for erysipeloid (13). Moreover, the P. irritans is implicated in the spread of diseases histori- cally associated with X. cheopis (14). The hu- man female flea can lay more than 400 eggs in her lifetime. The ability of pupae and “co- coons” adults to lie dormant for a year or more can confound efforts at flea control. It does not kill adult fleas, but it does prevent repro- duction. Adverse reactions flea bites appear as tense, pruritic, urticarial papules on exposed ar- eas including the hands, forearms, and lower legs. Excoriation with secondary impetiginiza- tion is common. Vesicular and bullous flea bite reactions occur and can be quite dramatic. His- tologic examination reveals a wedge-shaped der- mal infiltrate that surrounds both venules and arterioles. Eosinophils are common. Epidermal necrosis, spongiosis, and intraepidermal or sub- epidermal bullae may be seen. The human flea is distributed throughout the world and has been recorded in a wide range of mammal hosts in- cluding humans, domestic dogs, cats, pigs, badg- ers (Meles meles) and foxes (Vulpes vulpes) (15). This species is also medically important as a possible vector of flea-borne diseases such as tularemia, murine typhus, and tapeworm (16). In different parts of the world, for isolat- ing and identifying P. irritans, different char- acters have been used. Out of the genus Pulex, the species P. irritans is the most abundant in the world and in Iran (17). The results of the morphological studies indicated that out of the http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 196–205 A Azarm et al.: Pulex irritans on … 203 http://jad.tums.ac.ir Published Online: Sep 30, 2022 1053 fleas, 74 specimens belonged to P. irri- tans. In a similar study for investigating ecto- parasite of dogs and cats in Kurdistan Prov- ince west of Iran and bordered with the north- east of Iraq country, this species was isolated with Ctenocephalides felis and C. canis but with a lower percentage of frequency (18). Moreo- ver, in a review by Maleki-Ravassan et al. (19) on ectoparasite of dogs in different parts of Iran, P. irritans species and two species of C. felis and C. canis were isolated from hosts. In ad- dition, another study, which was conducted on dogs in Shiraz, had similar results (20). In a study in 2015 for identifying ectoparasite on sheep in Kurdistan Province bordered with the east of Iraq country, 1323 specimens of P. ir- ritans were isolated from hosts (21). In addi- tion, Sharifdini et al. 2021 were collected P. ir- ritans from seven raccoons from Guilan Prov- ince, northern Iran (22). In comprehensive re- search by Barutzki and Schaper (23) in 2000 for identifying ectoparasite of dogs and cats in Germany, out of 1508 cats and 2653 dogs, C. felis, C. canis, and P. irritans were isolated. Al- ready, there are about 2500 species and sub- species of fleas in about 220 genera, but only relatively, few are important human pests. In this study, the result of the morphological anal- ysis showed that the identified flea was P. ir- ritants. The ITS1 gene sequence obtained in this study plus the phylogenetic tree, and high se- quence homology with other available data in GenBank, confirm and support the result of our morphological study. Conclusion Pulex irritans is one of the most important ectoparasites of humans, which transmits var- ious pathogens (including bartonellosis and rick- ettsioses) and causes skin sensitivity and irrita- tion by biting humans and animals. This flea is transmitted from the cats and dogs body to hu- mans and can bite humans. To achieve control of parasitic skin diseases of P. irritans in de- veloping countries, local epidemiology has to be better understood. Studies in Iran on epide- miology and the medical significance of P. ir- ritans in different areas of animals are limited. Molecular approaches, although in their infan- cy, are now providing a better understanding of the biology of ectoparasites and will become cornerstones for prevention and control in the future. Therefore, this study was conducted in order to accurately identify this vector based on morphological and molecular characteris- tics. Morphological studies show that there is no moral root border in this flea and the first part of the spermatheca is not differentiated. In this study, P. irritans was not caught in the body of female cats, which is not important from the point of view of disease epidemiology because both male and female fleas are able to feed on blood and transmit the disease to cats, dogs, and humans. The specific primers used in this study amplified the ITS1 regions of about 900bp. It is suggested that to find out the dis- tribution of P. irritans in male and female cats, as well as the possibility of P. irritans being a vector among cats, dogs and human, more stud- ies should be conducted at the country level. Acknowledgements The authors would like to thank all per- sonals who work in department of medical En- tomology Tarbiat Modares University, Tehran, Iran for their helping to do this research. Ethical considerations The protocols were conducted in this study followed the guidelines of the institutional eth- ical 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. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Sep 2022, 16(3): 196–205 A Azarm et al.: Pulex irritans on … 204 http://jad.tums.ac.ir Published Online: Sep 30, 2022 References 1. Zurita A, Callejón R, De Rojas M, Cutillas C (2019) Origin, evolution, phylogeny and taxonomy of Pulex irritans. Med Vet En- tomol. 33(2): 296–311. 2. Perez-Martinez L, Venzal JM, Gonzalez- Acuna D, Portillo A, Blanco JR, Oteo JA (2009) Bartonella rochalimae and oth- er Bartonella spp in fleas, Chile. Emerg Infect Dis. 15(7): 1150–1152. 3. Lareschi M, Venzal JM, Nava S, Mangold AJ, Portillo A, Palomar-Urbina AM, Oteo Revuelta JA (2018) La pulga humana Pulex irritans (Siphonaptera: Pulicidae) en el noroeste argentino, una investigación de Bartonella y Rickettsia spp. Rev Mex Biodivers. 89(2): 375–381. 4. Fontalvo MC, Favacho ARdM, Araujo AdC, Santos NMd, Oliveira GMBd, Aguiar DM, Lemos ERSd, Horta MC (2017) Bar- tonella species pathogenic for humans in- fect pets, free-ranging wild mammals and their ectoparasites in the Caatinga biome, Northeastern Brazil: a serological and mo- lecular study. Braz J Infect Dis. 21(3): 290–296. 5. Foley P, Foley J, Sándor A, Ionică A, Ma- tei I, D’Amico G, Gherman C, Domșa C, Mihalca A (2017) Diversity of flea (Siphonaptera) parasites on red foxes (Vul- pes vulpes) in Romania. J Med Entomol. 54(5): 1243–1250. 6. 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. 7. Kumsa B, Abiy Y, Abunna F (2019) Ecto- parasites infesting dogs and cats in Bish- oftu, central Oromia, Ethiopia. Vet Par- asitol Reg Stud Reports. 15: 100263. 8. Mohebali M, Malmasi A, Khodabakhsh M, Zarei Z, Akhoundi B, Hajjaran H, Azarm A (2017) Feline leishmaniosis due to Leishmania infantum in Northwest Iran: The role of cats in endemic areas of vis- ceral leishmaniosis. Vet Parasitol Reg Stud Reports. 9: 13–16. 9. Hernández-Urbina CF, Vital-García C, Ávila AME, Colima AG, Sánchez-Olivas MP, Clemente-Sánchez F (2020) First report of Siphonaptera parasites in Canis latrans in the Flora and Fauna Protection Area, Médanos de Samalayuca Chihuahua, Mex- ico. Vet Parasitol Reg Stud Reports. 20: 100379. 10. Harimalala M, Ramihangihajason T, Ha- rimanana R, Girod R, Duchemin JB (2021) Illustrated Morphological Keys for Fleas (Siphonaptera) in Madagascar. J Med En- tomol. 58(4): 1701–1716. 11. Marrugal A, Callejón R, De Rojas M, Hala- jian A, Cutillas C (2013) Morphological, biometrical, and molecular characteriza- tion of Ctenocephalides felis and Cten- ocephalides canis isolated from dogs from different geographical regions. Parasitol Res. 112(6): 2289–2298. 12. Darvishi MM, Youssefi MR, Changizi E, Lima RR, Rahimi MT (2014) A new flea from Iran. Asian Pac J Trop Dis. 4: 85– 87. 13. Sanusi ID, Brown EB, Shepard TG, Graf- ton WD (1989) Tungiasis: report of one case and review of the 14 reported cases in the United States. J Am Acad Derma- tol. 20: 941–944. 14. Hamzaoui EL, Zurita B, Cutillas A, Parola C (2020) Fleas and flea-borne diseases of North Africa. Acta Trop. 211: 105627. 15. Anstead GM (2020) History, rats, fleas, and opossums: the ascendency of flea-borne typhus in the United States, 1910–1944. Trop Med Infect Dis. 5(1): 37. 16. Bourne D, Craig M, Crittall J, Elsheikha H, Griffiths K, Keyte S, Merritt B, Rich- ardson E, Stokes L, Whitfield V (2018) Fleas and flea-borne diseases: biology, http://jad.tums.ac.ir/ https://pubmed.ncbi.nlm.nih.gov/?term=Sanusi+ID&cauthor_id=2654224 https://pubmed.ncbi.nlm.nih.gov/?term=Brown+EB&cauthor_id=2654224 https://pubmed.ncbi.nlm.nih.gov/?term=Shepard+TG&cauthor_id=2654224 https://pubmed.ncbi.nlm.nih.gov/?term=Grafton+WD&cauthor_id=2654224 https://pubmed.ncbi.nlm.nih.gov/?term=Grafton+WD&cauthor_id=2654224 J Arthropod-Borne Dis, Sep 2022, 16(3): 196–205 A Azarm et al.: Pulex irritans on … 205 http://jad.tums.ac.ir Published Online: Sep 30, 2022 control and compliance. Companion Ani- mal. 23(4): 204–211. 17. Seidy S, Tavassoli M, Malekifard F (2022) Pyrethroids resistance in Pulex irritans and Ctenocephalides canis in west and northwest Iran. Vet Res Forum. 13(4): 529–535. 18. Yakhchali M, Bahramnejad K (2015) A survey of Pulex irritans (Linnaeus 1758, Siphonaptera: Pulicidae) infestation in sheep and residential areas in Kurdistan Province, Iran. Iran J Vet Sci Technol. 7(1): 40–47. 19. Maleki-Ravasan N, Solhjouy-Fard S, Beaucournu JC, Laudisoit A, Mostafavi E (2017) The fleas (Siphonaptera) in Iran: diversity, host range, and medical im- portance. PLoS Negl Trop Dis. 11(1): e0005260. 20. Shoorijeh SJ, Ghasrodashti AR, Tamadon A, Moghaddar N, Behzadi MA (2008) Seasonal frequency of ectoparasite infes- tation in dogs from Shiraz, Southern Iran. Turk J Vet Anim Sci. 32(4): 309–313. 21. Tavassoli M, Ahmadi A, Imani A, Ahmadia- ra E, Javadi S, Hadian M (2010) Survey of flea infestation in dogs in different ge- ographical regions of Iran. Korean J Par- asitol. 48(2): 145–149. 22. Sharifdini M, Norouzi B, Azari-Hamidian S, Karamzadeh N (2021) The first record of ectoparasites of raccoons (Procyon lotor) (Carnivora, Procyonidae) in Iran. Persian J Acarol. 10: 41–54. 23. Barutzki D, Schaper R (2003) Endopara- sites in dogs and cats in Germany 1999– 2002. Parasitol Res. 90 Suppl 3: S148– 150. http://jad.tums.ac.ir/