High prevalence of vector-borne pathogens in domestic and wild carnivores in Iraq

High prevalence of vector-borne pathogens in domestic and wild carnivores in Iraq

Acta Tropica 197 (2019) 105058 Contents lists available at ScienceDirect Acta Tropica journal homepage: www.elsevier.com/locate/actatropica High pr...

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Acta Tropica 197 (2019) 105058

Contents lists available at ScienceDirect

Acta Tropica journal homepage: www.elsevier.com/locate/actatropica

High prevalence of vector-borne pathogens in domestic and wild carnivores in Iraq

T



Domenico Otrantoa, , Roberta Iattaa, Gad Banethb, Maria Alfonsa Cavaleraa, Angelica Biancoc, Antonio Parisic, Filipe Dantas-Torresd, Vito Colellaa,e, Audrey C. McMillan-Colef, Bruno Chomelg a

Dipartimento di Medicina Veterinaria, Università degli Studi di Bari, 70010, Valenzano, Italy School of Veterinary Medicine, Hebrew University of Jerusalem, Rehovot, Israel c Istituto Zooprofilattico della Puglia e della Basilicata, 70017, Bari, Italy d Department of Immunology, Aggeu Magalhães Institute, Oswaldo Cruz Foundation (Fiocruz), 50670-420, Recife, Brazil e Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, 3010, Parkville, Australia f DLA Troop Support Europe and Africa, 67657, Kaiserslautern, Germany g Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, 95616, Davis, CA, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Anaplasma platys Babesia sp. MML Babesia vulpes Cats Dogs Foxes Hepatozoon canis Hepatozoon felis Iraq Jackals Vector-borne disease

Vector-borne diseases (VBDs) of domestic and wild carnivores are of major public health concern both in industrialized and developing countries, especially in poor socioeconomic settings. War-torn areas specifically suffer from absence of veterinary surveillance of VBDs, resulting in lack of scientific knowledge on this topic. To investigate occurence and prevalence of several vector-borne pathogens (VBPs) in some carnivore species from Iraq, blood samples (n = 397) were obtained from 190 canids [97 stray dogs (Canis familiaris), 55 jackals (Canis aureus) and 38 red foxes (Vulpes vulpes)] and 207 stray cats (Felis catus) collected during a feral animal control and zoonotic disease surveillance program in several United States military bases in Iraq. The presence of Babesia spp., Hepatozoon spp., Ehrlichia spp., Anaplasma spp., Dirofilaria spp. and Leishmania spp. DNA was molecularly investigated. Out of 397 animals tested, 176 (44.3%; 95% CI: 39.5–49.2%) were positive for at least one pathogen with the highest prevalence in foxes (73.7%; 95% CI: 58–85%), followed by jackals (54.5%; 95% CI: 41.5–67%), dogs (38.1%; 29.1–48.1%) and cats (39.1%; 95% CI: 32.7–45.9%). Up to five pathogens were diagnosed in dogs. Hepatozoon canis was the most prevalent VBP in jackals (49.1%; 95% CI: 36.4–61.9%), foxes (47.3%; 95% CI: 32.5–62.7%) and dogs (33%; 95% CI: 24.4–42.8%), whereas Hepatozoon felis was the only species detected in cats (39.1%; 95% CI: 32.7–45.9%). A species of Babesia related to but different from Babesia lengau and designated as Babesia sp. MML was detected in six foxes (15.8%; 95% CI: 7.4–30.4%) and in one jackal (1.8%; 95% CI: 0.3–9.6%). This finding suggested the existence of a new species in the genus Babesia as inferred by molecular and phylogenetical analysis. Further, Babesia vulpes was identified only in two foxes (5.3%; 95% CI: 1.5–17.3%). All samples were negative for Leishmania spp. and Ehrlichia spp. Co-infection with H. canis and Babesia spp. was the most prevalent (5/176, 2.8%, i.e., 4 foxes and 1 jackal), followed by H. canis and Dirofilaria immitis (1/176, 1.3%, i.e., in 1 jackal), H. canis and Dirofilaria repens or Acanthocheilonema reconditum (1/176, 1.3%, i.e., in one dog, each). Data presented fill gaps into knowledge of VBPs in dogs, cats and wild canids in Iraq, indicating that different pathogens circulate amongst animal populations living in the same areas, possibly sharing the same tick vectors. Large-scale surveys are urgently needed to further assess VBPs distribution in Iraq and establish preventative strategies in domestic animals to minimize the risk of infection for animals and humans.

1. Introduction

(Otranto et al., 2009a, 2009b; Colwell et al., 2011; Dantas-Torres et al., 2012). In particular, some canine vector-borne pathogens (VBPs) may represent a public health issue both in industrialized and developing countries where pet populations have steadily increased in recent decades (Dantas-Torres and Otranto, 2014, 2016). In certain

Vector-borne diseases (VBDs) of domestic and wild carnivores are of emerging importance in human and veterinary medicine due to the zoonotic potential of bacterial, protozoan and helminthic pathogens ⁎

Corresponding author at: Dipartimento di Medicina Veterinaria, Università degli Studi di Bari, 70010, Valenzano, Bari, Italy. E-mail address: [email protected] (D. Otranto).

https://doi.org/10.1016/j.actatropica.2019.105058 Received 13 May 2019; Received in revised form 5 June 2019; Accepted 7 June 2019 Available online 08 June 2019 0001-706X/ © 2019 Elsevier B.V. All rights reserved.

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Table 1 Targeted pathogens and list of primers used in this study. Pathogen

Target gene

Primers

Sequence (5′–3′)

Fragment length (bp)

Reference

Babesia spp.

18S rRNA

Gubbels et al. (1999)

18S rRNA

660bp

Inokuma et al. (2002)

Ehrlichia/Anaplasma spp.

16S rRNA

345bp

Martin et al. (2005)

Filarioids

cox1

648bp

Otranto et al. (2011)

Leishmania spp.

kDNA minicircle

GAGGTAGTGACAAGAAATAACAATA TCTTCGATCCCCTAACTTTC ATACATGAGCAAAATCTCAAC CTTATTATTCCATGCTGCAG GGTACCYACAGAAGAAGTCC TAGCACTCATCGTTTACAGC TGATTGGTGGTTTTGGTAA ATAAGTACGAGTATCAATATC AACTTTTCTGGTCCTCCGGGTAG ACCCCCAGTTTCCCGCC

460 bp

Hepatozoon spp.

RLB-F RLB-R Hep-F Hep-R EHR-16SD EHR-16SR NTF NTR LEISH-1 LEISH-2

120bp

Francino et al. (2006)

2.2. Ethics statement

circumstances, such as in poor socioeconomic settings, in war-torn areas and in the presence of political upheaval, absence of preventive strategies has concurred to increase the incidence of these infections (Otranto et al., 2017a, 2017b). Wild and domestic carnivores share many species of parasitic arthropod and associated pathogens, thus enhancing the maintenance and circulation of VBPs in territories where they live in sympatry (Otranto et al., 2015a, 2015b). Furthermore, there is no systematic surveillance and control of infectious diseases in wild carnivores, besides rabies (Pastoret and Brochier, 1999). Therefore, data on VBPs in wild carnivores in several countries is often limited to case reports or case series obtained under national programs for wildlife conservation (Otranto et al., 2015a). In war-torn territories, deployed military personnel may be exposed to many VBDs, which are often not endemic in their areas of origin (Dunton and Sargent, 2009). For example, 8.2% of the soldiers of the Austrian Armed Forces was exposed to Leishmania spp. infection after being deployed in peacekeeping missions in Bosnia and Herzegovina (Obwaller et al., 2018), and some years later a large proportion (16.7%) of the canine population from the latter country was reported as being exposed to or infected by L. infantum (Colella et al., 2019). In Iraq, VBPs and tick infestation have been mainly documented in livestock (Omer et al., 2007; Khamesipour et al., 2018). Meanwhile, besides reports on Bartonella spp. infection (Chomel et al., 2012; Switzer et al., 2013), limited information is available on VBPs of domestic and wild carnivores in Iraq. The latter animals are well known for their free-roaming behaviour and for living in proximity to human dwellings in the country (Switzer et al., 2013), thus increasing likelihood of contact between domestic animals and humans. The aim of this study was to investigate the presence and prevalence of several VBPs, in some domestic and wild carnivore species from Iraq, using samples collected during Operation Iraqi Freedom, in order to fill gaps in the knowledge of VBPs in the country.

All animals were sedated prior to blood collection and humanely treated prior to euthanasia, in accordance with the rules of the ethic committee of the US Army feral animal control and zoonotic disease surveillance program, the University of California, Davis (UCD) Animal Use and Care Committee (Protocol # 12668 originally approved on March 8, 2007) and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. 2.3. DNA extraction, PCR protocols and sequencing DNA was extracted from 200 μl aliquots of EDTA-treated blood samples using a commercial purification kit (QIAampDNA Blood & Tissue, Qiagen, Hilden, Germany), according to the manufacturer’s instructions. All samples were tested for the presence of DNA of species of the genera Acanthocheilonema, Anaplasma, Babesia, Dirofilaria, Ehrlichia, Hepatozoon and Leishmania. Molecular detection of Babesia spp., Hepatozoon spp., Ehrlichia spp. and Anaplasma spp., and Dirofilaria spp. was performed by conventional PCR using primers targeting partial 18S rRNA, 16S rRNA and cytochrome c oxidase subunit 1 (cox1) genes, respectively (Table 1; Gubbels et al., 1999; Inokuma et al., 2002; Martin et al., 2005; Otranto et al., 2011). PCR amplicons were visualized by capillary electrophoresis using a QIAxcel® DNA screening gel cartridge (Qiagen) on a QIAxcel system (Qiagen), as per the manufacturer’s instructions. A QX DNA Size Marker (Qiagen) was used to size PCR products. A QX Alignment Marker (Qiagen), which consisted of 15 bp and 3000 bp fragments, was injected onto the cartridge with each sample. The PCR product sizes were then determined by QIAxcel Screen Gel 1.4.0 software (Qiagen). The amplicons were purified and sequenced in both directions using the same primers as for PCR, employing the Big Dye Terminator v.3.1 chemistry in a 3130 Genetic Analyzer (Applied Bio-systems, Foster City, CA, USA). Sequences were edited and analysed using the Geneious software version 9.0 (Biomatters Ltd., Auckland, New Zealand) (Drummond et al., 2011) and molecular identification of selected species achieved by sequence comparison with those available in the GenBank database by the Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi) Presence of Leishmania spp. DNA was investigated using a real-time PCR assay by amplification of a 120 bp fragment of the kDNA minicircle as described elsewhere (Francino et al., 2006). For all conventional and real-time PCR tests, positive and negative controls were included, being represented by DNA of pathogen-positive or negative (i.e. naïve dog) blood samples, respectively.

2. Materials and methods 2.1. Sample collection The samples herein tested were analysed for Bartonella spp. in previous studies (Chomel et al., 2012; Switzer et al., 2013). In brief, blood samples were collected in EDTA tubes between February and December 2008 under the US Army feral animal control and zoonotic disease surveillance program in various geographical zones of Iraq. Samples (n = 397) were obtained from 190 canids [97 (51.1%) stray dogs (Canis familiaris), 55 (28.9%) golden jackals (Canis aureus) and 38 (20%) red foxes (Vulpes vulpes)] and 207 stray cats (Feliscatus) captured on several US military bases throughout Iraq. Blood samples were frozen and shipped to the Unit of Parasitology at the Department of Veterinary Medicine, University of Bari (Italy). Categorical age, sex, and location data were recorded for each animal.

2.4. Phylogenetic analysis For phylogenetic analyses, 18S rRNA representative sequences-type of Hepatozoon spp. and Babesia spp. and the corresponding sequences available from GenBank database were included. Phylogenetic 2

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Dirofilaria repens or Acanthocheilonema reconditum (1/176, 1.3%, i.e., in one dog, each). Hepatozoon canis was the most prevalent tick-borne pathogen species among jackals (49.1%; 95% CI: 36.4–61.9%), foxes (47.3%; 95% CI: 32.5–62.7%) and dogs (33%; 95% CI: 24.4–42.8%), whereas H. felis was the only species detected in cats (39.1%; 95% CI: 32.7–45.9%). The distribution of animals positive to Hepatozoon spp. in Baghdad and Northern, Western and Southern Iraq is shown in Table 2. Babesia sp. (related to but different from Babesia lengau and previously designated as Babesia sp. MML) was detected in six foxes (15.8%; 95% CI: 7.4–30.4%) and one jackal (1.8%; 95% CI: 0.3–9.6%), whereas Babesia vulpes was identified in two foxes (5.3%; 95% CI: 1.5–17.3%) (Table 3). All samples were negative for Leishmania spp. and Ehrlichia spp. No statistically significant association was found between positivity to H. canis and host species infected (P = 0.095), whereas Babesia sp. MML was more prevalent in red foxes than in jackals (P = 0.012). Consensus sequences of the 18S rRNA gene analysed displayed 99–100% nucleotide identity with sequences available in GenBank. Four sequence types (ST1-4) of partial 18S rRNA gene were identified for H. canis (Table 4), with the most representative (38.9%) designated as ST3 present in dogs (80%) and in jackals (20%). Conversely, ST1 was mainly identified in jackals (82.3%) compared to foxes (17.4%), whereas ST2 was evenly distributed in jackals (41.2%), dogs (47%) and less in foxes (11.8%). Accordingly, the BLAST analysis of other pathogens revealed 99–100% nucleotide identity to the following nucleotide sequences: H. felis (100%; GenBank accession nos. JQ867388), Babesia sp. MML (100%; KJ956782), Babesia vulpes (100%; KT223483, KT580785, KM115977), D. immitis (99%; LC107816, KR870344), D. repens (99%; MF695085, KX265049), A. reconditum (99%; JF461456) and Anaplasma platys (99%; KX792089). The molecular identification of all representative 18S rRNA sequence-type for Hepatozoon spp. and Babesia sp. MML was supported by the distinct separation of species-specific clades inferred from the phylogenetic analyses (Figs. 1 and 2). In particular, the ML tree grouped all representative sequence-type of H. canis in three monophyletic clades, supported by high bootstrap value (i.e., 98%), to the exclusion of other Hepatozoon spp. (Fig. 1). Similarly, sequence-type of H. felis was resolved as sister clades of the H. felis group and as paraphyletic clade respective to other Hepatozoon spp. (Fig. 1). The 18S rRNA Babesia sequence-type clustered with Babesia sp. MML2014 and, as a sister clade, with B. lengau (bootstrap value of 99%) to the exclusion of a large clade including other Babesia spp. (Fig. 2). Representative 18S rRNA sequence-types of Hepatozoon spp. and Babesia spp. here generated were deposited in GenBank under accession numbers from MK957183 to MK957189.

Table 2 Number and percentage of animal species positive to Hepatozoon spp. according to their sex, age and location. Hepatozoon canis Variables

Sex Male Female No info Age Young Adult No info Location Baghdad North Iraq West Iraq South Iraq No info

Hepatozoon felis

Jackal (n = 55)

Fox (n = 38)

Dog (n = 97)

Cat (n = 207)

Pos/Tot (%)

Pos/Tot (%)

Pos/Tot (%)

Pos/Tot (%)

20/43 (46.5) 5/8 (62.5) 2/4 (50)

9/20 (45) 9/18 (50) 0

17/43 (39) 13/32 (40.6) 2/22 (9.1)

53/127 (41.7) 23/73 (31.5) 5/7 (71.4)

6/13 (46.1) 18/36 (50) 3/6 (50)

4/9 (44.4) 14/29 (48.3) 0

1/10 (10) 29/60 (48.3) 2/27 (7.4)

4/10 (40) 71/188 (37.8) 6/9 (66.7)

18/40 (45) 4/9 (44.4) 4/5 (80) 0 1/1 (100)

9/15 (60) 0/3 9/20 (45)

28/77 (36.4) 3/15 (20) 0/3 1/2 (50)

54/125 (43.2) 11/55 (20) 16/27 (59.3) 0

relationships were inferred using the Maximum Likelihood (ML) method based on the Tamura 3-parameter model (Tamura, 1992) and Gamma distribution used to model evolutionary rate differences among sites (+G) selected by best-fit model (Nei and Kumar, 2000). Evolutionary analyses were conducted on 8000 bootstrap replications using the MEGA6 software (Tamura et al., 2013). Homologous sequence from Adelina bambarooniae and Babesia bovis, respectively for Hepatozoon spp. and Babesia spp., were used as outgroups (accession numbers AF494058; EF601930). 2.5. Statistical analysis Exact binomial 95% confidence intervals (CI) were established for proportions. The Chi-square and Fisher’s exact tests were used to compare proportions, with a probability P-value < 0.05 regarded as statistically significant. Analyses were done using GraphPad Prism version 8.0.0 (GraphPad Software, San Diego California, USA). 3. Results Data on sex, age and location of sampled animals are reported in Table 2 along with number and percentage of positivity for Hepatozoon canis and Hepatozoon felis, the most frequently detected pathogen species. Out of 397 animals tested, 176 (44.3%; 95% CI: 39.5–49.2%) were positive for at least one pathogen with the highest prevalence detected in foxes (73.7%; 95% CI: 58–85%), followed by jackals (54.5%; 95% CI: 41.5–67%), dogs (38.1%; 95% CI: 29.1–48.1%) and cats (39.1%; 95% CI: 32.7–45.9%) (Table 3). Dogs showed the highest diversity in arthropod-transmitted pathogens with up to five pathogens diagnosed (Table 3). Co-infection with H. canis and Babesia spp. was the most prevalent (5/176, 2.8%, i.e., 4 foxes and 1 jackal), followed by H. canis and Dirofilaria immitis (1/176, 1.3%, i.e., in 1 jackal), H. canis and

4. Discussion A very high percentage (44.3%) of the carnivores tested was positive for at least one VBP, with Leishmania spp. and Ehrlichia spp. not being detected in any of the examined samples. Although no data on the distribution of ticks infesting wild and domestic animals is available in Iraq, these results suggest that these ectoparasites are well distributed throughout the examined areas and that wild carnivores play an

Table 3 Number (percentage) of animals positive to vector-borne pathogens according to animal species. Host species (n)

Jackal (55) Red fox (38) Dog (97) Cat (207) Total (397)

Total no. of animal species infected with each pathogen (%)

Overall vector-borne pathogens

Hepatozoon spp.

Babesia spp.

Anaplasma platys

Dirofilaria spp.

Acanthocheilonema reconditum

27 (49.1) (Hc) 18 (47.3) (Hc) 32 (33.0) (Hc) 81 (39.1) (Hf) 158 (39.8)

1 (1.8) (Bsp. MML) 8 (21) (2Bv, 6 Bsp. MML) – – 9 (2.3)

– – 1 (1.0) – 1 (0.2)

2 (3.6) (Di) – 2 (2.1) (1 Di, 1 Dr) – 4 (1.0)

– 2 (5.3) 2 (2.1) – 4 (1.0)

Hc: Hepatozoon canis; Hf: Hepatozoon felis; Bv: Babesia vulpes; Bsp: Babesia sp. MML; Di: Dirofilaria immitis; Dr: Dirofilaria repens. 3

30 (54.5) 28 (73.7) 37 (38.1) 81 (39.1) 176 (44.3)

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Table 4 Prevalence of sequence type, GenBank™ accession number and percentage of nucleotide identity of partial 18S rRNA gene sequences detected in 77 wild and domestic animals positive to Hepatozoon canis. Pathogen

Sequence type

Hepatozoon canis ST1 ST2 ST3 ST4

Jackals

Foxes

n. (%) 14 (82.3%) 7 (41.2%) 6 (20)

3 (17.4) 2 (11.8)

Dogs

TOT (n = 77)

Accession number and nucleotide identity percentage

8 (47) 24 (80)

17 17 30 13

KX712126 – 100% MK091088– 100% MG254618– 100% MF142765– 100%

13 (100)

(22.1) (22.1) (38.9) (16.9)

preventative measures against infection by VBPs(Alho et al., 2018). A large proportion of animals was positive to pathogens of the genus Hepatozoon, with H. canis as the only species identified in Iraqi jackals, red foxes and dogs (Baneth, 2011). The prevalence of H. canis detected in jackals (49.1%) and red foxes (47.3%) is high and similar to that recently reported in a study conducted in Israel (i.e., jackals 46%, red foxes 43%; Margalit Levi et al., 2018). Only H. felis, which is the prevalent species infecting cats (Baneth et al., 2013), was identified in cats from Iraq, but not H. canis or Hepatozoon silvestris, which have also been sporadically reported in these animals (Jittapalapong et al., 2006; Baneth et al., 2013; Díaz-Regañón et al., 2017; Hodžić et al., 2017; Giannelli et al., 2017). The H. felis infection proportion recorded in the cat population tested (81/207; 39%) was very high and rather unique considering the prevalence of 5.1% (10/196) reported in a previous epidemiological survey conducted in southern Italy (Giannelli et al., 2017). Such a high prevalence of H. felis infection could indicate a high proportion of tick infestation in cats from Iraq, though the way of infection, including the potential vector of H. felis, is currently unknown. The existence of a single ST of H. felis in Iraq is similar to what was detected in cats from Spain and Israel (Baneth et al., 2013), but differed from two studies carried out in

important role in the maintenance of these VBPs. Hepatozoon canis was the most prevalent pathogen in the animal population examined in Iraq (39.8%) followed by Babesia spp. (2.3%) (Table 3). The occurrence of both pathogens suggests that tick infestation by Rhipicephalus sanguineus sensu lato (the recognized vector for both agents – Dantas-Torres, 2008; Latrofa et al., 2014) is prevalent in domestic and wild carnivores from this country. In fact, the presence of R. sanguineus s.l. ticks on a dog from Iraq was ascertained long time ago (Machattie and Chadwick, 1930) and a more recent study confirmed its presence on both dogs and foxes in this country (Shubber et al., 2014). Hepatozoon spp. and Babesia spp. are prevalent in wild and domestic canids worldwide (Otranto et al., 2015a), but limited data is available in areas of the Middle East where cultural and social factors often impair field work on dogs and cats and, consequently, limit scientific information on pathogens they may carry. For instance, in Qatar a large diversity of VBPs was only recently reported in domestic dogs (i.e. A. platys, Babesia gibsoni, B. vogeli, E. canis, H. canis, Mycoplasma spp. and Leishmania spp.) and cats (B. felis, B. vogeli, “Candidatus Mycoplasma haemominutum”, E. canis, Mycoplasma haemofelis and Leishmania) (Alho et al., 2017; Lima et al., 2019). These results highlighted the lack of awareness of VBPs distribution in the country and the need to establish

Fig. 1. Phylogenetic relationship of Hepatozoon canis sequence types (i.e. ST1, ST2, ST3, ST4) and Hepatozoon felis detected in this study to other Hepatozoon spp. based on a partial sequence of the 18S rRNA gene. Adelina bambarooniae was used as outgroup. Sequences are presented by GenBank accession number, host species and country of origin.

4

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Fig. 2. Phylogenetic relationship of Babesia vulpes and Babesia sp. MML-2014 detected in this study to other Babesia spp. based on a partial sequence of the 18S rRNA gene. Homologous sequence from Babesia bovis was used as outgroup. Sequences are presented by GenBank accession number, host species and country of origin.

fit with those by Margalit Levi et al. (2018), which indicated that Babesia sp. MML was the most prevalent agent in red foxes (19%). In addition, the 18S rRNA nucleotide identity of Babesia sp. MML with those deposited by Margalit Levi et al. (2018), along with the peculiar phylogenetic and nucleotide differences of all other Babesia sp. suggest the probable existence of a new Babesia species circulating in wild carnivores, which deserves further investigations. Although this is a first report in Iraq, the finding of A. platys in a dog is not surprising, considering the worldwide distribution of this pathogen. In addition, although the vector competence of R. sanguineus s.l. for A. platys has never been ascertained (Dantas-Torres et al., 2013), the geographical distribution of A. platys is commonly associated to that of R. sanguineus s.l., which are vectors of the most prevalent B. vogeli and H. canis, with the latter pathogen detected in this study. Conversely, the negative results for E. canis, which is also transmitted by R. sanguineus s.l., suggest a low prevalence or even the absence of this agent in the study areas. The detection of a D. immitis-positive dog agrees with a single previous finding of infection in three out of 20 dogs examined in the Baghdad area (Tarish et al., 1986) and suggests that this pathogen is endemic in the examined region. This data is further corroborated by the detection of D. immitis in two (3.6%) jackals, which represents the first report for this host species in Iraq. A high prevalence of D. immitis in jackals has been reported from Bulgaria (9.6%; Kirkova et al., 2011), Hungary (7.4%; Tolnai et al., 2014), and Serbia (12.7%; Penezić et al., 2014), and it indicates that this species plays a role in the maintenance of this pathogen’ life cycle, although its epidemiological role needs to be better defined. Dirofilaria repens was also diagnosed in a dog for the first time in Iraq. Both D. immitis and D. repens develop into the adult stage in dogs and in some wild canids (e.g., coyotes, jackals, wolves, foxes; reviewed in Otranto et al., 2015b) producing abundant and persistent microfilaremia in the bloodstream of infected animals

Italy, in which up to four distinct 18S rRNA gene sequences were detected (Giannelli et al., 2017; Otranto et al., 2017a, 2017b). This could result from a larger number of tick species infesting cats in Italy (i.e., R. sanguineus s.l., Rhipicephalus pusillus, Rhipicephalus turanicus, and Ixodes ventalloi; Otranto et al., 2017a, 2017b) than in cats from Iraq. However, the lack of data about ticks infesting cats in Iraq prevents drawing any definitive conclusion. Conversely, the four H. canis 18S rRNA STs identified in positive jackals (n = 55), foxes (n = 38) and dogs (n = 97) suggest some degree of genetic variability within this protozoan species in Iraq. The finding of ST3 in dogs (80%) and jackals (20%), and ST2 in dogs (47%), jackals (41.2%) and, to a lesser extent, in foxes (11.8%) suggests a circulation of this STs within the population of wild and domestic canids. Finally, ST1 was detected only in jackals (82.3%) and in red foxes (17.4%) and ST4 in the latter species, which possibly indicates a circulation limited to wildlife. The high genetic variability of H. canis in the examined population has already been recorded in different hosts and geographic regions (Najm et al., 2014) as well as in tick vectors (Latrofa et al., 2014) and it could be due to the low host specificity of this protozoon, which is a well-recognized biological feature for this parasite genus (Smith, 1996). In addition, many tick species feed on both dogs and wild canids (e.g., R. sanguineus s.l. and R. turanicus, Walker et al., 2000) possibly explaining the circulation of this pathogen in sympatric populations of carnivores (Ortuño et al., 2008; Baneth, 2011; Latrofa et al., 2014; Maia et al., 2014; Giannelli et al., 2016). Babesia spp. have already been detected in a range of wild canid species, including jackals (Margalit Levi et al., 2018; Sukara et al., 2018), suggesting a potential role as natural reservoirs and indirect sources of infection for domestic dogs (Otranto et al., 2015a). In the present study B. vulpes was identified in two red foxes (5.2%) but not in jackals, whereas the prevalence of Babesia sp. MML was significantly higher in red foxes (15.8%) than in jackals (1.8%). These results nicely 5

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(McCall et al., 2008). Under the above circumstances, wild canids could play an epidemiological role mainly because they are not protected by any preventative strategies (Otranto and Deplazes, 2019). Finally, A. reconditum was reported for the first time in Iraq in both red foxes (5.3%) and dogs (2.1%), but not in jackals. These data are similar to previous investigations conducted in Romania, where A. reconditum DNA was found only in one red fox (0.33%) but in none of the jackals examined in two consecutive studies (Ionică et al., 2016, 2017). In particular, even though samples have not been recently collected, living conditions of dogs and cats in Iraq are very likely not to have changed in the recent times. Data presented fill some gaps in the knowledge of VBPs in dogs, cats and wild canids in Iraq and report, for the first time, the occurrence of i) A. platys, H. canis, A. reconditum and D. repens in dogs; ii) H. canis and D. immitis in jackals; iii) H. canis, B. vulpes and A. reconditum in red foxes; and iv) H. felis in cats in this country. In addition, results support the existence of a new Babesia sp. (i.e. Babesia sp. MML) in both red foxes and jackals, which is genetically different from all the others including the closely related B. lengau. Finally, the occurrence of the same pathogen species and STs of H. canis in dogs and wild carnivores (e.g., foxes and jackals) indicate that these VBPs circulate amongst animal populations living in the same areas and share the same tick vectors. Large-scale surveys are urgently needed to further assess VBPs distribution in Iraq and establish preventative strategies in domestic animals, in order to minimize the risk of infection for animals and humans.

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