Canine vector-borne pathogens from dogs and ticks from Tamil Nadu, India

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Canine vector-borne pathogens from dogs and ticks from Tamil Nadu, India

Ranju Ravindran Santhakumari Manoj Conceptualizationmethodologyinvestigationformal and molecular data analy Roberta Iatta Supervisionmolecular data analysiswriting-review and editing , Maria Stefania Latrofa Supervisionmolecular data analysisvalidationwriting-review and editing , Loredana Capozzi writing-review and editing , Muthusamy Raman Sample collectionwriting-review and editing , Vito Colella writing-review and editing , Domenico Otranto Conceptualizationsupervisionwriting-review and editing PII: DOI: Reference:

S0001-706X(19)31145-3 https://doi.org/10.1016/j.actatropica.2019.105308 ACTROP 105308

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

26 August 2019 16 December 2019 16 December 2019

Please cite this article as: Ranju Ravindran Santhakumari Manoj Conceptualizationmethodologyinvestigationformal Roberta Iatta Supervisionmolecular data analysiswriting-review and editing , Maria Stefania Latrofa Supervisionmo Loredana Capozzi writing-review and editing , Muthusamy Raman Sample collectionwriting-review and editing , Vito Colella writing-review and editing , Domenico Otranto Conceptualizationsupervisionwriting-review and editing Canine vector-borne pathogens from dogs and ticks from Tamil Nadu, India, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105308

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Highlights 

The presence of canine vector-borne pathogens in feral dogs and ticks from Tamil Nadu (South India) was molecularly investigated.



Of 230 dogs examined, the 99.6% were infested by ticks with the 98.3% of ticks ideatified as Rhipicephalus sanguineus sensu lato.

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Out of 230 dogs, 156 dogs (67.8%) were positive to at least one pathogen with Hepatozoon canis being the most prevalent pathogen (37.8%) detected.

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The occurrence of Brugia malayi, Anaplasma phagocytophilum and Dirofilaria sp. “honkongensis” is reported for the first time from Tamil Nadu.

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Canine vector-borne pathogens from dogs and ticks from Tamil Nadu, India

Ranju Ravindran Santhakumari Manoja, Roberta Iattaa, Maria Stefania Latrofaa, Loredana Capozzib, Muthusamy Ramanc, Vito Colellaa,d, Domenico Otrantoa,e*

a

Dipartimento di Medicina Veterinaria, Università degli Studi di Bari, 70010 Valenzano, Italy

b

Istituto Zooprofilattico della Puglia e della Basilicata, Putignano, Italy

c

Translational Research Platform for Veterinary Biologicals, 2nd Floor, Central University

Laboratory Building, TANUVAS, Madhavaram Milk Colony, Chennai - 600051, Tamil Nadu, India. d

Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, 3010

Parkville, Australia e

Department of Pathobiology, Faculty of Veterinary Science, Bu-Ali Sina University,

Hamedan, Iran

*Corresponding author: Dipartimento di Medicina Veterinaria, Università degli Studi di Bari, 70010,

Valenzano,

Bari,

Italy.

Tel

+39

[email protected] (D. Otranto).

Abstract 2

080

4679944/9839;

E-mail

address:

Canine vector-borne diseases (CVBDs) pose a major health problem in dogs globally, with the potential to cause zoonoses, in particular in developing countries where scientific knowledge on the topic is minimal. Blood samples and ticks were collected from stray dogs in Tamil Nadu, South India to assess the prevalence of CVBD-causing pathogens (Anaplasma spp., Babesia spp., Ehrlichia spp., Hepatozoon spp., filarioids and Leishmania spp.) among host and vector populations. Of the 230 dogs examined, 229 (99.6%) were infested by ticks (mean intensity, 5.65) with Rhipicephalus sanguineus sensu lato and Rhipicephalus haemaphysaloides being morphologically identified in the 98.3% and 1.7% of the infested dogs, respectively. Overall, the 67.8% (n=156) of dogs was positive for at least one pathogen with Hepatozoon canis being the most prevalent (37.8%) followed by Anaplasma platys (22.6%), Ehrlichia canis (16.1%) Babesia vogeli (10%), Anaplasma phagocytophilum (0.4%) and Babesia gibsoni (0.4%). Two filarioids (1 each of Dirofilaria sp. “honghongensis” and Brugia malayi, 0.4%) were diagnosed among sampled animals. Coinfection with H. canis and A. platys was the most prevalent (8.3%, P = 0.00001), whilst all animals scored negative for Leishmania spp. Out of 295 ticks analysed, 215 R. sanguineus s.l. (75.4%) and 8 R. haemaphysaloides (88.9%) were positive for at least one pathogen with H. canis as the predominant pathogen (42.5%), followed by A. platys (33.8%), E. canis (16.9%), B. vogeli (3.8%) and A. phagocytophilum (0.3%). Fifty-six dogs (35.9%) harboured the same pathogen as the respective tick specimens, while 29 dogs (18.6%) had a different pathogen. Thirteen sequence types (STs) were identified for H. canis, with ST2 (49.4%) as the most representative sequence in dogs and ST1 (73.5%) in ticks. Similarly, seven STs were found for Anaplasma spp. (i.e., five for A. platys, one for A. phagocytophilum and one for Anaplasma sp.), with ST2 as the most representative sequence type in dogs (70.6%) and ST3 (52.5%) in ticks for A. platys. Only one ST was identified for B. vogeli, B. gibsoni, E. canis, D. sp. “hongkongensis” and B. malayi. Regular surveillance and adoption of adequate

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treatment and control measures are needed to reduce the risk of disease-causing pathogens in stray dogs and of pathogens with zoonotic potential. Keywords: Canine vector borne diseases, dogs, ticks, Rhipicephalus sanguineus s.l., Anaplasma platys, Anaplasma phagocytophilum, Babesia vogeli, Babesia gibsoni, Hepatozoon canis, Ehrlichia canis, India, Leishmania infantum

1. Introduction Canine vector borne diseases (CVBDs) are a group of diseases transmitted by several arthropod vectors, including fleas, biting and secretophagous flies, mosquitoes, sand flies and, especially, ticks (Otranto et al., 2009). In the past two decades the distribution of CVBDs has been investigated mainly in industrialized countries of the northern hemisphere, whereas data about the occurrence and the impact of these infections in developing countries is minimal (Maggi and Kramer, 2019). India has a population of about 118 million dogs, though no reliable information is available on the exact number of stray dogs in this country (Wallace et al., 2017). Scientific data indicate that the gross domestic product per capita is, at global level, inversely proportional to the percentage of stray dogs making the burden of zoonotic parasites linked to poverty (Otranto et al., 2017). For long time, capture-neutervaccinate-release (CVNR) programs were endorsed by the WHO in India (Jackaman and Rowan, 2007). Nonetheless, during economic crisis these programs often do not reach a large proportion of stray dogs (Otranto et al., 2017), making these animals suitable reservoirs of several parasites of zoonotic concern (Traub et al., 2014; Sudan et al., 2015). At these conditions, untreated animals play an important role in the maintenance of certain tick population such as the brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.), representing a risk for the transmission of zoonotic tick-borne pathogens (Otranto et al., 4

2010; Dantas-Torres et al., 2012). Indeed, R. sanguineus s.l. is the most common tick species parasitizing dogs of India (Raut et al., 2006; Abd Rani et al., 2011; Sahu et al., 2013) followed by Rhipicephalus haemaphysaloides, Rhipicephalus microplus, Haemaphysalis longicornis and Haemaphysalis bispinosa (Sahu et al., 2013; Augustine et al., 2017). All of these tick species act as vectors for pathogens causing babesiosis, ehrlichiosis, hepatozoonosis and anaplasmosis (Abd Rani et al., 2011; Augustine et al., 2017; Geurden et al., 2018). In particular, babesioses by Babesia vogeli and Babesia gibsoni are evenly distributed in India due to the presences of their vectors R. sanguineus s.l. and H. longicornis, respectively (Abd Rani et al., 2011). Among Ehrlichia spp., Ehrlichia canis is the most common in canine populations of tropics and sub tropics (Shaw et al., 2001). Anaplasmosis is a prevalent disease affecting both humans and animals (Demma et al., 2005; Nicholson et al., 2010), with Anaplasma phagocytophilum and Anaplasma platys being potentially zoonotic (Maggi et al., 2013; Arraga-Alvarado et al., 2014; Vlahakis et al., 2018). Rhipicephalus sanguineus s.l. is the putative vector of A. platys (Inokuma et al., 2000), a pathogen which may infect humans (Maggi et al., 2013; Arraga-Alvarado et al., 2014), whilst Ixodes spp. act as vector of A. phagocytophilum (Parola et al., 2005). Although, Ixodes spp. have been reported from the temperate zones of Europe, Asia and North America, (Parola et al., 2005), few studies described the presences of different Ixodes species (i.e., Ixodes acutitarsus and Ixodes ricinus) in different parts of India (Kaul et al., 1990; Ronghang et al., 2016). Canine hepatozoonosis is a tick-borne disease prevalent in various regions of India (Chhabra et al., 2013) with prevalence, at blood smear examination, of 11.23% for Hepatozoon canis in Tamil Nadu (Vairamuthu et al., 2014). Apart from tick borne diseases, canine dirofilarioses are prevalent in India (Abd Rani et al., 2010) and an increasing number of Dirofilaria immitis and Dirofilaria repens infection have also been reported in human patients (Sabu et al., 2005). Leishmania donovani and Leishmania infantum cause visceral leishmaniasis (VL) in

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humans worldwide. Whilst L. donovani is anthroponotic, L. infantum is the main etiologic agent of zoonotic canine leishmaniosis (Costa, 2011; Ribeiro et al., 2018: Colella et al., 2019). In India, it is still not clear the role of dogs in the occurrence of cases of human leishmaniasis. Global occurrence of ticks and their ability to cause diseases in dogs and transmit zoonotic pathogens warrant the regular screening of dogs for tick infestation. As dogs live in close proximity to humans, tick collection is advocated in order to screen both dogs and ticks for the presence of zoonotic tick-borne pathogens (Shaw et al., 2001; Baneth, 2004; Otranto et al., 2014). Due to the limited number of international literature available on CVBDs in feral/stray dogs from India (Irwin et al., 2004; Abd Rani et al., 2010), this study aimed to assess the occurrence of ticks, pathogens they may carry and CVBD-causing agents in dogs from Tamil Nadu, India.

2. Materials and methods 2.1. Sampling procedures From October to December 2018, a total of 230 free-ranging dogs were visited as a part of CVNR programme (Fig. 1) and none of dogs exhibited apparent clinical signs. Dogs were located in four different areas of Chennai, Tamil Nadu (i.e., Pulianthope and Vepery in North Chennai - 13° 8' 39.9948'' N and 79° 53' 38.4180'' E ; Velachery and Adyar in South Chennai - 12° 50' 3.0264'' N and 79° 42' 13.1184'' E) and their categorical age, sex and breed data were recorded. Each dog was checked for ticks and those found within 10 minutes were collected and placed in labelled tubes and individualized per dog. Ticks were identified at species level based on morphological keys (Walker et al., 2005; Keirans, 2009). Ticks (~20%) were randomly sampled from each tick species and from each infested animal, giving 6

preference to the engorged tick stage (i.e. unengorged ticks were selected if engorged ones were not available). About 2 ml of blood was collected from the saphenous vein of the dogs, transferred to K3 EDTA coated tubes and stored at -80°C until further processing. 2.2. Ethics statement Sampling of dogs was performed following guidelines provided by the committee for the purpose of Control and Supervision of Experiments on Animal (CPCSEA), Department of Animal Husbandry and Dairying, Ministry of Agriculture and Farmers Welfare, Government of India (Prevention of Cruelty to Animals Act, 1960).

2.3. DNA isolation from ticks and dog blood, molecular analysis by PCR and sequencing Genomic DNA was isolated from individual ticks of each species obtained from individual dogs using QIAamp DNA mini QIAcube kit (QIAGEN, Hilden, Germany) and from whole blood using GenUP DNA Kit (Biotechrabbit, Germany), following the manufacturer’s instructions. All blood samples were tested for the presence of Babesia spp., Hepatozoon spp., Ehrlichia spp., Anaplasma spp., Leishmania spp. and filarioids, whereas ticks were processed for all mentioned blood parasites, except for filarioids and Leishmania spp. Molecular detection of Babesia spp., Hepatozoon spp., Ehrlichia spp., Anaplasma spp. and filarioids was performed by conventional PCR (cPCR) using primers targeting partial 18S rRNA, 16S rRNA, and cytochrome c oxidase subunit 1 (cox1) and 12SrRNA, respectively (Table 1). Single PCR reaction was used for the simultaneous detection of Babesia spp./Hepatozoon spp. and Ehrlichia spp./Anaplasma spp. Individual species-specific PCRs were performed (Olmeda et al., 1997; Inokuma et al., 2002) in the positive samples to assess the co-infections with more than one parasite species. DNA from engorged tick specimens

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were diluted 1:5 to avoid any inhibition of the amplification process. Amplified PCR products were visualized by gel-electrophoresis in 2% agarose gel containing Gel Red® nucleic acid gel stain (VWR International PBI, Milan, Italy) and the same was documented in Gel Logic 100 gel documentation system (Kodak, New York, USA). The PCR products were purified and sequenced in both directions using the same forward and reverse primers, employing the Big Dye Terminator v.3.1 chemistry in a 3130 Genetic analyzer (Applied Biosystems, California, USA) in an automated sequencer (ABI-PRISM 377). Nucleotide sequences were edited, aligned and analysed using Geneious platform version 9.0 (Biomatters Ltd., Auckland, New Zealand) (Kearse et al., 2012) and compared with available sequences in the GenBank using Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi). The detection of Leishmania spp. DNA was investigated using a real-time PCR (qPCR) assay by the amplification of a short fragment (120 bp) of the kinetoplastid DNA (kDNA) as described elsewhere (Francino et al., 2006). For all PCR runs, DNA of pathogen-positive and negative blood samples served as control. 2.4. Phylogenetic analysis Representative sequences-types of the 18S rRNA of Hepatozoon spp. and Babesia spp., 16S rRNA of Ehrlichia spp. and Anaplasma spp., and of cox1 and 12S rRNA of Brugia spp. were included along with sequences available in the GenBank database for phylogenetic analyses. Phylogenetic relationships of Hepatozoon spp., Babesia spp., Ehrlichia spp., Anaplasma spp. and of Brugia spp. were inferred using the Maximum Likelihood (ML) method based on the Tamura 3-parameter (Tamura, 1992) with Gamma distribution (+G) of evolutionary rate differences among sites and on Hasegawa-Kishino-Yano models, respectively, selected by best-fit model (Nei and Kumar, 2000). Evolutionary analysis was conducted on 8000 8

bootstrap replications using the MEGA6 software (Tamura et al., 2013). Homologous sequences from Adelina bambrooniae, Babesia bovis, Wolbachia pipientis, Wuchereria bancrofti respectively for Hepatozoon spp., Babesia spp., Ehrlichia spp./Anaplasma spp. and for Brugia spp. were used as outgroups (Accession numbers AF494058; EF601930; AF179630; AJ544844; AJ271612). 2.5. Statistical analysis Prevalence (proportion of hosts infested by ticks and pathogens on the total population and of tick stages positive for a given pathogen) and tick infestation intensity (arithmetic mean number of ticks on the infested dogs) were assessed. Statistical analysis was done using StatLib software. Exact binomial 95% confidence intervals (CI) were established for proportions. The Chi-square test with a probability of P-value <0.05 was considered statistically significant.

3. Results Of the 230 dogs examined, 229 (99.6%; 95% CI: 97.51-99.9) were infected by ticks with a mean intensity of 5.6 (95% CI: 5.2-6.1). From a total of 1294 ticks collected, 1230 (95%) were adults (i.e., 644 males and 586 females), 62 (4.8%) nymphs and 2 larvae. Ticks were morphologically identified as R. sanguineus s.l. (98.3%) and R. haemaphysaloides (1.7%) (Fig. 2), being six animals (2.6%) coinfested with both species. Data on sex, age and breed of sampled animals are reported in Table 2 along with number and percentage of positivity for CVBD-causing pathogens. Out of 230 dogs tested, 156 dogs (67.8%; 95% CI: 61.5-73.7) were positive for at least one pathogen with H. canis being the most prevalent species (37.8%; 95% CI: 31.7-44.3). Two filarioids were detected (i.e., 9

Dirofilaria sp. “hongkongensis” and B. malayi, 0.4%; 95% CI: 0.03-0.02). Co-infection with H. canis and A. platys was the most prevalent and statistically significant association (8.3%, n = 19, P = 0.00001), followed by H. canis and E. canis (7.4%, n = 17), B. vogeli and A. platys (1.7%, n = 4), B. vogeli and E. canis (0.9%, n = 2), H. canis and D. sp. “hongkongensis”, B. vogeli and Anaplasma sp., B. gibsoni and A. platys, A. platys and B. malayi, B. vogeli, H. canis and E. canis (0.4% each, n = 1) (Table 2). All samples tested were negative for Leishmania spp. No Statistically significant association was found between the positivity of pathogens and sex (P = 0.17), age (P = 0.21) and breed (P = 0.22). Out of 295 molecularly tested ticks, 215 R. sanguineus s.l. (75.4%; 95% CI: 70.0-80.2) and 8 R. haemaphysaloides (88.9%; 95% CI: 55.6-99.4) were infected with at least one pathogen. Rhipicephalus sanguineus s.l. ticks harboured H. canis, B. vogeli, E. canis, A. platys, A. phagocytophilum and Anaplasma sp., whereas R. haemaphysaloides was positive for all of those pathogens except for A. platys and A. phagocytophilum. Hepatozoon canis was the predominant pathogen detected in ticks (42.5%, 95% CI: 34.9-50.3) (Table 3). No significant statistical association was observed among different life stages of ticks for haemoprotozoans (P = 0.18). Fifty-six dogs (35.9%; 95% CI: 28.4-43.8) harboured the same pathogen as that detected in the vector, whilst 29 (18.6%; 95% CI: 13.0-25.5) had a different pathogen. Sixty-six dogs (42.3%; 95% CI: 34.5-50.3) were positive for at least one pathogen and had negative ticks, whereas 40 dogs scored negative for pathogens and had ticks positive for pathogens (25.6%; 95% CI: 19.1-33.2). Representative sequence types (STs) for each pathogen displayed 97-100% nucleotide identity with those available in GenBank database (Table 4). Out of 110 Anaplasma spp. sequences, four herein referred as Anaplasma sp., displayed nucleotide identity (99.2%) with

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both A. platys and A. phagocytophilum, whereas two with A. phagocytophilum (i.e., 99.6% of identity, KF569909) and 98 with A. platys (99.6% of identity, MK814421). Among them, seven representative ST were found for Anaplasma spp. (i.e., ST2- ST6 for A. platys, ST1 for A. phagocytophilum and ST7 for Anaplasma sp.) (Table 4), with the most representative sequence ST2 (70.6%) in dogs and ST3 (52.5%) in ticks for A. platys (Table 4). Thirteen STs were identified for H. canis, with ST2 (49.4%) as the most representative sequence in dogs and ST1 (73.5%) in ticks. Only one ST was identified for B. vogeli, B. gibsoni, E. canis, A. phagoyctophilum, D. sp. “hongkongensis” and B. malayi (Table 4). The molecular identification of representative STs for H. canis, Babesia spp., E. canis, Anaplasma spp. and B. malayi was supported by the distinct separation of species-specific clades inferred from the phylogenetic analyses (Fig. 3-6). In particular, the ML tree of H. canis grouped all representative STs in a large clade supported by good bootstrap value (i.e., 88%), to the exclusion of other species of Hepatozoon (Fig. 3). STs of Babesia spp. clustered as two distinct paraphyletic clades including B. vogeli and B. gibsoni, respectively, to the exclusion of a large clade of other Babesia spp. (Fig. 4). The ML tree of Anaplasma spp. and E. canis included the representative STs in three robust clades (Fig. 5). In particular, the ST of E. canis clustered in the clade including those of same species from different geographic areas. Similarly, the ST1 of A. phagocytophilum clustered in the clade including sequences of the same species, whilst the ST7 of Anaplasma sp. formed a monophyletic clade with that of A. phagocytophilum, with the exclusion of other STs (i.e. 2-6) which were included in the large clade of A. platys (Fig. 5). The cox1 and 12S rRNA ML trees of Brugia spp. grouped the STs of B. malayi, for both genes respectively, in a large clade supported by a high bootstrap value (i.e., up to 80%), to the exclusion of other species of Brugia (Fig. 6A, B). Representative STs here generated have been deposited in GenBank under accession numbers from MN648675-MN648681 for Anaplasma spp., MN556943 for B. gibsoni, MN700646 for 11

B. vogeli, MN551053 and MN564741 for B. malayi, MN564742 for D. sp. “hongkongensis”, MN630202 for E. canis and MN628317-MN628329 for H. canis. 4. Discussion Data from the current study suggest that ticks and tick-borne pathogens are highly prevalent among stray dogs in South India, therefore posing a major risk to humans. The hot and humid tropical climate conditions of South India, especially in Tamil Nadu, represent an ideal environment for the survival and propagation of ticks and tick-borne pathogens (Patra et al., 2018). Indeed, this geographical area is characterized by a moderate winter, with an average temperature of 26-29°C and relative humidity of 76-79%, which allowed to record two peaks of tick abundance from January to March and from June to November (Vathsala et al., 2008). Rhipicephalus sanguineus s.l. was the most prevalent tick in the examined dog population (i.e., 99.1%), being the main species in tropical and sub-tropical regions of India and South Asia (Baneth et al., 2001; Raut et al., 2006; Abd Rani et al., 2011; Sahu et al., 2013; Zhang, 2017). This is mostly due to the strict affiliation of this tick species to canids and to its ability in surviving a large array of environmental conditions (Dantas-Torres et al., 2013). High vector prevalence among dogs lead to a high circulation of pathogens among them (67.8%) much higher than that previously reported in South India (i.e., from 10.5% to 16.3%; Senthil kumar et al., 2009; Selvaraj et al., 2010; Vairamuthu et al., 2014), though the comparison among results is difficult due to the different screening techniques (i.e., conventional microscopy vs PCR) used, category of dog involved (i.e., owned vs street dogs) and the size of canine population sampled. Even though strain-related variation in the vector competence of R. sanguineus s.l. ticks cannot be ruled out (Latrofa et al., 2014), this tick species has been demonstrated or is suspected to act as vector for several pathogens including rickettsial (i.e., E. canis, A. platys)

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and piroplasmids (i.e. B. vogeli), which may be transmitted simultaneously within a single blood meal (Dantas-Torres and Otranto, 2015; Galay et al., 2018). This might explain the high prevalence (i.e., 27%) of co-infected animals with more than one pathogen. Hepatozoon canis was the most frequent pathogen (37.8%), with a much higher prevalence than previously reported by microscopic examination, in Tamil Nadu (11.23%; Vairamuthu et al., 2014) and from other parts of India (from 3 to 9%; Chhabra et al., 2013; Sahu et al., 2013). This difference in prevalence of infection for H. canis could be explained by several factors, including the low sensitivity of the microscopic examination, the type of dogs screened, the sample size and the time of screening of infection, which may increase the possibility of false negative results when comparing with PCR testing (Otranto et al., 2011). The prevalence of Babesia spp. from this study (i.e., 10.4%) was lower than that in Tamil Nadu by cPCR (31%, Abd Rani et al., 2011; Azhahianambi et al., 2018), Kerala (57.5%, Augustine et al., 2017), South India (28.6%, Singh et al., 2016), North India (15.45%, Mittal et al., 2019). These difference in the prevalence of the infection could be due to the disparity in number of dogs tested (Augustine et al., 2017, n = 80; Abd Rani et al., 2011, n = 525), selection of clinically suspected dogs (Azhahianambi et al., 2018, Augustine et al., 2017) and geographical area of sample collection from North India (Abd Rani et al., 2011, Singh et al., 2016)..The prevalence of rickettsial pathogens, A. platys (19.6%) and E. canis (15.2%) among the sampled population is similar to that previously recorded from Tamil Nadu (A. platys: 16.2%, Bhoopathy et al., 2017; E. canis: 22.6%, Azhahianambi et al., 2018). Anaplasma phagocytophilum was detected from one dog. Even though there are reports of serological prevalence (6.09%) of A. phagocytophilum among the pet dog population of North East India (Borthakur et al., 2014), to our knowledge this is the first report of this pathogen among dogs from South India. Though, Ixodes spp. (i.e., I. ricinus in Europe; Ixodes scapularis in Eastern US; Ixodes pacificus in Western US; Ixodes persulcatus in Asia) are considered the main

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vector of A. phagocytophilum (Stuen et al., 2013), this pathogen has also been detected molecularly in R. sanguineus s.l from Egypt (Ghafar et al., 2012), Brazil (Santos et al., 2013), and France (Chastagner et al., 2013). The detection of A. phagocytophilum DNA both in dogs and R. sanguineus s.l. indicates the circulation of this pathogen in the sampling area. Though D. repens is the commonest zoonotic Dirofilaria spp. in southern part of India, a closely related D. sp. “hongkongensis” from humans, jackals and dogs (Pradeep et al., 2019) have also been reported, therefore supporting our findings and confirms the existence of this filarioid species in South India. To our knowledge, this is the first report from the state of Tamil Nadu. Human lymphatic filariasis is endemic in India with 90% of the cases caused by Wuchereria bancrofti while most of the remaining cases by B. malayi (Khan, 2018). National filarial programme for the elimination of lymphatic filariasis was launched in 1955, following which a global programme to eliminate lymphatic filariasis (GPELF) in 1999 to facilitate the national programme (Sabesan et al., 2010). Despite the admirable progress made by the Indian governamental policy towards the elimination of lymphatic filariasis, the target of 2015 has not been met and the programme has been extended up to 2020 (Khan, 2018). No data of infection of B. malayi have been previously recorded in a dog population of Tamil Nadu, though in a neighbour state, Kerala, the existence of B. malayi in dogs has been reported (Chirayath et al., 2017). The lower prevalence of dog positive for B. malayi may indicate a low circulation of this pathogen in Tamil Nadu, which is also reflected by the absence of human cases of brugian filariasis in this area. However, survey on a large canine population needs to be carried out in this part of India, since the pathogen is still present in the country despite the rigorous efforts to aim at the 2020 elimination (Sabesan et al., 2010). Pathogens detected from R. sanguineus s.l. and R. haemaphysaloides ticks, which were also collected from dogs PCR negative for pathogens, include H. canis, B. vogeli, E. canis and Anaplasma sp., whereas A. platys and A. phagocytophilum was only detected in the first

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species. Indeed, R. sanguineus s.l. is a well-known vector of several canine pathogens such as H. canis, B. vogeli and E. canis but its vector competence for A. platys and A. phagocytophilum has not been proven (Dugat et al., 2017; Dantas-Torres et al., 2013). Results herein presented suggest that R. sanguineus s.l. may be involved in the transmission cycle of many pathogens (potentially including A. phagocytophilum) though further investigations such as, transmission experiments, should be carried out to prove the vector competence of these tick species. B. gibsoni is transmitted by H. longicornis in Asia (Jain Jose et al., 2018). Though this tick was not detected in the current study, one dog was positive for B. gibsoni. This could indicate the participation of Rhipicephalus spp. ticks in the transmission of this pathogen species though it should also be considered the direct especially in fighting dogs (Birkenheuer et al., 2005). Molecular analysis revealed that 25.6% of ticks were positive for the same pathogen as that of the host and 18.6% for a different pathogen. Pathogen transmission by ticks depends upon a variety of factors, such as the duration of feeding time, pathogen load, extent of tick tissue involved and infection at the time of blood feeding (Otranto, 2017). In 42.3% of cases, the host was positive for pathogen, but the ticks collected on the same individual were negative. This could be due to the fact that ticks may had recently attached and therefore they had not acquired the pathogen from the host (Richards et al., 2017). Sequence analysis revealed the circulation of a variety of STs for H. canis (n =13) and A. platys (n = 5) among dog and tick populations while, pathogens like B. gibsoni. B.vogeli, E. canis, A. phagocytophilum, Anaplasma sp., D. sp. “hongkongensis”and B. malayi had only one ST. Previous reports identified three different strains of A. platys in dogs (de La Fuente et al., 2006). This suggests high genetic variability within H. canis and A. platys, which correlates with the records in different hosts and geographic regions (Vojta et al., 2009; Najm et al., 2014) and R. sanguineus tick (Latrofa et al., 2014). The high genetic variability of H.

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canis, may be attributed to its low host specificity, which is a well-recognised biological feature of this protozoon (Smith, 1996). Furthermore, the high genetic diversity and different ecotypes of A. phagocytophilum reported, and the broad host-spectrum (i.e., ruminants, rodents, insectivores, carnivores, birds and reptiles) (Jahfari et al., 2014; Stigum et al., 2019), may explain the high number of STs herein detected. Indeed, the ubiquitous distribution of these parasites and their vectors possibly explain the circulation of these parasites among different carnivores, thereby contributing for its high genetic variability (Ortuno et al., 2008; Baneth, 2001; Latrofa et al., 2014; Maia et al., 2014; Giannelli et al., 2016; de La Fuente et al., 2006; Otranto et al., 2019). Further molecular analyses on more than one target genes are needed in order to confirm this genetic variability of the Anaplasma spp. herein observed.

5. Conclusions Despite the world-wide distribution of H. canis and A. platys, limited information is available on the genetic diversity of these parasites. Similarly, the vectorial role of R. sanguineus s.l. for A. platys and A. phagocytophilum has not been thoroughly explored. These data fill gaps in the knowledge of canine vector-borne pathogens among stray dogs in South India including the first report of B. malayi, A. phagocytophilum and D. sp. “hongkongensis” from the state of Tamil Nadu. High prevalence of CVBD-causing pathogens advocate for regular surveillance and adoption of adequate treatment and control measures to reduce the risk of disease-causing pathogens in stray dogs.

Conflict of interest The authors declare no competing interest.

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Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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detection and characterization of zoonotic Anaplasma species in domestic dogs in Lusaka, Zambia. Ticks and Tick Borne Dis. 9(1), 39-43. doi: 10.1016/j.ttbdis.2017.10.010. Vojta, L., Mrljak, V., Ćurković, S., Živičnjak, T., Marinculić, A., Beck, R., 2009 Molecular epizootiology of canine hepatozoonosis in Croatia. International journal for parasitology 39(10), 1129-1136. doi.org/10.1016/j.ijpara.2009.02.007. Walker, J.B., Keirans, J.E., Horak, I.G., 2005. The genus Rhipicephalus (Acari Ixodidae): a guide to the brown ticks of the world. Cambridge University Press. Cambridge. Wallace, R.M., Undurraga, E.A., Blanton, J.D., Cleaton, J., Frank, R., 2017. Elimination of dog mediated human rabies deaths by 2030: Needs assessment and alternatives for progress based on dog vaccination. Front. Vet. Sci. 4, 9. doi: 10.3389/fvets.2017.00009. eCollection 2017. Zhang, J., Liu, Q., Wang, D., Li, W., Beugnet, F., Zhou, J., 2017. Epidemiolodical survey of tick and tick borne pathogens in peg dogs in South eastern China. Parasite 24(35). doi: 10.1051/parasite/2017036.

29

Table 1. Primers and target genes of pathogens investigated Parasite

Primer

Target gene

Product

Cyclic condition

References

size(bp) Babesia spp/

RLBF: 5′-GAGGTAGTGACAAGAAATAACAATA-

Hepatozoon

3′

spp.

Babesia spp.

18S rRNA

460

95°C-600 sec, 95°C-30 sec, 52°C-30 sec (x40),

Gubbels et al. (1999)

72°C-60 sec, 72°C- 420 sec

RLBR: 5′-biotin TCTTCGATCCCCTAACTTTC-3′

Piro-A: 5′-AATACCCAATCCTGACACAGGG-3′

18S rRNA

410

PiroB: 5′-TTAAATACGAATGCCCCCAAC-3′

Hepatozoon

HepF: 5′-ATACATGAGCAAAATCTCAAC-3′

canis

HepR: 5′-CTTATTATTCCATGCTGCAG-3′

Ehrlichia spp./

EHR16SD: 5′-GGTACCYACAGAAGAAGTCC-

Anaplasma spp.

3′EHR16SR: 5′-TAGCACTCATCGTTTACA GC-3

95°C-600 sec, 95°C-30 sec, 62°C-30 sec (x35),

Olmeda et al. (1997)

72°C-30 sec, 72°C- 420 sec

18S rRNA

660

95°C-600 sec, 95°C-30 sec, 60°C-30 sec (x35),

Inokuma et al. (2002)

72°C-60 sec, 72°C- 300 sec

16S rRNA

345

95°C-120 sec, 94°C-60 sec, 54°C-30 sec (x40), 72°C-30 sec, 72°C- 300 sec

30

Martin et al. (2005)

Leishmania

LEISH1: 5′-AACTTTTCTGGTCCTCCGGGTAG-3′

spp.

LEISH2: 5′- ACCCCCAGTTTCCCGCC-3′

KDNA

120

50°C-2 min, 95°C-10 min, 95°C-15 sec (x40), 60°C-

Francino et al. (2006)

1 min

TaqMan MGB probe 5′-AAAAATGGGTGCAGAAAT-3′

Blood microfilariae

NTF: 5′-TGATTGGTGGTTTTGGTAA-3′

cox1

648

95°C-600 sec, 95°C-60 sec, 50°C-60 sec (x40),

Otranto et al. (2011)

72°C-60 sec, 72°C-420 sec

NTR: 5′-ATAAGTACGAGTATCAATATC-3′ 12sf: 5′-GTTCCAGAATAATCGGCTA-3′ 12sR: 5′-ATTGACGGATG(AG)RTTTGTACC-3′

95°C-600 sec, 95°C-60 sec, 50°C-60 sec (x40), 12s rRNA

450

72°C-60 sec, 72°C-420 sec Casiraghi et al., (2004)

31

Table 2. Prevalence of infection among sampled dogs with CVBD pathogens according to sex, age and breed Category

Total no. of

Babesia spp.

dogs positive

Hepatozoon

Ehrlichia

canis

canis

Anaplasma spp.

for CVBD Sex

No. (%)

Pos/Total (%) Pos/Total (%) Pos/Total (%) Pos/Total (%)

Male

85 (75.2)

14/113 (12.4)

44/113 (38.9)

18/113 (15.9)

29/113 (25.7)

Female

71 (60.7)

10/117 (8.5)

43/117 (36.8)

19/117 (16.2)

23/117 (19.7)

≤1 Year

7 (87.5)

1/8 (12.5)

3/8 (37.5)

1-5 Years

107 (65.2)

19/164 (11.6)

6-15 Years

42 (72.4)

4/58 (6.9)

30/58 (51.7)

13/58 (22.4)

8/58 (13.8)

Pure

3 (30)

1/10 (10)

1/10 (10)

0

0

Cross

7 (50)

1/14 (7.1)

3/14(21.4)

5/14 (35.7)

0

Non

146 (70.8)

22/206 (10.7)

83/206 (40.3)

32/206 (15.5)

53/206 (25.7)

Age 3/8 (37.5)

54/164 (32.9) 21/164 (12.8)

1/8 (12.5) 34/164 (20.7)

Breed

determined

32

Table 3. Screening of CVBD pathogens in different life stages of tick vector R. sanguineus s.l.

Stage of Tick

No. of tick

No. of ticks

No. of ticks positive for pathogen

screened by

positive for

PCR

CVBD

phagocytophilum

pathogen

and Anaplasma sp.

H. canis

B.vogeli

E. canis

A. platys

A.

Engorged female

77

32 (41.6)

14 (8.75)

2 (1.3)

3 (1.9)

5 (3.1)

-

Semi engorged female

109

92 (84.4)

23 (14.4)

2 (1.3)

9 (5.6)

31 (19.4)

3 (1.9)

Un engorged female

34

32 (94.1)

8 (5)

1 (0.6)

5 (3.1)

4 (2.5)

-

Engorged male

69

62 (89.9)

23 (4.4)

1 (0.6)

10 (6.3)

13 (8.1)

2 (1.3)

Engorged nymph

5

5 (100)

-

-

-

1 (0.6)

-

Total

294

223 (75.85)

68 (42.5)

6 (3.8)

27 (16.9)

54 (33.75)

5 (3.1)

*Figures in parenthesis indicate percentage

33

Table 4. Prevalence of sequence type, GenBank accession number and percentage of nucleotide identity of partial sequences of the 18S rRNA,16S rRNA, cox1 and 12S rRNA genes detected in dogs and ticks positive to Hepatozoon spp./Babesia spp. and Ehrlichia spp./Anaplasma spp. and for filaroids.

Pathogen (Total)

H. canis (n = 152)

Sequence

Dogs

Ticks

Total

Accession number and nucleotide

type

n (%)

n (%)

n (%)

identity percentage

STI

20 (28.6)

50 (71.4)

70 (50.7)

MK645966 – 100%

ST2

43 (97.7)

1 (2.3)

44 (28.9)

MH615006 – 100%

ST3

13 (65)

7 (35)

20 (13.2)

MK091088 – 100%

ST4

1 (20)

4 (80)

5 (3.3)

MK091088 – 99.79%

ST5

1 (100)

1 (0.7)

MK091088 – 99.57%

ST6

1 (100)

1 (0.7)

KX712126 - 99.79%

ST7

1 (100

1 (0.7)

MK091088 – 99.79%

ST8

4 (100)

4 (2.6)

KX712126 - 100%

ST9

1 (100)

1 (0.7)

KX712126 - 99.57%

ST10

2 (100)

2 (1.3)

MK645965 – 100%

34

ST11

1 (100)

1 (0.7)

MK091088 – 99.57%

ST12

1 (100)

1 (0.7)

MK091088 – 99.57%

ST13

1 (100)

1 (0.7)

LC331054 – 100%

B.gibsoni (n = 1)

ST1

1 (100)

1 (3.4)

MG604346 – 100%

B. vogeli (n = 28)

ST2

23 (82.1)

5 (17.9)

28 (96.56)

MH100721 – 100%

E. canis (n = 64)

ST1

37(57.8)

27 (42.2)

64 (58.2)

MK138377 – 100%

A. phagocytophilum (n= 1)

ST1

1(50)

1(50)

2 (1.8)

AY776165 – 99.62%

A. platys (n = 105)

ST2

36 (100)

36 (32.7)

MK814421 – 99.62%

31 (100)

31 (28.2)

MK814421 – 99.62%

ST3 ST4

2 (22.2)

7 (77.8)

9 (8.2)

MK814421 – 99.62%

ST5

5 (22.7)

17 (77.3)

22 (20)

MK814421 – 99.22%

ST6

5 (83.3)

1 (16.7)

6 (5.5)

MK814421 – 99.62%

4 (100)

4 (3.6)

MK814421; KF569909 – 99.62%

Anaplasma sp. ( n= 4)

ST7

D. sp. “hongkongensis” (n = 1)

ST1

1 (100)

1 (100)

KX265050 – 100%

B. malayi (n = 1)

ST1

1 (100)

1 (100)

AF538716 – 97.43%

35

KP760317 – 98.67%

36

Figure legend

Fig. 1. Map of India highlighting the collection area.

Fig. 2. Distinctive characteristics of Rhipicephalus sanguineus and Rhipicephalus haemaphysaloides. R. haemaphysaloides: Sickle shaped adanal plate (a), comma shaped

37

spiracle broad throughout its length (b), narrowly U-shaped genital aperture (c); R. sanguineus: subtriangular adanal plate distinctly broad in posterior aspect (a), spiracle elongated throughout the length with narrow dorsal prolongation (b), broadly U-shaped genital aperture (c).

38

Fig. 3. Phylogenetic relationship of Hepatozoon canis sequence types (i.e. ST1 – ST13) detected in this study to other Hepatozoon spp. based on a partial sequence of the 18S rRNA gene. Evolutionary analysis was conducted on 8000 bootstrap replications using Maximum Likelihood method. Adelina bambarooniae was used as outgroup. Sequences are presented by GenBank accession number, host species and country of origin.

39

Fig. 4. Phylogenetic relationship of Babesia spp. sequence types (Babesia gibsoni - ST1 and Babesia vogeli – ST2) detected in this study to other Babesia spp. based on a partial sequence 40

of the 18S rRNA gene. Evolutionary analysis was conducted on 8000 bootstrap replications using Maximum Likelihood method. Babesia bovis was used as outgroup. Sequences are presented by GenBank accession number, host species and country of origin.

41

Fig. 5. Phylogenetic relationship of Anaplasma spp. sequence types (Anaplasma platys and Anaplasma phagocytophilum ST1 – ST7) and Ehrlichia spp. sequence detected in this study 42

to other Anaplasma spp. and Ehrlichia spp. based on a partial sequence of the 16SrRNA gene. Evolutionary analysis was conducted on 8000 bootstrap replications using Maximum Likelihood method. Wolbachia pipientis was used as outgroup. Sequences are presented by GenBank accession number, host species and country of origin.

Fig. 6. Phylogenetic relationship of Brugia malayi sequence types of cox1 (A) and 12SrRNA (B) detected in this study to other Brugia spp. Evolutionary analysis was conducted on 8000 bootstrap replications using Maximum Likelihood method. Wuchereria bancrofti were used as outgroups. Sequences are presented by GenBank accession number, host species and country of origin.

43

Graphical abstract

44