Screening of ectoparasites from domesticated dogs for bacterial pathogens in Vientiane, Lao PDR

Abstract Arthropod‐borne diseases are widespread worldwide and are a complex interaction between animals, humans and ectoparasites. The understanding of the diversity and epidemiology of organisms transmitted by arthropod vectors, and the role of hosts and vectors in transmission of infections remain limited in Lao PDR. What knowledge does exist is primarily focused on more rural regions of the country. This study screened ectoparasites from domestic dogs in Vientiane city for the presence of bacterial pathogens of zoonotic importance. A total of 3,511 arthropod vectors were collected from 112 dogs. Vectors collected were Rhipicephalus sanguineus ticks, Ctenocephalides felis felis and Ctenocephalides felis orientis fleas and Heterodoxus spiniger lice. A sub‐sample of vectors from each dog was analysed by PCR to identify the potential bacteria. From 129 vector pools, Rickettsia spp. was detected in 6.7% (7/105) pools of ticks, 86.4% (19/22) pools of fleas and both pools of lice. Sequencing analysis confirmed Rickettsia felis in 13 flea pools and one louse pool and Rickettsia asembonensis in six flea pools. Anaplasmataceae was identified in 14.3% (15/105) tick pools and 100% (22/22) flea pools. Sequencing revealed the presence of Anaplasma platys in ticks and Wolbachia pipientis in fleas. Leptospira spp. was detected in one tick and one louse pool, and Brucella spp. was detected in 12.4% (13/105) tick pools. All samples were negative for Bartonella spp., Coxiella burnetii and Borrelia burgdorferi. This is the first study providing evidence of R. asembonensis in fleas in Laos. Results from this study show arthropods are potential vectors to transmit zoonotic infection in Vientiane city, suggesting humans are at risk of zoonotic infections in the city.

pathogens of both veterinary and clinical importance (Leitner, Wali, Kincaid, & Costero-Saint Denis, 2015). Excellent examples are fleas and ticks, which are able to transmit a number of bacteria, many of which have been identified as emerging vector-borne diseases (Colwell, Dantas-Torres, & Otranto, 2011). There has been a rise in prevalence of tick-borne diseases in both clinical and veterinary settings in recent decades. The incidences have been attributed to Ixodidae ticks (hard ticks) as mode of transmission between animals and humans (Vannier & Krause, 2012). Spotted fever group rickettsia (SFGR) are commonly documented in humans who have had contact with animals and those who have reported exposure to arthropods. For instance, Rickettsia felis is the most widespread zoonotic pathogen transmitted by fleas (Assarasakorn et al., 2012;Kernif et al., 2012;Troyo et al., 2016;Varagnol et al., 2009), yet the number of actual cases is suspected to be much greater due to non-reporting and misdiagnosis.
Screening of ectoparasites and vectors in Southeast Asia has identified a number of important pathogens. Ctenocephalides felis fleas from cats in Bangkok, Thailand, were found to harbour a variety of Bartonella species including B. henselae, B. clarridgeiae and B. koehlerae, whilst in north-eastern Thailand, strains similar to B. elizabethae, B. rattimassiliensis, B. rochalimae and B. tribocorum were also reported (Assarasakorn et al., 2012;Billeter et al., 2013). There were various agents identified in ticks from the Thai-Myanmar border and Vietnam which include Anaplasma spp., Ehrlichia spp. and Rickettsia spp. (Parola et al., 2003).
In Lao People's Democratic Republic (Lao PDR), ticks from Khammouan Province carry Rickettsia japonica and Rickettsia tamurae, two rickettsia species of known clinical importance, as well as Borrelia spp., Anaplasma spp. and Ehrlichia spp. .
All these genera contain species of zoonotic relevance. Rickettsia typhi was successfully identified in fleas from cats and dogs in Phou Khao Khouay National Park, approximately 100 km from Vientiane (Varagnol et al., 2009), whilst Rickettsia spp., R. felis and B. clarridgeiae have been identified in fleas from dogs in northern Laos (Kernif et al., 2012). As yet no extensive survey has been carried out in Vientiane, the capital of Lao PDR which is a very different environment from the rest of the country. Compared to elsewhere, Vientiane is a highly urbanized environment and is the economic hub of the country, with a population of 700,000 to 1,000,000 people.
Population density is high compared to Laos as a whole (6,000/km 2 versus 130/km 2 ). The higher population density, along with pet ownership and a large population of stray dogs and cats, makes the potential for vector transmission of zoonotic pathogens an important aspect of public health.
Considering these aspects, the following study screened ectoparasite vectors from domestic dogs in Vientiane for the presence of bacterial pathogens of zoonotic importance.

| ME THODS
Arthropods, including ticks, fleas and lice, were collected from dogs brought to eight veterinary clinics in Vientiane capital. Basic information was collected for each dog, including reason for visit and overall health. Arthropods were morphologically identified (Hopkins & Rothschild, 1953;Price & Graham, 1997;Walker, Keirans, & Horak, 2000;Yamaguti, Tipton, Keegan, & Toshioka, 1971) and pooled according to species, life stage, sex (adult ticks only) and the individual animal they were retrieved from.

| RE SULTS
In total, 3,511 arthropods were collected from 112 dogs in Vientiane city. Ticks (n = 3,151) were collected from 105 dogs, and all were identified as Rhipicephalus sanguineus (brown dog tick). Adults Impacts • Domestic dogs in Lao PDR harbour a large number of arthropods including ticks, fleas and lice, which are known to be able to transmit bacterial pathogens of human and veterinary importance.
• A number of bacterial species were identified within these vectors, many of which are known to be able to cause disease in humans and animals.
• Due to the high number of domestic and feral dogs in Vientiane, and the large number of vectors, there is a high risk of transmission of these pathogens between dogs and humans within the city.
accounted for 74.5% (43.0% of adults were female), whilst nymphs and larvae accounted for 24.8% and 0.7%, respectively. On aver- Co. burnetii, whilst tick pools were negative for Bo. burgdorferi (summarized in Table 1

| D ISCUSS I ON
Although data on the presence of zoonotic pathogens in arthropod vectors in Lao PDR exists, much of this is related to rural areas (Kernif et al., 2012;Taylor et al., 2016;Varagnol et al., 2009)  from dogs in northern Laos (Kernif et al., 2012). Heterodoxus spiniger has been previously identified in Laos (Beaucournu, Jouan, & Menier, 2001).
Rickettsia felis and R. asembonensis were detected in fleas and lice. In Laos, R. felis has been found in fleas collected from domesticated dogs outside of Vientiane (Varagnol et al., 2009) and has been confirmed as a human pathogen in Laos (Dittrich et al., 2014;Mayxay et al., 2015;Phongmany et al., 2006) suggesting potential interactions between humans and vectors. Rickettsia asembonensis has been mapped worldwide (Maina et al., 2019) and is confirmed in Southeast Asia, including Thailand and Malaysia (Low et al., 2017;Odhiambo, Maina, Taylor, Jiang, & Richards, 2014). This study reports the first detection, to our knowledge, of R. asembonensis in Laos. Although R. asembonensis is closely related to R. felis, little is known of its pathogenicity to humans although there is some molecular evidence (gltA and ompB sequences) of infection in humans and monkeys in Malaysia (Tay, Koh, Kho, & Sitam, 2015). Similar to many reports that fleas are the most common vector to be infected with R. asembonensis, this study only identified this agent in Ct. felis (Kocher et al., 2016;Maina et al., 2016;Oteo et al., 2014;Silva et al., 2017;Troyo et al., 2016). Other arthropods have been found to be infected with R. asembonensis including Pulex simulans and Amblyomma ovale from dogs and Rhipicephalus microplus from cows (Troyo et al., 2016); yet, ticks and lice were negative for R. asembonensis in our sample; and further investigation is needed to confirm the distribution of this agent in the country.
This study presented evidence of Leptospira spp. in Rh. sanguineus and H. spiniger. Canine leptospirosis has been demonstrated in a number of studies, suggesting a risk of human infection (Gay, Soupe-Gilbert, & Goarant, 2014;Weekes, Everard, & Levett, 1997).
There are also questions as to whether Leptospira spp. can be vector transmitted (Wojcik-Fatla et al., 2012) although it is likely that ticks and lice here fed off an infected dog and it is the contaminated blood meal that is being detected.
A number of tick pools were suspected to be Brucella spp. positive, although this could not be confirmed by sequence analysis.
Transmission of Brucella spp. is via contact exposure to infected animals or by environmental contamination with infected fluids, although a number of studies have suggested the possibility of arthropod-borne transmission (Kosoy & Goodrich, 2018 (Hansford, Pietzsch, Cull, & Medlock, 2010;Rust & Dryden, 1997), control of arthropod vectors on domestic animals is both a veterinary and public health importance in the city of Vientiane, requiring greater attention in clinical diagnosis, treatment and prevention strategies.

ACK N OWLED G EM ENTS
The authors wish to thank the veterinary surgeons for their generous support and assistance with sample collection. We also thank Mjr Silas Davidson, Dr Takhampunya Ratree and colleagues at AFRIMS for their assistance in the identification of fleas and lice, and Dr Kristin Mullins, University of Maryland, for the Bartonella spp. qPCR.
The authors also thank Dr Paul Newton (University of Oxford) for his advice and assistance during the study. This study was accepted by the National Animal Health Laboratory, Lao PDR.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.