An overview of rickettsiae in Southeast Asia: Vector-animal-human interface

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An overview of rickettsiae in Southeast Asia: vector-animal-human interface Van Lun Low , Tiong Kai Tan , Jing Jing Khoo , Fang Shiang Lim , Sazaly AbuBakar PII: DOI: Reference:

S0001-706X(19)31433-0 https://doi.org/10.1016/j.actatropica.2019.105282 ACTROP 105282

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Acta Tropica

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16 October 2019 24 November 2019 24 November 2019

Please cite this article as: Van Lun Low , Tiong Kai Tan , Jing Jing Khoo , Fang Shiang Lim , Sazaly AbuBakar , An overview of rickettsiae in Southeast Asia: vector-animal-human interface, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105282

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Highlights 

This review summarizes the infections of Rickettsiae, including the newly discovered regional species in vectors, humans and animals in Southeast Asia.

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Issues on some unidentified Rickettsiae that elicit immune responses and production of antibodies that were cross-reactive with the antigens used are discussed.



Knowledge gaps which required attention are also identified in this review.

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An overview of rickettsiae in Southeast Asia: vector-animal-human interface

Van Lun Lowa*, Tiong Kai Tanb, Jing Jing Khooa, Fang Shiang Lima & Sazaly AbuBakara,c a

Higher Institution Centre of Excellence (HICoE), Tropical Infectious Diseases Research and Education Centre (TIDREC), University of Malaya, Kuala Lumpur, Malaysia b

Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia c

Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

*Corresponding author Email addresses: [email protected], [email protected]

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ABSTRACT Rickettsioses are emerging, and re-emerging diseases caused by obligate intracellular arthropod-borne bacteria that infect humans and animals worldwide. Various rickettsiae such as Orientia, Rickettsia, Anaplasma and Ehrlichia have been circulated in companion, domesticated and wild animals through bites of infected ticks, fleas, lice or mites. This review summarizes the infections of rickettsiae, including the newly discovered regional species Rickettsia thailandii, Candidatus Rickettsia sepangensis, Candidatus Rickettsia johorensis, Candidatus Rickettsia laoensis, Candidatus Rickettsia mahosotii, Candidatus Rickettsia khammouanensis, Candidatus Anaplasma pangolinii, and other novel genotypes in vectors, humans and animals in Southeast Asia. Issues on some unidentified rickettsiae that elicit immune responses and production of antibodies that are cross-reactive with the antigens used are discussed. Knowledge gaps which required attention are also identified in this review. Keywords: Infectious diseases, tropical, vector-borne, zoonosis, rickettsia, Anaplasmataceae, scrub typhus, spotted fever, typhus fever

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1. Introduction Rickettsioses are emerging and re-emerging infectious diseases caused by the obligate intracellular Gram-negative bacteria. They are mainly transmitted through bites of infected arthropod vectors, including ticks, fleas, lice and mites (Portillo et al., 2015). Rickettsioses represent one of the major causes of morbidity and mortality in various parts of Southeast Asia (Acestor et al., 2012) even though in general they are still very much under-appreciated. Rickettsial infections ranked fourth among the causes of systemic febrile diseases in returned travellers from Southeast Asia, after malaria, dengue and infectious mononucleosis (Freedman et al., 2006; Aung et al., 2014). It ranked as the second most frequently reported infections for non-malarial febrile illnesses in Southeast Asia, just after the mosquito-borne dengue virus infection (Acestor et al., 2012). In this review, we summarize the rickettsial infections in vectors, host animals, and humans in Southeast Asia (Table 1). Rickettsial diseases primarily include scrub typhus, spotted fever, typhus fever, anaplasmosis, ehrlichiosis, and the least studied trematode-borne neorickettsiasis. Notes on each disease are described as follows.

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1.1 Scrub typhus Scrub typhus is endemic in the Asia-Pacific region, spanning from Afghanistan to China, Korea, the islands of the southwestern Pacific, and northern Australia (Kelly et al., 2009). This is a life-threatening illness affecting approximately one billion people, and it is one of the most under-diagnosed and under-reported febrile illnesses requiring hospitalization in Asia (World Health Organization, 1999; Watt and Parola, 2003; Wangdi et al., 2019). Bacterium Orientia tsutsugamushi is the primary causative agent of scrub typhus hosted by rodents (Paris et al., 2013) and vectored by the chigger mites of the genus Leptotrombidium (Acari: Trombiculidae) (Luce-Fedrow et al., 2018). Candidatus Orientia chuto and other Orientia-like spp. are also known to infect humans. A novel isolate Ca. O. chuto was first detected from an Australian tourist returning from Dubai, in the United Arab Emirates (UAE), who developed acute scrub typhus (Izzard et al., 2010). Subsequently, several strains closely related Ca. O. chuto were reported in rodents in Asia (including Southeast Asia) and West Africa (Cosson et al., 2015). Further, a case of scrub typhus–like illness was also reported in a man who was bitten by terrestrial leeches in Chile, and the infectious agent recovered was closely related, but not identical to the O. tsutsugamushi and Ca. O. chuto in Asia (Balcells et al., 2011). Nevertheless, the role of the terrestrial leeches in transmitting this disease agent deserves additional investigation. 1.2 Typhus group Typhus group of rickettsioses is divided into (1) murine typhus (also known as endemic typhus) and (2) epidemic typhus. The former is a flea-bone disease caused by Rickettsia typhi, whereas the latter is a louse-borne disease caused by Rickettsia prowazekii. Endemic typhus is prevalent worldwide especially in warm climate and coastal areas including Southeast Asia, with seroprevalence rates ranged from 3-36% (Peniche Lara et al., 2012). In contrast, 5

epidemic typhus is primarily transmitted by the body louse Pediculus humanus corporis, and typically occurs during cold-weather months in the Americas and Africa (Baxter, 1996). The mortality rate can reach 10 to 30% for epidemic typhus, whereas endemic typhus usually runs a milder course, with 1 to 4% mortality rates, subject to the use of appropriate antibiotics (Raoult et al., 1997; Civen and Ngo, 2008). 1.3 Spotted fever Spotted fever group rickettsiae (SFGR) comprises more than 20 different species that are globally distributed (Parola et al., 2013). A spotted fever is typically a tick-borne disease (Robertson and Wisseman Jr, 1973), but subsequently flea and mites have also been incriminated as the vectors of several SFGRs, including the flea-borne Rickettsia felis and R. felis-like organisms such as Rickettsia asembonensis and Candidatus Rickettsia senegalensis (Yazid Abdad et al., 2011), and the mite-borne rickettsialpox caused by Rickettsia akari (Akram and Tyagi, 2017). Rocky Mountain spotted fever (RMSF) is a life-threatening disease caused by Rickettsia rickettsii, which is transmitted by infected ticks. It is the most common rickettsial disease in the American continent and being the most malignant human infection that can be fatal even in otherwise previously healthy young people (Dantas-Torres, 2007). Other common spotted fevers include the Mediterranean spotted fever (Rickettsia conorii and other related species), Flinders Island spotted fever (Rickettsia honei), Siberian tick typhus (Rickettsia sibirica), African tick-bite fever (Rickettsia africae), Queensland tick typhus (Rickettsia australis), Japanese spotted fever (Rickettsia japonica), (Far-Eastern spotted fever (Rickettsia heilongjiangensis), aneruptive fever (Rickettsia helvetica) and rickettsialpox (R. akari) (Brouqui et al., 2007; Parola et al., 2013). In Southeast Asia, R. felis, R. honei (previously known as Thai tick typhus TT-118), R. japonica and R. helvetica were reported in febrile returned travellers (Aung et al., 2014). Particularly, R. felis and R. honei are of importance (Aung et al., 2014). The previously reported murine typhus infections may 6

have been caused by R. felis because of their clinical similarities (Legendre and Macaluso, 2017). Rickettsia honei was first isolated from Ixodes and Rhipicephalus ticks in northern Thailand and it is endemic to Thailand and possibly other parts of Southeast Asia.

1.4 Anaplasmosis Anaplasmosis is an acute febrile tick-borne disease caused by several species of Anaplasma. Particularly, Anaplasma phagocytophilum is of public health importance because it is the pathogen that causes human granulocytic anaplasmosis (HGA). Cases of HGA were reported worldwide but remain unrecognized mainly because of its usually asymptomatic infection (Dumler et al., 2005). On the other hand, bovine anaplasmosis caused by Anaplasma marginale occurs in tropical and subtropical regions of the Americas, Europe, Africa, Asia and Australia (Aubry and Geale, 2011). Its potential socioeconomic impact often leads to trade restrictions both locally and internationally (Goodger et al., 1979; Reinbold et al., 2010). Canine anaplasmosis caused by A. phagocytophilum or Anaplasma platys can lead to acute febrile illness in dogs with lethargy and inappetence (Sainz et al., 2015), but in most cases, dogs naturally infected with these bacteria do not exhibit clinical illness as indicated by widespread serological evidence of exposure in endemic areas (Carrade et al., 2009). The zoonotic potential of A. phagocytophilum is of health concern because it can infect domestic and companion animals which have close contact with humans (Stuen et al., 2014), hence, can result in human infections. 1.5 Ehrlichiosis Ehrlichiosis is a flu-like illnesses caused by Ehrlichia bacteria that infect humans and mammals via tick bite. Tick-borne canine ehrlichiosis is primarily caused by Ehrlichia canis

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and transmitted by the brown dog tick Rhipicephalus sanguineus s.l. (Skotarczak, 2003). Ehrlichia chafeensis and Ehrlichia ewingii have also been found to infect dogs and both are emerging zoonoses that cause human monocytotropic ehrlichiosis (HME) and human ehrlichiosis ewingii (HEE), respectively (Ismail et al., 2010). The geographic distribution of E. chaffeensis and E. ewingii in dogs has been associated with HME or HEE infections (Beall et al., 2012), further supporting the zoonotic transmission of these pathogens. Nevertheless, E. ewingii appears to be less virulent than E. chaffeensis (Angelakis and Raoult, 2017). Ehrlichia canis, Ehrlichia ruminantium and a recently described species, Ehrlichia muris eauclairensis are also occasionally infect humans (Allsopp et al., 2005; Ismail et al., 2010; Pritt et al., 2017). 1.6 Neorickettsiasis Neorickettsia has been relatively little studied in comparison to other groups of rickettsiae. This genus is commonly found in digeneans, the parasitic flatworms that transmit the organisms vertically within the life stages as well as horizontally to the animal or human host (Dittrich et al., 2015b). Three species of Neorickettsia have been found to be pathogenic, namely Neorickettsia sennetsu, Neorickettsia helminthoeca, Neorickettsia risticii. Infection of N. sennetsu in humans is a neglected cause of fever in Asia (i.e., Japan, Malaysia, Laos and Thailand) (Vaughan et al., 2012; Dittrich et al., 2015a). Neorickettsia helminthoeca causes salmon poisoning disease, an acute, febrile, fatal disease of dogs, and endemic in the Pacific Northwest of the United States of America and Canada (Headley et al., 2011). Neorickettsia risticii, the causative agent of Potomac horse fever, infects horses in the United States and Canada (Durán and Marqués, 2016). 2. Rickettsiae in Southeast Asia

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An overview of rickettsiae in Southeast Asia is summarized in Table 1. Rickettsial species or their closely related strains that have been found to infect ectoparasites, animals and/or humans are reviewed. Other related infections associated with ectoparasites such as bartonellosis, borreliosis and Q fever are not within the scope of this current review.

2.1 Cambodia In a 3-year cross-sectional prospective observational study, O. tsutsugamushi DNA was detected at a relatively low prevalence (3.7%) among patients with acute undifferentiated febrile patients in rural Cambodia, in comparison to other infectious agents such as Plasmodium, Leptospira, influenza and dengue viruses (Mueller et al., 2014). Isolation of O. tsutsugamushi from the patients in Cambodia (and also Vietnam and Thailand) revealed high genetic diversity of this pathogenic bacterium as revealed by the 56-kDa gene and multilocus sequence typing (MLST) analyses (Duong et al., 2013b; Wongprompitak et al., 2015). Notably, the Cambodian isolates were clustered into five major groups, including Karp, JG-v, Kato/TA716, TA763 and Gilliam (Duong et al., 2013b), and all were genetically unique (a specific haplotype for each locus, for each strain), even with patients coming from the same region (Duong et al., 2013a). This could complicate development of diagnostics and vaccines targeting O. tsutsugamushi for the region. Orientia tsutsugamushi infections in rodents and mite vectors in Cambodia have been under-reported, precluding an understanding of the transmission dynamics among humans, rodents and mites. More recently, an unknown species of Rickettsia was detected at a very low frequency (0.2%) in febrile patients in rural Cambodia (Mueller et al., 2014). Whether the morbidity is caused by murine typhus or spotted fever remains unexplored. However, murine typhus caused by R. typhi was serologically diagnosed in travellers who returned from Cambodia 9

(Walter et al., 2012) and among patients with unknown febrile illness (13.4%) in Cambodia (Kasper et al., 2012). Rickettsia felis (10.9%) on the other hand, was well-reported in dogs in Cambodia (Inpankaew et al., 2016), hence its zoonotic potential could not be disregarded. There is a possibility that antibodies developed against murine typhus cross-reacted with that of R. felis resulting in some of these cases to be attributed to the flea-borne spotted fever (Inpankaew et al., 2016). In addition to R. felis, Inpankaew et al., (2016) also reported the presence of E. canis in dogs at a higher frequency (21.8%) in comparison to other vector borne pathogens such as Dirofilaria immitis, Hepatozoon canis and Mycoplasma haemocanis. As to the trematode-borne neorickettsiasis, a new Neorickettsia sp. was detected in a freshwater needlefish in Cambodia. However, the natural life cycle of this species is still unknown because the trematode was not found in these positive fish (Seng et al., 2009). At the point of writing, there is as yet no well-documented reports of infection with rickettsial agents in vectors in Cambodia. 2.2 Indonesia Scrub typhus and murine typhus are both endemic in Indonesia. Seroprevalences of O. tsutsugamushi and R. typhi were reported at varying frequencies on many of the islands of the archipelago (Liat and Hadi, 1986; Richards et al., 1997; Gasem et al., 2009). Murine typhus was of particularly conspicuous, with many documented cases imported into other countries from Indonesia (Parola et al., 1998; Takeshita et al., 2010; Walter et al., 2012; Kato et al., 2014). Seroconversion for O. tsutsugamushi and R. typhi was also observed among the Indonesian military personnel following their peace keeping operations in Cambodia (Corwin et al., 1997).

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Evidence of O. tsutsugamushi and R. typhi in rodents has been established. Rattus rattus, R. tanezumi, R. norvegicus and Suncus murinus were the potential hosts of murine typhus while scrub typhus had a wider range of hosts which include R. tiomanicus, R. sabanus, R. tanezumi, R. norvegicus, R. whiteheadi and Maxomys sp. (Richards et al., 1997; Ibrahim et al., 1999; Richards et al., 2002; Widjaja et al., 2016). The DNA of O. tsutsugamushi and R. typhi was detected in their primary vectors, Leptotrombidium mite and Oriental rat flea Xenopsylla cheopis, collected from small mammals, respectively (Barbara et al., 2014; Widjaja et al., 2016). In addition to fleas, the DNA of R. typhi was also found in Poliplax lice and Laelaps mites, suggesting their potential role in the transmission cycle (Widjaja et al., 2016). The cat flea Ctenocephalides felis is the primary vector of R. felis worldwide, but related reports have not been documented in Indonesia. Instead, the DNA of R. felis and unidentified Rickettsia was detected in X. cheopis, suggesting this flea species as a potential vector for the flea-borne spotted fever in Indonesia (Jiang et al., 2006; Barbara et al., 2014). Rickettsia felis or closely related species was also detected in Poliplax lice, Laelaps and Haemogamassus mites, and Haemaphysalis, Ixodes and Rhipicephalus ticks through a realtime PCR assay (Widjaja et al., 2016). Further DNA sequencing will be beneficial in confirming the presence of R. felis, R. asembonensis, Ca. R. senegalensis or other closely related strains in these arthropods. Other spotted fever rickettsiae such as R. conorii and R. rickettsii in human (Richards et al., 2003), and R. honei (TT-118), R. rickettsii and R. conorii in rodents (Ibrahim et al., 1999; Widjaja et al., 2016) were reported based on serological tests. Occurrence of R. rickettsii, the causative agent of Rocky Mountain spotted fever, in this region raises uncertainty as to whether this result represents a true R. rickettsii infection or simply cross-

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reactivity with another related pathogens. Cross-reactivity between R. rickettsii and R. felis may have occurred in the serological assay because the DNA of R. felis was detected in most of the samples (Widjaja et al., 2016). Antibody blocking assays, western blotting or crossadsorption assays should be performed to complete the identification of the circulating agents; however, these experiments are not generally performed during surveillance studies. Occurrence of Anaplasmataceae is under-investigated because there are only a few known studies reporting these infectious agents in Indonesia. The human infection of agent closely related to E. chaffeensi based on serological test was reported in a small and isolated island in Indonesia (Richards et al., 2003). In animals, occurrences of E. canis and Anaplasma sp. were reported in dogs (Hadi et al., 2016; Erawan et al., 2017). The Asian palm civet (Paradoxurus hermaphroditus) was also found infected with Anaplasma sp. (Putri et al., 2012).

2.3 Laos Scrub typhus and murine typhus are common causes of previous undiagnosed fever in Laos. The spatial distribution of antibodies for these two diseases was radically different in that the murine typhus was more prevalent in urbanized areas while scrub typhus was common in remote areas of Laos (Vallée et al., 2010). Scrub typhus appeared to be more prevalent than murine typhus (Phongmany et al., 2006; Mayxay et al., 2013; Dittrich et al., 2015b). Previous studies demonstrated a highly diverse of O. tsutsugamushi including the discovery of new genotypes and revealed evidence for geographical structuring and local clonal expansion within Laos (Parola et al., 2008; Phetsouvanh et al., 2015).

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Exposure of R. helvetica, R. felis, R. conorii, R. tamurae AT1 was reported in patients with clinical symptoms (Mayxay et al., 2013). Genetic analysis, however, is required to confirm the presence of these SFGR because they may represent unidentified Rickettsia species that are closely related to them. Detection of R. felis with molecular techniques was reported for the first time in patients from Laos, suggesting that this species as possible agent causing human infections (Phongmany et al., 2006; Mayxay et al., 2013). Surveys of rickettsial agents have been conducted in a wide range of arthropod vectors in Laos. Rickettsia felis was detected in the cat fleas C. orientis and C. felis, and dog flea C. canis (Varagnol et al., 2009; Kernif et al., 2012). Three regional novel species were reported in Haemaphysalis and A. testudinarium ticks, namely Candidatus Rickettsia laoensis (R. massiliae group), Candidatus Rickettsia mahosotii (R. rickettsii group) and Candidatus Rickettsia khammouanensis (R. helvetica group) (Taylor et al., 2016). Occurrence of the novel genotype closely related to R. helvetica is of concern because there has been serologic evidence for its role as a human pathogen in Laos (Phongmany et al., 2006). The humaninfective R. japonica and R. tamurae were also reported in H. hysticis and A. testudinarium, respectively (Taylor et al., 2016), suggesting the potential role of these tick species as the vectors of spotted fever in Laos. Limited information is currently available for the incidence of anaplasmosis and ehrlichiosis in Laos. An unknown Ehrlichia species was reported in Haemaphysalis tick but further characterization was not performed (Taylor et al., 2016). Nevertheless, detection of the other closely related member, N. sennetsu, using molecular and serological approaches and the high seroprevalence (17%) of antibodies against this organism suggested that sennetsu neorickettsiosis is a common infection in Laos (Newton et al., 2009). The DNA of N. sennetsu was detected in human (Dittrich et al., 2015a) and a fish Anabas testudineus (Newton et al., 2009) suggesting possible epidemiological link between the fish and human. 13

2.4 Malaysia Scrub typhus is a public health concern with an estimated annual incidence of 18·5% (Tay et al., 2000). It was reported among many febrile patients with antibody prevalence that ranged from 0.8% in East Malaysia (Taylor et al., 1986) to 73% in West Malaysia since early 1970s (Cadigan Jr et al., 1972). Recent seroprevalence of 36% was reported in different Orang Asli (aborigines of Peninsular Malaysia) populations (Tay et al., 2013). Earlier studies suggested that those engaged in agricultural activities were likely to have been affected (Tay et al., 2000; Tay et al., 2013). Forest rats commonly found in Malaysia such as Rattus sabanus, Rattus tiomanicus, Rattus argentiventer, Sundamys muelleri and Leopoldamys sabanus were the hosts for trombiculid mites which can serve as the reservoir and vector for O. tsutsugamushi (Muul et al., 1977; Tay et al., 1998). Earlier studies also incriminated Leptotrombidium delicense, Leptotrombidium arenicola and Leptotrombidium akamushi as potential vectors of scrub typhus in Malaysia (Traub and Wisseman Jr, 1968). However, recent study failed to detect O. tsutsugamushi DNA in L. delicense mites collected from a wide range of small mammals in Malaysia (Azima et al., 2013). The role of L. delicense in recent transmission of scrub typhus cannot be excluded but other potential vectors should also be taken into consideration in the vector surveillance programs. Murine typhus or previously known as urban typhus of Malaya was first recognised in in 1926. The occurrence of the disease was sporadic and uncommon with a reported annual country incidence of 36-155 from the years 1968-1974 (Brown et al., 1977). Since then, an increasing trend of this disease occurrence was reported from 1991-1997 (Sekhar and Devi, 2000). Rickettsia typhi DNA was detected in a patient in Malaysia (Kho et al., 2016). It is not known, however, how the patient could have been infected. It could have been from the house rat Rattus rattus diardii which was most often found positive for R. typhi, followed by the domiciliated or semi domiciliated species Rattus exulans, Rattus rattus jalorensis and S. 14

murinus. In an earlier study, bird in a secondary forest-scrub habitat was also found seropositive to murine typhus (Marchette, 1966). Earlier experimental study further showed that the transmission of R. typhi can take place from rat to rat vectored by the rat flea Xenopsylla cheopis (Lewthwaite et al., 1936). A large-scale serosurvey conducted among 1596 febrile patients in Malaysia showed that the SFGR appeared much more widespread than murine typhus and scrub typhus (Tay et al., 2000). Higher prevalence of R. honei (70.7%) in wild rodents Rattus spp. and Tupais glis in comparison to R. typhi (2.4%) and O. tsutsugamushi (4.9%) was observed (Tay et al., 1998). The seroprevalence of R. honei in humans, however, varied widely from 1.7% in urban blood donors, 42.5% in rural febrile patients, to 57.3% in rural healthy individuals (Tee et al., 1999; Tay et al., 2000; Tay et al., 2002b). Nevertheless, a recent study reported a contrasting infection pattern, in which lower prevalence of R. honei (20.6%) was found in comparison to the murine typhus (27.9%) (Tappe et al., 2018). Whether these results are attributed to a recent infection or a reflection of past outbreaks remain unanswered. Further, cross-reactivity of the antibodies with other SFGRs could not be excluded. A recent study also revealed an approximately 50% of the Orang Asli and animal farm workers were seropositive against R. conorii (Kho et al., 2017). However, it could be an unknown Rickettsia which elicits antibodies cross-reacting with R. conorii antigens. In an earlier study, it was shown that H. conigera ticks were reactive to the fluorescein isothiocyanate−labeled antibodies to R. conorii (Tay, 1996), suggesting the potentially role of this tick species in transmitting pathogen. Rickettsia felis or R. felis-like organisms (Rickettsia sp. RF2125, Rickettsia sp. RF31, and R. asembonensis) have been detected in the cat fleas C. felis and C. orientis and the 15

brown dog tick R. sanguineus in Malaysia (Mokhtar and Tay, 2011; Low et al., 2017). Recent studies using molecular detection method suggested the presence of Rickettsia sp. RF2125 (R. asembonensis) in blood samples of a Malaysian febrile patient and cynomolgus monkeys in Malaysia, suggesting the zoonotic potential of transmission (Tay et al., 2015; Kho et al., 2016). Further, a closely related strain of R. raoultii was suggested as the aetiological agent for rickettsioses in two febrile patients in Malaysia. This pathogen was detected in R. sanguineus, R. microplus and Haemaphysalis ticks infesting peri-domestic animals (Kho et al., 2017). Oher closely related strains of R. raoultii were also reported in wild rats captured in Malaysia (Tay et al., 2014b) and Amblyomma spp. parasitizing wild snakes (Kho et al., 2015). Other SFGRs closely related to R. heilongjiangensis, R. tamurae, R. sp. TCM1, R. sp. LON13 and R. hulinensis were detected in tick samples in Malaysia (Kho et al., 2017). The newly discovered Candidatus Rickettsia sepangensis and Candidatus Rickettsia johorensis were also reported for the first time in A. varanense and A. helvolum ticks parasitizing wild snakes in Malaysia (Kho et al., 2015). The pathogenicity of these novel bacteria, however, deserves additional research efforts. In a recent study, Koh et al., (2018) reported that 6.9% of the indigenous people were seropositive for Anaplasma phagocytophilum, however, they may have antibodies reacting with the antigens of other Anaplasma species. Occurrences of A. platys, A. phagocytophilum, A. marginale, A. capra, Candidatus A. camelii and Candidatus A. boloense were reported in dogs, livestock and wildlife in Malaysia (Pong and Nik-Him, 2012; Rahman et al., 2012; Mokhtar et al., 2013; Tay et al., 2014a; Tay et al., 2015; Koh et al., 2016; Koh et al., 2017; Konto et al., 2017; Ola-Fadunsin et al., 2017; Koh et al., 2018). Notably, a novel species of

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Anaplasma (Candidatus Anaplasma pangolinii) closely related to A. bovis and A. phagocytophilum, was reported in pangolins (Manis javanica) (Koh et al., 2016). Several tick species have been shown to contain DNA of Anaplasma spp. For example, A. platys and/or A. phagocytophilum in R. sanguineus, H. bispinosa, Haemaphysalis. sp. and Dermacentor sp; A. bovis in R. sanguineus and Haemaphysalis sp; and A. marginale in R. microplus and Haemaphysalis sp. (Koh et al., 2017; Koh et al., 2018; Low et al., 2018). Occurrences of A. centrale and A. ovis in Malaysia are worthy of further investigation though a genotype identical to those of A. marginale/A.centrale/A. ovis was detected in cattle and R. microplus tick (Tay et al., 2014a). A strain of Anaplasama closely related to Candidatus Cryptoplasma californiense which was first reported in I. pacificus tick in California, was also detected in a Haemaphysalis tick in Malaysia (Koh et al., 2018). A recent study reported high antibodies reacting with E. chaffeensis antigens among the Orang Asli (34.3%) and animal farm workers (29.9%). Ehrlichia chaffeensis DNA was not detected from any tick vectors, instead, Ehrlichia sp. strain EBm52, E. mineirensis and Candidatus E. shimanensis were detected in R. microplus and H. bispinosa collected from cattle (Koh et al., 2017). Nevertheless, in a separate study, DNA of Ehrlichia sp. closely related to E. chaffeensis was detected in R. sanguineus ticks (Koh et al., 2015). Canine ehrlichiosis caused by E. canis is of veterinary concern in Malaysia. Its DNA prevalence was consistently higher than A. platys (61% against 38%) in dogs in Malaysia (Mokhtar et al., 2013; Nazari et al., 2013; Koh et al., 2015; Konto et al., 2017; Mohammed et al., 2017; Low et al., 2018). Molecular evidence of E. canis in the R. sanguneius was also reported (Koh et al., 2015; Low et al., 2018), hence, the role of this tick species in ehrlichiosis transmission in Malaysia could not be disregarded.

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The agent of neorickettsiasis, N. sennetsu was reported in Malaysia. Neorickettsia sennetsu was identified from patients with fevers of unknown origin in Malaysia via propagation in primary canine blood monocyte cultures (Vaughan et al., 2012).

2.5 Myanmar Rickettsial diseases in Myanmar are still very much under-reported, though there was a report of imported scrub typhus contracted in Myanmar. A Japanese who had been exposed to mosquito and tick bites while in Myanmar had antibodies for O. tsutsugamushi serotype Gilliam upon returning to Japan (Matsumura and Shimizu, 2009). Subsequently, murine typhus was also recognized among French travelers returning from Myanmar (Walter et al., 2012), suggesting the presence of both scrub typhus and murine typhus in Myammar. There were also a few reports of rickettsial infections from the border regions of Myanmar and her neighboring countries. Soldiers serving the Indo-Myanmar border region have reportedly been infected with scrub typhus (Biradar et al., 2015). In addition, scrub typhus, murine typhus, and other SFGRs including R. helvetica, R. conorii and the first Asian case of R. felis infection in humans were documented at the Thai-Myanmar border areas (Parola et al., 2003b; Ellis et al., 2006; McGready et al., 2010; Brummaier et al., 2017). Nevertheless, most of the studies were based on serological methods, thus it is not possible to confirm the above-mentioned Rickettsia species as the etiological agents in this region unless molecular or cross-absorption studies are performed. Two genotypes related to R. felis were identified in the dog flea C. canis and cat flea C. felis (Parola et al., 2003c). Two unknown Rickettsia spp. were also found in Dermacentor auratus and Dermacentor sp. in this area, but the pathogenic role of these bacteria has yet to

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be confirmed (Parola et al., 2003a). In the same area, Ehrlichia sp. strain EBm52 was recovered from R. microplus. Three Anaplasma spp. were also detected in ticks, including Anaplasma sp. strain AnDa465 from D. auratus; Anaplasma sp. strain AnHl446 from H. lagrangei, and Anaplasma sp. strain AnAj360 from A. javanense (similar to Candidatus A. pangolinii reported from Malaysia) (Parola et al., 2003a). 2.6 Philippines Both scrub typhus and murine typhus are present in the Philippines, but their presence is still under-recognized and in general treated as fever of unknown origin. Earlier studies showed that murine typhus (up to 23%) was more prevalent than scrub typhus (up to 16%) (Cross and Basaca-Sevilla, 1981; Camer et al., 2003). Cases of murine typhus in French travelers returning from the Philippines were reported (Walter et al., 2012). On the other hand, scrub typhus has been known to occur in the islands with the chigger mites associated with rodents in sylvan or less disturbed areas (Cross and Basaca-Sevilla, 1981). Antibodies react with R. japonica antigens were detected in blood samples of dogs and rodents, and humans in the Philippines (Camer et al., 2000; Camer et al., 2003). Evidence of spotted fever rickettsial agents in arthropod vectors, however, is still scarce. Rickettsia felis is the only known spotted fever agent found in the cat flea C. felis in the Philippines. (Wolf and Reeves, 2012). In contrast to the typhus fever, Anaplasmataceae agents have been well-documented. Anaplasma platys and E. canis in dogs; and A. marginale in cattle, and their respective ticks (i.e, R. sanguineus and R. microplus) were reported in recent years (Baticados and Baticados, 2011; Ybañez et al., 2012; Ybanez et al., 2012; Ybanez, 2013; Ybañez et al., 2013; Corales et al., 2014; Ybanez, 2014; Ochirkhuu et al., 2015; Ybañez et al., 2015; Ybañez et al., 2016; Adao et al., 2017). Anaplasma centrale, the less commonly reported species across Southeast 19

Asia, was detected in a R. microplus tick (Ybanez, 2013). Occurrences of A. ovis and A. phagocytophilum in the Philippines nonetheless remained to be confirmed using longer fragment of the 16S rRNA gene or other genetic markers (Ybañez et al., 2013). Neorickettsia sp. in the two Paralecithodendrium spp. of bats from the Philippines was reported for the first time based on PCR and sequencing evidence (Greiman et al., 2017). 2.7 Singapore Scrub typhus and murine typhus were notifiable diseases in Singapore until 1976 (Loh et al., 1996). Several earlier reports confirmed the presence of scrub typhus in Singapore and investigation of Rattus rattus diardi rats for the vector mites suggested that most of the rats were infested with L. delicense, from which O. tsutsugamushi was recovered (Lawley, 1957; Kurup et al., 2013). While murine typhus has been thought to be a disease of the past (Ong et al., 2001), with improved diagnostics capability, murine typhus has been increasingly implicated as among the cause of febrile illnesses in Singapore (Chen et al., 2001; Ong et al., 2001). Other forms of rickettsial diseases such as the spotted fever group and Anaplasmataceae to date have not been reported (Loh et al., 1996), but it could have been under-diagnosed and treated simply as pyrexia of unknown origin. 2.8 Thailand In 1999-2014, scrub typhus (up to 16%) appeared to be more prevalent in Thai febrile patients compared to murine typhus (up to 5%) (Aung et al., 2014). Travel-associated scrub typhus is of particular concern because most of the travellers have been mainly infected in Thailand, followed by a few cases in Vietnam, Malaysia, and the Philippines (Ericsson et al., 2004). 20

Rodents and associated chigger mites, L. delicense were found PCR positive for O. tsutsugamushi, but strains recovered from chiggers differed from those of rodent hosts, indicating high genetic variation of O. tsutsugamushi strains circulating in the survey areas (Rodkvamtook et al., 2018). Strikingly, the presence of Orientia sp. that is closely related to Ca. O. chuto was also found in the spleens of rodents in Thailand (Cosson et al., 2015). In addition to Leptotrombidium spp., DNA of O. tsutsugamushi was also detected in the mite genera Ascoschoengastia, Blankaartia, Gahrliepia, and Lorillatum (Takhampunya et al., 2018). As for murine typhus, R. norvegicus was incriminated as an important reservoir species of rodents in various types of habitats in Thailand (Siritantikorn et al., 2003; Chareonviriyaphap et al., 2014). Its common ectoparasite X. cheopis was found positive for R. typhi (Chareonviriyaphap et al., 2014). Further, dual exposure of R. typhi and O. tsutsugamushi in R. norvegicus was also documented (Chareonviriyaphap et al., 2014), highlighting the important role of this rodent species in the transmission of rickettsial diseases in Thailand. The Thai tick typhus strain TT-118, (now known as R. honei), originally isolated from a mixed pool of Ixodes sp. and Rhipicephalus sp. from R. rattus in northern Thailand (Robertson and Wisseman Jr, 1973), has now been found in I. granulatus (Kollars Jr et al., 2001) and wild rats (Okabayashi et al., 1996), and it is a human-infective pathogen (Jiang et al., 2005; Sangkasuwan et al., 2007). Rickettsia japonica, R. felis and R. helvetica were also found to infect humans in Thailand (Parola et al., 2003a; Fournier et al., 2004; Gaywee et al., 2007; Takada et al., 2009; Edouard et al., 2014). Rickettsia japonica was detected in H. hysticis ticks (Takada et al., 2009) and B. indica rats (Okabayashi et al., 1996), whereas R. felis (and closely related

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genotypes, including R. asembonensis) was reported in cats and fleas C. felis, C. orientis and C. canis (Foongladda et al., 2011; Phoosangwalthong et al., 2018). Seroprevalences of R. rickettsii, R. prowazekii, and R. canada were documented in dogs in Thailand (Suksawat et al., 2001), but seroreactivity to Rickettsia sp. antigens may have occurred, particularly R. prowazekii, and R. canada which have not been reported in Asia. Other SFGRs closely related to R. raoultii, R. bellli, R. tamurae, R. monaensis, R. montana, and Rickettsia spp. were also reported in a wide range of tick species in Thailand (Hirunkanokpun et al., 2003; Doornbos et al., 2013; Malaisri et al., 2015; Sumrandee et al., 2016). Notably, a new species of Rickettsia (R. thailandii) was found in I. granulatus (Kollars Jr et al., 2001). Anaplasma platys and E. canis are the common Anaplasmataceae agents reported in dogs and R. sanguineus ticks in Thailand (Pinyoowong et al., 2008; Foongladda et al., 2011; Liu et al., 2016; Saeng-Chuto et al., 2016; Kaewmongkol et al., 2017). Interestingly, a first report of A. platys in a naturally infected domestic cat from Thailand was also documented (Salakij et al., 2012). Anaplasma bovis was detected in H. lagrangei and H. obesa ticks, whereas a closely related strain of A. platys was also found in H. lagrangei and D. auratus ticks (Sumrandee et al., 2014). As for bovine anaplasmosis, buffaloes and cattle in Thailand were also infected with A. marginale (Saetiew et al., 2015a; Saetiew et al., 2015b; Jirapattharasate et al., 2017) Based on immunofluorescence antibody testing, Thai dogs were seroreactive to E. chaffeensis, E. canis, E. equi and E. risticii, but only the DNA of E. canis was amplified, supporting the hypothesis of cross-reactivity among Ehrlichia species (Suksawat et al., 2001). Nevertheless, antibodies reacting with E. chaffeensis antigens were detected in Thai patients 22

with malaria-like fever, suggesting the existence of E. chaffeensis or an antigenically related organism in Thailand (Heppner et al., 1997; Suksawat et al., 2001). Previous study showed a 4% seroprevalence of N. sennetsu in patients in Thailand but only one sample was PCR positive for N. sennetsu, suggesting the presence of organisms closely related to N. sennetsu (Vaughan et al., 2012). This hypothesis was supported by a recent study which reported the two distinct genotypes of Neorickettsia species in digeneans from Thailand (Greiman et al., 2017). 2.9 Timor-Leste There is merely one known study reporting human ehrlichiosis in the country. An American aid worker who worked for one year in Timor-Leste experienced multiple tick bites over a 1week period up until 4 days before clinical illness onset. The combination of serological, clinical and laboratory findings supported a diagnosis of human monocytic ehrlichiosis (i.e., E. chaffeensis) (Burke et al., 2015). 2.10 Vietnam Scrub typhus was a well-known disease during the Vietnam war, in which 10% of the febrile American soldiers were positive to O. tsutsugamushi (Deller and Russell, 1967). Additionally, scrub typhus antibodies were also found in military dogs owned by the US armed forces after serving in Vietnam (Alexander et al., 1972). Scrub typhus in Vietnam has continued to receive attention when a French traveler who visited Vietnam was infected with scrub typhus (Thiebaut et al., 1997). This disease persists until today; cases have been occurred throughout the year, with incidence highest in the summer (Nadjm et al., 2014; Hamaguchi et al., 2015; Wongprompitak et al., 2015; Le Viet et al., 2017; vu Trung et al., 2017).

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Murine typhus cases were also reported during and before the 1960s, and since then the first diagnosis of murine typhus in Vietnam after the 1960s was reported in a Japanese traveler who visited Ho Chi Minh City (Azima et al., 2013). Subsequently, murine typhus cases have been commonly diagnosed in urban rather than rural populations (Hamaguchi et al., 2015; vu Trung et al., 2017). SFGR has been little studied in Vietnam, though a low antibody prevalence against R. conorii was reported recently (vu Trung et al., 2017). Antibodies to R. canada were reported in military dogs after serving in Vietnam (Alexander et al., 1972), but its possibility on the cross-reactivity with other Rickettsia spp. cannot be excluded since R. canada is not endemic in Asian region. Anaplasma marginale and A. centrale were reported in cattle in Vietnam (Ha et al., 1997; Nguyen et al., 1999; Geurden et al., 2008). There is an absence of study to date regarding other Anaplasma species in Vietnam. Pathogens in insect vectors have been under-studied in Vietnam. Only two species of ticks in Vietnam were found to contain the DNA of Ehrlichia species. Haemaphysalis hystricis collected from wild pigs has was infected with Ehrlichia sp. and Ehrlichia chaffeensis (Parola et al., 2003a) whereas R. sanguineus was infected with E. canis (Nguyen et al., 2018). Stellantchasmus falcatus and Saccocoelioides sp. were the fish digenean species known to harbor Neorickettsia spp. in Vietnam (Greiman et al., 2017).

3. Perspectives 3.1 Vector

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Taxonomy of insect vectors has been a major obstacle in combating vector-borne diseases. Identifications of insect vectors particularly ticks, fleas and mites are challenging due to their highly variable morphological characters, incomplete description of their life stages, or the presence of species complex (Dantas-Torres, 2010; Barker and Walker, 2014; Nava et al., 2015; Kumlert et al., 2018). The presence of morphologically similar but genetically distinct species (cryptic species) further complicates the assessment of species boundaries and vector identification because different lineages of vectors may present variable susceptibility levels to pathogen infections (Burlini et al., 2010; de Castro Demoner et al., 2013). As far as zoonotic transmission is concerned, R. sanguineus and R. microplus are the two well-known species complexes that have gained research attention worldwide due to their widespread distribution and close contact with humans. Rhipicephalus microplus consists of at least three genetically distinct lineages, including a novel lineage discovered from Malaysia (Low et al., 2015), which was subsequently detected in Myanmar (Roy et al., 2018). On the other hand, R. sanguineus comprises three lineages: southeast European lineage, southern lineage, and the northern lineage which is represented by Malaysia, Singapore and Thailand (Low & Prakash, 2018). Genetic lineages of both species remain uncharacterized in most of the countries in Southeast Asia. The cat flea C. felis and its congeners C. orientis are the common species infesting companion dogs and cats in Asia (Azrizal-Wahid et al., 2019). Unfortunately, both species are often misidentified due to their similar morphological characters. Previous study reported high occurrence of R. asembonensis (previously known as genotype RF2125) but absence of R. felis in C. orientis in India, suggesting a specific vector-endosymbiont adaptation and coevolution of this organism in C. orientis. Similar findings have been observed in Malaysia in which the C. orientis was accurately identified via a PCR approach (Low et al., 2017). There is an urgent need to closely re-examine the previously identified so-called C. felis that 25

has been known to harbor R. asembonensis in Southeast Asia. Verifying the true vector responsible for the transmission of R. asembonensis is of paramount importance because this species was found to infect humans and monkeys, for the first time, in Malaysia (Tay et al., 2015; Kho et al., 2016). It is also worth noting that the extremely tiny mites often make inspection and identification difficult. In certain circumstances, mites were not identified up to the species level because they need to be cleared and slide-mounted, which do not allow for DNA isolation (Takhampunya et al., 2018). Although glossaries of morphological terminology and established identification keys of some mite species are available, but they are mostly confined to larval stage (Kumlert et al., 2018). Molecular identification is the alternative approach to resolve the issue of mite identification but depositing the correct reference sequences onto the database is the prerequisite, including those of endemic species. Same practices should also be applied on tick ‒ a long debated issue among the acarologists around the world.

3.2 Animal Taxonomic status of certain rodent species, especially the Southeast Asian black rats, has also been long questioned. There has been no solid agreement on how many species should be recognized within the black rat complex, and there have been at least three cryptic species living in sympatry in this region (Pages et al., 2013). Identification of etiology associated with disease outbreak and assessment of the effectiveness of the prevention program could be obstructed since different rodent species have been shown to influence their mite population density (Kim et al., 2010; Rodkvamtook et al., 2018).

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Serological testing has been a gold standard for diagnosis of infectious diseases in animals (and humans), however, cross-reactivity with other disease agents is its major limitation. As discussed earlier, seroprevalences of R. prowazekii and R. canada were documented in dogs in Southeast Asia (Suksawat et al., 2001), but the presence of both pathogens is doubtful because they are only endemic in the Americas. On the other hand, the commercial rapid test kit, SNAP 4Dx Plus Test has been widely used by the veterinarians to detect the antibodies to A. platys/A. phagocytophilum and E. canis/E. ewingii. Nevertheless, accurate species identification cannot be performed, though E. ewingii has not been reported in this region. The presence of both A. platys and A. phagocytophilum, however, has been reported in dogs (Koh et al., 2015; Koh et al., 2017; Low et al., 2018), making it challenging to determine the epidemiology of the human-infective A. phagocytophilum. 3.3 Human For scrub typhus, the exact etiological agents are well-recognized and characterized with a large body of findings based on serological and molecular findings (Parola et al., 2008). In contrary, most studies of rickettsial infections in humans caused by Rickettsia, Anaplasma and Ehrlichia are dependent on serological methods using antigens generated from rickettsial strains which are not endemic to this part of the world. Due to the antigenic cross-reactivities between closely related rickettsial species, findings from these studies could at most indicate the presence of rickettsial species belonging to an antigenic or genetic group, such as typhus group or SFGR. For instance, positive findings in serological assays using antigen preparations from R. conorii and R. rickettsii could only indicate the exposure to spotted fever Rickettsia, since these pathogens are not known to occur in this region and therefore unlikely to be the etiological agents to human infections here (Richards et al., 2003). This is also evident in animal surveillance studies, in which animals that are serologically positive to

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non-endemic Rickettsia, such as R. rickettsii, were later found to carry a different but genetically related Rickettsia species (i.e., R. felis) (Widjaja et al., 2016). Molecular diagnostic methods are rarely used in the clinical setting for diagnosis of rickettsial infections as many molecular methods, such as real-time polymerase chain reactions and gene sequencing, require highly specialized equipment and trained personnel that may not be available in resource-poor healthcare or diagnostic facilities. Therefore, the exact rickettsial species causing human infections are rarely known since serological methods based on antigenic cross-reactivities to non-endemic strains are most widely used in the clinical settings (Ellis et al., 2006; Punjabi et al., 2012; Hamaguchi et al., 2015). A limited number of studies, nevertheless, successfully identified the actual rickettsial agents causing human infections in this region by molecular techniques (Jiang et al., 2005; Sangkasuwan et al., 2007; Vaughan et al., 2012; Kho et al., 2016). Rickettsia felis (Kho et al., 2016), Rickettsia sp. RF2125 or R. asembonensis (Kho et al., 2016), R. raoultii (Kho et al., 2016), R. honei TT-118 (Jiang et al., 2005), R. japonica (Gaywee et al., 2007) and N. sennetsu (Vaughan et al., 2012) are some of the rickettsial species detected from human patients in this region. These findings, together with the continuous discovery of novel rickettsial agents from animal hosts or ectoparasite vectors, could be informative for the design of new serological assays with improved sensitivity and specificity to the rickettsial species endemic to this region. 4.0 Conclusion Rickettsiae appear to be endemic across Southeast Asia and have increasingly been recognized as causes of febrile illnesses in travellers upon returning to their respective home countries. However, the unavailability or under-reported information on rickettsial infections in Brunei Darussalam, Timor-Leste and Myanmar necessitate immediate research to be

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carried out. In a wider perspective, a need of new paradigm or choice to control rickettsial infection is undeniably urgent. Particularly, identifying strategies and measures for disease and vector control, and improved diagnostic tools are the efforts to be bound to combat these endemic diseases in Southeast Asia. conflic of interest None. Funding This study was financially supported by the University of Malaya research grant (RP021C16SUS) and Higher Institution Centre of Excellence (HICoE) program (MO002-2019).

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Table 1. An overview of rickettsial infections in Southeast Asia Group Scrub typhus

Species Orientia tsutsugamushi

Infecting vector/ trematode MITE Leptotrombidium delicense L. chiangraiensis L. scutellare L. arenicola L. imphalum L. fletcheri L. akamushi Ascoschoengastia Blankaartia Gahrliepia Lorillatum

Animal host Dog Rodent

Human Yes

Distribution Cambodia

Indonesia

Laos

Malaysia

Myanmar Philippines Thailand

Vietnam

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References Duong et al., 2013a; Duong et al., 2013b Mueller et al., 2014 Wongprompitak et al., 2015 Liat and Hadi, 1986 Richards et al., 1997; Richards et al., 2003 Widjaja et al., 2016 Phongmany et al., 2006 Parola et al., 2008 Vallée et al., 2010 Phommasone et al., 2013 Phetsouvanh et al., 2015 Traub and Wisseman Jr, 1968 Cadigan Jr et al., 1972 Taylor et al., 1986 Muul et al., 1977 Tay et al., 1998; Tay et al., 2000; Tay et al., 2002b; Tay et al., 2013 Azima et al., 2013 Tee et al., 1999 Mohamed et al., 2016 Matsumura and Shimizu, 2009 Traub and Wisseman Jr, 1968 Cross and Basaca-Sevilla, 1981 Chareonviriyaphap et al., 2014 Wongprompitak et al., 2015 Rodkvamtook et al., 2018 Takhampunya et al., 2018 Deller and Russell, 1967 Alexander et al., 1972 Thiebaut et al., 1997 Nadjm et al., 2014 Hamaguchi et al., 2015 Wongprompitak et al., 2015 Le Viet et al., 2017 vu Trung et al., 2017

Indo-Myanmar Thai-Myanmar

Murine typhus

Orientia sp. Rickettsia typhi

Unknown FLEA Xenopsylla cheopis

Unknown

Unknown

Thailand

Biradar et al., 2015 Parola et al., 2003b Ellis et al., 2006 McGready et al., 2010 Brummaier et al., 2017 Cosson et al., 2015 Walter et al., 2012 Richards et al., 1997; Richards et al., 2003 Parola et al., 1998 Gasem et al., 2009 Takeshita et al., 2010 Kato et al., 2014; Widjaja et al., 2016 Phongmany et al., 2006 Vallée et al., 2010 Walter et al., 2012 Phommasone et al., 2013 Dittrich et al., 2015b Lewthwaite et al., 1936 Marchette, 1966 Brown et al., 1977 Tee et al., 1999 Tay et al., 2000 Kho et al., 2016 Camer et al., 2003 Walter et al., 2012 Cross and Basaca-Sevilla, 1981 Walter et al., 2012 Loh et al., 1996 Chen et al., 2001 Ong et al., 2001 Aung et al., 2014 Chareonviriyaphap et al., 2014 Azuma et al., 2006 Hamaguchi et al., 2015 Ellis et al., 2006 McGready et al., 2010 Sumrandee et al., 2014

Rodent

Yes

Indonesia

Ibrahim et al., 1999

Rodent Rodent

Yes

Thailand Cambodia Indonesia

MITE Laelaps LOUSE Polyplax

Laos

Malaysia

Myanmar Philippines Singapore

Thailand Vietnam Thai-Myanmar Spotted fever

Rickettsia bellii Rickettsia conorii?

TICK Amblyomma varanense Unknown

47

Thai-Myanmar

Richards et al., 2003 Phongmany et al., 2006 Tay, 1996 Kho et al., 2017 vu Trung et al., 2017 Parola et al., 2003a Inpankaew et al., 2016 Jiang et al., 2006 Barbara et al., 2014 Widjaja et al., 2016 Varagnol et al., 2009 Kernif et al., 2012 Mayxay et al., 2013 Mokhtar and Tay, 2011 Kernif et al., 2012 Tay, 2013; Tay et al., 2015 Kho et al., 2016 Khoo et al., 2016 Wolf and Reeves, 2012 Foongladda et al., 2011 Edouard et al., 2014 Phoosangwalthong et al., 2018 Parola et al., 2003

TICK Haemaphysalis spp. H. bispinosa Rhipicephalus microplus R. sanguineus Unknown

Unknown

Unknown

Malaysia

Kho et al., 2017

Unknown

Yes

TICK Ixodes sp. Ixodes granulatus Rhipicephalus sp.

Rodent

Yes

Laos Thailand Thai-Myanmar Malaysia

Phongmany et al., 2006 Fournier et al., 2004 Parola et al., 2003b Tay et al., 1998; Tay et al., 2000; Tay et al., 2002a Tee et al., 1999 Tappe et al., 2018 Robertson and Wisseman Jr, 1973 Okabayashi et al., 1996 Kollars Jr et al., 2001 Jiang et al., 2005

Laos Malaysia

Rickettsia felis -like (including R. asembonensis)

FLEA Ctenocephalides felis C. orientis Xenopsylla cheopis

Cat Dog Macaque Rodent

Yes

Vietnam Thai-Myanmar Cambodia Indonesia

Laos MITE Laelaps sp. Polyplax sp. Haemogamasus

Malaysia

TICK Ixodes sp. Haemaphysalis sp. Rhipicephalus sp. R. microplus R. sanguineus

Rickettsia heilongjiangensis-like

Rickettsia helvetica

Rickettsia honei

Philippines Thailand

Thailand

48

Rickettsia hulinensis

Sangkasuwan et al., 2007 Kho et al., 2017

TICK Haemaphysalis spp. TICK Haemaphysalis hysticis

Unknown

Unknown

Malaysia

Dog Rodent

Yes

Laos Malaysia Philippines Thailand

Rickettsia monacensis-like

TICK Amblyomma integrum A. testudinarium Hyalomma lagrangei

Unknown

Unknown

Thailand

Taylor et al., 2016 Kho et al., 2018 Camer et al., 2000; Camer et al., 2003 Okabayashi et al., 1996 Gaywee et al., 2007 Takada et al., 2009 Malaisri et al., 2015

Rickettsia motana-like

TICK Hyalomma bispinosa TICK Haemaphysalis spp. H. bispinosa Rhipicephalus microplus R. sanguineus Amblyomma helvolum A. varanense Unknown

Unknown

Unknown

Thailand

Malaisri et al., 2015

Rodent

Yes

Malaysia

Tay et al., 2014b Kho et al., 2015; Kho et al., 2018 Doornbos et al., 2013 Sumrandee et al., 2014

Dog Rodent

Yes

Indonesia

TICK Haemaphysalis spp. Hyalomma bispinosa H. lagrangei Amblyomma integrum A. testudinarium TICK Ixodes granulatus TICK Amblyomma testudinarium TICK Haemaphysalis ornithophila TICK Haemaphysalis ornithophila

Unknown

Yes

Thailand Laos

Rickettsia japonica (including Rickettsia sp. TCM1 & Rickettsia sp. LON-13)

Rickettsia raoultii-like

Rickettsia rickettsii?

Rickettsia tamurae (including Rickettsia tamurae-like & Rickettsia sp. AT1 type strain)

Rickettsia thailandii Rickettsia sp. ATT Rickettsia sp. HOT1 Rickettsia sp. HOT2

Thailand

Malaysia Thailand

Richards et al., 2003 Widjaja et al., 2016 Suksawat et al., 2001 Phongmany et al., 2006 Taylor et al., 2016 Kho et al., 2015 Malaisri et al., 2015

Unknown

Unknown

Thailand

Kollars Jr et al., 2001

Unknown

Unknown

Thailand

Hirunkanokpun et al., 2003

Unknown

Unknown

Thailand

Hirunkanokpun et al., 2003

Unknown

Unknown

Thailand

Hirunkanokpun et al., 2003

49

Rickettsia sp. Kagoshima6

Rickettsia sp. RDla420 Rickettsia sp. RDla440 Candidatus Rickettsia johorensi Candidatus Rickettsia khammouanensis Candidatus Rickettsia laoensis Candidatus Rickettsia mahosotii

Anaplasma

Candidatus Rickettsia sepangensis Anaplasma bovis

Anaplasma capra Anaplasma centrale Anaplasma marginale

TICK Amblyomma testudinarium Haemaphysalis sp. TICK Dermacentor auratus TICK Dermacentor sp. TICK Amblyomma helvolum A. varanense TICK Haemaphysalis sp. TICK Haemaphysalis sp. TICK Amblyomma testudinarium Haemaphysalis sp. TICK Amblyomma varanense TICK Amblyomma varanense Haemaphysalis bispinosa H. langrangei H. obesa H. shimoga Rhipicephalus sanguineus Unknown TICK Rhipicephalus microplus TICK Haemaphysalis spp. Rhipicephalus microplus

Unknown

Unknown

Laos

Taylor et al., 2016

Unknown

Unknown

Thai-Myanmar

Parola et al., 2003a

Unknown

Unknown

Thai-Myanmar

Parola et al., 2003a

Unknown

Unknown

Malaysia

Kho et al., 2015

Unknown

Unknown

Laos

Taylor et al., 2016

Unknown

Unknown

Laos

Taylor et al., 2016

Unknown

Unknown

Laos

Taylor et al., 2016

Unknown

Unknown

Malaysia

Kho et al., 2015

Bear Goat Monkey

Yes

Malaysia

Kho et al., 2015 Koh et al., 2016; Koh et al., 2017; Koh et al., 2018 Tay et al., 2015 Malaisri et al., 2015 Sumrandee et al., 2016 Parola et al., 2003a Koh et al., 2018 Ybañez et al., 2013 Ha et al., 1997 Pong and Nik-Him, 2012 Rahman et al., 2012 Koh et al., 2017; Koh et al., 2018 Ola-Fadunsin et al., 2017 Ybañez et al., 2013 Ochirkhuu et al., 2015 Saetiew et al., 2015a; Saetiew et al., 2015b Jirapattharasate et al., 2017 Ha et al., 1997 Nguyen et al., 1999

Thailand

Cattle Cattle

Unknown Unknown

Buffalo Cattle

Unknown

Thai-Myanmar Malaysia Philippines Vietnam Malaysia

Philippines Thailand

Vietnam

50

Anaplasma phagocytophilum

TICK Rhipicephalus sanguineus

Anaplasma platys (including Anaplasma sp. strain AnDa465, strain AnAj360, strain AnH1446)

TICK Dermacentor auratus Haemaphysalis bispinosa H. langrangei Rhipicephalus sp. Rhipicephalus sanguineus

Cattle Deer Dog Lizards Cat Dog

Yes

Malaysia

Unknown

Malaysia

Dog Toddy cat Buffalo Cattle Deer Cattle Deer Pangolin

Unknown

Thai-Myanmar Indonesia

Unknown

Malaysia

Mokhtar et al., 2013 Koh et al., 2016; Koh et al., 2018 Low et al., 2018 Konto et al., 2017 Ybañez et al., 2012; Ybanez, 2013; Ybañez et al., 2016 Adao et al., 2017 Pinyoowong et al., 2008 Foongladda et al., 2011 Salakij et al., 2012 Liu et al., 2016 Saeng-Chuto et al., 2016 Sumrandee et al., 2016 Kaewmongkol et al., 2017 Parola et al., 2003a Malaisri et al., 2015 Hadi et al., 2016 Kho et al., 2018

Unknown

Malaysia

Kho et al., 2018

Unknown

Dog

Unknown

Malaysia Thai-Myanmar Cambodia Indonesia

Kho et al., 2015 Parola et al., 2003a Inpankaew et al., 2016 Hadi et al., 2016 Erawan et al., 2017 Nazari et al., 2013 Koh et al., 2015 Konto et al., 2017 Mohammed et al., 2017 Low et al., 2018 Baticados and Baticados, 2011 Ybañez et al., 2012; Ybanez, 2014; Ybañez et al., 2015

Philippines

Thailand

Ehrlichia

Anaplasma sp.

Unknown

Candidatus Anaplasma boleense

Unknown

Candidatus Anaplasma camelii Candidatus Anaplasma pangolinii Ehrlichia canis

Unknown TICK Amblyomma javanense TICK Rhipicephalus sp. R. sanguineus

Geurden et al., 2008 Koh et al., 2015; Koh et al., 2017; Koh et al., 2018

Malaysia

Philippines

51

Unknown

Vietnam Indonesia Malaysia Timor-Leste Thailand Vietnam Malaysia

Corales et al., 2014 Adao et al., 2017 Pinyoowong et al., 2008 Foongladda et al., 2011 Liu et al., 2016; Saeng-Chuto et al., 2016 Kaewmongkol et al., 2017 Nguyen et al., 2018 Richards et al., 2003 Koh et al., 2018 Burke et al., 2015 Suksawat et al., 2001 Parola et al., 2003a Koh et al., 2016

Unknown

Unknown

Malaysia

Koh et al., 2018

Unknown

Unknown

Laos

Taylor et al., 2016

Unknown

Unknown

Unknown

Unknown

Malaysia Thailand Vietnam Malaysia

Kho et al., 2017; Kho et al., 2018 Parola et al., 2003a Parola et al., 2003a Koh et al., 2018

Needlefish

Unknown

Cambodia Philippines Thailand Vietnam

Seng et al., 2009 Greiman et al., 2017 Greiman et al., 2017 Greiman et al., 2017

Fish

Yes

Laos

Newton et al., 2009 Dittrich et al., 2015a Vaughan et al., 2012 Vaughan et al., 2012 Bhengsri et al., 2016

Thailand

Ehrlichia chaffeensis (including Ehrlichia sp. strain EHh324)

TICK Haemaphysalis hystricis

Dog

Yes

Ehrlichia chaffeensis-like

TICK Rhipicephalus microplus TICK Rhipicephalus microplus TICK Haemaphysalis sp. TICK Haemaphysalis hystricis Rhipicephalus microplus TICK Rhipicephalus microplus TREMATODE Lecithodendrium sp. Paralecithodendrium sp. Saccocoelioides sp. S. lizae Stellantchasmus falcatus TREMATODE Digenea

Unknown

Ehrlichia mineirensis Ehrlichia sp.

Neorickettsia

Ehrlichia sp. strain EBm52 (including Ehrlichia sp. strain EHh317) Candidatus Ehrlichia shimanensis Neorickettsia sp.

Neorickettsia sennetsu

Malaysia Thailand

*Animal host herein refers to those infected with rickettsiae, but not the host of ectoparasites.

52