DISCUSSION
There have been a notable change in the epidemiology of rickettsial spotted fever globally and these could be attributed to warmer temperatures and increased relative humidity that have occurred in recent times due to cycles of drought and rains. These cycles stimulates increased physiological processes that in turn lead to rapid growth and ultimately accelerated reproduction. Other factors that could be responsible for the observed notable changes are;-i) infringement of humans into ecological territories that are ticks reserves as a result of increased human population, ii) increased human contact with ticks which are reservoirs and transmitters of tick-borne SFGR pathogens due to increased outdoor activities, iii) improved global trade and travels that have facilitated the distribution of tick vectors and their suitable animal hosts in a short space of time, iv) as well as migration of animals including birds which have the capacity to introduce exotic ticks and their pathogens into new territories.
Genetic materials of R. africae , R. parkeri and R. tamurae were the three SFGR that were detected amongst the ticks genera collected in this study. These three species are closely related and they are the etiologic agents of African and American tick bite fever that are very prevalent in the sub-Saharan African, United States of America and Brazil and rickettsiosis in Japan respectively (Bogovic et al., 2016). African tick bite fever is generally transmitted byAmblyomma ticks which servers as its host and wild rodents are it reservoir from which it is transmitted to it humans through ticks bite (Bogovic et al., 2016).
Even though rickettsial diseases are found globally, there is no one single tick-borne rickettsial diseases that is found all over the world rather they are restricted to a given geographical regions and are transmitted by ticks inhabiting the given region. Majority of the populace living in the sub-Saharan Africa might be seropositive toR. africae but hardly do they succumb to African tick bite fever as it is with travelers to the endemic regions of Africa. Sero-prevalence of R. africae in Cameroon is between 11.9% - 51.8% while in Senegal it ranges between 21.4% -51% (Ndip et al., 2004; Mediannikov et al., 2010; Consigny et al., 2005). In a group of 940 travelers to South Africa, majority (27%) of them had flulike symptoms as a result of contacting R. africae the etiologic agent of African tick-bite fever upon returning from their travel (Prabhu et al., 2011). Also Prabhu et al., (2011), reported a seroprevalence of 51.7% among inpatients identified with febrile fever who were tested for acute SFGR and TGR in Moshi, Tanzania. May June Thu1Unit of Risk Analysis and Management, Hokkaido University Research Center for Zoonosis Control, N 20 W 10, Kita-ku, Sapporo, 001-0020 Japan
2Laboratory of Parasitology, Faculty of Veterinary Medicine, Graduate School of Infectious Diseases, Hokkaido University, N 18 W 9, Kita-ku, Sapporo, 060-0818 Japan
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Yongjin Qiu

3Hokudai Center for Zoonosis Control in Zambia, School of Veterinary Medicine, University of Zambia, P. O. Box 32379, Lusaka, Zambia
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Keita Matsuno

4Laboratory of Microbiology, Faculty of Veterinary Medicine, Graduate School of Infectious Diseases, Hokkaido University, N 18 W 9, Kita-ku, Sapporo, 060-0818 Japan
5Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, N 18 W 9, Kita-ku, Sapporo, 060-0818 Japan
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Masahiro Kajihara

6Division of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, N 20 W 10, Kita-ku, Sapporo, 001-0020 Japan
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Akina Mori-Kajihara

6Division of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, N 20 W 10, Kita-ku, Sapporo, 001-0020 Japan
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Ryosuke Omori

7Division of Bioinformatics, Hokkaido University Research Center for Zoonosis Control, N 20 W 10, Kita-ku, Sapporo, 001-0020 Japan
8Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Saitama, 332-0012 Japan
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Naota Monma

9Department of Infection Control, Fukushima Medical University, 1 Hikarigaoka, Fukushima, 960-1295 Japan
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Kazuki Chiba

10Fukushima Institute for Public Health, 16-6 Mitouchi Houkida, Fukushima, 960-8560 Japan
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Junji Seto

11Yamagata Prefectural Institute of Public Health, 1-6-6 Toka-machi, Yamagata, 990-0031 Japan
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Mutsuyo Gokuden

12Kagoshima Prefectural Institute for Environmental Research and Public Health, 11-40 Kinko cho, Kagoshima, 892-0835 Japan
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Masako Andoh

13Laboratory of Veterinary Public Health, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065 Japan
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Hideo Oosako

14Kumamoto Prefectural Institute of Public-Health and Environmental Science, Uto-shi, Kumamoto, 869-0425 Japan
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Ken Katakura

2Laboratory of Parasitology, Faculty of Veterinary Medicine, Graduate School of Infectious Diseases, Hokkaido University, N 18 W 9, Kita-ku, Sapporo, 060-0818 Japan
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Ayato Takada

5Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, N 18 W 9, Kita-ku, Sapporo, 060-0818 Japan
6Division of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, N 20 W 10, Kita-ku, Sapporo, 001-0020 Japan
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Chihiro Sugimoto

5Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, N 18 W 9, Kita-ku, Sapporo, 060-0818 Japan
15Division of Collaboration and Education, Hokkaido University Research Center for Zoonosis Control, N 20 W 10, Kita-ku, Sapporo, 001-0020 Japan
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Norikazu Isoda

1Unit of Risk Analysis and Management, Hokkaido University Research Center for Zoonosis Control, N 20 W 10, Kita-ku, Sapporo, 001-0020 Japan
5Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, N 18 W 9, Kita-ku, Sapporo, 060-0818 Japan
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Ryo Nakao

2Laboratory of Parasitology, Faculty of Veterinary Medicine, Graduate School of Infectious Diseases, Hokkaido University, N 18 W 9, Kita-ku, Sapporo, 060-0818 Japan
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The majority of data on ATBF cases that have been documented to date have been obtained from tourists returning from countries to which it is endemic, most from the Southern Africa countries like Botswana, South Africa, and Zimbabwe (Nilssona et al., 2017; Prebhu et al., 2011; Bogovic et al., 2016; Socolovschi et al., 2007). Bogovic et al. (2016) reported a case of ATBF in a Slovenian traveler returning from Uganda. The 29-year-old Slovenian man without underlying illnesses sought care after returning from a two weeks visit to Uganda for fever, chills, pains and complained of a tick bite a day prior to departing the country. Lorusso et al. (2013) were the first people to report aboutR. africae in ticks in Uganda where R. conorii had previously been reported by Socolovschi et al. (2007) as being prevalent. Similarly, Angerami et al. (2018) reported ATBF on a Brazilian who visited South Africa upon his return to Brazil. He had eschar and symptoms characteristics of ATBF which was confirmed by both immunological and molecular diagnostic methods to be infected byR. africae. African tick bite fever had also been reported for the first time by Harrison et al. (2016) on an Austrian traveler to East Africa who acquired the disease through tick bite during a visit to Tanzania. ATBF is generally a mild disease and to date, there has not been any reported deaths attributed to infection of R. africae.However, just like R. parkeri rickettsiosis, the disease caused by R. africae is often associated with an inoculation eschar at the spot of attachment of the tick vector. Usually, the symptoms associated with ATBF normally appear many days after the development of the eschar and they are usually that of fever, headache, myalgia, regional lymphadenopathy and generalized rash in about 50% of the cases.
Amblyomma variegatum tick has been reported to be the vector ofR. africae with a prevalence of 97.1% in Uganda (Nakao et al., 2013). However, Waner et al. (2014), reported finding the DNA ofR. africae in Hyalomma detritum tick collected from a wild boar in Israel indicating that the spotted fever group rickettsia is not limited in distribution to the African continent nor to a given host tick. Similarly, Yssouf et al. (2014) reported the detection of R. africae in 90% of A. variegatum , 1% of R. appendiculatus and 2.7% of Rhipicephalus (Boophilus) microplusin study ticks collected from locally domesticated animals in the Union of the Comoros, as well as in 77.14% in A. variegatum ticks obtained from cattle imported into the country. Also, Maina et al. (2014) reported the detection of R. africae –genotype DNA in 92.6% of adult A. variegatum ticks collected from domestic ruminants in Kenya even though they found no evidence of the pathogen in blood specimens in the domestic animals sampled. R. africaegenetic materials have been detected by PCR from different species of ticks belonging to Amblyomma, Rhipicephalus, Hyalomma genera in several African countries such as Mali, Senegal, Guinea, Liberia, Sudan, Democratic Republic of Congo, Cameroon, Nigeria, Niger, Kenya and Burundi (Parola et al., 2013; Bogovic et al., 2016) and these reports are in consonant with our finding as the DNA of R. africae was detected in the different genera of ticks that we assessed.
R. parkeri a member of the spotted fever group rickettsia is the etiologic agent of American tick bite fever that is prevalent in the South and North America continents is transmitted by Amblyomma species. The spotted fever disease associated with the organism is characterized by eschar related ailments in humans which are similar to symptoms of Rocky Mountain spotted fever. The index rickettsiosis spotted fever case caused by R. parkeri was first recognized by Paddock et al., (2004) and ever since then; numerous cases have been identified and reported in many southeastern states of the USA (Kimita et al., 2016; Paddock et al., 2008). Cowdry, (1993), was the first to describe the finding of the organism in the tissues and eggs of female A. maculatum ticks that were collected in Jackson County, Missouri. However, Parker et al. (1963), isolated the organism for the first time from Gulf Coast ticks that were collected in South eastern Texas and ever since then, R. parkeri a SFG rickettsiae have been frequently detected in A. maculatum. However, R. parkerihas been detected in other tick species other than A. maculatumas Williamson et al., (2010), reported the detection of its DNA inD. variabilis in ticks removed from persons in Texas, USA.R. parkeri infections in dogs and cows have been described in southeastern United States. Infection of humans by R. parkeri in most cases is associated with a necrotic eschar at the point of inoculation after several days of an infected tick bite and it is usually with a low grade to moderate fever that is very similar to RMSF though less in severity. Some of the symptoms associated with R. parkeri rickettsioses are fever, inoculation eschar, macules or papules rashes, vesicles or pustules, petechiae on palms or soles, headache, myalgias, sore throat, lymphadenopathy, diarrhea, nausea or vomiting (Paddock et al., 2008). No case of R.parkeri rickettsiosis have however been reported in Central America though A. maculatum is widely distributed throughout the region even though a mild eschar-related rickettsiosis that is very akin to R. parkeririckettsiosis have been reported in a traveler who returned from Honduras (Paddock et al., 2008). Since the first human disease case caused by R. parkeri was reported by Paddock et al. (2004), numerous cases of rickettsioses caused by R. parkeri have been reported among persons residing in the ecological range of the vector tick A. maculatum in the USA. Infections and eschar associated illness with R. parkeri have been frequently reported in several Latin American countries such as Argentina, Brazil, and Uruguay, and the organism has been detected in A. tristeticks (Romer et al., 2011). For the first time, here we report the detection of genetic material of R. parkeri in the African continent and the epidemiological implications are not well known. However, because it has been documented as a human pathogen, its involvement in human cases in the study sites may not be unlikely as it may probably have gone undetected. We observed discordant phylogenetic assignments of the omp A and omp B genes of sample 188 as they were found to cluster with Candidatus_Rickettsia EU27216.1 and R. parkeri KY113110 respectively in Figs 4 and 5 and this was shown to be so with nucleotide and amino acid alignments as shown in Figures 2A to D.
Rickettsia sp. strain Ga-Seema is an incompletely described rickettsial that was detected from three fed adult maleRhipicephalus simus ticks collected from two donkeys in 2014 in Hlahlagane, Limpopo Province, South Africa by Halajian et al. (2018) which has not been reported previously and its pathogenic potential is currently unknown.
R. tamurae infection according to Imaokaa et al. (2013) is associated with symptoms such as mild local inflammatory signs like swellings, erythema, redness heat and pain. Symptoms of R. tamurae infection mimics cellulitis with increased serum titers of antibody against the organism (Halajian et al., 2018). Unlike most SFGR, infection with R.tamurae is not associated with high fever, generalized rash, lymphadenopathy as it is often seen in other spotted fever rickettsioses. R.tamurae was first isolated from A. testudinarium ticks in Japan and has the wild boar and domestic pigs as it primary host although it can also infest deer, cattle, other ungulates and domestic livestock as well as humans (Halajian et al., 2018; Imaokaa et al., 2011; Motoi et al., 2013). R. tamurae has been isolated from the skin biopsy specimen from wild boars and also in ticks (Halajian et al., 2018). It was previously thought to be non-pathogenic to humans until it was reported in human cases in Japan (Halajian et al., 2018) as well as in Laos where its involvement in spotted fever case was documented after a patient tested seropositive for the organism (Gaowa et al., 2013). Phylogenetic analyses of theomp A and omp B sequences of sample 209 assigned them asR. africae and R. tamurae respectively and homology search confirmed that omp A sequence is R. africae while theomp B sequence had 100% similarity with R. tamurae . We performed nucleotide and amino acid sequences alignments with the two sequences as shown in Figures 3A-D, the omp B showed complete homology with R. tamurae indicating that sample B209 is most highly R. tamurae in the omp B gene region while theomp A was closely related with R. africae . We are not sure if recombination did occur in the two genes in question. Further study like full genome sequencing is needed to elucidate this observation.R. tamurae has been associated with different Amblyommaspp. as reported by Blanco (Blanco et al.,2017) who detected the pathogen in screened nymphs of A. ovale tick collected from small mammals such as wild rodents and marsupials in Brazil while a recent report stated its detection in a Haemaphysalis megaspinosa tick (Blanco et al., 2017). However, this is the first report of R. tamurae- like pathogen, the agent of SFG rickettsiosis in Japan and some Far East Asian countries in A. variegatum tick collected from cattle in the African continent.
Ticks as well as the diseases they transmit have an ecological range constrained by animal host diversity, movement and climatic factors. The current rapid expansion of ticks and the diseases they transmit into new ecological niches can be attributed to increased mobility of pets and animal migration over long distances. Due to climate change, new and favorable niches are being created thus making the spread of ticks very rapid over a wide range of ecological zones (Nooroong et al., 2018). These might be possible factors why there is an increasing spread of ticks and tick-borne diseases globally. For example, in Germany,Dermacentor reticulatus has spread to over a large part of the country along with babesiosis that they transmit (Phongmany et al., 2006) just as the ecological range of Ixodes ricinus the agent that transmit anaplasmosis and Lyme borreliosis has extended greatly in Sweden of recent (Gaowa et al., 2013). Similarly, D. veriabilisthe host and vector of RMSF has spread to the North-East of United States of America (Berglund et al., 1995). The current climate change has been reported to be the reason for these observable spread of ticks and their vectored pathogens. With the change in global climate, increased interaction of humans with ticks and expansive global trade in animals as well as the migratory nature of animals (Heile et al., 2006; Berglund et al., 1995; CDC, 2019; Palomar et al., 2012; Sparagano et al., 2015), it is not unlikely that ticks-borne pathogens could be easily introduced into new ecological niches thus fueling the global spread of tick-borne diseases.