Wide distribution of Mediterranean and African spotted fever agents and the first identification of Israeli spotted fever agent in ticks in Uganda

Rickettsia microorganisms are causative agents of several neglected emerging infectious diseases in humans transmitted by arthropods including ticks. In this study, ticks were collected from four geographical regions of Uganda and pooled in sizes of 1–179 ticks based on location, tick species, life stage, host, and time of collection. Then, they were tested by real-time PCR for Rickettsia species with primers targeting gltA, 17kDa and ompA genes, followed by Sanger sequencing of the 17kDa and ompA genes. Of the 471 tick pools tested, 116 (24.6%) were positive for Rickettsia spp. by the gltA primers. The prevalence of Rickettsia varied by district with Gulu recording the highest (30.1%) followed by Luwero (28.1%) and Kasese had the lowest (14%). Tick pools from livestock (cattle, goats, sheep, and pigs) had the highest positivity rate, 26.9%, followed by vegetation, 23.1%, and pets (dogs and cats), 19.7%. Of 116 gltA-positive tick pools, 86 pools were positive using 17kDa primers of which 48 purified PCR products were successfully sequenced. The predominant Rickettsia spp. identified was R. africae (n = 15) in four tick species, followed by R. conorii (n = 5) in three tick species (Haemaphysalis elliptica, Rhipicephalus appendiculatus, and Rh. decoloratus). Rickettsia conorii subsp. israelensis was detected in one tick pool. These findings indicate that multiple Rickettsia spp. capable of causing human illness are circulating in the four diverse geographical regions of Uganda including new strains previously known to occur in the Mediterranean region. Physicians should be informed about Rickettsia spp. as potential causes of acute febrile illnesses in these regions. Continued and expanded surveillance is essential to further identify and locate potential hotspots with Rickettsia spp. of concern.


Introduction
Arthropod vectors transmit many pathogens to humans including Rickettsia which is a bacteria responsible for multiple emerging infectious diseases globally [1][2][3]. Clinical presentations of rickettsioses differ by Rickettsia group; the Spotted Fever Group (SFG) transmitted by ticks and the Typhus Group (TG) transmitted by fleas and lice. Seroprevalence rates for Rickettsia spp. vary widely and range from 8-10% in the East African region and <1-37% worldwide [4][5][6][7][8]. These pathogens are increasingly associated with undifferentiated febrile illnesses in humans potentially resulting in severe illness and/or death [9][10]. Several cases of SFG rickettsioses have been reported in international travellers returning to their home countries, particularly from endemic regions in sub-Saharan Africa and southeast Asia [11][12][13][14]. Historically, these diseases have been poorly studied in sub-Saharan Africa where the largest burden of disease exists, particularly in indigenous populations [15].
Rickettsioses manifest with non-specific signs such as fever, severe headache, skin rash and general malaise, which is often misdiagnosed as other febrile illnesses or viral diseases. Confirmation of a rickettsial infection requires direct molecular detection or serological testing to detect antibodies, potentially leading to a false negative results if testing occurs too early in the bacterial infection before antibodies are generated [12,[16][17]. Moreover, proper testing requires expensive equipment and reagents and relies on skilled laboratory technicians complicating the ability of countries with constrained resources to test for Rickettsia infections. Cases of febrile illnesses are often over diagnosed as malaria and later proven otherwise by more sensitive and specific PCR assays [18]. Distinguishing Rickettsia from other pathogenic agents early allows for timely treatment and informs any necessary public health measures.
Uganda is home to multiple medically relevant arthropod vectors including ticks, fleas, and mites. In two regions of the country, the northeast and southwest, diverse species of ticks have been recovered from both animals and the environment carrying medically relevant human and animal pathogens [19][20]. We recently reported the abundance and distribution of seven tick species of the Rhipicephalus, Haemaphysalis, and Amblyomma genera with rickettsial pathogens detected in the Ugandan cattle corridor [21]. Tick-borne Rickettsia spp., R. africae, R. conorii, and R. massiliae, of human relevance are prevalent in Uganda [22][23][24].
Rickettsia africae is the etiologic agent of African tick-bite fever (ABTF) transmitted predominantly by Amblyomma tick species. Infection with R. africae is the most common tick-borne bacterial zoonosis reported in travelers returning from sub-Saharan Africa [6,11]. Rickettsia conorii, mainly transmitted by Rh. sanguineus tick species, is endemic in the Mediterranean and causes Mediterranean spotted fever (MSF) [10]. Israeli spotted fever (ISF), a disease similar to but more severe than MSF, is caused by the subspecies R. conorii israelensis. Rickettsia conorii israelensis is also transmitted by Rh. sanguineus (sensu lato) [22]. Although these diseases are generally mild and manifest with the common characteristics of rickettsioses, infection often results in hospitalizations and delays the diagnosis of potentially co-infected febrile illnesses [6,22].
Tick population territories have changed significantly in the past decade largely due to anthropogenic and environmental changes resulting from climate change [10,[25][26]. An increase in densities of medically important vectors and pathogens is commonly associated with emergence of disease in humans, representing a major public health concern. This poses a risk to the 58% of Ugandans who derive their livelihoods through livestock keeping, predominantly kept on open grazing [27]. Limited knowledge about the ticks associated with SFG rickettsia transmission, their frequencies, and geographic range in Uganda creates challenges in designing appropriate control measures. While there is evidence of widespread Rickettsia spp. present throughout sub-Saharan Africa, there is limited data about the species and frequency in Ugandan ticks [21]. Therefore, it is essential to characterise ticks and their associated Rickettsia pathogens to better inform control strategies and contribute to our understanding of tick-borne diseases in Uganda.

Study sites
Ticks were collected from homesteads in five districts [Jinja (Eastern Uganda), Kampala (Capital of Uganda), Kasese (Western Uganda), Gulu (Northern Uganda) and Luwero (Central Uganda)] (Fig 1). The selected districts are considered major economic hubs in their respective regions and are geographically and culturally diverse, with high levels of economic heterogeneity. The source of the ticks were livestock (cattle, goats, sheep, pigs), companion animals (dogs and cats), chicken, and from the grass environment, collected between April 2017 and September 2018. Ticks collected from animals were picked with forceps and preserved in 70% ethanol. The ticks from vegetation were collected by dragging a 1m 2 white flannel cloth on vegetation midmorning (after dew drop) around homesteads where ticks from animals were collected. Depending on the size of area available around the homesteads, an approximate area covering at least 100m 2 (10 x10m or 100m long transect) were sampled as previously described [28][29]. Five transects about 2 meters apart were covered by a slow pace of dragging, checking the cloth every 15-20 paces. Ticks that attached to the cloth were picked with fine-tipped forceps (tweezer) and also preserved in 70% ethanol.

Tick pools
Ticks were identified to the species level using morphologic taxonomic keys [30] under a stereomicroscope. A total of 5,790 ticks were sorted into 471 pools (1-179 ticks per pool, average 12.3) according to tick species, host, collection area, date of collection and developmental stage. There were 306 pools (5,408 ticks) from livestock, 138 pools (677 ticks) from companion animals, 1 pool (1 tick) from a chicken, and 26 pools (64 ticks) from vegetation. The pools were placed in Eppendorf tubes containing RNA later (Sigma Life Science, Darmstadt, Germany) and disrupted using sterile disposable pestles attached to a motorized grinder (HLD-12, Ryobi, China). Ticks were then homogenized by passing them through 20-gauge needles, with homogenate then stored at -80˚C until DNA extraction.

DNA extraction and PCR
Total DNA was extracted from the tick homogenates using the Qiagen DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. Every batch of samples were extracted alongside two positive and two negative controls. All 471 tick pool DNA samples were screened for SFG Rickettsia spp. with primers amplifying the 74-bp citrate synthase (gltA) gene as previously described [31][32]. The primers (CS-F (5-TCGCAAATGTT-CACGGTACTTT-3) and CS-R (5-TCGTGCATTTCTTTCCATTGTG-3) were used with the Platinum Quantitative PCR SuperMix-UDG (ThermoFisher Scientific) PCR kit. Briefly, the qPCR conditions involved initial incubation of the reactions at 50˚C for 2 min, then denaturation at 94˚C for 2 min followed by 45 cycles of two-step amplification at 94˚C for 15 sec and 60˚C for 1 min in a 7500 Real-Time PCR System (Applied Biosystems, US). Only positive samples from the screening were subsequently tested for 115-bp segment of the 17kDa gene and ompA genes using the primers and methods previously described to confirm the initial PCR results [31]. For every run, two positive and two negative controls were set. The first two wells set were negative controls, followed by the templates (samples) and then the positive controls were set last (in the last wells). In between the templates, new pipette tips were used with regular changes of gloves. Rickettsia conorii DNA (provided by Walter Reed Army Institute of Research, Silver Spring, MD) was used as a positive control and ultrapure water as a negative control.

Sequencing and phylogenetic analysis
A 539 base pair amplicon for 17kDa and a 650 base pair amplicon for ompA gene was amplified as previously described [33] using Platinum taq (Thermo Fisher Scientific). Cycling conditions for ompA amplification were 95˚C for 2 min and 45 cycles of 95˚C for 30 sec, 42˚C for 35 sec, and 60˚C for 2 min. Cycling conditions for 17kDa amplification were 95˚C for 2 min and 45 cycles of 95˚C for 30 sec, 57˚C for 60 sec, and 72˚C for 2 min. PCR products were resolved on a 2% agarose gel and purified using the QIAquick PCR Purification Kit (Qiagen). Samples were sequenced on the SeqStudio (Thermo Fisher Scientific) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) according to the manufacturer's recommendations. Forward and reverse reads were aligned using CLC Genomics Workbench (Qiagen) and a consensus sequence for each gene was generated for BLAST analysis. Sequences of 17kDa and ompA genes and references from GenBank were imported and aligned in Geneious Prime 2022.11.0.14.1. The sequences were MAFFT aligned and exported to MEGA 10.2.6 [34] where maximum likelihood trees were created at 1,000 bootstrap iterations.

Mapping
Descriptive maps showing the collection sites were created in QGIS 3.28 [35]. The Uganda district shapefile is available at https://data.unhcr.org/en/documents/details/83043.

Statistical analysis
The probability of Rickettsia spp. detection from the pooled tick samples was estimated using detection rates; maximum likelihood estimation (MLE) and minimum infection rate (MIR) by collection district and tick species. Both MLE and MIR estimates and their corresponding confidence intervals were calculated accounting for individual pool sample sizes using the CDC's Mosquito Surveillance Software (https://www.cdc.gov/westnile/resourcepages/ mosqSurvSoft.html). A Pearson chi-squared test was used to detect any differences between the distributions of outcomes in different groups, with a p-value of <0.05 considered significant. Data were analyzed using STATA software, version 16.1 (StataCorp, College Station, TX).

Distribution of tick species by collection sites
Five tick species were identified from the collections across the five districts. Rhipicephalus genera ticks accounted for over half of collections from each district. The most abundant tick species was Rh. appendiculatus which constituted 30.6% of all tick pools collected, followed by Rh. decoloratus (28.2%) and the least collected tick by pool count was Rh. sanguineus (0.8%). Rhipicephalus sanguineus was collected from dogs in three districts (Gulu, Jinja, and Kampala) whereas the other four species (Rh. appendiculatus, Rh. decoloratus, A. variegatum and H. elliptica) were found on animals and environment in all the districts (Fig 2). The tick species variation per district was significant (χ 2 = 32.88, df = 20, p = 0.035). Seventy-nine tick pools could not be fully identified because they contained larvae and nymphs with incomplete body parts.

Rickettsia spp. identified by nucleotide sequences and phylogenetic analysis
Of the 86 tick pools positive from gltA and 17kDa, 48 pools of purified PCR amplicons were successfully sequenced. The nucleotide sequences obtained from 17kDa (~539 base pairs) and ompA (~650 base pairs) were compared to those available on NCBI GenBank database by BLASTn analyses. Sequence identity and phylogenetic trees are presented in Table 2

PLOS NEGLECTED TROPICAL DISEASES
subsp. israelensis was identified in one nymph pool from a cat. Rickettsia africae were recovered from all animal types excluding cats, with R. conorii detected in ticks removed from cattle, goats, dogs, and a cat. Rickettsia asembonensis was found in livestock, grass, and a dog and R. helvetica was detected in Rh. appendiculatus collected from the environment.
Ticks collected from all livestock species had similar sequences for R. africae, R. conorii, and R. asembonensis based on the 17kDa gene. The two sequences from the environmentally collected ticks were unique compared to previously published sequences. Based on the 17kDa sequences from this study, tick pools from Gulu, Jinja, Luwero and Kampala had identical homology with R. africae strain PELE, which was isolated from a human traveller in Brazil.   Values less than 70% were excluded from the tree. The legend shows the tick species from which the Rickettsia spp. shown in this tree were detected. The legend shows the source of isolation by tick species and tick host by symbol. If multiple source icons appear next to an accession number, the pool of ticks came from more than one source. *One sample, MZ545592.1 was isolated from human serum and not from a tick. All GenBank accession numbers beginning with OP were sequenced in this study.
https://doi.org/10.1371/journal.pntd.0011273.g003 Values less than 70% were excluded from the tree. The legend shows the tick species from which the Rickettsia spp. shown in this tree were detected. The legend shows the source of isolation by tick species and tick host by symbol. If multiple source icons appear next to an accession number, the pool of ticks came from more than one source. *One sample, MZ545592.1 was isolated from human serum and not from a tick. All GenBank accession numbers beginning with OP were sequenced in this study.

Discussion
A relatively high pool positivity rate (24.6%) for Rickettsia spp. was detected in this study. This Rickettsia spp. positivity is comparable to a similar study in Kenya that demonstrated 25% Rickettsia spp. prevalence in tick pools collected from livestock and camels in dispersed pastoral communities [36]. The highest positive pool detection rates among tick species in our study were in A. variegatum (48.8%), Rh. decoloratus (27.8%) and Rh. appendiculatus (21.6%). These ticks feed predominantly on cattle, sheep, goats and large wild ruminants [20]. There is evidence of up to 97% prevalence of R. africae in A. variegatum collected from cattle in Eastern parts of Uganda [32]. Similar to other findings from sub-Saharan Africa, our study confirms that Rickettsia spp. are likely present across Uganda where animals reside, posing a risk to over half of the Ugandan population that derive their livelihoods from animals [27,[37][38][39]. Rickettsia-positive ticks were found in every district, with higher MLEs and pool-positivity rates observed in northern and eastern regions of Uganda suggesting potential hotspots for Rickettsia spp. infections that need to be further investigated. The relative variation in infection rates by district could be explained by differences in livestock populations and intensity of acaricide use. Farms in central and western Uganda are more likely to have dairy cattle and use more acaricides compared to livestock farms in northern and eastern Uganda, which predominantly have indigenous cattle [40][41]. Acaracide usage could account for the greater tick-species diversity in Gulu as opposed to the other districts. Areas that spray acaracides on their livestock can reduce the number and diversity of susceptible tick species because of selective pressure. However, tick species that are resistant to acaricides, Amblyomma spp. and Rhipicephalus spp., are likely to persist in Uganda [42]. Amblyomma spp. are the major vector for R. africae in sub-Saharan Africa and are likely harboring many Rickettsia pathogens in northern Uganda with the highest MLE result. Potential resistance in this species may contribute to the maintenance of the highly prevalent R. africae in the northern region. Rhipicephalus ticks are the most prevalent in Uganda on livestock and were the most common tick genus collected from every district [41]. There have been detections of acaracide resistant Rhipicephalus spp. in northern Uganda brought by livestock movements [43]. The continued development of acaracide resistance in this genus would pose a large threat in Uganda as all Rickettsia spp. detected in this study were present Livestock trading could lead to the movement of ticks, potentially with acaracide resistance, across district or country borders.
While livestock rearing increases the risk for SFG rickettsia, another big industry in Uganda at risk for tick-borne diseases is tourism. One major tourism area in the Kasese district, Queen Elizabeth National park, has an estimated 34,000 visitors annually [44]. This environment is conducive to wildlife and domestic encounters increasing the risk for human contact with infected ticks. Specifically, ATBF has been reported in a Slovenian traveller returning from southwestern Uganda [14]. Surprisingly, Kasese district has the lowest MLE, given the large number of tourists visiting the area. Additional sampling should be done in this district to understand the risk of Rickettsia spp. to tourists as R. africae was not detected in this region, likely because only two A. variegatum ticks were collected. Travelers should be aware of potential illnesses associated with pathogens present in the region.
Multiple Rickettsia sequences (R. africae and R. conorii) from this study had high homology to sequences causing human illness in Brazil and Iran emphasizing the importance of monitoring these pathogens in Uganda. The R. africae strain, PELE, caused ATBF leading to hospitalization in a traveler from South Africa and was identical to 17kDa sequences from this study from multiple districts [11]. Of concern is the R. conorii subsp. israelensis sequence from Gulu district that matched a fatal human case from Iran [45]. The discovery of these pathogens in the most abundant tick species on livestock [41] poses a high risk of potential illness to Ugandans.
As the first report of R. africae in Uganda in Rh. appendiculatus and Rh. decoloratus in all collection districts aside from Kasese, there is a need for more surveillance of Rickettsia spp. among Rhipicephalus ticks. Especially because this study confirmed these two Rhipicephalus spp. are the most abundant livestock ticks in Uganda and they are the most multi-acaricide resistant ticks on animal farms [20,30]. This study also presents the first detection of R. conorii in Rh. appendiculatus and Rh. decoloratus ticks in Uganda, which could lead to MSF, especially since a common transmission route is contact with domestic animals [22]. Another causative agent of febrile illness, R. helvetica, was found for the first time in Uganda in Rh. appendiculatus and was unique to other published sequences. Interestingly, this tick was collected from vegetation and not from livestock. Rickettsia asembonensis was detected in two tick species, Rh. appendiculatus and Rh. decoloratus, but it is mostly flea borne. It has occasionally been detected in ticks with limited information about its pathogenicity in humans [46]. Additional studies on Rhipicephalus genera would be beneficial to understanding the scope of these Rickettsia spp. in these ubiquitous vectors across Uganda.

Limitations
Ticks were collected from five districts in Uganda to represent the four regions. However, the results from these districts may not be representative of Rickettsia spp. found within the entire respective region, so careful consideration was taken when extrapolating the results. Additionally, a limited number of ticks (64/5790) were collected from the vegetation and analyzed in 26/471 pools so minimal environmental conclusions were made in this study. Ticks were identified solely using morphology thus limiting the confidence of species identification. By pooling ticks, the MLE was immeasurable when 100% of tick pools were positive and MIR was immeasurable when 0% of tick pools were positive and this was noted in Table 2 using dashes (-). The PCR targets were designed for one species, Rickettsia, so coinfection was not assessed.

Conclusions
This is the first major study using targeted gene sequencing for Rickettsia spp. covering diverse ecological zones of Uganda. The detection of Rickettsia spp. in every surveyed district and in multiple tick species highlights the need to monitor the threat of rickettsial disease in these regions and develop rapid diagnostic tests. This was the first detection of the ISF agent in ticks in Uganda and the first identification of ATBF and MSF causative agents in Rh. appendiculatus and Rh. decoloratus ticks in Uganda. Clinicians must be informed of circulating Rickettsia spp. endemic to Uganda to timely and effectively detect, treat, and prevent human illness. Further tick-borne pathogen surveillance and seroprevalence studies are essential in Uganda to further characterize Rickettsia spp. which threaten Ugandans, travelers, and public health.