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Article

Ehrlichia Species in Dromedary Camels (Camelus dromedarius) and Ruminants from Somalia

by
Aamir M. Osman
1,2,3,*,
Ahmed A. Hassan-Kadle
2,4,
Marcos R. André
5,
Flávia C. M. Collere
1,
Amir Salvador Alabí Córdova
5,
Fabiano Montiani-Ferreira
1,6,
Thállitha S. W. J. Vieira
7,
Abdalla M. Ibrahim
2,4,
Abdulkarim A. Yusuf
4,8,
Rosangela Z. Machado
5,* and
Rafael F. C. Vieira
1,9,10,*
1
Vector-Borne Diseases Laboratory, Department of Veterinary Medicine, Universidade Federal do Paraná, Curitiba 80035-050, Brazil
2
Somali One Health Centre, Abrar University, Mogadishu P.O. Box 25, Somalia
3
Department of Animal Health and Veterinary Services, Ministry of Livestock, Forestry, and Range, Mogadishu, Somalia
4
Abrar Research and Training Centre, Abrar University, Mogadishu P.O. Box 25, Somalia
5
Vector-Borne Bioagents Laboratory (VBBL), Department of Pathology, Reproduction and One Health, Faculty of Agrarian and Veterinary Sciences, São Paulo State University (FCAV/UNESP), Jaboticabal CEP 14884-900, Brazil
6
Department of Veterinary Medicine, Universidade Federal do Paraná, Curitiba 80035-050, Brazil
7
Department of Chemistry, The University of North Carolina at Charlotte, Charlotte, NC 28223, USA
8
Department of Slaughterhouses, Somali Meat Company, Mogadishu, Somalia
9
Center for Computational Intelligence to Predict Health and Environmental Risks (CIPHER), The University of North Carolina at Charlotte, Charlotte, NC 28223, USA
10
Department of Epidemiology and Community Health, The University of North Carolina at Charlotte, Charlotte, NC 28223, USA
*
Authors to whom correspondence should be addressed.
Pathogens 2025, 14(1), 65; https://doi.org/10.3390/pathogens14010065
Submission received: 11 December 2024 / Revised: 24 December 2024 / Accepted: 9 January 2025 / Published: 13 January 2025
(This article belongs to the Section Bacterial Pathogens)
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Abstract

:
Ehrlichioses, caused by Ehrlichia species, are tick-borne diseases (TBDs) that affect animals and humans worldwide. This study aimed to investigate the molecular occurrence of Ehrlichia spp. in 530 animals (155 Dromedary camels, 199 goats, 131 cattle, and 45 sheep) in the Benadir and Lower Shabelle regions of Somalia. Blood DNA samples were tested for PCR targeting dsb and sodB genes of Ehrlichia spp. and PCS20 and map1 genes of E. ruminantium. The obtained sequences were submitted for phylogenetic analyses. Ehrlichia spp. were detected in 26.4% (140/530) of animals by dsb-PCR, with the highest prevalence in dromedary camels (54.8%), followed by cattle (29.8%), goats (7.0%), and sheep (4.4%). Dromedary camels, cattle, and goats had significantly higher infection odds compared to sheep (p < 0.05). Among dsb-PCR-positive samples, 76.9% (30/39) of cattle tested sodB-positive, while other species were negative. E. ruminantium was detected in 13.7% (18/131) of cattle by pCS20-PCR, but none were positive for the map1 gene. Phylogenetic analysis confirmed E. minasensis in camels, sheep, and goats and E. ruminantium in cattle, marking the first molecular evidence of E. minasensis in dromedary camels, sheep, and goats globally, and E. ruminantium in cattle from Somalia. These findings emphasize the need for further research on its economic and public health impact.

1. Introduction

Ehrlichioses are tick-borne diseases (TBDs) caused by at least six bacterial species of the genus Ehrlichia, namely E. canis, E. chaffeensis, E. ewingii, E. muris, E. ruminantium, and E. minasensis [1,2]. These diseases affect animals and humans worldwide, representing a growing public health concern [3].
Heartwater disease, caused by E. ruminantium, is a notifiable disease that is listed by the World Organization for Animal Health (WOAH) [4]. It is primarily transmitted by Amblyomma ticks and affects domestic and wild ruminants across sub-Saharan Africa and Caribbean islands. Goats and sheep are more susceptible than cattle, posing significant economic concerns [2,4,5]. Therefore, comprehensive attention should be given to its impact on both wildlife and domestic livestock.
Livestock play a significant economic role in Somalia as they serve as a vital source of food, a form of currency, and a means of generating export earnings. In the Lower Shabelle region, the predominant farming system is the traditional mixed-farming system. However, the development of livestock in these systems has been hindered by various factors, including TBDs [6]. Additionally, challenges such as poor livestock nutrition and management practices, as well as the lack of tailored disease control strategies that are adapted to the unique characteristics of these systems, further contribute to the limitations faced in livestock development. Addressing these challenges and implementing effective disease control measures are crucial for improving the overall health and productivity of livestock in Somalia’s traditional mixed farming systems.
Globally, ehrlichioses have been extensively studied in dogs, but they have been mostly neglected in other animal species. In ruminants and dromedary camels, few studies have described the presence of Ehrlichia spp. Notable instances include cattle from Canada (Ehrlichia spp.) [7], Brazil (E. minasensis) [8,9,10], France (E. minasensis) [11], Pakistan (Ehrlichia sp. (Multan) and E. minasensis) [12], Ethiopia (E. ruminantium and E. minasensis) [13], South Africa (E. canis and Ehrlichia spp.) [14], and Kenya (E. minasensis) [15]. Additionally, small ruminants from the Caribbean islands (E. canis) [16], China (E. canis and Ehrlichia spp.) [17], Kenya, Gambia, Senegal, South Africa, Sudan, Uganda, and Malawi (E. ruminantium) [18], Italy (E. canis) [19], and Malawi (E. canis) [20], as well as dromedary camels from Kenya (‘Candidatus Ehrlichia regneryi’; E. ruminantium) [21,22] and Saudi Arabia (E. canis related strains) [23], have also been identified as hosts for Ehrlichia spp.
The distribution of ehrlichiosis is related to the environmental requirements of the tick vectors [24]. Molecular evolution analysis suggests that E. minasensis likely originated from highly variable strains of E. canis [1], and it has been reported in various locations, including Canada [7], Brazil [8], France [11], Pakistan [12], Ethiopia [13], South Africa [14], and Kenya [15]. It has also been detected in several tick species, including Rhipicephalus sp., Hyalomma sp., and Amblyomma sp., suggesting that several tick species may vector this agent [11,12,25,26]. In Somalia, six tick species, namely Rhipicephalus pulchellus, Hyaloma truncatum, Hyaloma dromedarii, Ambyomma gemma, Ambyomma lepidum, and Ambyomma variegatum, have been commonly identified and widespread [27,28]. However, no survey on Ehrlichia infection has been reported to date. Accordingly, this study aimed to perform a comprehensive molecular survey for Ehrlichia sp. in ruminants and dromedary camels from Somalia.

2. Material and Methods

2.1. Study Area and Design

A cross-sectional study was carried out between December 2018 and March 2022 in the Benadir (2.1065° N, 45.3933° E) and Lower Shabelle Regions (1.8670° N, 44.5502° E), Somalia. For this purpose, a non-probabilistic convenience sampling was performed. A total of 131 cattle (Bos taurus indicus), 199 goats (Capra aegagrus hircus), 45 sheep (Ovis aries), and 155 dromedary (Camelus dromedarius) blood samples were collected by jugular venipuncture. Animals were physically restrained, and blood samples (5 mL) were collected by venipuncture of the jugular vein using vacuum tubes containing EDTA (BD Vacutainer®, Franklin Lakes, NJ, USA). A total of 125 µL of blood samples from 104 dromedaries, 199 goats, and 45 sheep were applied on filter paper (FTA® card, Whatman®, GE Healthcare, Buckinghamshire, United Kingdom), while EDTA blood samples from 131 cattle and 51 dromedaries were collected and stored at −20 °C until molecular analysis. Samples were retrieved from previous studies [28,29].

2.2. DNA Extraction

DNA was extracted from dried blood spots (DBS) on filter paper (FTA® card, Whatman®, GE Healthcare, Buckinghamshire, United Kingdom) from 104 dromedary camels, 199 goats, and 45 sheep, and from 200 μL of whole blood from 131 cattle and 51 dromedary camels using a commercial kit (IndiMag® Pathogen Kit, Qiagen for Indical Bioscience, Leipzig, Germany), according to the manufacturer’s instructions.
For DBS, three discs were removed from each sample using a three mm Punch (Harris Uni-Core™, Qiagen, Hilden, Germany) and placed into clean RNase/DNase-free 1.5 mL microtubes. Pre-treatment was performed by adding 200 µL of Phosphate-buffered saline (PBS) to the macerated FTA discs using an 18-gauge needle and heating for 10 min at 96 °C. Filter paper discs without blood and RNase/DNase-free water were used in parallel as a negative control to monitor cross-contamination. DNA samples were stored at −20 °C before PCR assays.
EDTA blood samples from 131 cattle and 51 dromedaries stored in DNase/RNase-free microtubes were subjected to DNA extraction using the aforementioned commercial kit, according to the manufacturer’s recommendations. Ultra-pure water was used as negative controls to monitor cross-contamination in each batch of extraction.

2.3. Polymerase Chain Reactions (PCR) Assays

Conventional PCR for the mammal endogenous gene glyceraldehyde-3-phosphate dehydrogenase (gapdh) was performed in all samples to monitor DNA extraction [30]. DNA samples were initially screened by a nested-PCR assay based on the 349 bp of the disulfide bond formation protein gene (dsb) gene from Ehrlichia spp., as previously described [31]. Additionally, to further molecularly characterize Ehrlichia spp., PCR-positive samples were subjected to a PCR assay targeting 300 bp of the sodB gene [32]. DNA of E. canis (Jaboticabal strain) was used as a positive control, and ultrapure nuclease-free water was used as a negative control in all analyses.
The screening for E. ruminantium for cattle DNA was performed using a semi-nested PCR targeting a fragment of 280 bp of ribonuclease III (pCS20) gene, as previously described [33]. For further molecular characterization, E. ruminantium-positive samples were subjected to a semi-nested PCR assay to amplify a fragment of 720–738 bp of the map1 gene of E. ruminantium [33]. DNA of E. ruminantium obtained from naturally infected cattle from Mozambique [34] and ultrapure nuclease-free water were used as positive and negative controls, respectively.
The amplified products were subjected to electrophoresis in 1.8% agarose gels stained with 1% ethidium bromide (Life Technologies®, Carlsbad, CA, USA) for 50 min at 90 V. The gels were photographed under ultraviolet light using Image Lab Software (Thermo Fisher Scientific®).

2.4. DNA Sequencing and Phylogenetic Analyses

Twenty-seven positive PCR products of the expected size for each assay were purified using the Promega Wizard® PCR and Gel Clean-Up and sequenced in both directions using the same PCR primers (forward and reverse) by Sanger sequencing [35].
A maximum likelihood phylogenetic tree was performed by incorporating sequences from the present study and relevant sequences sourced from the GenBank® database (http://www.ncbi.nlm.nih.gov/genbank accessed on 15 March 2024). To ensure the quality of electropherograms and generate consensus sequences, Geneious prime 11.1.3 software was employed. The consensus sequences were subjected to multiple alignments with the sequences selected from GenBank® using MAFFT online software (https://mafft.cbrc.jp/alignment/server/ accessed on 16 March 2024). The best-fit model of nucleotide substitution was determined using jModeltest v.2.1.10 [36] and was set as GTR +G based on the Akaike Information Criterion (AIC). The phylogenetic tree was visualized with FigTree software version 1.4.4.

2.5. Statistical Analysis

Data analyses were performed with SPSS Statistics software® (IBM Corp, Armonk, NY, USA, version 26). The chi-square test was used to evaluate significant differences in the infection rate of different species of animals. Odds ratios (ORs), 95% confidence intervals (95% CI), and p-values were calculated separately for each variable, and the results were considered significant when p ≤ 0.05. Data were compiled and analyzed in Epi Info™ software, version 7.2.3.1 (Centers for Disease Control and Prevention, CDC, USA).

3. Results

The gapdh gene was consistently amplified from all DNA samples. Overall, 140/530 (26.4%, 95% CI: 22.7–30.4%) animals tested positive for Ehrlichia sp. by the dsb-PCR assay. Notably, a high occurrence of Ehrlichia sp. was observed in dromedary camels (85/155; 54.8%). Among the cattle analyzed, 39/131 (29.8%) tested Ehrlichia-positive, while 4/199 (7.0%) goats and 2/45 (4.4%) sheep (Table 1). Dromedary camels (OR: 26.1, p < 0.001, χ2 = 36.0), cattle (OR: 9.1, p < 0.001, χ2 = 12.1), and goats (OR: 1.6, p = 0.81, χ2 = 0.4) were more likely to be infected with Ehrlichia sp. than sheep.
Further analysis using sodB gene-based PCR revealed that out of the 39 Ehrlichia-positive cattle by the dsb-PCR assay, 30 (76.9%) also tested positive for the sodB gene. However, samples from dromedary camels, goats, and sheep that tested Ehrlichia-positive by the dsb-PCR assay tested negative by the sodB-PCR assay (Table 1).
Twelve out of the thirty-nine (30.8%) Ehrlichia-positive cattle were infested by ticks. However, no significant association was observed between Ehrlichia sp. positivity and the presence of ticks (OR = 1.5; 95% CI: 0.6–3.4; p = 0.48). Conversely, for dromedary camels, 23/85 (27.1%) identified as Ehrlichia-positive animals were infested by ticks, with an association between Ehrlichia sp. positivity and tick infestation (OR = 0.16; 95% CI: 0.08–0.32; p < 0.001). It is noteworthy that there was no association between Ehrlichia positivity and the presence of ticks in goats and sheep.
Moreover, 18 out of 131 (13.7%, 95% CI: 8.3–20.8) cattle tested positive for the pCS20 gene of E. ruminantium. Notably, all positive samples subjected to the E. ruminantium map1 gene assay tested negative. Among the 18 animals testing positive for E. ruminantium, 11 were concurrently infested by ticks. A statistically significant association was observed between E. ruminantium positivity in cattle and the presence of ticks (OR = 6.7; 95% CI: 2.3–9.4; p = 0.001).
In total, 28 amplicons were sequenced: eighteen from cattle (nine Ehrlichia sp. dsb genes, eight Ehrlichia sodB genes, and one E. ruminantium PCS20 gene), four from goats (all Ehrlichia sp. dsb genes), one from sheep (Ehrlichia sp. dsb gene), and five from dromedary camels (all Ehrlichia sp. dsb gene). The BLASTn results for all agents and target genes are shown in Table 2.
The phylogenetic analysis based on the Ehrlichia dsb gene (390 bp alignment) was performed using sequences detected in cattle, goats, sheep, and dromedary camels and selected sequences of Ehrlichia sp. obtained from the GenBank® database. The obtained sequences from studied animals grouped with Ehrlichia sp., (GenBank® KT314243, MT135766, KX595269) and E. minasensis (GenBank® JX629808) sequences detected in ticks from Czech Republic and Brazil are shown in Figure 1.
The phylogenetic analysis based on the Ehrlichia sodB gene (300 bp alignment) was performed using sequences detected in cattle and selected sequences of Ehrlichia sp. obtained from the GenBank® database. The obtained sequences from studied animals grouped separately are shown in Figure 2.
The phylogenetic analysis based on the E. ruminantium pCS20 gene (280 bp) was performed using sequences detected in cattle and selected sequences of E. ruminantium obtained from GenBank® confirmed the BLASTn analysis. The obtained sequences from studied animals placed within the East and South African clades are shown in Figure 3.

4. Discussion

Ehrlichioses are regarded as one of the most economically important TBDs in sub-Saharan Africa and Caribbean islands [2,5]. However, the current economic consequences of ehrlichioses in Somalia remain undetermined, primarily due to the absence of knowledge regarding the occurrence, distribution, and genetic diversity of their causative agents. In this study, we present the first molecular identification of Ehrlichia spp. in ruminants and dromedaries from Somalia. To detect the prevalence and spatial distribution of Ehrlichia spp., we employed molecular assays targeting Ehrlichia spp. based on both the dsb and sodB genes, along with the species-specific identification of E. ruminantium based on the pCS20 gene specifically for cattle.
The highest prevalence for Ehrlichia was observed in dromedaries (54.8%), followed by cattle (29.8%), goats (7%), and sheep (4.4%), respectively. This prevalence differs from previous investigations, such as those conducted in the Caribbean islands, where 385 cattle (19.7%), 340 sheep (13.2%), and 376 goats (3.5%) were positive in a PCR targeting the 16S rRNA gene [17]. In China, a lower prevalence was reported, with 1830 cattle (3.6%), 111 sheep (1.8%), and 270 goats (1.1%) positive in a PCR targeting the 16S rRNA gene [17]. Consistently, our study aligns with Zhang [16] and Qiu [17], reporting consistency in ruminants, where cattle had the highest prevalence. Notably, a study sampling 196 cattle, 200 sheep, and 198 goats from Sudan and using a PCR based on a 16S rRNA gene demonstrated a complete absence of Ehrlichia sp. positivity [37]. Factors such as geographic location, climatic conditions, presence of tick vectors, and population dynamics could contribute to these observed differences in prevalence rates.
The prevalence for Ehrlichia in cattle found herein (29.8%) differs from previous investigations, such as the study conducted in apparently healthy dairy cattle in Kenya, where 3.3% of the 306 blood DNA samples analyzed were positive in a PCR based on the 16S rDNA [15]. Conversely, a study in Brazil reported a higher prevalence, with 48.12% of 347 blood samples from beef cattle in the Brazilian Pantanal testing positive in a PCR targeting the dsb gene [8]. While our study detected E. minasensis and E. ruminantium in cattle, E. minasensis was identified in cattle from Brazil [8] and E. minasensis was reported in Kenya [15].
In this study, we observed occurrences of Ehrlichia in goats and sheep of 7% and 4.4%, respectively. The prevalence found in our study differs from that reported in other regions, such as the Caribbean islands, where sheep showed a prevalence of 13.2% and goats showed 3.5% [16]. Additionally, studies conducted in China reported a prevalence of 1.8% in sheep and 1.1% in goats [17], with both studies showing a higher prevalence in sheep than in goats. Herein, while E. minasensis was identified in both goats and sheep, the Caribbean study found E. canis, E. ovina, Ehrlichia sp. (BOV2010, UFMG-EV), and E. ruminantium, based on identities (98–100%) inferred from 16S rRNA and gltA genes [16]. A study from China reported E. canis in goats based on the gltA identity (98%) [17]. Studies in Malawi reported E. ruminantium and E. canis detection based on 16S rDNA and groEL genes [20]. Additionally, a study in Italy detected E. canis in sheep and goats based on the 16S rRNA gene [19].
Out of the 155 dromedaries’ blood samples analyzed, 54.8% were found positive for the Ehrlichia sp. dsb gene. This prevalence differs from previous investigations. For instance, a study conducted on 296 apparently healthy dromedaries in Kenya reported an occurrence of 14.5% for ‘Candidatus Ehrlichia regneryi’ using a PCR based on a 16S rDNA gene [21]. Similarly, a study in Saudi Arabia reported 26% positivity among 100 blood samples from apparently healthy dromedaries testing positive for ‘Candidatus Ehrlichia regneryi’ in a PCR targeting the 16S rRNA and groEL genes [23]. Herein, we identified E. minasensis based on the Ehrlichia sp. dsb gene, with identity ranging from 98.7 to 100%.
The observed variations in Ehrlichia prevalence among the studied animal populations in the present study compared to other studies underscore regional and species-specific differences. Factors like geographic variations, climate conditions influencing tick distribution, the health status of sampled populations, the genetic diversity of Ehrlichia strains, and differences in tick vectors and host species contribute to these inconsistencies. These multifaceted factors underscore the complexities of tick-borne diseases, emphasizing the need for comprehensive, context-specific investigations to understand Ehrlichia infection dynamics.
Subsequently, after confirming positive results for the Ehrlichia sp. dsb gene, further characterization was conducted by testing positive samples for the sodB gene. Of the 39 positive cattle samples, 30 (76.9%) were positive for the sodB gene, indicating a substantial prevalence of Ehrlichia spp. infection. However, samples from goats, sheep, and dromedaries that tested positive for Ehrlichia spp. based on the dsb gene were negative in the sodB gene-based assays. This disparity suggests potential variations in Ehrlichia strains or species infecting different hosts, underscoring the need for further molecular investigations to elucidate diversity and host-specificity. Alternatively, negative sodB results in other animals may be due to assay sensitivity, potentially indicating bacteremia levels below detection limits. Furthermore, partial dsb and sodB gene sequences from cattle showed inconsistencies in BLAST analysis, suggesting that these sequences may represent different regions of the dsb gene of Ehrlichia spp. It is also important to note that fewer sodB sequences are available in GenBank compared to dsb sequences, which may account for some of the observed discrepancies.
Recent studies have unveiled a novel pathogen, E. minasensis, initially reported in Canada [7] and Brazil [38] and subsequently identified in various countries, including Brazil [8], France [11], Pakistan [12], Ethiopia [13], South Africa [14], and Kenya [15]. Molecular analyses have revealed the genetic similarity of these strains, suggesting their evolution from a diverse clade within E. canis [39]. Phenotypic and genotypic differences from E. canis are attributed to the pathogen’s adaptation to new hosts including ruminants and tick vectors [16]. This study detected E. minasensis DNA in all animal species studied, including cattle, goats, sheep, and dromedaries. Notably, to the best of the authors’ knowledge, this study revealed, for the first time, the presence of E. minasensis in sheep, goats, and dromedaries globally. Such identification was based on Ehrlichia sp. dsb gene sequencing, exhibiting a high degree of identity (98.5–100%) and the phylogenetic positioning of the detected genotypes.
Ehrlichia minasensis has been identified in several tick species, including R. appendiculatus, R. evertsi eversi, R. sanguineus, Amblyomma sp. [14], R. microplus [40], and Hyalomma sp. [12]. The transstadial transmission of E. minasensis has been demonstrated specifically in R. microplus [26]. Considering that this pathogen was found in different tick species, including Amblyomma sp. and Hyalomma sp., which were found to be parasitizing the studied animals as previously reported for cattle by Ferrari [28], this emphasizes the need for further investigation into the role of these ticks in the transmission dynamics of E. minasensis and its prevalence in various livestock species. Moreover, E. minasensis infection in cattle, as demonstrated experimentally, presents clinical signs including fever, lethargy, anorexia, anemia, leukopenia, thrombocytopenia, and bleeding, with symptoms resembling chronic canine ehrlichiosis [41,42]. These findings underscore the importance of exploring its prevalence in different livestock species and understanding its impact on animal health.
In the present study, 13.7% of cattle were positive for E. ruminantium based on the pCS20 gene. This prevalence is notably consistent with findings in Maputo Province, Mozambique, where 15% of 210 cattle samples tested positive [34], and in Gambia, where 16.6% of 145 adult Amblyomma variegatum ticks collected from indigenous cattle were positive [33]. Conversely, a study in Kenya, utilizing RLB analysis on 453 calves, reported a considerably lower prevalence of 0.4% [43]. Similarly, in Ethiopia, 0.6% of 457 blood samples from cattle across four localities tested positive for the pCS20 gene of E. ruminantium [44]. The endemic stability of heartwater in most sub-Saharan African countries, as highlighted by Deem [45], contributes to generally low infection rates in ticks within the field [46]. Infected animals often remain asymptomatic, harboring the pathogen at minimal levels [44]. Given the intracellular localization of E. ruminantium predominantly in endothelial cells and its periodic presence in the bloodstream, the observed low prevalence rates based on PCR assays from blood samples align with expectations [47,48]. Notably, none of the samples positive for the pCS20 gene were positive in the PCR assays targeting the map1 gene, likely due to low bacteremia levels below the detection limit of the molecular assays. The pCS20 gene, specific to E. ruminantium, is more sensitive to detection, while the map1 gene is commonly used for diagnosing and characterizing various genotypes of the pathogen [49].
Heartwater occurs in wild and domestic ruminants where the tick vectors, primarily Amblyomma species, are present, predominantly in Africa. Amblyomma variegatum and Amblyomma hebraeum represent the two most important tick vectors associated with the distribution of disease in Africa [50]. In Somalia, A. variegatum has been identified in cattle, goats, sheep, and dromedaries [27,51], but A. hebraeum, to the best of our knowledge, has not been reported yet. Notably, the ticks collected from the studied animals did not include A. variegatum and A. hebraeum, suggesting the possible transmission of E. ruminantium by ticks other than these two species. Further investigation is necessary to understand the significance of this pathogen for cattle health and to identify the vectors responsible for its transmission in Somalia.
In this study, only one cattle sample was successfully sequenced for the E. ruminantium PCS20 gene. The partial sequence obtained showed a high identity of 98.5% with a sequence identified in A. variegatum ticks from Uganda (MK371032). Phylogenetic analysis of the PCS20 gene indicated that the positive case in this study closely resembled E. ruminantium isolated from A. gemma ticks collected in Ethiopia (GU644448), placing our sequences within the East and South Africa clade.
Ehrlichia minasensis and E. ruminantium are the only species of Ehrlichia known to naturally infect cattle [18]. The cattle sampled in this study showed a 16.3% rate of co-infection by E. minasensis and E. ruminantium. The coexistence of these pathogens may contribute to heightened disease complexity and severity, potentially influencing clinical manifestations, immune responses, and overall health outcomes in the infected cattle [13]. This underscores the necessity of considering multiple Ehrlichia spp. in the diagnostic and management strategies for diseases affecting cattle populations. Further research is needed to clarify the specific interactions between E. minasensis and E. ruminantium, shedding light on their implications for animal health.
It is important to acknowledge a key limitation regarding the construction of the phylogenetic tree. Due to sequencing constraints, we were only able to successfully sequence one sample for the E. ruminantium PCS20 gene. While this limitation narrows the scope of our analysis, it also underscores the need for further research with larger sample sizes to provide a more comprehensive understanding. Nonetheless, our study’s focus on exploring the genetic diversity within E. ruminantium remains a notable strength, pointing toward paths for future research to explore deeper into the evolutionary dynamics of this pathogen within our target population.
In conclusion, this study represents a significant step toward understanding the prevalence and genetic diversity of Ehrlichia sp. in ruminants and dromedaries from the Lower Shabelle region of Somalia. The detection of Ehrlichia sp. in 26.4% of the sampled animals underscores the substantial prevalence of this infection in the studied population. This is the first report of E. minasensis in cattle from Somalia and the first report of this agent in sheep, goats, and dromedaries worldwide. Furthermore, the identification of E. ruminantium in 13.7% of the samples adds valuable insights into the epidemiology of heartwater disease in Somalia.

Author Contributions

Conceptualization, A.M.O., A.A.H.-K., M.R.A., T.S.W.J.V., R.Z.M. and R.F.C.V.; methodology, all authors collected the data and carried out the methodology; funding acquisition, A.A.H.-K., M.R.A., T.S.W.J.V., R.Z.M. and R.F.C.V.; data analysis, A.M.O., M.R.A., T.S.W.J.V., R.Z.M. and R.F.C.V.; validation, M.R.A., T.S.W.J.V., R.Z.M. and R.F.C.V.; original draft preparation, A.M.O.; writing, A.M.O.; review and editing, M.R.A. and R.F.C.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Process #2021/12198-6), Abrar University (grant no. #AURG04012017), UFPR (grant no. #23075.042760/2021-59), and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) Productivity Fellowship conceived to MRA (Process CNPq nº 303701/2021-8) and RZM (Process #302961/20194).

Institutional Review Board Statement

The animal study protocol was approved by the ethical committee of Abrar University, Somalia (reference number for dromedary, sheep, and goats: AU/ARTC/EC/10/2018; for cattle: AU/ARTC/EC/06/2019).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data underpinning the conclusions of this study can be obtained from the corresponding authors upon reasonable request.

Acknowledgments

This study is part of a PhD degree for Aamir M. Osman at the Universidade Federal do Paraná (UFPR). Aamir M. Osman and Flavia Collere were sponsored by a fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) at the time of this study. Ahmed A. Hassan-Kadle acknowledges The World Academy of Sciences (TWAS), UNESCO, and the Islamic Development Bank (IsDB) for their support through the IsDB-TWAS postdoctoral fellowship programme in Sustainability Sciences (grant no. 15/2020) at the Universidade Federal do Paraná, Brazil, during this research. The authors also thank Indical Bioscience for having provided DNA extraction kits for this research.

Conflicts of Interest

The authors have no competing financial interests to declare.

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Pathogens 14 00065 g001
Figure 1. Phylogenetic tree inferred by using maximum likelihood inference and GTR+G evolutionary model based on an alignment of 390 bp of the dsb gene. The sequences detected are highlighted in red in the present study. Ehrilichia muris was used as the outgroup.
Figure 1. Phylogenetic tree inferred by using maximum likelihood inference and GTR+G evolutionary model based on an alignment of 390 bp of the dsb gene. The sequences detected are highlighted in red in the present study. Ehrilichia muris was used as the outgroup.
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Pathogens 14 00065 g002
Figure 2. Phylogenetic tree inferred by using maximum likelihood inference and GTR+G evolutionary model based on an alignment of 300 bp of the sodB gene. The sequences detected are highlighted in red in the present study. The numbers at the nodes correspond to posterior probability values higher than 50% accessed with 1000 replicates. A. marginale was used as outgroups.
Figure 2. Phylogenetic tree inferred by using maximum likelihood inference and GTR+G evolutionary model based on an alignment of 300 bp of the sodB gene. The sequences detected are highlighted in red in the present study. The numbers at the nodes correspond to posterior probability values higher than 50% accessed with 1000 replicates. A. marginale was used as outgroups.
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Pathogens 14 00065 g003
Figure 3. Phylogenetic tree inferred by using maximum likelihood inference and GTR+G evolutionary model based on an alignment of 280 bp of the E. ruminantium PCS20 gene. The sequences detected are highlighted in red in the present study. The numbers at the nodes correspond to posterior probability values higher than 50% accessed with 1000 replicates. E. canis was used as the outgroup.
Figure 3. Phylogenetic tree inferred by using maximum likelihood inference and GTR+G evolutionary model based on an alignment of 280 bp of the E. ruminantium PCS20 gene. The sequences detected are highlighted in red in the present study. The numbers at the nodes correspond to posterior probability values higher than 50% accessed with 1000 replicates. E. canis was used as the outgroup.
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Table 1. Prevalence of Ehrlichia spp.
Table 1. Prevalence of Ehrlichia spp.
dsb GenesodB Gene
Animal Species+/n95% CI+/n95% CI
Dromedary85/15554.8 (46.7–62.8%)0/850
Cattle39/13129.8%, (22.1–38.4%)30/3976.9 (60.7–88.9%)
Goat4/1997.0% (3.9–11.5%)0/40
Sheep2/454.4% (0.5–15.2%)0/20
Table 2. Percentage of BLASTn-associated identity of sequences of Ehrlichia spp. detected in ruminants and dromedary camels from Somalia.
Table 2. Percentage of BLASTn-associated identity of sequences of Ehrlichia spp. detected in ruminants and dromedary camels from Somalia.
AgentsAnimal IDAccession NumberTarget GeneQuery CoverE-ValueIdentityGenBank® Accession Number
Ehrlichia sp. Cattle 3PQ041218dsb100%2 × 10−16199.4%E. minasensis collected from Rhipicephalus microplus from Panama (OR711253)
Ehrlichia sp. Cattle 19PQ041219dsb98%3 × 10−16499.1%E. minasensis collected from Bos Taurus from Australia (MH500007)
Ehrlichia sp.Cattle 56PQ041220dsb100%2 × 10−165100%E. minasensis collected from Rhipicephalus sp. from Pakistan (OQ627010)
Ehrlichia sp.Cattle 65PQ041221dsb100%2 × 10−166100%E. minasensis collected from Bos Taurus from Philippines (LC641910)
Ehrlichia sp.Cattle 72PQ041222dsb100%1 × 10−15899.4%E. minasensis collected from Rhipicephalus microplus from Panama (OR711253)
Ehrlichia sp.Cattle 82PQ041223dsb100%4 × 10−16799.1%E. minasensis collected from Bradypus variegatus from Brazil (MT212415)
Ehrlichia sp.Cattle 91PQ041224dsb100%3 × 10−16499.7%E. minasensis collected from Rhipicephalus microplus from Panama (OR711253)
Ehrlichia sp. Cattle 97, 113PQ041225, PQ041226dsb98%6 × 10−16699.4%E. minasensis collected from Bos Taurus from Australia (MH500007)
Ehrlichia sp. Goat 109PQ084567dsb100%4 × 10−13799.6%E. minasensis collected from Bradypus variegatus from Brazil (MT212415))
Ehrlichia sp. Goat 125PQ084568dsb96%099.3%E. minasensis collected from Bradypus variegatus from Brazil (MT212415)
Ehrlichia sp. Goat 135PQ084569dsb94%099.5%E. minasensis collected from Rhipicephalus microplus from Czech Republic (JX629808)
Ehrlichia sp. Goat 151PQ084570dsb100%8 × 10−18898.9%E. minasensis collected from Bradypus variegatus from Brazil (MT212415)
Ehrlichia sp. Sheep 40PQ084571dsb100%1 × 10−161100%E. minasensis collected from Rhipicephalus microplus from Czech Republic (JX629808)
Ehrlichia sp.Dromedary FTA 60PQ084562dsb100%2 × 10−114100%E. minasensis collected from Rhipicephalus sp. from Pakistan (OQ627010)
Ehrlichia sp.Dromedary FTA 61PQ084563dsb100%1 × 10−151100%E. minasensis collected from Rhipicephalus sp. from Pakistan (OQ627010)
Ehrlichia sp.Dromedary FTA 62PQ084564dsb100%2 × 10−159100%E. minasensis collected from Bos Taurus from Australia (MH500007)
Ehrlichia sp.Dromedary DNA 17PQ084565dsb100%9 × 10−16499.4%E. minasensis collected from Rhipicephalus sp. from Pakistan (OQ627010)
Ehrlichia sp.Dromedary DNA 50PQ084566dsb100%3 × 10−15398.7%E. minasensis collected from Bos Taurus from Philippines (LC641910)
Ehrlichia sp. Cattle 3, 19OR545662, OR5456653sodB100%2 × 10−7485.4%Ehrlichia sp. strain H7 collected from Capybara from Brazil (MW816653)
Ehrlichia sp. Cattle 56OR545664sodB100%1 × 10−7785.4%Ehrlichia sp. strain H7 collected from Capybara from Brazil (MW816653)
Ehrlichia sp. Cattle 65OR545665sodB100%4 × 10−5783.8%E. ewingii collected from dog from USA (KC778986)
Ehrlichia sp. Cattle 72, 82OR545666, OR545667sodB99%3 × 10−6883.4%Ehrlichia sp. strain H7 collected from horse from USA (KJ434180)
Ehrlichia sp. Cattle 91OR545668sodB100%7 × 10−7083.4%E. ruminantium collected from Senegal (DQ647026)
Ehrlichia sp. Cattle 113OR545669sodB99%3 × 10−6983.1%E. ruminantium collected from Senegal (DQ647026)
E. ruminantiumCattle 79PP911082PCS20100%4 × 10−13298.5%E. ruminantium collected from Amblyomma variegatum from Uganda (MK371032)
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Osman, A.M.; Hassan-Kadle, A.A.; André, M.R.; Collere, F.C.M.; Córdova, A.S.A.; Montiani-Ferreira, F.; Vieira, T.S.W.J.; Ibrahim, A.M.; Yusuf, A.A.; Machado, R.Z.; et al. Ehrlichia Species in Dromedary Camels (Camelus dromedarius) and Ruminants from Somalia. Pathogens 2025, 14, 65. https://doi.org/10.3390/pathogens14010065

AMA Style

Osman AM, Hassan-Kadle AA, André MR, Collere FCM, Córdova ASA, Montiani-Ferreira F, Vieira TSWJ, Ibrahim AM, Yusuf AA, Machado RZ, et al. Ehrlichia Species in Dromedary Camels (Camelus dromedarius) and Ruminants from Somalia. Pathogens. 2025; 14(1):65. https://doi.org/10.3390/pathogens14010065

Chicago/Turabian Style

Osman, Aamir M., Ahmed A. Hassan-Kadle, Marcos R. André, Flávia C. M. Collere, Amir Salvador Alabí Córdova, Fabiano Montiani-Ferreira, Thállitha S. W. J. Vieira, Abdalla M. Ibrahim, Abdulkarim A. Yusuf, Rosangela Z. Machado, and et al. 2025. "Ehrlichia Species in Dromedary Camels (Camelus dromedarius) and Ruminants from Somalia" Pathogens 14, no. 1: 65. https://doi.org/10.3390/pathogens14010065

APA Style

Osman, A. M., Hassan-Kadle, A. A., André, M. R., Collere, F. C. M., Córdova, A. S. A., Montiani-Ferreira, F., Vieira, T. S. W. J., Ibrahim, A. M., Yusuf, A. A., Machado, R. Z., & Vieira, R. F. C. (2025). Ehrlichia Species in Dromedary Camels (Camelus dromedarius) and Ruminants from Somalia. Pathogens, 14(1), 65. https://doi.org/10.3390/pathogens14010065

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