Molecular detection of Ehrlichia ruminantium in ticks from ruminants during the 2021 Rift Valley fever outbreak in Mananjary, Madagascar

Abstract

Ehrlichia ruminantium, the causative agent of heartwater, is a tick-borne pathogen affecting livestock in Africa and the Caribbean. This disease is transmitted primarily by Amblyomma variegatum ticks and poses a significant threat to animal health. In Madagascar, the prevalence of E. ruminantium remains poorly documented. During a Rift Valley fever (RVF) outbreak in Mananjary, Madagascar (April–May 2021), we conducted a field study to assess the circulation of vector-borne pathogens in ticks collected from ruminants. Ticks were morphologically identified, and DNA was extracted for quantitative PCR targeting the pCS20 gene of E. ruminantium. Statistical analyses were performed to explore associations between tick infection status, ruminant health, and infestation levels. A total of 332 ticks were collected from 25 ruminants. The tick species identified included Rhipicephalus microplus (51.5%) and Amblyomma variegatum (48.2%). E. ruminantium DNA was detected in 5.1% (17/332) of ticks, consisting of 16 A. variegatum and one R. microplus, with the majority being male. No association was observed between ruminant clinical signs and the presence of infected ticks. This study provides the first molecular evidence of E. ruminantium circulation in ticks from Madagascar during an RVF outbreak. Our findings emphasize the need for improved disease surveillance and integrated tick control strategies to mitigate the impact of heartwater on livestock.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00436-025-08508-x.

Background

Heartwater, also known as cowdriosis, is a tick-borne disease affecting domestic and wild ruminants. It is caused by Ehrlichia ruminantium (E. ruminantium), an intracellular bacterium transmitted by ticks of the genus Amblyomma (Bath et al. 2005). Ticks likely become infected for life while feeding on infected animals (Bath et al. 2005). Amblyomma variegatum (A. variegatum), one of the most important vectors of E. ruminantium and the second most invasive tick worldwide after Rhipicephalus microplus, a one-host species, is also among the most important ticks in Africa (Madder et al. 2011; Nyangiwe et al. 2018), alongside A. hebraeum, which is endemic to over 30 countries (Pfäffle et al. 2013). Cattle and large mammals host all stages of A. variegatum and R. microplus, whereas birds and carnivores host only the immature stages of A. variegatum (Barre and Uilenberg 2010; Oyen and Poh 2024). Although E. ruminantium has been extensively studied, other Ehrlichia species have also been reported in Madagascar; Ehrlichia canis, E. ewingii, and E. muris were detected for the first time in R. microplus ticks collected from cattle (Matysiak et al. 2016).

Heartwater is endemic to tropical and subtropical regions, posing a major threat to livestock farming and is often fatal. It is considered the second most significant tick-borne disease in Africa, after East Coast Fever (Allsopp 2015). In areas of high transmission, repeated exposure to infected A. variegatum ticks often leads to sustained immunity in animals, resulting in decreased mortality (WOAH 2021). Conversely, in low-transmission regions, the disease is more severe and potentially fatal. Symptoms of heartwater include fever exceeding 41 °C, often but not always anorexia, lethargy, rapid breathing, and nervous signs (van Amstel et al. 1988; Saimo et al. 2001). Autopsy often reveals the presence of clear yellow fluid in the thorax and pericardium, which may result from increased capillary permeability, although the precise mechanisms remain unclear (Van der Merwe et al.1987; Camus et al. 1996). These symptoms often overlap with other diseases, such as Rift Valley fever (RVF), which is characterized by fever, lethargy, anorexia, massive abortions, and hepatic lesions. The neurological signs and hydrothorax seen in heartwater help to differentiate them, but accurate diagnosis is essential, particularly in regions where both diseases coexist (Van der Merwe et al. 1987; Camus et al. 1996).

This study focuses on the molecular detection of E. ruminantium DNA in ticks collected from ruminants during a multidisciplinary field survey conducted in April–May 2021 in the district of Mananjary, Madagascar, in response to an RVF epizootic epidemic.

Methods

Tick collection and classification

The team collected 332 ticks from 25 cattle (Bos indicus) between April 26 and May 5, 2021, in the district of Mananjary, Madagascar, as part of an investigation related to an outbreak of RVF (Harimanana et al. 2024; Tantely et al. 2024). We preserved the ticks in 70% ethanol and identified them to species level based on morphological characteristics (Walker et al. 2014) and assessed for engorgement using features such as body size and coloration. We recorded the age, sex, geographical location, and health status of each ruminant—both up to 2 months before sampling and at the time of sampling. Epidemiologists administered a structured questionnaire to the owners/herders, and they documented the health status of the animals based on clinical observations, which included symptoms and clinical signs, as informed by veterinarians (Supplement Tables 1 and 2). Additionally, we supplemented the data with the diagnostic results for RVF.

Table 1.

Characteristics of ruminants and ticks according to the presence of Ehrlichia ruminantium DNA in ticks

	E. ruminantium detected in ticks	E. ruminantium not detected in ticks	p value
Total number of ruminants N = 25	7*	18£
Sex			1
Female	5	11
Male	2	7
Age			0.27
Median	2	7
Range	1–12	0.6–15
Symptoms at collection			0.67
Absent	6	14
Present	1	4
Symptoms prior collection			0.2
Absent	4	5
Present	3	13
Total number of ticks N = 332
Number of ticks per animal			0.08
Median	11	8
Range	9–56	2–25
Tick species			< 0.001
Amblyomma variegatum	16	144
Rhipicephalus microplus	1	170
Unidentified		1
Sex of ticks			< 0.001
Female	1	168
Male	10	87
Undetermined	6	60
Status of engorgement			0.12
Engorged	7	203
Non-engorged	10	111
Undetermined	0	1

Fisher exact test for categorical data, Wilcoxon rank-sum test for numerical data

^*The number of ruminants from which at least one Ehrlichia ruminantium-positive tick was collected

^£The number of ruminants from which no single Ehrlichia ruminantium-positive tick was collected

DNA extraction

We extracted DNA from all the ticks using the commercial QIAMP 96 QIAcube HT Kit (Qiagen, Venlo, Netherlands) on a Qiacube instrument (Qiagen, Venlo, Netherlands), following the manufacturer’s recommendations, but included an additional mechanical disruption step using a micro pestle to ensure efficient cell lysis and sample homogenization (Halos et al. 2004; Crowder et al. 2010), followed by vortexing for enhanced mixing and transferred to a tube containing 180 µL of ATL buffer included in the kit, followed by the addition of 20 µL of proteinase K, and incubated overnight at 56 °C. After centrifugation (14,500 g for 1 min), 200 µL of supernatant was mixed with 200 µL of AL buffer and 200 µL of absolute ethanol. A 400 µL aliquot of the mixture was then transferred into the automated system. We included an extraction negative control using molecular biology-grade water in each extraction series. DNA was eluted in 200 µL of AE buffer and stored at − 20 °C until use.

qPCR amplification

We performed qPCR targeting pCS20 following a previously published method (Cangi et al. 2017). In brief, each reaction was performed in a final volume of 20 μL containing 5 μL of DNA as template. The reaction mixture contained 0.4 µM (forward and reverse) Sol1 primer, 0.6 μM Sol1 probe with FAM fluorophore, 4 μL of 5X HOT FIREPol Probe qPCR Mix Plus (Solys Biodine, Tartu, Estonia), and 8.2 μL of molecular grade water. Positive control DNA from E. ruminantium (p53 Gardel strain) was provided by the Centre for Research and Surveillance on Vector-borne Diseases in the Caribbean, WOAH Reference Laboratory for Heartwater, F-97170 Petit-Bourg, Guadeloupe, France. Negative controls contained all qPCR components except the template DNA. Each qPCR run included also the negative extraction control. We performed amplifications on the CFX96 Touch Real-Time PCR Detection System (BioRad, Hercules, CA, USA) using the following protocol: initial denaturation at 95 °C for 15 min, followed by 45 cycles of 95 °C for 20 s (denaturation) and 60 °C for 1 min (annealing).

Data analysis

We described the characteristics of the ruminants and ticks (Supplementary data 1 and 2). The status of infection by E. ruminantium of ticks and ruminants carrying the ticks was compared using the Fisher test for categorical data and Wilcoxon Rank-Sum Test for numerical data (Nussbaum 2024), with the free R and RStudio software (version 3.6.1) (R Core Team 2024 and RStudio Team 2020). The statistical significance was assessed at the 0.05 level.

Results and discussions

We studied a total of 25 ruminants from three localities (Anosimparihy, Ambohimiarina II, and Antsenavolo; Fig. 1) during a period when a Rift Valley fever (RVF) outbreak had been declared in the region. The animals ranged from 7½ months to 15 years old, and 16 were females. Up to 2 months before tick collection, nine animals appeared healthy, while the remaining 16 exhibited clinical signs such as eye problems, anorexia, diarrhea, asthenia, fever, or hypersalivation. Two animals had experienced abortions. At the time of tick collection, 11 animals had recovered, and five were still sick. None of the animals died (Table 1 and Supplement Table 1). We did not observe any clear differences in the results between sick and healthy animals.

Fig. 1.

Map of the Mananjary district illustrating the location of the municipalities of origin of the sampled ruminants

We collected 332 ticks, including 171 Rhipicephalus microplus, 160 Amblyomma variegatum, and one unidentified tick (Table 1). Most R. microplus ticks were engorged females (145/155), while all males (16) were non-engorged. We could determine the gender of 96/160 A. variegatum ticks; they were primarily males (82), and half of the female A. variegatum were engorged (7/14). The number of ticks collected per ruminant ranged from 2 to 56 based on their level of infestation.

Among the tick species examined, A. variegatum exhibited a significantly (p < 0.001) higher pathogen detection rate (16/160) compared to R. microplus (1/171), with one additional positive sample identified among ticks of undetermined species (Table 1). The known biological traits of A. variegatum, including its longer feeding duration and broader host range, may enhance its potential as a vector by increasing opportunities for pathogen uptake and transmission (Barre and Uilenberg 2010). Conversely, R. microplus, despite being more abundant in the sampled population, demonstrated a markedly lower infection rate, potentially reflecting species-specific differences in host specificity, ecological niche, or physiological capacity to support pathogen replication (Matysiak et al. 2016). These findings underscore the need for species-focused vector surveillance, particularly in ecological settings where both tick species are sympatric.

In parallel, tick sex also emerged as a factor influencing pathogen detection. Male ticks showed a significantly (p < 0.001) higher infection rate (10/97) relative to females (1/169), with ticks of undetermined sex showing intermediate positivity (6/66) (Table 1). This pattern may be attributable to behavioral and physiological differences between sexes. Male ticks are known to exhibit intermittent feeding behavior and may attach to multiple hosts in search of mates, thereby increasing their exposure to infected hosts (Bartosik et al. 2019). In contrast, females generally engage in a single, prolonged feeding event to support egg production, which may reduce their likelihood of encountering and acquiring pathogens. These sex-specific differences in feeding ecology and host interaction likely influence vector-pathogen dynamics and warrant further investigation within the context of tick-borne disease epidemiology (Randolph 2008).

Finally, 17 out of 332 (5.1%) ticks tested positive for E. ruminantium by qPCR, which is lower than the previously reported variations (11.2–40.9%) in E. ruminantium positivity rate within vector populations (1). The lower positivity rate may reflect differences in environmental factors, tick density, and vector or host susceptibility across regions (Esemu et al. 2011).

All the infected ticks, except for one, belonged to the species A. variegatum. All A. variegatum were male ticks or of unidentifiable gender, the only R. microplus infected tick was an engorged female (Table 1).

Engorged ticks are typically more likely to test positive for E. ruminantium, as they have ingested larger volumes of blood, increasing the probability of detecting the pathogen. However, it is important to recognize that the presence of pathogen DNA in these ticks does not necessarily indicate a true infection or replication within the tick tissues. Rather, the detection may reflect residual DNA originating from the blood meal taken from an infected host, without colonization of the tick’s salivary glands or midgut (Peter et al. 1995). This distinction is essential when interpreting infection rate and assessing the actual vector competence of a given tick species. Further studies involving dissection and pathogen detection in specific tick organs are necessary to confirm whether the pathogen survives, replicates, and is transmitted by the tick. Failure to differentiate between transient carriage and true infection may lead to overestimations of vector capacity.

Thus, both engorged and non-engorged ticks can play a role in the transmission dynamics of E. ruminantium. Consequently, detecting the pathogen in non-engorged ticks suggests trans-stadial transmission and that even immature stages of the ticks may contribute to the overall pathogen burden in the environment (Mnisi et al. 2022).

We detected infected ticks on seven out of 25 (28%) animals, which carried between nine and 56 ticks. Between one and five ticks per animal were infected, yielding a tick positivity rate of 8–33% per animal. The higher positivity rate in ticks collected from certain animals suggests potential clustering of infection risk (Mtshali et al. 2015). However, a key limitation of this study is the absence of qPCR testing on the host animals themselves. This limitation restricts our ability to distinguish between transient pathogen carriage and actual colonization of the tick tissues. Future investigations should include parallel testing of both ticks and their hosts to more accurately assess vector-pathogen dynamics and clarify the role of host infection status in influencing tick positivity (Cangi et al. 2017). Yet no association was observed between tick numbers, clinical symptoms of ruminant, and tick infection status. This suggests that tick burden alone is not a reliable indicator of clinical severity or E. ruminantium infection. Various factors, such as host immune response, tick species, and pathogen load or virulence, could influence the clinical outcome of these tick-borne infections.

The ruminants carrying infected ticks ranged from 1 to 12 years old, with a median age of 2 years; five of them were females. Infection among younger animals may be possibly due to their lower exposure to E. ruminantium compared to older animals in enzootic settings (Faburay et al. 2007; Nasirian 2022). At the time of tick collection, four ruminants were healthy (Peter et al. 1998), two had recovered from symptoms such as anorexia and abortion, and one still exhibited ocular opacity (“white eyes”). Infected ticks were found on both healthy and symptomatic animals, but the clinical signs are not pathognomonic for heartwater. Therefore, the role of symptomatic and asymptomatic animals in the epidemiology of the disease remains unclear without further diagnostic confirmation.

In conclusion, this study provides the first molecular evidence of E. ruminantium infection in A. variegatum ticks collected during the 2021 RVF outbreak in Mananjary, Madagascar. While the primary objective was to investigate the RVF epidemic, the detection of E. ruminantium highlights the concurrent risk of heartwater in regions facing multiple vector-borne disease threats. This underscores the importance to remain vigilant for co-circulating pathogens in outbreak settings. The clinical overlap between RVF and heartwater also reinforces the critical role of molecular diagnostic tools for differential diagnosis in endemic areas.

Our findings support the need for integrated tick and disease surveillance systems in Madagascar. Control strategies should prioritize continuous monitoring of tick populations, context-adapted acaricide application, and promotion of tick-resistant livestock breeds. Future research should focus on characterizing the genetic diversity of E. ruminantium strains in Madagascar, as strain variability may influence pathogenicity, transmission dynamics, and the development of effective vaccines. Moreover, incorporating ecological and seasonal predictors of tick infection prevalence will be essential to designing targeted, evidence-based interventions for heartwater control.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

MANR, AH, NFR, TTR, TC: Conceptualizarion, Data curation, Formal analysis, Methodology, Project administration, Resources, Writing—original draft, Writing—review & editing. MLT, VR, ANH, SFA, LR, JI, NNR, JTR, RG, PD, VL, RVR, DA: Investigation, Methodology, Project administration, Resources, Writing—review & editing.

Funding

The field and the laboratory works were funded by the U.S. Agency for International Development (USAID) under the Research, Innovation, Surveillance and Evaluation (RISE) program (Cooperative Agreement #72068719 CA0001). The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the USAID.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval

This study was conducted as part of the investigation of the 2021 Rift Valley fever (RVF) outbreak, carried out by the Ministry of Health through the Directorate of Health Surveillance, Epidemiological Surveillance, and Response (DVSSER), in collaboration with the Institut Pasteur de Madagascar. An authorization for the investigation was issued by the Ministry of Public Health under reference number No. 619 MSANP/SG.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Clinical trial number

Not applicable.