Introduction

Ehrlichiae are obligately intracellular gram-negative tick-transmitted bacteria that primarily infect monocytes or granulocytes and are responsible for diseases of human and veterinary importance worldwide (Maeda et al. 1987; Anderson et al. 1991; Buller et al. 1999). Agents such as Ehrlichia chaffeensis and Ehrlichia ewingii, which cause human infections of varying severity, are considered to be emerging tick-borne zoonoses transmitted by Amblyomma americanum (Walker 1998). The diagnosis of these infections is largely based on the combined evaluation of clinical signs and laboratory and epidemiological data (Olano et al. 2003). Unfortunately, most physicians are unfamiliar with human ehrlichiosis, and the disease is not routinely considered in the clinical and laboratory diagnosis of undifferentiated flu-like febrile illness, particularly outside the known geographical distribution of the primary vector, A. americanum.

Ehrlichia chaffeensis and E. ewingii are two pathogens previously considered to be geographically limited to North America. However, the detection of E. chaffeensis reactive sera from humans and animals in other parts of the world such as Brazil, Mexico and Korea, where A. americanum, is not found suggests that E. chaffeensis may be prevalent in other geographical regions (Heppner et al. 1997; Gongora-Biachi et al. 1999; Machado et al. 2006; Heo et al. 2002; Kim et al. 2003; Park et al. 2003; Calic et al. 2004), and the agent has been detected in other tick species such as Haemaphysalis longicornis and Ixodes persulcatus (Cao et al. 2000; Lee et al. 2005). Ehrlichia canis and E. ewingii DNA has been detected in dogs presenting at local veterinary clinics in Cameroon, indicating that multiple Ehrlichia species are present and suggesting that a tick vector other than A. americanum is responsible for transmission (Ndip et al. 2005). Moreover, DNA from multiple Ehrlichia species (E. canis, E. ewingii, and E. chaffeensis) were detected in unengorged R. sanguineus ticks obtained from these dogs (Ndip et al. 2007). Findings reported by others, also suggests that the agents of human ehrlichiosis are present on the African continent. A serologically confirmed case of E. chaffeensis infection acquired in Mali has been reported (Uhaa et al. 1992). Furthermore, dogs examined in South Africa had antibody titers to E. chaffeensis higher than that for E. canis, suggesting that E. chaffeensis actually elicited the antibody response (Pretorius and Kelly 1998). Others have suggested that human Ehrlichial infections occur infrequently in the African continent (Brouqui et al. 1994), but the existence and epidemiology of human ehrlichioses in Africa are still undetermined.

The recent emergence and increased recognition of diseases caused by tick-transmitted Ehrlichiae and the recent discoveries of rickettsial species in areas and of tick species that were previously thought to be uninfected by these agents have suggested that these agents may have wider distribution than the United States. This stimulated our interest to investigate the presence of these obligately intracellular bacteria in Cameroon. We report herein, a high E. chaffeensis prevalence in R. sanguineus ticks collected during a field collection from a group of kennel-confined dogs in Limbe, Cameroon.

Materials and methods

Tick collection

A total of 63 (21 from dog 1; 14 from dog 2; 8 from dog 3; 11 from dog 4; 9 from dog 5) adult incompletely engorged R. sanguineus ticks were collected from five heavily infested dogs (two adults [dogs 1 and 2] and three juveniles [dogs 3–5]) in a kennel from the Atlantic coastal city of Limbe (4°2′N, 9°19′E), Cameroon. Consent to collect blood from the dogs was denied by the owner. Care was taken to minimize discomfort to the animals. Ticks were surface sterilized by washing three times in 70% ethanol, stored in 1.5-ml vials containing the same concentration of ethanol at 4°C until transported to University of Texas Medical Branch, Galveston, TX.

Isolation of DNA from ticks

Before DNA extraction, ticks were each rinsed three times with sterile phosphate-buffered saline (PBS) to remove any residual ethanol. Ticks were cut into small pieces with separate sterile scissors and homogenized with a separate sterile micropestle in a sterile 1.5 ml microtube. The DNA was extracted using the DNeasy Tissue Kit (Qiagen) following the manufacturer’s instructions for isolation of DNA from animal tissues. The DNA was quantified in a digital spectrophotometer at 260 nm wavelength (Perkin Elmer MBA 2000). Purified DNA was stored at 4°C until used as template for PCR amplifications.

PCR detection of Ehrlichia species in ticks

All ticks were individually processed and examined by multicolor realtime PCR to detect the presence of E. canis, E. chaffeensis, or E. ewingii DNA in ticks. DNA (~250 ng) from each tick was added to individual reactions (25 μl) and the PCR product (378 bp) amplified using conditions, primers, and probes as previously described (Doyle et al. 2005). Plasmids containing the dsb gene of E. chaffeensis, E. canis or E. ewingii were included with each run as positive controls in addition to negative control reactions without DNA template. PCR was performed in 96-well plates using an iCycler iQ multicolor real-time PCR detection system equipped with the appropriate filter sets and analyzed with iQ software version 3.1 (BioRad Laboratories).

DNA sequencing

PCR amplicons were purified using EXOSAP-IT (USB Corporation, Cleveland, Ohio) according to the manufacturer’s instructions and sequenced directly with the same primers used for PCR on an ABI automated sequencer (UTMB Protein Chemistry Laboratory). The BLAST program (National Center for Biotechnology Information, Bethesda, MD) was used to compare dsb sequences in order to determine the species and genotype.

Results

Detection of Ehrlichia DNA

Real-time PCR detected Ehrlichial DNA in 38 (60%) of the 63 R. sanguineus ticks. Real-time PCR with specific probes to detect E. chaffeensis, E. canis, and E. ewingii confirmed the high prevalence of E. chaffeensis DNA in the tick samples. E. chaffeensis was detected in 33 (56%) ticks, E. canis DNA was detected in four (6%) ticks, and one tick was coinfected. E. ewingii DNA was not detected in this group of ticks. Amplicons (378 bp) amplified (Fig. 1) and identified by real-time PCR as E. canis or E. chaffeensis were sequenced and found to be identical to North American strains AF403710 and AY403711, respectively.

Fig. 1
figure 1

Polymerase chain reaction-amplified products from Rhipicephalus sanguineus ticks. Amplification of a 378-basepair (bp) product of the dsb protein gene and electrophoresis on a 1.5% agarose gel. Lane 1, 100-bp molecular weight marker; lane 2, positive control Ehrlichia canis plasmid DNA; lanes 3 and 4, negative controls; lanes 5–7 and 18, negative samples of DNA extracted from R. sanguineus ticks; lanes 8–17, positive samples of DNA extracted from individual R. sanguineus ticks

Full size image

Discussion

Ehrlichia chaffeensis is a gram-negative bacterium that infects monocytes causing the zoonosis, human monocytic ehrlichiosis (HME), an important emerging tick-borne disease that was first reported in the United States in 1987 (Maeda et al. 1987; Anderson et al. 1991). Since then, many cases have been reported to the Centers for Diseases Control, and the number of cases diagnosed each year has risen (Paddock and Childs 2003). In this study, we identified two Ehrlichial species in these ticks, E. chaffeensis (56%) and E. canis (6%). While R. sanguineus is the known vector of E. canis worldwide, previous studies have demonstrated that E. chaffeensis is maintained in nature through a cycle involving white-tailed deer (Odocoileus virginianus) as the cardinal reservoir host and the lone star tick, A. americanum (Ewing et al. 1995; Anderson et al. 1993; Lockhart et al. 1997). However, our findings of a high of prevalence of E. chaffeensis in R. sanguineus ticks collected dogs in one kennel suggests that this pathogen not only circulates in Cameroon, but also that other tick vectors and/or reservoirs may be implicated in its transmission in other parts of the world where A. americanum is not present. This study adds to the increasing evidence that the pathogen is not geographically restricted to the continental United States. Growing evidence of E. chaffeensis infection in humans and other mammalian hosts such as dogs and deer in other continents is extending the known range of E. chaffeensis (Chahan et al. 2005; Heppner et al. 1997; Gongora-Biachi et al. 1999; Machado et al. 2006; Heo et al. 2002; Kim et al. 2003; Calic et al. 2004). E. chaffeensis has also been detected in other tick species such A. testudinarium ticks from southern China (Cao et al. 2000) and in H. longicornis ticks from Korea (Lee et al. 2005) although their role in transmission of the pathogen has not been investigated.

This study identified R. sanguineus as a probable vector of E. chaffeensis in Cameroon. Recently, we examined 92 R. sanguineus ticks collected from 51 dogs from different sites in the Mount Cameroon region and found that these ticks were infected with three Ehrlichial species, E. canis, E. chaffeensis, and E. ewingii, the most prevalent being E. canis (Ndip et al. 2007). However, the present finding of a high prevalence of E. chaffeensis in ticks collected from dogs from one kennel suggests that E. chaffeensis has increased transmission efficiency where infection and reinfection potential is higher. A high number of E. chaffeensis infections and coinfections in kennel-confined dogs where R. sanguineus was the predominant tick vector has been reported, but the Ehrlichia species in these ticks was not determined (Kordick et al. 1999).

Rhipicephalus sanguineus are also important vectors of other tick-borne human pathogens, notably R. conorii, R. rickettsii (Mexico and Arizona) and E. canis and Babesia canis, the later three being pathogens of veterinary importance (Walker et al. 2003). These have three developmental stages, with the larvae and nymphal stages capable of feeding on humans (Walker et al. 2003). Therefore, the high prevalence of E. chaffeensis observed in these ticks suggests that this vector could potentially transmit this agent to humans in this region. The new discoveries of rickettsial agents in unexpected tick vectors seem to be on the rise. Recently, R. rickettsii, the agent of Rocky Mountain spotted fever known to be transmitted by Dermacentor spp., was detected in R. sanguineus ticks in Arizona (Demma et al. 2005).

Based on these findings, it is most likely that the exact distribution of E. chaffeensis and its vectors outside North America is not yet understood and warrants further investigation. In Cameroon, efforts will be concentrated on isolating Ehrlichiae from suspected tick vectors and vertebrate hosts since isolation is the gold standard for diagnosis of any infectious disease and comprehensive characterization of these pathogens can only be done upon isolated organisms. Due to the potential health risk to the human population, it is also critical to examine the role of these pathogens in undifferentiated febrile illness. Although E. chaffeensis is not yet recognized as a problem in the tropics, the situation is likely to change if greater attention is paid to the possibility.