Advancements In Understanding and Diagnosing Canine Ehrlichiosis: A Comprehensive Review

1. Introduction

Canibe ehrlichiosis, caused by the obligate intracellular, pleomorphic bacterium of Ehrlichia, primarily Ehrlichia canis, is a significant concern within veterinary and public health [1]. This tick-borne zoonosis mainly affects canines and poses a risk to other animals, including humans [2]. The prevalence of E. canis infection is notable in areas with tropical or temperate climates, where tick proliferation is influenced by environmental factors [3,4] alongside ecological and socioeconomic elements [5]. Upon infection, the disease may manifest acutely, subclinically, or chronically, displaying diverse clinical signs such as fever, anorexia, anemia, thrombocytopenia, hemorrhages, lymphadenopathy, splenomegaly, uveitis, and polyarthritis [6]. Diagnosis of canine ehrlichiosis presents a challenge, necessitating a combination of direct and indirect methods, each with limitations in sensitivity, specificity, availability, and cost [7].

Treatment of the disease typically relies on antibiotics, primarily doxycycline, albeit effectiveness and affordability vary [8,9]. Despite treatment efforts, prevention of canine ehrlichiosis hinges on tick control and immunoprophylaxis, yet a commercially available vaccine remains elusive [10,11]. Consequently, canine ehrlichiosis stands as a zoonotic threat to public and animal health, demanding an integrated "One Health" approach for mitigation. This article aims to review the scientific literature on canine ehrlichiosis, exploring future perspectives and recent advances in genomics, proteomics, and artificial intelligence technologies to enhance diagnostic accuracy, identify therapeutic targets and biomarkers, and develop preventive strategies on a global scale.

2. Ehrlichia and Its Morphology

Ehrlichiosis stems from intracellular bacteria belonging to the genus Ehrlichia, encompassing six species: E. canis, E. chaffeensis, E. ewingii, E. muris, E. ruminantium, and E. mineirensis [1,12]. While E. chaffeensis, E. muris, and E. ewingii have been linked to ehrlichiosis in humans [2], reports indicate that E. ewingii and E. chaffeensis can infect dogs [4,13]. Thus, ehrlichiosis presents in two manifestations in both species: Canine Monocytic Ehrlichiosis (CME), primarily caused by E. canis, and Canine Granulocytic Ehrlichiosis (CGE), induced by E. ewingii [4]. Human Monocytic Ehrlichiosis (HME) involves E. chaffeensis, while E. ewingii contributes to Human Granulocytic Ehrlichiosis (HGE) alongside other agents like A. phagocytophilum and N. sennetsu [14]. Morphologically, Ehrlichia exhibits two distinct forms throughout its life cycle: an infectious, non-replicating form and a non-infectious form that undergoes binary fission for replication. Intracellular bacteria employ various mechanisms via surface-expressed proteins, facilitating functions such as adhesion to host cell membranes, nutrient absorption, cell signaling, and evasion [15,16]. They display morphological variations, encased in a thin, undulating outer membrane, featuring a narrow periplasmic space, and lacking a capsular layer. Moreover, Ehrlichia avoids the expression of lipopolysaccharide (LPS) and peptidoglycan on its surface, evading recognition by human leukocyte receptors and vector hemocytes, thereby thwarting elimination [17,18].

3. Tick Biological Cycle

The R. sanguineus tick serves as the primary biological vector for transmitting E. canis to dogs, although other tick species also possess this capability [3,19]. Consequently, dogs function as the principal host for R. sanguineus, concurrently acting as reservoirs due to prolonged bacteremia [20,21]. However, R. sanguineus infests other hosts, including small mammals and large animals, such as humans [19].

The transmission of canine ehrlichiosis initiates when a tick feeds on a previously infected host, thereby acquiring E. canis [2,22]. Throughout its biological cycle, the tick progresses through four life stages: egg, larva, nymph, and adult. While larvae, nymphs, and adults partake in blood meals and can acquire E. canis, only nymphs and adults can transmit the pathogen. Larvae, having not been previously exposed to the pathogen, cannot transmit it. Consequently, transstadial transmission, where the pathogen persists across the various life stages of the tick, is feasible, but transovarian transmission does not occur [2,22]. Subsequently, during the nymph and adult stages of its life cycle, the tick may transmit the bacteria to other hosts during subsequent blood meals.

The prevalence of E. canis typically peaks during warmer periods of the year in tropical and subtropical regions, correlating with the tick's life cycle (Figure 1).

4. Epidemiology of Canine Ehrlichiosis

Canine ehrlichiosis exhibits a widespread global distribution, particularly prevalent in regions with tropical and subtropical climates [23]. Noteworthy studies shed light on the disease's prevalence across various continents.

Laboso and colleagues conducted a comprehensive assessment spanning 2016 to 2021 in Africa, analyzing 400 samples from Kenya and Tanzania using the IDEXX SNAP 4Dx™ Plus test. Results unveiled a seropositivity of 31% (29/245) in Kenya and a surprising 69% (63/155) in Tanzania, underscoring the significant dissemination of ehrlichiosis in the region [24]. In contrast, a study in Europe by Guadalupe et al. over five years (2016-2020), covering samples from 35 countries, revealed an intriguing trend. Despite an increase in the number of tests conducted, there was a decline in Ehrlichia spp. Positivity, with seropositivity dropping from 4.3% to 3.4% [25].

In Latin America, evidence from Argentina indicates E. canis infection prevalence ranging between 13.5% and 37.5% in symptomatic dogs. In Paraguay, a study in domestic dogs reported a seroprevalence of 23.5%, accompanied by a molecular prevalence of 11.8%. Similarly, in Peru, seroprevalence was recorded at 16.5% for 140 dogs [10].

Previous studies in Brazil highlighted variability in canine ehrlichiosis seroprevalence, which is associated with different regions of the country. Values ranged from 62.8% in asymptomatic dogs to 78% in symptomatic dogs, with the molecular prevalence of E. canis ranging from 15% to 88%. For instance, Aguiar et al. observed a lower prevalence of 24.8% among 161 dogs from rural areas compared to 37.9% among 153 dogs from urban areas in the municipality of Monte Negro. Additionally, studies reported prevalence ranging from 5% to 16% in Rio de Janeiro and 27.6% in São Paulo, suggesting a significantly higher incidence of canine ehrlichiosis in urban areas compared to rural regions [26,27]. Conversely, seroprevalence in Brazil's southern region consistently appears low. Further insights come from Pereira and colleagues, reporting a molecular prevalence of 42.5% in dogs in the state of Mato Grosso Pantanal Norte [29]. Similarly, studies have depicted varying E. canis seropositivity rates, including 12.2% among 327 samples from dogs in the Amazon region, 13.7% among 153 dogs from the coastal area of Ceará state, and 59.1% among 264 dogs from the Midwest [30,31,32].

5. Pathogenesis

Upon tick infection, E. canis dissemination begins within the epithelial cells of the intestine, progressing to the tick's hemocytes and salivary gland cells [33]. Transmission to dogs occurs when infected ticks feed on blood, transferring the infective form of E. canis and their salivary secretions, which contain molecules facilitating pathogen acquisition and transmission, alongside exhibiting anticoagulant and anti-inflammatory properties [6,8].

Within the vertebrate host, the bacteria infiltrate monocytes, forming intracellular aggregates known as "morulas." Multiplication transpires within the phagolysosome and vacuoles of the host cell, affording isolation and protection from the immune system [34]. After 7 to 12 days post-infection, morulae are released into the bloodstream, infecting other cells. This interaction between infected and uninfected cells within blood vessels can incite vasculitis and perivascular migration of macrophages and lymphocytes. Predominant multiplication within macrophages and lymphocytes can lead to splenomegaly, hepatomegaly, and lymphadenopathy [8].

During host-pathogen interaction, various glycoproteins (GP) like GP19, gp36, gp140, and O-glycan, alongside outer membrane proteins such as P28/OMP and TRP 120, are pivotal for growth, accentuating the significance of these immunogenic outer membrane-associated proteins for pathogen replication within macrophages both in vitro and in vivo [12,15,35,36]. Studies conducted by McBride and colleagues underscore the importance of the TRP 120 protein in mediating E. canis and host cell interaction, influencing adhesion and internalization by phagosomes [36,37]. This capability enables bacteria to modulate the host's immune response, diminishing reactive oxygen species production and impeding host cell apoptosis. Additionally, the gp200 gene plays a critical role in immune response modulation, facilitating immune system evasion [38] and the potential to differentiate distinct pathogen strains [39].

The onset of the immune response, from initial exposure to symptom manifestation in canine ehrlichiosis, encompasses an incubation period spanning 8 to 20 days [8]. Subsequently, the disease progresses through acute, chronic, and subclinical phases, with the latter potentially persisting for several years without evident symptoms, underscoring the complexity and variability of E. canis infection within the host.

6. Immune Response

Both cellular and humoral immune responses are pivotal in defense against E. canis. CD4+ and CD8+ T cells play significant roles in the cellular immune response, crucial for resisting infections caused by this bacterium [40]. During E. canis infection, CD4 T lymphocytes secrete cytokines like IFN-γ and TNF-α, which can modulate the inflammatory response or confer protection to the host [2]. The protective immune response involves IFN-γ and Th1 secretion. However, components of tick saliva act as host immunomodulators during blood meals, reducing the production of IL-9, IL-2, and IL-4 in Th1 cells stimulated by IFN-γ.

Additionally, Castro and colleagues observed differences in MHCII molecule expression between lymphoid tissues and inflammatory infiltrates in organs of dogs with CME, along with an increase in the IgG2 subclass and a decrease in IgG1 and IgE [9,41]. Following E. canis infection, immunoglobulin release occurs in the blood circulation. IgA appears approximately four to seven days later and IgM around 15 days later, while initial IgG levels are relatively low. IgG titers (Figure 2) notably increase as the infection progresses, predominantly comprising the IgG2 subclass during both acute and convalescent phases [7,42,43].

However, the pathogen also exhibits immune system evasion mechanisms during infection. Two studies noted a decrease in MHC II expression, suggesting an evasion mechanism by E. canis [34,41]. Another observed evasion mechanism is the absence of LPS in E. canis, which, upon infecting leukocytes, aids their circulation through the vascular system, facilitating bacterial spread throughout the host's body and potentially enhancing survival in cellular vacuoles [18].

Figure 2. Antibody response about E. canis infection and time.

Figure 2. Antibody response about E. canis infection and time.

7. Clinical Signs

Canine monocytic ehrlichiosis (CME) presents as a polysystemic disease with clinical or subclinical manifestations [43,44], categorized as (A) Acute Phase: This initial stage, lasting 15 to 30 days, typically exhibits more pronounced symptoms. Bacteria multiply in mononuclear cells during this phase, spreading within the host. Clinical signs include fever, loss of appetite and weight, petechiae and/or cutaneous ecchymosis, lymphadenopathy and/or splenomegaly, muscle weakness, and vomiting [43,44]. (B) Chronic Phase: This phase resembles an autoimmune condition impacting the host's immune system. Symptoms mirror those of the acute phase but are more severe. It represents a prolonged and persistent stage of the infection, with intensified symptoms such as glomerulonephritis, nephrotic syndrome, bone marrow suppression leading to pancytopenia, and susceptibility to secondary infections [44,45]. (C) Subclinical Phase: Characterized by asymptomatic presentation for 6 to 9 weeks or persistent absence of clear clinical signs over several years. Nevertheless, it may present with non-regenerative anemia, leukopenia, and thrombocytopenia [8,46]. Proteins associated with evasion, adherence, DNA repair, and efflux pump influence pathogenesis and bacterial virulence [47]. Variability in E. canis strain virulence can lead to differing disease severities. This broad range of clinical manifestations underscores the complexity of E. canis infection.

8. Diagnosis

Diagnosing canine ehrlichiosis entails amalgamating clinical and epidemiological data with direct or indirect methods to confirm the suspicion, guiding appropriate treatment, and epidemiological control, which is crucial for both veterinary and public health, given the zoonotic nature of ehrlichiosis [8].

Microscopy: Microscopic analysis enables the identification of E. canis bacteria in various clinical samples, including peripheral blood, bone marrow, and biological fluids. Confirmation of canine ehrlichiosis relies on detecting microcolonies or morulae in the cytoplasm of specific cells, like monocytes or leukocytes [23]. This method is most effective during the acute phase, although its sensitivity diminishes in chronic and subclinical phases. Expertise is required to differentiate between morulae and other tissue structures [8].

Molecular: Polymerase chain reaction (PCR) assays are increasingly utilized due to their ability to detect minute amounts of genetic material with high precision, providing evidence of active infection and superior sensitivity to conventional microscopy [11,23]. Despite the wide use of PCR, discrepancies between PCR and microscopic results underscore the need for cautious interpretation [51]. Quantitative real-time PCR (qPCR) and multiplex PCR assays represent advancements in enhancing sensitivity and enabling simultaneous detection of multiple Ehrlichia species [52]. However, some studies report lower sensitivity than conventional PCR [53,54].

Serology: Serological tests, including indirect immunofluorescence reaction test (IFAT) and enzyme-linked immunosorbent assay (ELISA), detect antibodies against E. canis immunoreactive proteins [53,55]. Commercial tests for both methods are available, with varying sensitivities and specificities compared to the "gold standard" IFAT [56,57,58]. Limitations exist in serological tests, particularly in distinguishing acute from chronic infection and defining previous exposure, prompting research into new protein targets for improved diagnosis [44,59].

Recently, an innovative study combined statistics artificial intelligence (AI) and machine learning (ML) techniques to identify biomarkers by analyzing the transcriptome of patients with cardiovascular diseases (CVD). Eighteen transcriptomic biomarkers were identified with 96% accuracy [60]. This approach may contribute to the early diagnosis of tick-borne diseases.

In summary, a multifaceted approach combining different diagnostic methods is essential for accurate diagnosis, treatment, and control of canine ehrlichiosis.

9. Future Outlook and Recent Advances

Advancements in genomics and proteomics have facilitated the identification of E. canis antigens, enhancing our comprehension of host-pathogen interactions in this complex zoonosis. The pathogen's ability to disrupt the host's immune response poses diagnostic challenges, but screening antigenic protein epitopes with automated systems has improved research outcomes.

Genetic variability in bacteria, a consequence of mutation and genetic recombination, presents challenges for diagnosis and vaccination. Studies combining genomics, bioinformatics, and immunological screening have identified novel immunoreactive proteins from E. chaffeensis and E. canis, potentially serving as therapeu tic targets and predictive biomarkers [61,62].

Comparative genomics [63,64] has elucidated intraspecific variability in Ehrlichia bacteria, aiding in understanding the genetic basis of different clinical manifestations. Markers like the "TEDSVSAPA" repeat motif found in Australian trp36 sequences offer insights for phylogenetic and epidemiological studies of E. canis.

Immune response molecules are increasingly utilized as disease biomarkers [65]. CXCL13, for instance, shows promise in diagnosing Lyme neuroborreliosis, with studies comparing assay methods to enhance sensitivity and specificity. Additionally, innovative approaches combining statistics, artificial intelligence (AI), and machine learning (ML) techniques have identified transcriptomic biomarkers for cardiovascular diseases (CVD), potentially enabling early diagnosis of tick-borne diseases.

These advancements underscore the multidisciplinary efforts to improve diagnostics, understand disease mechanisms, and develop targeted interventions for canine ehrlichiosis and related conditions.