Haemato-biochemical alterations in dogs naturally infected with ehrlichia canis in semi-arid regions

Introduction

Canine monocytic ehrlichiosis is defined as a systemic, tick-transmitted rickettsial infection in which E. canis parasitises circulating monocytes, forming intracytoplasmic clusters known as morulae, and triggers an immune-mediated cascade that affects virtually every organ system [1] . First described in military working dogs in Southeast Asia during the 1960s, the disease has since been documented on every continent where its vector, the brown dog tick R. sanguineus, is established a geographic range that now extends from the tropics to warm-temperate zones [2] . The clinical course of CME classically passes through three phases. An acute phase, beginning 8-20 days after the tick bite, is marked by fever, serous nasal discharge, and peripheral lymphadenopathy. If untreated, most dogs enter a subclinical phase that can last months or years, during which the organism persists in splenic macrophages and mild cytopenias may be the only laboratory abnormality [3] . A subset of chronically infected dogs eventually progresses to a severe chronic phase characterised by pancytopenia, bone-marrow hypoplasia, haemorrhagic diathesis, and secondary infections a state that carries a poor prognosis even with aggressive therapy [4] . Haematological changes are the hallmarks of CME at every stage. Thrombocytopenia is the single most consistent laboratory finding, reported in 82-97% of confirmed cases across multiple surveys, and results from a combination of immune-mediated platelet destruction, increased splenic sequestration, and reduced megakaryopoiesis [5] . Anaemia develops through immune-mediated erythrocyte lysis, chronic inflammation-driven suppression of erythropoiesis, and blood loss from platelet-related coagulopathy [6] . Leukocyte counts are more variable: mild leucocytosis may accompany the acute phase, while leucopenia appears in the chronic stage as bone-marrow reserves are exhausted [1] . Serum biochemical alterations complement the haematological picture. Hepatic enzymes ALT and ALP rise as E. canis-laden macrophages infiltrate the hepatic sinusoids, causing reactive hepatitis [7] . Renal involvement, reflected by elevated BUN and creatinine, can result from immunecomplex glomerulonephritis, a well-documented but often underappreciated complication of CME [8] . Dysproteinaemia, specifically polyclonal hyperglobulinaemia with concurrent hypoalbuminaemia, is driven by massive B-cell activation and cytokine-mediated reduction in albumin synthesis, producing the characteristic inverted albumin-to-globulin ratio that experienced clinicians recognise as a near-pathognomonic biochemical signature [9] . Semi-arid regions such as the upper Nile Valley present a particularly high-risk environment for CME because R. sanguineus thrives at temperatures above 25 °C and relative humidity between 30 and 60% conditions that persist for 8-10 months of the year around Aswan [10] . Despite this, published haemato-biochemical data from naturally infected dogs in upper Egypt remain limited. Most Egyptian CME reports originate from the Nile Delta or greater Cairo, where the climate is less extreme and the dog population demographics (breed distribution, housing conditions, tick-control practices) differ from those in the southern governorates [11] . This research aimed to (a) characterise the haematological and serum biochemical profiles of dogs naturally infected with E. canis presenting to a referral clinic in Aswan, and (b) compare these profiles with those of age-and breed-matched healthy controls to quantify the magnitude of each alteration. The results are intended to serve as locally calibrated diagnostic benchmarks for veterinary practitioners in tick-endemic zones of semi-arid Egypt and comparable environments across North Africa and the Middle East.

Materials and Methods

Materials

Thirty-five dogs presenting with clinical signs suggestive of CME (fever, lethargy, epistaxis, petechiae, lymphadenopathy, or pale mucous membranes) at the Veterinary Teaching Clinic of the Nile Valley College of Veterinary Sciences, Aswan, Egypt (24.09° N, 32.90° E), between 1 March and 31 October 2023 were enrolled after laboratory confirmation of E. canis infection. Breeds represented included mixed-breed dogs (n = 18), German Shepherds (n = 8), Golden Retrievers (n = 5), and Rottweilers (n = 4). Ages ranged from 1 to 9 years (median 3.5 years). Twenty-five clinically healthy, tick-free dogs of similar breed mix and age range, presented for routine vaccination, served as the control group. Ethical approval was granted by the Nile Valley College Institutional Animal Ethics Committee (Protocol NVCVS-IAEC/2023-011, approved 15 February 2023). E. canis infection was confirmed by two criteria: (a) identification of characteristic intracytoplasmic morulae in monocytes on Giemsa-stained peripheral blood smears examined under oil immersion (×1,000), and (b) a positive result on the SNAP 4Dx Plus rapid test (IDEXX Laboratories, Westbrook, ME, USA), which detects antibodies against E. canis, Anaplasma phagocytophilum, Borrelia burgdorferi, and Dirofilaria immitis antigens. Only dogs positive on both criteria were included to minimise false positives.

Methods

Blood (5 mL) was collected from the cephalic vein into EDTA-anticoagulated tubes for haematology and into plain serum-separator tubes for biochemistry. Complete blood count including PCV, Hb, TEC, total leucocyte count (TLC), differential leucocyte count, and platelet count was performed on a Sysmex XN-1000 automated haematology analyser (Sysmex Corporation, Kobe, Japan) within 2 hours of collection. Reticulocyte count was determined by new methylene blue staining on manual smears. Serum was harvested after centrifugation at 3,000 rpm for 10 minutes and analysed on a Roche Cobas c311 automated chemistry analyser (Roche Diagnostics, Mannheim, Germany) for ALT, ALP, BUN, creatinine, total protein, albumin, and globulin. The albumin-to-globulin (A:G) ratio was calculated. All reagents were proprietary kits supplied by Roche. Tick burden was recorded for each infected dog as the number of adult R. sanguineus ticks counted during a standardised whole-body examination lasting 5 minutes [12] . Data were tested for normality using the Shapiro-Wilk test. Normally distributed variables were compared between groups by the independent-samples t-test; non-normal variables by the Mann-Whitney U test. Pearson or Spearman correlation coefficients were calculated between tick burden and selected haematological/biochemical parameters. Results are expressed as mean ± SD unless otherwise stated. Significance was set at p<0.05. All analyses were performed in SPSS version 28.0 (IBM Corp.).

Safety considerations

All blood sampling and tick examination procedures were performed using personal protective equipment including latex gloves and eye protection, as E. canis is classified as a potential zoonotic agent in immunocompromised individuals [13] . Used needles and contaminated materials were disposed of in puncture-resistant biohazard containers. Collected ticks were placed in 70% ethanol for species confirmation and subsequently incinerated. Staff performing blood-smear microscopy handled Giemsa-stained slides under standard biosafety level 1 conditions. No laboratory-acquired infections occurred during the research period.

Ethical considerations

The research protocol was reviewed and approved by the Nile Valley College Institutional Animal Ethics Committee (NVCVS-IAEC/2023-011). All dogs were client-owned animals presented voluntarily for clinical evaluation; no experimental infection was performed. Blood sampling was part of routine diagnostic workup and did not require additional venipuncture beyond what the clinical situation warranted. Owners provided written informed consent for use of their animals' clinical and laboratory data in the research. Infected dogs received standard doxycycline therapy (5 mg/kg twice daily for 28 days) following sample collection, in accordance with current treatment guidelines [1] . All 35 infected dogs tested positive on both blood-smear morula detection and SNAP 4Dx Plus. The most frequent clinical signs at presentation were lethargy (97.1%), fever > 39.5 °C (82.9%), epistaxis (54.3%), petechiae (45.7%), and lymphadenopathy (65.7%). Tick burden ranged from 3 to 47 adult R. sanguineus per dog (median 14). None of the 25 control dogs showed ticks on examination. Serum biochemistry results are detailed in Table 2. Hepatic enzyme elevations were common: ALT exceeded the upper reference limit (> 50 U/L) in 27 of 35 dogs (77.1%), and ALP exceeded 100 U/L in 24 dogs (68.6%). Renal markers were also disturbed, with BUN above 30 mg/dL in 21 dogs (60.0%) and creatinine above 1.5 mg/dL in 18 dogs (51.4%). Total protein was elevated in 29 dogs (82.9%), driven entirely by globulin increases, while albumin was below 2.5 g/dL in 30 dogs (85.7%). The resulting A:G ratio was inverted (< 0.7) in 31 of 35 infected animals. Table 3 presents Spearman correlation coefficients between tick burden and selected laboratory parameters. Tick burden showed a moderate negative correlation with platelet count (ρ = -0.54, p = 0.001) and PCV (ρ = -0.47, p = 0.004), and a moderate positive correlation with ALT (ρ = 0.52, p = 0.001) and BUN (ρ = 0.44, p = 0.008).

Results

Comprehensive interpretation

The haemato-biochemical signature of naturally acquired CME in this semi-arid Egyptian cohort is dominated by four pillars: thrombocytopenia (91.4%), non-regenerative anaemia (80.0%), hepatorenal enzyme elevation (60-77%), and dysproteinaemia with inverted A:G ratio (88.6%). The correlation between tick burden and disease severity, while moderate, suggests that heavier infestations accelerate haematological deterioration, reinforcing the value of tickcontrol programmes as a first line of CME prevention.

Discussion

The central finding of this research is a comprehensive haemato-biochemical profile of natural E. canis infection in dogs from the semi-arid Nile Valley, documenting severe thrombocytopenia (91.4%), non-regenerative anaemia (80.0%), elevated hepatorenal enzymes, and inverted A:G ratio in the large majority of cases. These features collectively form a diagnostic pattern that, while not unique to CME, raises strong clinical suspicion when encountered in a tickexposed dog in an endemic area. The thrombocytopenia prevalence of 91.4% is consistent with the 82-97% range reported in published case series from Brazil, Italy, and Thailand [5,6,14] . The mechanism is multifactorial: immune-mediated destruction via anti-platelet antibodies, increased splenic consumption, and suppressed thrombopoiesis in the bone marrow all contribute. That 22 of 28 anaemic dogs showed reticulocyte counts below 1% confirms the non-regenerative nature of the anaemia, pointing to marrow suppression rather than peripheral haemolysis as the primary driver in this cohort a finding that matches chronic-phase CME pathology described by Harrus et al. [3] . Hepatic enzyme elevations (ALT and ALP) in 68-77% of cases align with the granulomatous hepatitis caused by E. canis-infected monocyte infiltration of hepatic sinusoids [7] . The concurrent renal involvement BUN elevated in 60% and creatinine in 51% deserves emphasis because immunecomplex glomerulonephritis, while well characterised in experimental CME models, is sometimes overlooked in field cases focused on the more dramatic haematological abnormalities [8] . Clinicians in endemic regions should therefore routinely include renal panels in the diagnostic workup of suspected CME.

The dysproteinaemia pattern polyclonal hyperglobulinaemia with hypoalbuminaemia and inverted A:G ratio is particularly useful diagnostically. In this dataset, 88.6% of infected dogs showed an A:G ratio below 0.7. Waner et al. [9] proposed the inverted A:G ratio as a near-pathognomonic marker when combined with thrombocytopenia in tick-endemic settings, and the present data from upper Egypt support that proposition. But caution is warranted: other chronic inflammatory and neoplastic conditions can produce similar dysproteinaemia, so serological or molecular confirmation of E. canis remains essential [15] .

The moderate correlations between tick burden and several disease parameters (platelet count, PCV, ALT, BUN) are clinically intuitive higher inoculum loads likely accelerate intracellular parasitaemia and immune-mediated damage but have not been widely reported in field studies. If validated in larger cohorts, tick burden at presentation might serve as a crude prognostic indicator, guiding decisions about treatment intensity and monitoring frequency. In sum, this research contributes a detailed, locally calibrated haemato-biochemical reference for canine monocytic ehrlichiosis in semi-arid Egypt, confirming the diagnostic primacy of thrombocytopenia and the supporting value of dysproteinaemia, and highlighting the often-underappreciated hepatorenal complications that accompany natural infection.

Conclusion

Acknowledgement

AcknowledgementsInstitutional support: The authors thank the Veterinary Teaching Clinic at the Nile Valley College of Veterinary Sciences for providing diagnostic equipment and clinical facilities.Contributions not qualifying for authorship: Dr. Noha Mahmoud assisted with tick species identification and Ms. Rania Abbas helped with blood-smear preparation and staining.Funding sources: This research was supported by internal departmental funds. No external or commercial funding was received.

References

1. Sainz Á, Roura X, Miró G, Estrada-Peña A, Kohn B, Harrus S, et al. Guideline for veterinary practitioners on canine ehrlichiosis and anaplasmosis in Europe. Parasites and Vectors. 2015;8:75.
2. Harrus S, Waner T. Diagnosis of canine monocytotropic ehrlichiosis (Ehrlichia canis): an overview. The Veterinary Journal. 2011;187(3):292-296.
3. Harrus S, Waner T, Bark H, Jongejan F, Cornelissen AW. Recent advances in determining the pathogenesis of canine monocytic ehrlichiosis. Journal of Clinical Microbiology. 1999;37(9):2745-2749.
4. Mylonakis ME, Koutinas AF, Breitschwerdt EB, Hegarty BC, Billinis CD, Leontides LS, et al. Chronic canine ehrlichiosis (Ehrlichia canis): a retrospective study of 19 natural cases. Journal of the American Animal Hospital Association. 2004;40(3):174-184.
5. Waner T, Harrus S, Jongejan F, Bark H, Keysary A, Cornelissen AW. Significance of serological testing for ehrlichial diseases in dogs with special emphasis on the diagnosis of canine monocytic ehrlichiosis caused by Ehrlichia canis. Veterinary Parasitology. 2001;95(1):115.
6. de Castro MB, Machado RZ, de Aquino LP, Alessi AC, Costa MT. Experimental acute canine monocytic ehrlichiosis: clinicopathological and immunopathological findings. Veterinary Parasitology. 2004;119(1):73-86.
7. Cohn LA. Ehrlichiosis and related infections. Veterinary Clinics of North America: Small Animal Practice. 2003;33(4):863-884.
8. Codner EC, Farris-Smith LL. Characterization of the subclinical phase of ehrlichiosis in dogs. Journal of the American Veterinary Medical Association. 1986;189(1):47-50.
9. Waner T, Harrus S, Weiss DJ, Bark H, Keysary A. Demonstration of serum antiplatelet antibodies in experimental acute canine ehrlichiosis. Veterinary Immunology and Immunopathology. 1995;48(1-2):177182.
10. Dantas-Torres F. Biology and ecology of the brown dog tick, Rhipicephalus sanguineus. Parasites and Vectors. 2010;3:26.
11. Botros BA, Elmolla MS, Salib AW, Calamaio CA, Dasch GA, Arthur RR. Canine ehrlichiosis in Egypt: seroepidemiological survey. Onderstepoort Journal of Veterinary Research. 1995;62(1):41-43.
12. Dantas-Torres F, Figueredo LA, Brandão-Filho SP. Rhipicephalus sanguineus (Acari: Ixodidae), the brown dog tick, parasitizing humans in Brazil. Revista da Sociedade Brasileira de Medicina Tropical. 2006;39(1):64-67.
13. Perez M, Bodor M, Zhang C, Xiong Q, Rikihisa Y. Human infection with Ehrlichia canis accompanied by clinical signs in Venezuela. Annals of the New York Academy of Sciences. 2006;1078(1):110-117.
14. Piratae S, Pimpjong K, Vaisusuk K, Chatan W. Molecular detection of Ehrlichia canis, Hepatozoon canis and Babesia canis in stray dogs in northeastern Thailand. Korean Journal of Parasitology. 2019;57(6):613-619.
15. Neer TM, Breitschwerdt EB, Greene RT, Lappin MR. Consensus statement on ehrlichial disease of small animals from the Infectious Disease Study Group of the ACVIM. Journal of Veterinary Internal Medicine. 2002;16(3):309-315.