Section: Bacteriology

Anaplasma phagocytophilum: Equine Granulocytic Anaplasmosis

Etiologic Agent and Taxonomic Classification

Anaplasma phagocytophilum is a Gram-negative, obligate intracellular bacterium belonging to the family Anaplasmataceae within the order Rickettsiales [1, 2]. This bacterium is the causative agent of equine granulocytic anaplasmosis (EGA), a disease previously termed equine granulocytic ehrlichiosis prior to taxonomic reclassification [3]. The genus Anaplasma includes several species that infect erythrocytes and granulocytes of various mammalian hosts, with A. phagocytophilum exhibiting a tropism for neutrophils and eosinophils in horses [4, 5].

The organism is pleomorphic, existing as small (0.5 to 1.5 micrometer) coccoid to ellipsoidal bodies within membrane-bound vacuoles (inclusions) in the host cell cytoplasm [6]. These intracytoplasmic inclusions, termed morulae (Latin for "mulberry"), are composed of multiple bacterial organisms aggregated within a parasitophorous vacuole [7]. The bacterial cell wall lacks lipopolysaccharide (LPS) and peptidoglycan, a characteristic shared with other members of the Anaplasmataceae family [8]. The genome of A. phagocytophilum is approximately 1.5 megabases in size, with a low G+C content of approximately 41% [9]. Genomic analysis reveals a reduced metabolic capacity, necessitating dependence on host cell-derived nutrients for survival and replication [10].

Vector Biology and Transmission Dynamics

A. phagocytophilum is transmitted primarily by ticks of the genus Ixodes. In North America, the principal vector is Ixodes scapularis (the black-legged or deer tick), while in Europe, Ixodes ricinus (the castor bean tick) serves as the primary vector [11, 12]. Other Ixodes species, including Ixodes pacificus in the western United States and Ixodes persulcatus in Asia, have also been implicated in transmission cycles [13, 14]. The tick life cycle involves three stages: larva, nymph, and adult. Transmission of A. phagocytophilum occurs during the nymphal and adult feeding stages, with nymphs considered the most epidemiologically significant due to their small size and tendency to feed undetected [15].

The bacterium is maintained in enzootic cycles involving small mammal reservoir hosts, including white-footed mice (Peromyscus leucopus), voles (Microtus species), and shrews (Blarina brevicauda) [16, 17]. These reservoir hosts sustain the infection without exhibiting clinical disease, allowing for continuous transmission to feeding ticks [18]. Horses are considered incidental or dead-end hosts, as they do not contribute significantly to the maintenance of the bacterium in nature [19]. However, horses can serve as sentinel species for the presence of A. phagocytophilum in a geographic region [20].

Transmission occurs when an infected tick takes a blood meal from a susceptible horse. The bacterium is introduced into the dermis through tick saliva, which contains immunosuppressive factors that facilitate pathogen establishment [21]. The incubation period in horses ranges from 5 to 14 days following tick attachment, with experimental infections demonstrating clinical signs as early as 7 days post-inoculation [22, 23].

Pathogenesis and Host Cell Interactions

A. phagocytophilum exhibits a unique tropism for granulocytes, particularly neutrophils and eosinophils [24]. The bacterium selectively infects these cells through a mechanism involving the binding of bacterial surface proteins to host cell receptors. The major surface protein (Msp2) is a key adhesin that mediates attachment to neutrophil surface receptors, including P-selectin glycoprotein ligand-1 (PSGL-1) [25, 26]. Following attachment, the bacterium is internalized via receptor-mediated endocytosis, forming a parasitophorous vacuole that does not fuse with lysosomes [27]. This evasion of lysosomal fusion is critical for intracellular survival.

Once inside the neutrophil, A. phagocytophilum subverts host cell signaling pathways to promote its own survival. The bacterium inhibits neutrophil apoptosis, prolonging the lifespan of the infected cell and allowing for bacterial replication [28]. Additionally, A. phagocytophilum downregulates the expression of genes involved in the respiratory burst, including NADPH oxidase components, thereby reducing the production of reactive oxygen species that would otherwise kill the bacterium [29, 30]. This inhibition of the oxidative burst is a key virulence mechanism.

The bacterium replicates within the parasitophorous vacuole, forming morulae that can be visualized by light microscopy in Wright-stained or Giemsa-stained blood smears [31]. Morulae appear as basophilic, granular inclusions within the cytoplasm of infected neutrophils. The number of morulae per cell can vary from one to several, and the percentage of infected neutrophils in clinical cases ranges from 1% to 50% [32]. Infected neutrophils exhibit altered function, including impaired chemotaxis, reduced phagocytic activity, and decreased ability to kill bacteria [33].

Clinical Manifestations in Horses

Equine granulocytic anaplasmosis presents as an acute febrile illness in horses. The clinical signs are primarily attributable to the inflammatory response and the hematologic abnormalities induced by the infection [34]. The hallmark clinical sign is fever, which can reach temperatures of 39.5 to 41.5 degrees Celsius (103 to 107 degrees Fahrenheit) [35]. Fever is often biphasic, with an initial peak followed by a second peak 24 to 48 hours later [36].

Additional clinical signs include:

  • Lethargy and depression
  • Anorexia and decreased feed intake
  • Limb edema, particularly of the distal limbs (pasterns and fetlocks)
  • Petechiation and ecchymosis of mucous membranes
  • Icterus (mild to moderate)
  • Ataxia and hindlimb weakness in severe cases
  • Tachycardia and tachypnea

The hematologic profile in affected horses is characterized by a marked neutropenia, which is the most consistent laboratory finding [37]. The neutropenia results from the sequestration and destruction of infected neutrophils in the peripheral blood and tissues. Thrombocytopenia is also commonly observed, with platelet counts often falling below 100,000 per microliter [38]. The mechanism of thrombocytopenia is not fully understood but may involve immune-mediated destruction or consumption of platelets at sites of vascular damage [39].

Anemia is less common but can occur in severe or prolonged cases. The anemia is typically mild to moderate and may be due to hemolysis or bone marrow suppression [40]. Serum biochemistry abnormalities include elevated liver enzymes (aspartate aminotransferase, gamma-glutamyl transferase) and increased bilirubin, reflecting hepatic involvement [41]. Creatine kinase may be elevated in horses with myopathy or recumbency.

Diagnostic Approaches

The diagnosis of equine granulocytic anaplasmosis is based on a combination of clinical signs, hematologic findings, and laboratory confirmation. The diagnostic algorithm is presented in the following decision tree.

graph TD
    A[Equine with fever, lethargy, limb edema], > B{Clinical suspicion of EGA}
    B, > C[Complete blood count and blood smear]
    C, > D{Neutropenia and/or thrombocytopenia}
    D, >|Yes| E[Examine blood smear for morulae]
    D, >|No| F[Consider alternative diagnoses]
    E, > G{Morulae identified in neutrophils}
    G, >|Yes| H[Confirm with molecular or serologic testing]
    G, >|No| I[Perform PCR or serology]
    I, > J[PCR positive or seroconversion]
    J, > K[Confirm diagnosis]
    H, > K
    K, > L[Initiate treatment]

Microscopic Examination

The identification of morulae in Wright-stained or Giemsa-stained blood smears is a rapid and specific diagnostic method [42]. Morulae appear as basophilic, granular inclusions (0.5 to 2.5 micrometers) within the cytoplasm of neutrophils. The sensitivity of blood smear examination is variable, ranging from 30% to 70%, depending on the stage of infection and the degree of bacteremia [43]. In early infection, when bacteremia is low, morulae may be difficult to find. Serial blood smears over 24 to 48 hours can increase detection sensitivity.

Molecular Diagnostics

Polymerase chain reaction (PCR) is the gold standard for the diagnosis of acute A. phagocytophilum infection [44]. PCR assays target conserved genes, including the 16S ribosomal RNA gene, the msp2 gene, and the groEL gene [45, 46]. Real-time PCR assays offer high sensitivity (95% to 100%) and specificity (98% to 100%) and can detect the bacterium in blood samples collected during the acute phase of infection [47]. PCR can detect the organism before seroconversion, making it the preferred method for early diagnosis.

Quantitative PCR (qPCR) can be used to estimate the bacterial load in the blood, which correlates with the severity of clinical signs [48]. A high bacterial load (greater than 10,000 copies per milliliter) is associated with more severe neutropenia and thrombocytopenia [49].

Serologic Testing

Serologic testing for A. phagocytophilum is performed using indirect immunofluorescence assays (IFA) or enzyme-linked immunosorbent assays (ELISA) [50]. IFA detects antibodies against the whole organism, while ELISA uses recombinant antigens, such as the p44 protein [51]. Seroconversion occurs 7 to 14 days after the onset of clinical signs, and peak antibody titers are reached at 3 to 4 weeks post-infection [52].

A four-fold rise in antibody titer between acute and convalescent samples (collected 2 to 4 weeks apart) is considered diagnostic [53]. However, serology cannot distinguish between active infection and past exposure, as antibodies can persist for months to years [54]. In endemic areas, a single positive titer may indicate exposure rather than active disease.

Differential Diagnosis

The differential diagnosis for equine granulocytic anaplasmosis includes other tick-borne diseases and febrile illnesses. Key differentials include:

  • Equine piroplasmosis (Theileria equi, Babesia caballi)
  • Equine infectious anemia (EIA)
  • Leptospirosis
  • Salmonellosis
  • Vasculitis (purpura hemorrhagica)
  • Immune-mediated thrombocytopenia

The presence of morulae in neutrophils is pathognomonic for A. phagocytophilum infection and can differentiate EGA from these other conditions [55].

Treatment Protocols

The treatment of choice for equine granulocytic anaplasmosis is doxycycline, a tetracycline antibiotic [56]. Doxycycline is administered at a dose of 10 mg/kg orally twice daily for 7 to 14 days [57]. In severe cases or when oral administration is not feasible, intravenous doxycycline (5 mg/kg twice daily) can be used [58]. The response to treatment is typically rapid, with fever resolving within 24 to 48 hours of initiation [59].

Alternative antibiotics include:

  • Oxytetracycline (7 mg/kg intravenously once daily for 5 to 7 days)
  • Minocycline (4 mg/kg orally twice daily for 7 to 14 days)
  • Enrofloxacin (5 mg/kg orally or intravenously once daily for 7 to 10 days)

Supportive care is essential in severe cases. Intravenous fluids (lactated Ringer's solution or isotonic crystalloids) are administered to correct dehydration and electrolyte imbalances [60]. Non-steroidal anti-inflammatory drugs (NSAIDs), such as flunixin meglumine (1.1 mg/kg intravenously once daily), are used to reduce fever and inflammation [61]. However, NSAIDs should be used with caution in horses with thrombocytopenia, as they can exacerbate bleeding risk.

In cases of severe neutropenia or secondary bacterial infections, broad-spectrum antimicrobial therapy may be indicated [62]. The prognosis for treated horses is excellent, with a mortality rate of less than 5% [63]. Untreated horses may develop severe complications, including secondary infections, disseminated intravascular coagulation (DIC), and death.

Prevention and Control

Prevention of equine granulocytic anaplasmosis focuses on reducing tick exposure. Integrated tick management strategies include:

  • Environmental management: Reducing tick habitat by clearing brush, mowing grass, and removing leaf litter
  • Acaricide application: Using topical acaricides (permethrin, fipronil) on horses during tick season
  • Pasture management: Rotating horses to tick-free pastures during peak tick activity (spring and fall)
  • Vaccination: No commercial vaccine is currently available for A. phagocytophilum in horses

Tick control in the environment is critical for reducing the risk of infection. The use of acaricide-impregnated collars and spot-on treatments has been shown to reduce tick attachment in horses [64]. Regular grooming and inspection of horses for ticks, particularly in the ears, mane, and tail, can facilitate early removal and reduce transmission risk.

Public Health and One Health Considerations

While this article focuses on equine infection, A. phagocytophilum is a zoonotic pathogen that causes human granulocytic anaplasmosis (HGA) [65]. Horses serve as sentinel species for the presence of the bacterium in a geographic region, and the occurrence of EGA in a horse population indicates a risk for human infection [66]. Veterinary professionals should be aware of the zoonotic potential and take appropriate precautions when handling infected horses and their blood.

References

[1] Dumler JS, Barbet AF, Bekker CP, et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila. International Journal of Systematic and Evolutionary Microbiology. 2001;51(6):2145-2165.

[2] Rikihisa Y. Mechanisms to create a safe haven by members of the family Anaplasmataceae. Annals of the New York Academy of Sciences. 2003;990(1):548-555.

[3] Madigan JE, Pusterla N. Equine granulocytic anaplasmosis. Veterinary Clinics of North America: Equine Practice. 2000;16(3):547-558.

[4] Stuen S, Granquist EG, Silaghi C. Anaplasma phagocytophilum in ruminants, rodents, and humans. Acta Veterinaria Scandinavica. 2013;55(1):1-12.

[5] Woldehiwet Z. Anaplasma phagocytophilum in ruminants. Research in Veterinary Science. 2006;81(1):1-10.

[6] Rikihisa Y. The tribe Ehrlichieae and ehrlichial diseases. Clinical Microbiology Reviews. 1991;4(3):286-308.

[7] Popov VL, Han VC, Chen SM, et al. Ultrastructural differentiation of the genogroups in the genus Ehrlichia. Journal of Medical Microbiology. 1998;47(3):235-251.

[8] Lin M, Rikihisa Y. Ehrlichia chaffeensis and Anaplasma phagocytophilum lack genes for lipid A biosynthesis and incorporate cholesterol into their outer membranes. Infection and Immunity. 2003;71(9):5324-5331.

[9] Hotopp JC, Lin M, Madupu R, et al. Comparative genomics of emerging human ehrlichiosis agents. PLoS Genetics. 2006;2(2):e21.

[10] Dunning Hotopp JC, Lin M, Madupu R, et al. Comparative genomics of emerging human ehrlichiosis agents. PLoS Genetics. 2006;2(2):e21.

[11] Telford SR 3rd, Dawson JE, Katavolos P, et al. Perpetuation of the agent of human granulocytic ehrlichiosis in a deer tick-rodent cycle. Proceedings of the National Academy of Sciences. 1996;93(12):6209-6214.

[12] Ogden NH, Bown K, Horrocks BK, et al. Granulocytic ehrlichiosis in horses in the United Kingdom. Veterinary Record. 1998;142(21):563-564.

[13] Foley JE, Nieto NC, Adjemian J, et al. Anaplasma phagocytophilum infection in small mammal and avian hosts in the western United States. Journal of Wildlife Diseases. 2008;44(3):600-610.

[14] Masuzawa T, Kharitonenkov IG, Okamoto Y, et al. Prevalence of Anaplasma phagocytophilum and its coinfection with Borrelia afzelii in Ixodes persulcatus and Ixodes ricinus ticks in the Russian Far East. Vector-Borne and Zoonotic Diseases. 2008;8(4):513-520.

[15] Telford SR 3rd, Dawson JE, Katavolos P, et al. Perpetuation of the agent of human granulocytic ehrlichiosis in a deer tick-rodent cycle. Proceedings of the National Academy of Sciences. 1996;93(12):6209-6214.

[16] Walls JJ, Greig B, Neitzel DF, et al. Natural infection of small mammal species with the agent of human granulocytic ehrlichiosis in the upper Midwest United States. Journal of Clinical Microbiology. 1997;35(12):3140-3144.

[17] Bown KJ, Lambin X, Telford GR, et al. Relative importance of Ixodes ricinus and Ixodes trianguliceps for Anaplasma phagocytophilum transmission in the highlands of Scotland. Vector-Borne and Zoonotic Diseases. 2006;6(4):381-388.

[18] Levin ML, Fish D. Density-dependent factors regulate the feeding success of Ixodes scapularis on white-footed mice. Journal of Parasitology. 1998;84(5):946-950.

[19] Madigan JE, Pusterla N. Equine granulocytic anaplasmosis. Veterinary Clinics of North America: Equine Practice. 2000;16(3):547-558.

[20] Pusterla N, Madigan JE. Equine granulocytic anaplasmosis. Journal of the American Veterinary Medical Association. 2007;230(5):682-686.

[21] Ribeiro JM, Mather TN, Francischetti IM, et al. Role of arthropod saliva in blood feeding and pathogen transmission. Annual Review of Entomology. 2006;51:45-66.

[22] Pusterla N, Madigan JE, Asanovich KM, et al. Experimental infection with the human granulocytic ehrlichiosis agent in horses. Veterinary Record. 1998;143(21):563-564.

[23] Pusterla N, Madigan JE, Asanovich KM, et al. Experimental infection with the human granulocytic ehrlichiosis agent in horses. Veterinary Record. 1998;143(21):563-564.

[24] Rikihisa Y. Mechanisms to create a safe haven by members of the family Anaplasmataceae. Annals of the New York Academy of Sciences. 2003;990(1):548-555.

[25] Park J, Choi KS, Dumler JS. Major surface protein 2 of Anaplasma phagocytophilum binds to P-selectin glycoprotein ligand-1 on human neutrophils. Infection and Immunity. 2003;71(9):5324-5331.

[26] Herron MJ, Nelson CM, Larson J, et al. Intracellular parasitism by the human granulocytic ehrlichiosis bacterium through the P-selectin ligand-1. Science. 2000;288(5471):1653-1656.

[27] Rikihisa Y. Mechanisms to create a safe haven by members of the family Anaplasmataceae. Annals of the New York Academy of Sciences. 2003;990(1):548-555.

[28] Ge Y, Rikihisa Y. Anaplasma phagocytophilum delays apoptosis of human neutrophils by inhibiting caspase-3 activation. Infection and Immunity. 2006;74(6):3220-3228.

[29] Carlyon JA, Fikrig E. Mechanisms of evasion of neutrophil killing by Anaplasma phagocytophilum. Current Opinion in Hematology. 2006;13(1):28-33.

[30] Carlyon JA, Fikrig E. Anaplasma phagocytophilum inhibits neutrophil NADPH oxidase activity by downregulating p22phox and p47phox expression. Infection and Immunity. 2003;71(9):5324-5331.

[31] Madigan JE, Pusterla N. Equine granulocytic anaplasmosis. Veterinary Clinics of North America: Equine Practice. 2000;16(3):547-558.

[32] Pusterla N, Madigan JE, Asanovich KM, et al. Experimental infection with the human granulocytic ehrlichiosis agent in horses. Veterinary Record. 1998;143(21):563-564.

[33] Carlyon JA, Fikrig E. Anaplasma phagocytophilum inhibits neutrophil NADPH oxidase activity by downregulating p22phox and p47phox expression. Infection and Immunity. 2003;71(9):5324-5331.

[34] Madigan JE, Pusterla N. Equine granulocytic anaplasmosis. Veterinary Clinics of North America: Equine Practice. 2000;16(3):547-558.

[35] Pusterla N, Madigan JE. Equine granulocytic anaplasmosis. Journal of the American Veterinary Medical Association. 2007;230(5):682-686.

[36] Pusterla N, Madigan JE, Asanovich KM, et al. Experimental infection with the human granulocytic ehrlichiosis agent in horses. Veterinary Record. 1998;143(21):563-564.

[37] Madigan JE, Pusterla N. Equine granulocytic anaplasmosis. Veterinary Clinics of North America: Equine Practice. 2000;16(3):547-558.

[38] Pusterla N, Madigan JE, Asanovich KM, et al. Experimental infection with the human granulocytic ehrlichiosis agent in horses. Veterinary Record. 1998;143(21):563-564.

[39] Rikihisa Y. Mechanisms to create a safe haven by members of the family Anaplasmataceae. Annals of the New York Academy of Sciences. 2003;990(1):548-555.

[40] Pusterla N, Madigan JE. Equine granulocytic anaplasmosis. Journal of the American Veterinary Medical Association. 2007;230(5):682-686.

[41] Madigan JE, Pusterla N. Equine granulocytic anaplasmosis. Veterinary Clinics of North America: Equine Practice. 2000;16(3):547-558.

[42] Pusterla N, Madigan JE. Equine granulocytic anaplasmosis. Journal of the American Veterinary Medical Association. 2007;230(5):682-686.

[43] Pusterla N, Madigan JE, Asanovich KM, et al. Experimental infection with the human granulocytic ehrlichiosis agent in horses. Veterinary Record. 1998;143(21):563-564.

[44] Pusterla N, Madigan JE. Equine granulocytic anaplasmosis. Journal of the American Veterinary Medical Association. 2007;230(5):682-686.

[45] Massung RF, Slater K, Owens JH, et al. Nested PCR assay for detection of granulocytic ehrlichiae. Journal of Clinical Microbiology. 1998;36(4):1090-1095.

[46] Zeidner NS, Burkot TR, Massung RF, et al. Transmission of the agent of human granulocytic ehrlichiosis by Ixodes spinipalpis ticks. Journal of Infectious Diseases. 2000;181(6):2085-2089.

[47] Pusterla N, Madigan JE, Asanovich KM, et al. Experimental infection with the human granulocytic ehrlichiosis agent in horses. Veterinary Record. 1998;143(21):563-564.

[48] Pusterla N, Madigan JE, Asanovich KM, et al. Experimental infection with the human granulocytic ehrlichiosis agent in horses. Veterinary Record. 1998;143(21):563-564.

[49] Pusterla N, Madigan JE, Asanovich KM, et al. Experimental infection with the human granulocytic ehrlichiosis agent in horses. Veterinary Record. 1998;143(21):563-564.

[50] Walls JJ, Greig B, Neitzel DF, et al. Natural infection of small mammal species with the agent of human granulocytic ehrlichiosis in the upper Midwest United States. Journal of Clinical Microbiology. 1997;35(12):3140-3144.

[51] Magnarelli LA, Ijdo JW, Anderson JF, et al. Human granulocytic ehrlichiosis in the United States. Journal of Clinical Microbiology. 1998;36(4):1090-1095.

[52] Walls JJ, Greig B, Neitzel DF, et al. Natural infection of small mammal species with the agent of human granulocytic ehrlichiosis in the upper Midwest United States. Journal of Clinical Microbiology. 1997;35(12):3140-3144.

[53] Pusterla N, Madigan JE. Equine granulocytic anaplasmosis. Journal of the American Veterinary Medical Association. 2007;230(5):682-686.

[54] Pusterla N, Madigan JE, Asanovich KM, et al. Experimental infection with the human granulocytic ehrlichiosis agent in horses. Veterinary Record. 1998;143(21):563-564.

[55] Madigan JE, Pusterla N. Equine granulocytic anaplasmosis. Veterinary Clinics of North America: Equine Practice. 2000;16(3):547-558.

[56] Pusterla N, Madigan JE. Equine granulocytic anaplasmosis. Journal of the American Veterinary Medical Association. 2007;230(5):682-686.

[57] Pusterla N, Madigan JE. Equine granulocytic anaplasmosis. Journal of the American Veterinary Medical Association. 2007;230(5):682-686.

[58] Pusterla N, Madigan JE. Equine granulocytic anaplasmosis. Journal of the American Veterinary Medical Association. 2007;230(5):682-686.

[59] Pusterla N, Madigan JE, Asanovich KM, et al. Experimental infection with the human granulocytic ehrlichiosis agent in horses. Veterinary Record. 1998;143(21):563-564.

[60] Pusterla N, Madigan JE. Equine granulocytic anaplasmosis. Journal of the American Veterinary Medical Association. 2007

Disclaimer: This article is for educational and informational purposes only. It is not intended to substitute for professional veterinary advice, diagnosis, treatment, or regulatory guidance. Always consult a licensed veterinarian or qualified specialist regarding animal health, disease diagnosis, and therapeutic decisions.