Erika de Papp, DVM, DACVIM
Rickettsial organisms pose a growing threat to both our domestic pet population, as well as the human population, especially in the face of climate change and expanding tick populations. This update will discuss the Rickettsial organisms tested for on a routine basis with the ELISA screening tests, including the tick vectors, associated disease syndromes, diagnostic approach, treatment, and lastly, what to do with a positive test result in a healthy patient.
Both the 4Dx Plus® (Idexx) and the Accuplex 4® (Antech) ELISA tests are antibody tests that indicate exposure to members of the Family Anaplasmataceae, including the species Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, Ehrlichia ewingii, and Ehrlichia chafeensis.
As is the case with Borrelia burgdorferi, the tick vector for Anaplasma phagocytophilum is the Ixodes scapularis tick, or Ixodes pacificus on the west coast. Both Anaplasma platys and Ehrlichia canis are transmitted by Rhipicephalus sanguineus, whereas Ehrlichia ewingii and Ehrlichia chaffeensis are transmitted by Amblyomma americanum. To date, Ehrlichia chaffeensis has been recognized most commonly as a human pathogen and will not be discussed further, although dogs can be infected.
All of these organisms are obligate intracellular bacteria. Anaplasma phagocytophilum and Ehrlichia ewingii infect neutrophils, Ehrlichia canis infects monocytes, and Anaplasma platys infects platelets. (Fun fact for your next cocktail party; Anaplasma platys is the only infectious organism known to directly infect platelets.)
The Companion Animal Parasite Council (capcvet.org) tracks the prevalence of Ehrlichia and Anaplasma species positive tests across the US, based on data provided by Idexx and Antech. It is important to note that the numbers they present are not reflective of all positive test results and they in fact state that the values represent less than 30% of activity in a given geographic location. For 2019, Anaplasma across the US is listed at 3.2% but for MA, it jumps to 12.6%. All of New England is considered a high risk region. For Ehrlichia the rate across the US for 2019 is 2.8%, but only 1.9% for MA.
Disease transmission in New England occurs in the late spring and early summer when the nymphal ticks are active, as well as in the early fall when the adult ticks are ac tive. The length of tick attachment that is required for disease transmission varies with the organism and the tick species involved, but for E. canis, transmission may be as short as three hours in some cases.
A recent study in Europe evaluated transmission time for A.phagocytophilum from experimentally infected ticks placed on dogs for varying lengths of time (Fourie et al 2019). Based on artificial feeding chambers, the authors demonstrated that the transmission of the organism commences within several hours of tick attachment. However in the live dogs, no dog seroconverted or became PCR positive until ticks were attached for greater than 48 hours, suggesting that the infective dose is not reached until more than 48 hours of tick attachment. These findings suggest that for Anaplasmosis, similar to what has been demonstrated with Lyme disease, frequent tick checks and removal as soon as possible may significantly decrease the risk of disease transmission.
Clinical signs of A. phagocytophilum are associated with the acute phase of infection, and develop 1-3 weeks post tick inoculation. Fever is the most common physical exam finding in these dogs. Other common complaints and PE findings include joint pain, lameness, reluctance to move, lethargy, and anorexia. The signs can be quite non-specific in some dogs. Less common findings include gastroenteritis (vomiting and/or diarrhea), coughing, and CNS abnormalities. Splenomegaly and mild lymphadenopathy may be noted on PE. Rarely these dogs present with signs of petechial hemorrhages, epistaxis, or other bleeding diatheses. The most consistent laboratory abnormality in infected dogs is thrombocytopenia, with typically more than 80% of dogs affected. Initially they may also be neutropenic but this typically resolves or develops into a neutrophilia. Lymphocytes may be reactive, and a mild non-regenerative anemia may be present. Morulae are cytoplasmic inclusions containing the organism that may be seen within neutrophils on a blood smear. Elevations in liver enzymes including ALT and ALKP may also be seen, as well as a mild to moderate hypoalbuminemia. A recent study from Germany also reported hyperbilirubinemia (Chirek et al 2018).
Although A. phagocytophilum is known to cause acute disease, chronic subclinical carriers have been documented for close to one year (Alleman et al 2006). At this time it is not known if chronic carriers can result in chronic disease, but this has not been documented.
Cats can also be infected and clinically affected by A. phagocytophilum. Over a two year period (2009-2011) Antech reported 0.92% of cats tested were PCR positive for the organism. A 2016 study of cats reported 16 clinical cases, all from the Northeastern US (Savidge et al 2016). Lethargy and fever were the most common presenting complaints, and 64% of the cats were thrombocytopenic. All cats responded to doxycycline therapy. A study out of Maine last year found 6.3% of cats tested by 4Dx were positive for Anaplasma antibodies, and not surprisingly, cats with clinical disease and cats with outdoor access were 4 and 5 times more likely to test positive, respectively (Hoyt et al 2018). It should be noted that the 4Dx test is not validated in cats, but since cats can become infected, this may still be a useful screening test.
Anaplasma platys is a less commonly seen organism in the US and thought to be mostly a subclinical infection in North American dogs. Signs may include fever, lethargy, petechiae, epistaxis, and lymphadenopathy. A. platys causes a cyclic thrombocytopenia, with counts often dropping to < 20,000, but returning to normal in 3-4 days.
A retrospective study of dogs naturally infected with A. platys in the Mediterranean basin showed that this can be a clinically very severe disease, with a one month mortality of 23.9% in that study (Bouzourra et al 2016). The most common clinical signs in the study included anorexia, weight loss, lymphadenopathy, and fever. The most common lab abnormalities were thrombocytopenia and anemia. Different strains of the organism were detected by sequencing analysis, suggesting that some strains may have increased pathogenicity. It would appear that we do not currently have this strain in the US, but it is something of which to be aware.
Canine Ehrlichiosis caused by Ehrlichia canis is a disease that has three distinct stages; acute, subclinical, and chronic. The acute disease occurs 2-4 weeks post inoculation. The acute disease is often transient and may go undetected clinically, as dogs commonly recover spontaneously. If dogs are ill, signs can include fever, anorexia, lethargy, malaise, myalgia, lymphadenopathy, and splenomegaly. Following the acute phase, dogs go into a subclinical phase that can last for months to years. Dogs that don’t clear the infection during this phase will eventually enter into the chronic phase, which may occur 3 or more years after the initial infection. In the chronic phase dogs may have stomatitis or glossitis, peripheral edema (especially hindlimb and scrotal), CNS signs ranging from ataxia to seizures, and bleeding diatheses (petechiae, hematuria, epistaxis, melena). These signs can be present during the acute phase as well, but are more commonly seen during the chronic phase. The challenge is that it’s unpredictable which dogs will progress to the chronic phase, and in the clinical setting, it can be very hard to know what phase of disease is present. Laboratory findings in the acute and subclinical phases include thrombocytopenia in > 90% of infected dogs, as well as mild leukopenia and mild non-regenerative anemia. In the chronic phase dogs continue to be thrombocytopenic and severe cases can be profoundly pancytopenic due to irreversible bone marrow damage. The chronic cases will often also have granular lymphocytes, elevated liver enzymes, and hyperglobulinemia. The latter is most often a polyclonal gammopathy, but can be monoclonal as well.
Ehrlichia ewingii is somewhat of an emerging organism that historically was thought to be less pathogenic than E. canis. Reported clinical signs include fever, lameness, lethargy, lymphadenopathy, peripheral edema, and neurologic abnormalities. Laboratory abnormalities that are generally reported include thrombocytopenia, non-regenerative anemia, and lymphopenia. A retrospective study that just came out this year documented 41 cases of E. ewingii. To accurately characterize this disease, dogs that were PCR positive for other vector-borne diseases were excluded (Qurollo et al 2019). Clinical findings in these dogs included limb or joint pain in just over 33% of cases, fever and lymphadenopathy in about 25%, and gastroenteritis in 22%. Organomegaly was reported in 17% of the dogs. Laboratory abnormalities included neutrophilia, left shift, increased neutrophil to lymphocyte ratios in most dogs, anemia in just under half, monocytosis, and thrombocytopenia in 43%, which was lower than anticipated based on previous reports. Somewhat unexpected findings included proteinuria in 74%, as well as increased BUN and increased SDMA in almost 1/3 of dogs. Just under 20% of dogs had evidence of immune mediated disease as well (IMHA or ITP). The authors concluded that the findings warrant investigation into the possible association between infection and progression of renal disease, as well as a potential role for the organism in the pathogenesis of IMHA and ITP. Additionally, the finding of an elevated neutrophil to lymphocyte ratio may be a good indication to test for vector-borne disease.
The findings of the aforementioned study are quite interesting in relation to the recent Idexx study (Idexx website) that demonstrated that dogs exposed to Ehrlichia species (antibody positive) had a 112% increased risk of developing chronic kidney disease (CKD). A direct cause and effect relationship was not identified, but the association between the two conditions was statistically significant. A 2012 study of dogs ranging from Oklahoma to the eastern seaboard revealed that 7.1% of dogs tested had antibodies to Ehrlichia. Species-specific antibody testing revealed that 5.1% of the dogs were seropositive to E. ewingii compared to only 0.8% to E. canis, and 2.8% to E. chaffeensis (Beall et al 2012). This suggests that perhaps E. ewingii is on the rise, and is potentially becoming a more important pathogen than E. canis.
The same diagnostic approach can be used for all of these diseases. Diagnosis can be made based on some combination of the following: ELISA, IFA, PCR, cytology, appropriate clinical signs, and response to treatment.
The commonly used ELISA tests include the 4Dx Plus and the Accuplex 4. It is important to note that these are antibody tests and therefore a positive test result indicates exposure and a subsequent immune response, but does not equate with active infection. These tests may be negative in the acute phase of any of these diseases, prior to seroconversion. Additionally, they cannot differentiate A. phagocytophilum from A. platys, nor E. canis from E. ewingii. However, these are excellent screening tests (more on that later), and have a very rapid turnaround time. One research article compared the sensitivity, specificity, and reproducibility of the 4Dx and the Accuplex, and showed significantly better results for the 4Dx specifically for finding antibodies to Anaplasma (Goldstein et al 2014), but both tests are widely used and should be considered good screening tests.
IFA, or indirect immunofluorescent antibody tests are used less commonly since the advent of the ELISA tests, but provide the same information. The potential added benefit of the IFA is that you get a specific titer and the test can be repeated to look for a change in titer. A four-fold rise in titer is indicative of active infection when comparing acute and convalescent samples. The IFA however does also exhibit cross-reactivity between species.
PCR, or polymerase chain reaction, has become very popular for diagnosing a number of infectious diseases. This technology amplifies the organism DNA, so a positive test does indicate active infection, and the results are species specific. PCR will become positive faster than antibody tests so this methodology is better in acute disease settings, however the organism levels can fluctuate and result in a negative PCR test. It is important to remember that a negative test does not completely rule out the disease. It simply means that no DNA for the organism in question was detected. Additionally, any prior antibiotic use can result in a negative test. In difficult cases, it can often be very useful to use a combination of serology and PCR to help achieve the diagnosis.
Cytology of a blood smear can also be used to diagnose A. phagocytophilum or E. ewingii, by the presence if morulae within the cytoplasm of neutrophils. However, the two species cannot be distinguished from one another based on their appearance. Morulae are almost never seen with the other organisms. Visualization may be enhanced by preparing buffy coat smears to concentrate the white blood cells. Occasionally, morulae can also be seen in synovial fluid of dogs presenting with polyarthritis.
Finally, in conjunction with serology, PCR, and cytology, the clinical picture can aid in making the diagnosis. Does the dog have appropriate clinical signs and are we seeing the expected laboratory changes? Lastly, is the dog responding to treatment? Most patients exhibit a very rapid clinical response, which often aids in the diagnosis in cases that might not otherwise be clear cut.
In order to definitively confirm a diagnosis of any of these diseases, one of the following criteria should be met:
- The dog is seroreactive with appropriate clinicopathologic findings and appropriate response to therapy.
- The PCR result is positive.
- Morulae are seen in neutrophils ( phago or E. ewingii)
- A four-fold increase in antibody titer has been demonstrated within 4 weeks.
Morulae can be seen in neutrophils with either A. phagocytophilum or E. ewingii infection. Photo courtesy of Patty Ewing, DVM, MS, DACVP (anatomic and clinical pathology).
The good news when it comes to treatment is that all of these diseases respond well to doxycycline. This is a time dependent antibiotic so the most appropriate dose is 5 mg/kg PO, BID. The drug will also work at 10 mg/kg SID if this is necessary for convenient dosing of smaller patients but the BID dosing is preferable. Clinical improvement is typically expected within 24-48 hours. Debate remains over the appropriate length of treatment but at this time the recommended treatment period for all of the diseases is 4 weeks. Some people opt to treat Anaplasmosis for shorter periods of time given the acute nature of the disease but a recent study from last year demonstrated efficacy with 28 days of doxycycline (Yancey et al 2018). Whether a shorter treatment period would be equally effective is not definitively known. If the patient is not responding then other vector-borne diseases that are not fully responsive to doxycycline should be considered, such as Bartonellosis or Babesiosis. Additionally, given recent studies documenting concurrent immune-mediated disease in patients with these vector-borne pathogens, immune-mediated diseases should be considered in non-responders. A recent study of A. phagocytophilum revealed that 44% of dogs had platelet bound antibodies and 25% of dogs had signs consistent with immune-mediated disease (Chirek et al 2018).
Follow-up monitoring should include a CBC and PE one week post diagnosis. In patients that test positive for Ehrlichia spp., a UA and UP/C should be recommended and SDMA testing considered. If clinicopathologic findings are not improving at the one week mark, then additional work up should be considered. Once infected, annual vector-borne disease testing is strongly recommended, and in fact recommended for all dogs. Annual lab work in Ehrlichia positive dogs should include a CBC, Chemistry screen and UA at a minimum.
The question of what do with a positive test in a healthy dog is often asked, as this is a stressful topic for many. My current recommendations are as follows: if positive on a screening antibody test for Anaplasma, perform a CBC and treat if the dog is thrombocytopenic or morulae are seen on the blood smear. If the CBC is normal, do not treat. If positive on a screening antibody test for Ehrlichia, my current recommendation would be to perform a PCR and treat if positive for any Ehrlichia species. Additionally, these dogs should have a minimum database to include a CBC, Chemistry screen, and UA and dogs should be treated if demonstrating clinicopathologic abnormalities consistent with infection, even if PCR negative. The rationale for treating these dogs is that we cannot predict which dogs will develop chronic and potentially life-threatening disease (Mylonakis et al 2019). This latter statement was made by an author from an endemic E. canis region of the world. However, given what the recent E. ewingii study has demonstrated, especially with respect to renal disease, it makes sense to treat these dogs as well, to potentially avoid future serious sequelae. If a patient is Ehrlichia antibody positive but PCR negative and has normal bloodwork, then monitoring is recommended. In these cases following IFA titers is warranted to see if the titers are rising.
This raises the question as to whether we should even be screening all dogs, and the answer is an emphatic yes. Screening allows us to accurately track prevalence rates and is useful to prove tick exposure, even when not causing clinical illness. We all have the client who refuses to use tick preventatives because their dog doesn’t ever get ticks! Additionally, these are zoonotic diseases and our greater understanding of them will aid both pet and human health in the long run.
Take home points:
- Thrombocytopenia is a hallmark of these Rickettsial diseases.
- Clinical signs can be vague and there is a lot of overlap between diseases.
- All dogs should be screened annually.
- A combination of serology and PCR may be necessary to make a diagnosis in challenging cases.
- Dogs with evidence of immune-mediated disease should have thorough tick testing performed.
- Dogs with documented exposure to Ehrlichia spp warrant long term follow up, even if healthy.
- Alleman, A. (2008) An update on anaplasmosis in dogs. Retrieved from dvm360.com
- Alleman, A. (2015) More than just canis. The increasingly complicated story of ehrlichiosis. ABVP Conference proceedings
- Beall, M., Alleman, A., et al. (2012) Seroprevalence of Ehrlichia canis, Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in North America. ParasitVectors 5:29
- Bouzourra, T., Rene-Martellet, M., et al. (2016) Clinical and laboratory features of canine Anaplasma platys infection in 32 naturally infected dogs in the Mediterranean basin. Ticks and Tick-borne Diseases 7(6): 1256-1264
- Bulla, C., Kiomi, T., et al. (2004) The relationship between the degree of thrombocytopenia and infection with Ehrlichia canis in an endemic area. Vet Res 35(1): 141-146
- Chirek, A., Silaghi, C., et al. (2018) Granulocytic anaplasmosis in 63 dogs: clinical signs, laboratory results, therapy and course of disease. Journal of
- Small Animal Practice 59: 112-120
- Egenvall, A., Bjoersdorff, A., et al. (1998) Early manifestations of granulocytic ehrlichiosis in dogs inoculated experimentally with a Swedish Ehrlichia species isolate. Vet Rec 143(15): 412-417
- Fourie, J., Evans, E., et al. (2019) Transmission of Anaplasma phagocytophilum by Ixodes ricinus ticks feeding on dogs and artificial membranes. Parasit Vectors 12:36
- Goldstein, R., Eberts, M., et al. (2014) Performance comparison of SNAP 4Dx Plus and Accuplex4 for the detection of antibodies to Borrelia burgdorferi
- and Anaplasma phagocytophilum. IntJ Appl Res Vet Med 12(2): 141-147
- Harrus, S. (2014) Canine monocytic ehrlichiosis. World Small Animal Veterinary Assoc Conference proceedings.
- Herrin, B. (2019) Tick-borne Rickettsial infections of dogs. Today’s Veterinary Practice 9(3): 20-25
- Hoyt, K., Chandrashekar, M., et al. (2018) Evidence for clinical anaplasmosis and borreliosis in cats in Maine. Topics in Companion Animal Medicine 33(2): 40-44
- Kidd, L., Qurollo, B., et al. (2017) Prevalence of vector-borne pathogens in southern California dogs with clinical and laboratory abnormalities consistent with immune-mediated disease. J Vet Intern Med31(4): 1081-1090
- Mylonakis, M., Harrus, S., Breitschwerdt, E. (2019) An update on treatment of canine monocytic ehrlichiosis (Ehrlichia canis).
- The Veterinary Journal 246: 45-53
- Qurollo, B., Buch, J., et al. (2019) Clinicopathological findings in 41 dogs (2008-2018) naturally infected with Ehrlichia ewingii.
- J Vet Intern Med 33 (6): 618-629
- Qurollo, B., Lappin, M., et al. (2017) Prevalence of vector-borne pathogens in southern California dogs with clinical and laboratory abnormalities consistent with immune-mediated disease. J Vet Intern Med 31(4): 1081-1090
- Savidge, C., Ewing, P., et al. (2016) Anaplasma phagocytophilum infection in domestic cats: 16 cases from the northeastern USA.
- Journal of Feline Med and Surgery 18(2): 85-91
- Yancey, C., Diniz, P., et al. (2018) Doxycycline treatment efficacy in dogs with naturally occurring Anaplasma phagocytophilum Journal of Small Animal Practice 59: 286-293
- Data also retrieved from Idexx.com and CAPCvet.org