Testing for vector-borne pathogens in dogs: The best use of diagnostic panels


Serologic and PCR panels can help identify infection-so how do you choose which panel to use?

Vector-borne disease is an important cause of morbidity and mortality in dogs. Clinical findings include those commonly associated with idiopathic immune-mediated disease. Like vector-borne disease, the incidence of idiopathic immune-mediated disease can be seasonal.1,2 It has been hypothesized that many patients with immune-mediated disease actually have undiagnosed underlying infections with vector-borne and other infectious agents.

In addition to ruling out infection in patients with immune-mediated disease, vector-borne disease screening is also sometimes performed in healthy animals to detect subclinical infections and to screen blood donors and animals living with or interacting with immunocompromised people. Inappropriately declaring that infection has been ruled out because of a "negative tick panel" may adversely affect the health of dogs and people.


"Tick panels" and "vector-borne disease panels" give clinicians the ability to test blood for multiple agents and the presence of coinfection by using serology, polymerase chain reaction (PCR) assays, or both. Although these panels can be comprehensive, clinicians should remember that both PCR and serologic test results may be negative in the presence of infection, even if the assays are exquisitely sensitive.

It has been shown that using a combination of serology and PCR can substantially increase recognition of the presence of vector-borne disease.3,4 Familiarity with the epidemiology and pathophysiology of each organism helps determine which agents should be included and which type of testing—PCR or serology, or both—should be used.

This article reviews the concepts that help clinicians decide which organisms to include in screening, which type of test may have the highest sensitivity and specificity for a patient, and whether further testing for a particular organism is warranted in a particular case context. The form Vector-borne disease testing: Helpful questions to ask yourself summarizes the questions clinicians can ask to facilitate the testing process.


A patient's clinical findings, breed, geographic location, and travel history can help clinicians decide which organisms to include in testing.

Clinical findings

The nonspecific signs of malaise and gastrointestinal abnormalities are often reported in patients with vector-borne disease. Physical examination findings that more specifically suggest a vector-borne disease include but are not limited to

  • Fever

  • Evidence of disordered primary hemostasis (petechiation or mucosal bleeding and epistaxis due to vasculitis, thrombocytopenia, or thrombocytopathia)

  • Lymphadenomegaly

  • Ocular abnormalities (e.g. retinal hemorrhages, uveitis, chorioretinitis, perivascular infiltrates)

  • Splenomegaly

  • Arthralgia and joint effusion

  • Myalgia

  • Respiratory signs

  • Neurologic signs.

Clinicopathologic findings commonly associated with vector-borne disease include but are not limited to

  • Anemia

  • Thrombocytopenia

  • Hypoalbuminemia

  • Hyperglobulinemia

  • Proteinuria

  • Synovial fluid analysis results consistent with neutrophilic polyarthritis.

Although immune-mediated disease could theoretically be triggered by any infectious agent, it can be diagnostically useful to consider that some findings have been specifically associated with particular organisms in dogs. For example, Babesia species, Anaplasma phagocytophilum, Bartonella species, Ehrlichia canis, and hemotropic Mycoplasma species have been associated with immune-mediated hemolytic anemia (IMHA) in dogs.5-9 Therefore, it would be prudent to rule out infection with these agents in a dog with IMHA.

Further examples of signs associated with certain disease agents include vasculitis with Rickettsia, Ehrlichia, and Bartonella species; granulomatous or pyogranulomatous inflammation with Bartonella species; and hyperglobulinemia with Ehrlichia species and other organisms that cause chronic disease.7,10 Comprehensive summaries of clinical signs that have been associated with specific infections are available.11


Breed association can also help clinicians decide which organisms to include in diagnostic testing. For example, infection with Babesia gibsoni should be ruled out in American pit bull terriers with hemolytic anemia or thrombocytopenia.12,13 Commonly used serologic and PCR tests that target Babesia canis may have negative results in a dog infected with B. gibsoni. Knowing that B. gibsoni is prevalent in American pit bull terriers can help you direct appropriate testing.

Another example is that greyhounds are overrepresented among dogs infected with B. canis,13 presumably because of their exposure to Rhipicephalus sanguineus (the brown dog tick) in kennel environments. In addition to B. canis, E. canis would also be important to rule out in this or any dog breed exposed to kennels because both of these organisms are transmitted by R. sanguineus.

Additional examples include the fact that vector-borne disease is common in hunting breeds, and leishmaniasis is common in foxhounds in the United States. Thus, consideration of breed can help guide testing.

Geographic location and travel history

Geographic location and travel history are also important to consider when deciding which organisms to include in testing. For some organisms, the distribution of exposure is fairly geographically restricted. For example, A. phagocytophilum infection should be suspected in an acutely ill thrombocytopenic dog with arthralgia if that dog lives in or has traveled to the upper Midwest, Northeast, or Pacific Coast; whereas, Ehrlichia ewingii infection would be considered more likely if a dog lives in or has traveled to the south central or southeastern United States.14-16

Babesia conradae is an important differential diagnosis in dogs with IMHA that have lived in or traveled to southern California, but it has not been documented in other areas of the United States.17 In contrast, infection with E. canis or B. canis should be considered in a dog with compatible clinical signs in most parts of the United States, as these organisms are transmitted by R. sanguineus, a ubiquitous tick with an expanding geographic distribution.18-20 Summaries of common clinical signs and geographic distributions of infectious agents are available.11

Although geographic locale helps determine which agents should be included in testing, it is important to be aware the distribution of vectors and their associated infectious agents is expanding. For example, the incidence of Rocky Mountain spotted fever (RMSF) in people in the United States has recently expanded beyond the distribution of its historic tick vectors, Dermacentor variabilis and Dermacentor andersoni. Rhipicephalus sanguineus recently caused an outbreak of RMSF in people in a nonendemic area of Arizona.20 Retrospectively, it was shown that infection existed in the dog population before the fatal outbreak occurred in people.21 Thus, although it can help clinicians to decide what to include, geographic locale should not necessarily be used to restrict diagnostic testing.


Most serologic tests document infection indirectly by detecting antibodies to the infectious agent. Demonstrating the presence of antibody indicates exposure but not necessarily active infection. Therefore, it can be difficult to definitively attribute clinical signs to infection, particularly in endemic areas.

PCR directly detects an organism's nucleic acid sequences. Recently, advances in molecular biology have made PCR panels easy to perform and widely available. PCR makes it possible to directly test for the presence of multiple organisms with high sensitivity and specificity, assuming appropriate laboratory controls are in place. However, organisms are not always circulating in blood in high enough numbers to be present in a blood sample at the time clinical signs occur.

Considering whether the organism is likely to be circulating in blood at the time of presentation and the significance of a positive or negative test result can help determine whether PCR or serology will be more sensitive or specific in a patient. Table 1 summarizes this information for some common vector-borne disease agents that infect dogs.

Table 1: Some pathophysiologic considerations for determining which diagnostic test to use for select vector–borne disease agents*

Is the organism in the sample? The clinical sensitivity of PCR

In order for PCR results to be positive, the organism (or the nucleic acid sequence that is the target of the PCR) has to be present in the sample. Many current PCR assays have a high absolute sensitivity, meaning that they can reliably detect the presence of only a few organisms in a sample. Therefore, if organisms are circulating in the blood in adequate numbers at the time they cause clinical signs, it is very likely that a PCR test will detect infection.

A relatively large percentage of circulating blood cells are infected with organisms during the acute phase of infection with A. phagocytophilum, B. gibsoni, B. canis, and E. canis in dogs. Thus, PCR is a sensitive test in a dog acutely infected with these agents.10,22,23 PCR can also detect infection in dogs with very low levels of parasitemia.

However, it is important to be aware that these organisms do not always circulate in adequate numbers in peripheral blood to be detected using PCR. Babesia species and E. canis may circulate in blood in extremely low numbers or intermittently, particularly during chronic infection. Thus, although highly sensitive, PCR results are not positive in all actively infected patients.7,10,24 Performing serologic testing in addition to PCR and conducting tests on additional blood samples using PCR and serology can be important to facilitate a diagnosis of infection.

Some organisms never circulate in peripheral blood in high numbers. For example, Rickettsia rickettsii causes acute illness in dogs. Being endotheliotropic, these organisms circulate in relatively low numbers in peripheral blood at the time clinical signs are occurring. Although PCR can be very useful in confirming acute infection, PCR is not as sensitive as demonstrating a fourfold change in serologic titer.25

In dogs, Bartonella species circulate in blood in very low numbers. Furthermore, up to 50% of patients may be seronegative despite the presence of infection.7 To document infection with Bartonella species, sterilely collected samples of blood and other specimens can be cultured in enrichment media before performing PCR to increase sensitivity.7

Borrelia burgdorferi also does not circulate in peripheral blood in high numbers. Furthermore, clinical signs of Lyme borreliosis do not occur in experimentally infected dogs until two to six months after tick exposure.26 Consequently, serologic testing rather than PCR is routinely used to document exposure to B. burgdorferi.

What is the significance of a positive result?

PCR. Assuming appropriate laboratory controls are in place, a positive PCR result indicates active infection or the presence of circulating dead organisms. PCR assay results can be positive at the level of genus or species, depending on the specific DNA sequence that the primers target. Determining species may have implications with regard to treatment and prognosis, among other factors, so speciation of a positive result for a genus is important.

Serology. A positive serologic test result may indicate exposure to or active infection with a given organism or an antigenically related organism. Considering the epidemiology and pathophysiology of the organism can help determine the clinical significance of a positive result.

For example, a positive titer to B. burgdorferi in a Lyme disease-endemic area may or may not be evidence that infection is causing clinical signs. Anaplasma phagocytophilum and R. rickettsii typically cause acute disease in dogs. Persistent infection with A. phagocytophilum has been documented in experimentally infected dogs.27 Chronic infection has not been documented in naturally infected dogs, but it may occur.8,22 Chronic infection with R. rickettsii has not been documented in experimentally or naturally infected dogs, but chronic disease has been associated with exposure to another spotted fever group (SGF) Rickettsia, R. massiliae.28 Previous infection with either A. phagocytophilum or R. rickettsii may result in long-lived antibody titers.22,29

The clinical relevance of these observations is that if serologic test results are positive for one of these agents and clinical signs have been present for many months, coinfection with other vector-borne agents, the presence of cross-reacting antibodies, or the presence of other underlying disease should be considered as additional explanations for the clinical signs. This is particularly true if infection cannot be verified by PCR or there is inadequate response to treatment. In contrast, a positive titer to Babesia or Bartonella species or E. canis is more likely to be clinically significant in a patient with or without clinical signs because longstanding, subclinical infection is common in dogs naturally infected with these agents.7,10,12

Serologic cross-reactivity among species within a genus and among genera also affects the interpretation of a positive serologic test. For example, there is extensive serologic cross-reactivity among SFG Rickettsia. Rickettsia rickettsii is the organism used in most serologic testing for SFG Rickettsia in the United States. However, there are other species of SFG Rickettsia that infect people in the United States. It is likely that these organisms also infect dogs and induce disease.28,30 Furthermore, exposure to nonpathogenic SFG Rickettsia, some of which are common endosymbionts in ticks, may be a common cause of positive titers, particularly low and persistent titers, to R. rickettsii in dogs.31 Serologic cross-reactivity with SFG Rickettsia also appears to occur in dogs infected with Bartonella henselae.32

Cross-reactivity also occurs among other genera and species and must be considered when interpreting any positive serologic test.

What is the significance of a negative result?

PCR. If organisms circulate only transiently during a particular phase of infection or in low numbers in peripheral blood, even the most sensitive PCR test may not indicate that a dog is infected with a particular agent (see above "Is the organism in the sample? The clinical sensitivity of PCR" above). PCR results may also be negative because treatment has reduced the numbers of circulating organisms.

If the infecting species' DNA is not targeted by the assays' primers, PCR test results may also be negative. This can be a problem when PCR assays target a genus. All species within a genus may not necessarily be detected by the assay.

For example, B. conradae was discovered to be a cause of canine IMHA in southern California in the early 1990s.33 However, commercial diagnostic tests targeting this species only recently became available. Since then, reemergence of this organism as a cause of IMHA in southern California dogs has been described.7 It is important to verify with a laboratory that a species of interest for a particular patient is targeted by a given PCR before assuming infection is ruled out based on a negative PCR test result.

Serology. Like PCR tests, serologic testing on a single sample may produce negative results in the face of infection, if the organism causes clinical signs in the acute phase of disease, before antibodies are formed. For example, serology might not indicate exposure in dogs acutely infected with agents such as R. rickettsii, E. canis, Babesia species, and A. phagocytophilum, because clinical signs can sometimes develop before seroconversion during infection with these agents.10,12,22,29

Furthermore, for certain organisms such Bartonella and Babesia species, antibodies may not be detectable in infected patients even after seroconversion theoretically should have occurred.7,34 The absence of antibody has been theorized to be due to young age or immunosuppression in some patients, or, for Bartonella, antigenic differences may exist between organisms grown in vitro for immunofluorescence assay (IFA) testing and organisms infecting a patient.7,34 Importantly, up to 50% of dogs infected with Bartonella species may be seronegative.7

Sensitivity can also differ among types of serologic assays. Infrequently, in some dogs actively infected with E. canis, antibodies can be detected by using IFAs but not enzyme-linked immunosorbent assays. This may be related to differences in the nature of antigens used in the respective assays.35


Serologic and PCR panels can facilitate the diagnosis of vector-borne disease in dogs. Clinicians should consider the epidemiology and pathophysiology of each agent to guide testing.

Testing with both serology and PCR and testing more than one sample is often necessary to confirm the presence of infection. Testing using both serology and PCR in parallel can improve diagnostic sensitivity and is recommended.3,4 If only one modality (PCR or serology) is used initially, and the results are negative, stored samples can be accessed to test with an alternative methodology if both serum and EDTA samples are obtained at initial presentation. Furthermore, convalescent serologic testing can be considered to confirm acute infection.

Repeating PCR on initial or additional samples or using specialized techniques such as enrichment before PCR testing in the case of bartonellosis7 may help document infection in cases in which low numbers of circulating organisms is a concern.

Linda Kidd, DVM, PhD, DACVIM

College of Veterinary Medicine

Western University of Health Sciences

309 E. Second St.

Pomona, CA 91766

Shadie Ghaffari, DVM

VCA West Los Angeles Animal Hospital

1900 S. Sepulveda Blvd.

Los Angeles, CA 90025

Related Videos
merck leptospirosis panel
merck leptospirosis panel
merck leptospirosis panel
dvm360 Live! with Dr. Adam Christman
Vet Perspective parasitology discussion
Vet Perspective parasitology discussion
Vet Perspective
© 2023 MJH Life Sciences

All rights reserved.